WO2012068344A2 - Regulation of cathepsin l by its transcription factor dendrin - Google Patents

Abstract

Compositions modulating the expression, function or activity of cathepsin L. (CatL.) are effective as protective against kidney disease or disorders and in the treatment of patients with kidney disease or disorders. The agents can target various pathways or molecules involved in CatL expression, activity or function.

Description

REGULATION OF CATHEPSIN L BY ITS TRANSCRIPTION FACTOR DENDRIN

STATEMENT OF FEDERAL FUNDING

[001] This invention was made with federal funds by the National Institutes of Health, grant numbers R01 DK73495-01 and R01 D 64787. The U.S. government may have certain rights.

CROSS REFERENCE TO RELATED APPLICATIONS

[002] This application claims priority to U.S. provisional application No.: 61/414,782, filed November 17, 2010, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[003] Embodiments of the invention comprise compositions for modulating cathepsin L transcription and molecules regulated or associated with cathepsin L expression levels. Methods of treating kidney diseases or disorders are provided.

BACKGROUND

[004] Several hundred million people worldwide - about 1 in 15 adults- have some form of kidney damage, and every year, millions die prematurely of cardiovascular or renal

complications linked to chronic kidney disease (CKD). CKD often begins with urinary protein loss (proteinuria), an early sign of kidney injury that constitutes a risk factor for further progressive destruction of the kidney, a process that can last from weeks to several years.

Proteinuria stems from injury to podocytes, terminally differentiated cells that reside in the kidney glomeruli, the location of the renal filtration barrier. The function of podocytes is primarily based on their intricate structure, which consists of a cell body, major processes, and interdigitating foot processes (FPs), which are actin-driven membrane extensions. At the interface of adjacent FPs, a specialized intercellular junction known as the slit diaphragm (SD) is formed. Nephrin, a key structural and signaling transmembrane protein of the SD, recruits proteins such as podocin, CD2AP, and Nek to the podocyte membrane. One of the earliest events in the development of podocyte dysfunction is the disruption of FPs (referred to as FP effacement), which causes proteinuria, the first clinical sign in CKD. Once podocytes are injured, there are 2 possible outcomes: (a) proteinuria resolves and podocyte structure normalizes, or (b) renal function declines, resulting in progressive glomerular and consecutive tubular destruction. The latter outcome is characterized by an increased occurrence of podocyte apoptosis, a recognized event commonly observed during renal disease progression. Generally, the reason for heightened susceptibility of podocytes during proteinuria is a phenomenon that is not well understood.

SUMMARY

[005] This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

[006] Embodiments of the invention are directed to compositions modulating the expression, function or activity of cathepsin L (CatL). These agents are effective as protective against kidney disease or disorders and in the treatment of patients with kidney disease or disorders. The agents can target various pathways or molecules involved in CatL expression, activity or function. These include agents which modulate CD2-associated protein (CD2AP), Transforming Growth Factor-beta 1 (TGF-βΙ ), dendrin and the like.

[007] Methods of treatment of kidney diseases or disorders comprise administering to a patient in need thereof, a therapeutically effective amount of one or more agents that modulate CatL expression, function or activity. The agents are effective in the preventative or protection against kidney diseases or disorders in at risk patients.

[008] Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[009] Figures 1 A- 1 H show that the high level of TGF-β Ι induces expression of CatL. Figure 1 A is a graph showing the levels of Tgfbl, Ctsl, and control gene in glomeruli of TGF-β Ι Tg and WT animals. Figure I B is a scan of a photograph of H&E-stained sections of the renal cortex, showing increased CatL staining in TGF-β Ι Tg mice. Kidneys of WT and TGF-βΙ Tg mice were stained for CatL using anti-cytosolic CatL antibody, and nuclei were stained using DAPI. Figure 1 C is a graph showing CatL staining levels in sections from Figure I B. ***p < 0.001. Figure I D is a scan of a photograph showing H&E-stained sections showing dendrin localization in the nucleus of the podocytes in TGF-β Ι Tg animals (arrows). Kidneys of WT and TGF-βΙ mice were stained for dendrin using anti-dendrin antibody. Figure I E is a graph showing dendrin staining levels in sections from Figure I D. ***P < 0.001. Figures I F and 1 G are scans of photographs of H&E-stained sections showing increased CatL staining in glomeruli of 3- week-old Cd2ap^~/~ mice (arrows). Kidneys of WT and Cd2ap^~/~ mice were stained for CatL using anti-cytosolic CatL antibody, and nuclei were stained using DAPI. Figure 1 H is a scan of a photograph showing dendrin localization in glomeruli of WT and Cd2ap^~'^~ mice at different stages of development. WT 1 was used to stain podocyte nuclei (red). In the young glomerulus, dendrin (green) was localized at the membrane (ribbon staining pattern), without any

colocalization with WT1 . At 4 weeks, dendrin staining was still localized to the plasma membrane in WT mice, but was predominantly found in the nucleus co-staining with WT1 in Cd2ap^"/" mice. Scale bars: 20 μιη (Figures IB, I D, I F, and 1G); 30 μιη (Figure 1H).

[010] Figures 2A-2F shows that prolonged loss of CD2AP leads to expression of cytosolic CatL. Figure 2A show the actin cytoskeleton and FA organization in fully differentiated low- and high-7¾ ¾7 Cd2ap^~'^~ cells. Note the loss of well-defined stress fibers and dramatic increase in number of transverse. arcs in high-7g/¾7 Cd2ap^~/~ cells. FAs and F-actin were visualized with anti-paxillin antibodies and rhodamine-phalloidin, respectively. Figure I B Low-Tgfbl Cd2ap^~/~ podocytes could be transformed into high-Tgfbl Cd2ap^~/~ podocytes by high TGF-βΙ levels. WT and low-7g/¾7 Cd2ap^~'^~ passage cells were treated with 5 ng/ml TGF-βΙ in the media for 24 hours. Actin cytoskeleton was monitored by staining cells with rhodamine-phalloidin, and dendrin localization was monitored using anti-dendrin antibody (green). Figures 2C-2E mRNA levels for Tgfol (Figure 2C) and Ctsl (Figures 2D and E), determined by RT-PCR in podocytes. When indicated, cells were treated with 5 ng/ml TGF-β Ι for 24 hours or with 50 μΜ LPS for 24 hours. Podocytes expressing Actn4 mutant are also shown in Figure 2E. Figure 2F is a blot showing the subcellular fractionation of low- and high-7g/¾/ Cd2ap^~'^~ podocytes in isotonic sucrose. Total proteins from soluble (S) and particulate (P) fractions were probed for CatL and for the lysosomal protein markers Lamp-2 and mannosidase (Manno), and then analyzed by Western blotting. GAPDH was used as a loading control. Strong cytosolic CatL induction (asterisk) was observed in both soluble and pellet fractions of high- g Z>7 Cd2ap-/- podocytes. Scale bars: 20 ^'μιη.

[0111 Figures 3A-3G show that cytosolic CatL activity regulates actin cytoskeleton in

Cd2ap^{~ ~} cells. Figure 3A is a graph showing Ctsl mRNA levels, determined by RT-PCR, in high-7g/¾7 Cd2ap^~'^~ podocytes infected with different shRNA constructs (C2, C5, C6) downregulating endogenous CatL. Con, high-7¾/Z>7 Cd2ap^~/~ podocytes not infected with lentiviruses; Scr, cells infected with lentiviruses expressing a scrambled oligonucleotide. Figure 3B is a graph showing CatL levels in high-7¾/¾7 Cd2ap^~'^~ podocytes infected with lentiviruses expressing different shRNA constructs to downregulate CatL at relative volumes as indicated. Figure 3C is a graph showing the time course of CatL activity in high-7g/¾7 Cd2ap^~/~ podocytes and in high-7g/¾7 Cd2ap^"/~ podocytes infected with lentiviruses to downregulate CatL in the absence and presence of CA074 at neutral pH. Figure 3D is a graph showing the time course of CatL activity in the presence of CA074 in high-7g/¾7 Cd2ap^~/~ podocytes, high-Tgfbl Cd2ap-/- podocytes infected with lentiviruses to downregulate CatL, and WT podocytes at neutral pH. Figures 3E and 3F are blots showing the protein levels in WT cells, high-7g ¾7 Cd2ap^~'^~ podocytes, and high- 7g/¾7 Cd2ap^~/" podocytes treated with E64 or infected with lentiviruses to downregulate CatL (shR A-C6). Dyn, dynamin; Synpo, synaptopodin. GAPDH was used as a loading control. Figure 3G is a graph showing the quantitation of protein levels from Western blots in Figures 3E and 3F.

[012] Figures 4A and 4B show number of FAs within WT podocytes and high-7g ¾7

Cd2ap^~~/~ podocytes with CatL downregulation by diverse treatments. Figure 4A is a scan of a photograph showing the organization of the actin cytoskeleton and FAs in podocytes in which CatL was downregulated. FAs and F-actin were visualized with anti-paxillin and rhodamine- phalloidin, respectively. Figure 4B is a graph showing the quantitation representing

measurements of >50 cells shown in Figure 4A. Data are mean ± standard deviation. ***P < 0.001. Scale bars: 20 μιη.

[013] Figures 5A- 51 show that prolonged CD2AP loss leads to dendrin translocation into the nucleus. Figure 5A is a graph showing Ddn mRNA levels, determined by RT-PCR, in high- Tgft>l Cd2ap^~/" podocytes infected with different shRNA constructs (D2-D4) downregulating endogenous dendrin. Con, uninfected high- Tgfbl Cd2ap~^/~~ podocytes; Scr, high- TgfbJ Cd2ap^~ '^~ podocytes infected with lentiviruses expressing a scrambled oligonucleotide. Figure 2B is a blot showing dendrin levels in high- Tgfbl Cd2ap^{~ ~} podocytes infected with lentiviruses expressing the 2 most efficient shRNA constructs, D3 and D4. Figure 5C is a scan of a photograph showing the organization of the actin cytoskeleton and FAs in high- Tgjbl Cd2ap^~/~ podocytes with dendrin downregulation. FAs and F-actin were visualized with anti-paxillin and rhodamine-phalloidin, respectively. Figure 5D is a graph showing the number of FAs within WT and high- Tgfbl Cd2ap^{~ ~} podocytes with dendrin downregulation. Data (mean ± standard deviation) represent measurements of >50 cells in Figure 5C. ***p < 0.001 . Figure 5E is a graph showing Ctsl mRNA levels, determined by RT-PCR, in high- Tgfbl Cd2ap^~/~ podocytes with dendrin downregulation. Figure 5F is a blot showing the protein levels of CatL, dynamin, synaptopodin, RhoA, and GAPDH in high- Tgfbl Cd2ap^~/~ podocytes and cells infected with lentiviruses. Figure 5G is a graph showing the time course of CatL activity in high- Tgfbl Cd2ap^{~ ~} podocytes, high- Tgfbl Cd2ap^{~ ~} podocytes infected with lentiviruses, and low-7¾ Z>7 Cd2ap^~/~ podocytes at neutral pH. Figure 5H is a graph showing the loss of CD2AP rendered podocytes hypersensitive to proapoptotic signals, as shown by specific enrichment of mono- and oligonucleosomes released into the cytoplasm of WT and high- Tgfbl Cd2ap^~/" podocytes treated with different apoptosis inducers. Stauro, staurosporine; Actino D, actinomycin D; Angio II, angiotensin II. Figure 51 is a graph showing that TGF-p i-induced apoptosis was reversed by downregulation of CatL or dendrin in high- Tgfbl Cd2ap^~/~ podocytes. Scale bars: 20 μιη.

[014] Figures 6A-6E show that dendrin is a transcription factor of CatL. Figures 6A and 6B are graphs showing that nuclear dendrin induced transcription from the CatL promoter, but not from the CatB promoter. HEK 293 cells were cotransfected with pSEAP reporter vector containing the promoter of interest, dendrin, and Metridia luciferase to normalize for transfection efficiency. Mutant dendrin lacking its nuclear localization signal (dNLS) was ineffective.

Promoter activity and response to dendrin depended on a region between bp -1 ,215 and bp -339, as revealed by partial deletion promoter constructs. ***p < 0.001 . Figure 6C is a schematic representation showing an embodiment of a strategy used to identify the dendrin binding site within the CatL promoter. The promoter fragment between bp -1 ,21 5 and -339 was divided into 4 parts. EMSA with these unlabeled DNA fragments, visualized by SYBR green, revealed dendrin binding to fragment F, which was then further divided into overlapping fragments to fine-map the dendrin binding site. Figure 6D is a blot showing EMSA demonstrating specific dendrin binding to one of the biotin-labeled 60-bp oligonucleotides that were completely abolished by a 200-fold excess of unlabeled oligonucleotide. Figure 6E is a blot showing the 60- bp oligonucleotide that exhibited dendrin binding was divided into 3 overlapping 24-bp oligonucleotides to further narrow the dendrin binding site. Mutation of 3 nucleotides in the 5 ' region, but not in the central or 3' region, of the 24-bp oligonucleotide 4- 1 completely abolished dendrin binding, as demonstrated by the inability of a 200-fold excess of unlabeled mutated oligonucleotide to compete with the labeled WT oligonucleotide.

[015] Figures 7A-7I show that cytosolic CatL proteolytically processes CD2AP in podocytes. Figure 7A is an immunoblot showing cleaved CD2AP fragments tagged with N- terminal GFP. At pH 7.0, CD2AP was cleaved into a stable 55-kDa fragment (squares), as detected with anti-GFP antibody. The same fragment was detected with the N-CD2AP antiserum. This antiserum also detected a weak band corresponding to a 44-kDa fragment (triangle). Figure 7B is a schematic representation showing the match of cleavage fragments with predicted CatL cleavage site QPLGS (SEQ ID NO: 22). Figure 7C is a blot showing that deletion of the CatL cleavage site LSAAE (SEQ ID NO: 23) protected CD2AP from limited proteolysis into p32 (circle). Figure 7D is a schematic representation showing the match of p32 with predicted CatL cleavage site LSAAE (SEQ ID NO: 23). Figure 7E is a blot showing the CatL cleaved CD2AP-FLAG, yielding p32 (circle), detected by anti-C-CD2AP. Figure 7F is a blot showing WT Ctsl cleaved CD2AP in HEK 293 cells. Cytosolic CatL (CatL M l ) was sufficient to cleave CD2AP, yielding p32 (circle). These cleavage reactions were prevented by incubation of the cells with E64. Figure 7G are blots showing the co-immunoprecipitation of nephrin, synaptopodin, and dendrin from HEK 293 cells transfected with full-length CD2AP, N- terminal CD2AP, and p32. Figure 7H is a schematic representation showing the structural domains of CD2AP, together with major CatL cleavage sites, predicted sizes of resulting fragments from CatL digestion, and recognition sites of the antibodies used. SH3, src homology domain 3; PR, proline-rich motif; CC, coiled-coil domain. Figure 71 is a scan of photographs showing the immunofluorescent staining of kidney biopsies from patients with MCD and FSGS. N-terminal CD2AP was reduced only in progressive disease (i.e., FSGS). Scale bar: 30 μιη.

[016] Figures 8A and 8B are a schematic representation showing that the signaling between nucleus and cytoplasm of healthy(Figure 8A) and injured (Figure 8B) podocyte and SDs are mediated by CatL.

[017] Figures 9A-91 show that loss of CD2AP induces expression of cytosolic CatL. Figure 9A are scans of photographs showing dendrin localization in the cultured podocytes (green). Figure 9B are bar graphs depicting mRNA levels for CatL determined by RT-PCR in podocytes treated with TGF-βΙ for 24 hours. Figure 9C is a graph showing that CatL mRNA levels are upregulated only in CD2AP^{" "(H,gh TGFp)} podocytes. Bar graphs depict CatL mRNA levels in wild type (WT), CD2AP^{~ ~(High TGFp)} podocytes, podocytes in which synaptopodin (sypKD) and dynamin (DynKD) were downregulated using lentivirus. Figure 9D shows that mRNA for CatL has seven AUG codons. Translation initiation from the first AUG site yields pre-pro-CatL with a signal peptide that targets the protein to the endoplasmic reticulum (ER), and subsequently to the lysosome. Pre-pro-CatL is processed to become pro-CatL (~39 kD), which can either be delivered into the lysosomes, or can be secreted into the extracellular space. Alternatively, translation initiation from six downstream AUG sites results in short form of CatL (-32-34 kD) that is devoid of the signal peptide and therefore localizes in the cytoplasm, from which it can translocate into the nucleus by diffusion. Figure 9E is a blot showing the nuclear fraction from CD2AP-^/-^{(Lovv TGFp)} and CD2AP^{" "(High TGFp)} cells probed with anti-CatL antibody confirms the presence of cytosolic CatL in the nucleus. Figure 9F is a blot showing the total protein from wild type (Con) and CD2AP^{" "(H}'^{8h TGFp)} podocytes shows downregulation of dynamin, synaptopodin (synpo) and RhoA. Figures 9G-9I are graphs showing that the downregulation of dynamin, synaptopodin and RhoA are not transcriptionally regulated. Bar graphs depicts mRNA levels using RT-PCR for endogenous dynamin 2 (Figure 9G), RhoA (Figure 9H) and

synaptopodin (Figure 91) in wild type and CD2AP^{" "(High TGFp)} podocytes.

[018] Figures 10A- 10H show that cytosolic CatL regulated focal adhesion turnover in wild type podocytes. Figure 10A are bar graphs depicting the levels of CatL mRNA determined by RT-PCR in wild type podocytes infected with different shRNA constructs downregulating endogenous CatL (C2, C5, C6). Con, podocytes not infected with lentiviruses. Scr, cells infected with lentiviruses expressing a scrambled oligonucleotide. Figure 10B is a blot showing the CatL levels in podocytes infected with lentiviruses expressing different shRNA constructs to downregulate CatL. Notice that majority of CatL is the lysosomal form (25 kD). Figure I OC is a blot showing the protein levels in podocytes infected with lentiviruses expressing different shRNA constructs to downregulate CatL. Figure 10D is a blot showing the protein levels in podocytes treated with CatL inhibitor, E64 for 48 hours. Figure 10E are scans of photographs showing the organization of the actin cytoskeleton and FAs in podocytes in which CatL has been downregulated. FAs and F-actin were visualized with anti-paxillin and rhodamine-phalloidin, respectively. Figure 10F are bar graphs depicting number of FAs within the wild type podocytes and podocytes in which CatL was downregulated. Data represent measurements of >50 cells shown in Figure 10E. Figure 10G is a graph showing that the downregulation of cytosolic CatL in podocytes shifts the size of FAs toward more mature and super mature forms. Data represent measurements of >50 cells shown in Figure 10E. Figure 1 OH is a schematic diagram showing the possible role of dynamin, synaptopodin and R oA in regulating maturation of FAs in podocytes. This study indicates that cytosolic CatL specifically targets regulatory proteins involved in regulating turnover of the FAs. Thus, downregulation of dynamin and synaptopodin (and thus indirectly RhoA) leads to decrease in number and size of FAs, whereas loss of CatL leads to opposite effects.

[019] Figures 1 1 A-l 1 C show that the downregulation of CatL or dendrin cannot rescue hypersensitivity to different pro-apoptotic signals in CD2AP^{' ' H}'^{sh TGFp)} podocytes. Bar graphs representing the specific enrichment of mono and oligonucleosomes released into the cytoplasm of CD2AP^{" (H}'^{sh TGFp)} podocytes treated with different apoptotic inducers as indicated in the figure (Angio II: Angiotensin II; Stauro: Staurosporine; Actino D: Actinomycin D). CD2AP^{" ~} (Hⁱgh TGF ) _pocj_OCytes were treated with shRNA to downregulate CatL (C2 and C6), or dendrin (D3 and D4). CD2AP-^{/"(High TGFp)} podocytes were also treated with 2 doses of CatL inhibitor E64 (20 μΜ each) for 24 hours prior to starting the assay.

[020] Figure 12 is a blot showing the specificities of N- and C-terminal CD2AP antibodies detected by the immunoblots of HE 293 cells, which were transfected with full length, N- and C-terminal CD2AP (CON: untransfected).

[021] Figures 13A-13H show that CatL cleaves CD2AP in vivo. Figure 13A is an immunoblot for CD2AP in cultured podocytes that were exposed to lipopolysaccharides (LPS) for 24 h (CON: untreated). Figure 13B is an immunoblot of soluble (Glom-S) and pelleted (Glom-P) fractions of the glomeruli from wild type (WT) mice (Dyn: Dynamin, Synpo:

Synaptopodin). Figure 13C are scans of photographs showing the ilmmunofluorescent labeling of WT and cathepsin L knockout (CatL KO) mouse glomeruli against anti-N-CD2AP before and after LPS. Figure 13D is a graph showing the quantification of the staining intensity in Figure 13C using Image J software ( *P<0.05). Figure 13E is a graph showing the CatL activity in soluble fractions from isolated glomeruli of control (untreated) and LPS-treated mice. Figure 13F are scans of photographs showing that dendrin staining is unaltered in LPS treated WT mice with an exclusive extra-nuclear location. Figure 13G are blots showing that urine albumin analysis reveals that both WT and Dendrin knockout ( O) mice develop LPS-mediated proteinuria. Lanes were loaded with urine samples taken at different time points following LPS injection (1 : t=0, 2: 12 h, 3: 24 h, 4: 48 h, 5: 72 h, 6:96 h, 7: 7 days). Figure 13H are graphs showing the Effect of LPS on TGF-β Ι (middle panel) and CatL levels (bottom panel) in wild type (WT) podocytes and podocytes in which dendrin was downregulated (DenKD) using lentivirus (top panel). LPS induces upregulation of CatL in dendrin-independent manner.

DETAILED DESCRIPTION

[022] Embodiments of the present invention relate to compositions which regulate the cathepsin L (CatL) expression, function or activity. In particular embodiments, agents which modulate CatL expression, function or activity also encompass molecules that are involved in the regulation of CatL or any of the molecules associated with pathways leading to kidney disease, such as for example, TGF-βΙ , CD2-associated protein (CD2AP) or dendrin.

[023] Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details,

relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

[024] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene _ products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes or nucleic acid sequences are human.

[025] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Definitions

[026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[027] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, such that the description includes instances where the circumstance occurs and instances where it does not.

[028] The terms "determining", "measuring", "evaluating", "detecting", "assessing" and "assaying" are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute. "Assessing the presence of includes determining the amount of something present, as well as determining whether it is present or absent.

[029] As used herein "proteinuria" refers to any amount of protein passing through a podocyte that has suffered podocyte damage or through a podocyte mediated barrier that normally would not allow for any protein passage. In an in vivo system the term "proteinuria" refers to the presence of excessive amounts of serum protein in the urine. Proteinuria is a characteristic symptom of either renal (kidney), urinary, pancreatic distress, nephrotic syndromes (i.e., proteinuria larger than 3.5 grams per day), eclampsia, toxic lesions of kidneys, and it is frequently a symptom of diabetes mellitus. With severe proteinuria general hypoproteinemia can develop and it results in diminished oncotic pressure (ascites, edema, hydrothorax).

[030] As used herein, the terms "podocyte disease(s)" and "podocyte disorder(s)" are interchangeable and mean any disease, disorder, syndrome, anomaly, pathology, or abnormal condition of the podocytes or of the structure or function of their constituent parts.

[031] As used herein "a patient in need thereof refers to any patient that is affected with a disorder characterized by proteinuria. In one aspect of the invention "a patient in need thereof refers to any patient that may have, or is at risk of having a disorder characterized by proteinuria.

[032] As used herein, the term "agent" is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition. The term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the invention at any stage during clinical trials, during pre-trial testing, or following FDA-approval.

[033] As used herein the phrase "diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[034] As used herein the phrase "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The term "detecting" may also optionally encompass any of the above. Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease. It should be noted that a "biological sample obtained from the subject" may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.

[035] "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. "Treatment" may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy. Accordingly, "treating" or "treatment" of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human or other mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[036] As defined herein, a "therapeutically effective" amount of a compound (i.e., an effective dosage) means an amount sufficient to produce a therapeutically (e.g., clinically) desirable result. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compounds of the invention can include a single treatment or a series of treatments. A. "prophylactically effective amount" may refer to the amount of an agent sufficient to prevent the recurrence or spread of kidney diseases or disorders, particularly proteinuria, or the occurrence of such in a patient, including but not limited to those predisposed to kidney disease, for example those genetically predisposed to kidney disease or previously exposed to environmental factors, such as for example, alcohol or infectious organisms such as hepatitis virus. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease. Further, a prophylactically effective amount with respect to an agent of the invention means that amount of agent alone, or in combination with other agents, that provides a prophylactic benefit in the prevention of disease.

[037] The term "sample" is meant to be interpreted in its broadest sense. A "sample" refers to a biological sample, such as, for example; one or more cells, tissues, or fluids (including, without limitation, plasma, serum, whole blood, cerebrospinal fluid, lymph, tears, urine, saliva, milk, pus, and tissue exudates and secretions) isolated from an individual or from cell culture constituents, as well as samples obtained from, for example, a laboratory procedure. A biological sample may comprise chromosomes isolated from cells (e.g., a spread of metaphase chromosomes), organelles or membranes isolated from cells, whole cells or tissues, nucleic acid such as genomic DNA in solution or bound to a solid support such as for Southern analysis, RNA in solution or bound to a solid support such as for Northern analysis, cDNA in solution or bound to a solid support, oligonucleotides in solution or bound to a solid support, polypeptides or peptides in solution or bound to a solid support, a tissue, a tissue print and the like.

[038] Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject. Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.

[039] The phrase "specifically binds to", "is specific for" or "specifically immunoreactive with", when referring to an antibody refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies. For example, an antibody "specifically binds" or "preferentially binds" to a target or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein.

[040] As used herein, the term "oligonucleotide specific for" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene.

[041] As used herein, the terms "oligonucleotide," "siRNA," "siRNA oligonucleotide," and "siRNA's" are used interchangeably throughout the specification and include linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.

Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.

[042] The oligonucleotide may be "chimeric," that is, composed of different regions. In the context of this invention "chimeric" compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties. The desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cel lular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties. The chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above.

[043] The oligonucleotide can be composed of regions that can be linked in "register," that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers. The spacers are intended to constitute a covalent "bridge" between the regions and have in preferred cases a length not exceeding about 100 carbon atoms. The spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophors etc.), being lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.

[044] As used herein, the term "monomers" typically indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphornates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.

[045] In the present context, the terms "nucleobase" covers naturally occurring nucleobases as well as non-naturally occurring nucleobases. It should be clear to the person skilled in the art that various nucleobases which previously have been considered "non-naturally occurring" have subsequently been found in nature. Thus, "nucleobase" includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Illustrative examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N₄,N₄- ethanocytosin, N₆,N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5- fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat. No. 5,432,272. The term "nucleobase" is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleobases are adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.

[046] As used herein, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Romberg and Baker, DNA Replication, 2nd Ed.

(Freeman, San Francisco, 1992).

[047] "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid. Res., 1997, 25(22), 4429- 4443, Toulme, J. J., Nature Biotechnology 19: 17- 18 (2001); Manoharan M., Biochemica et Biophysica Acta 1489: 1 17-139 (1999); Freier S., M., Nucleic Acid Research, 25:4429-4443

(1997) , Uhlman, E., Drug Discovery & Development, 3: 203-213 (2000), Herdewin P.,

Antisense & Nucleic Acid Drug Dev., 10:297-310 (2000)); 2'-0, 3'-C-linked [3.2.0]

bicycloarabinonucleosides (see e.g. N. Christiensen., et al, J. Am. Chem. Soc, 120: 5458-5463

(1998) . Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.

[048] As used herein, the term "gene" means the gene and all currently known variants thereof and any further variants which may be elucidated.

[049] As used herein, "variant" of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

[050] The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type target gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. [051] The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.

[052] As used herein, the term "mRNA" means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which may be elucidated.

[053] By "desired RNA" molecule is meant any foreign RNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include antisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA.

[054] By "antisense RNA" is meant a non-enzymatic RNA molecule that binds to another RNA (target RNA) by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi et al., 1991 Annu. Rev. Biochem. 60, 631 -652).

[055] RNA interference "RNAi" is mediated by double stranded RNA (dsRNA) molecules that have sequence-specific homology to their "target" nucleic acid sequences (Caplen, N. J., et al, Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001)). In certain embodiments of the present invention, the mediators of RNA-dependent gene silencing are 21 -25 nucleotide "small interfering" RNA duplexes (siRNAs). The siRNAs are derived from the processing of dsRNA by an RNase enzyme known as Dicer (Bernstein, E., et al., Nature 409:363-366 (2001 )). siRNA duplex products are recruited into a multi-protein siRNA complex termed RISC (RNA Induced Silencing Complex). Without wishing to be bound by any particular theory, a RISC is then believed to be guided to a target nucleic acid (suitably mRNA), where the siRNA duplex interacts in a sequence-specific way to mediate cleavage in a catalytic fashion (Bernstein, E., et al, Nature 409:363-366 (2001 ); Boutla, A., et al, Curr. Biol 1 1 : 1776- 1780 (2001 )). Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are well known in the art and that will be familiar to the ordinarily skilled artisan. Small interfering RNAs for use in the methods of the present invention suitably comprise between about 0 to about 50 nucleotides (nt). In examples of nonlimiting embodiments, siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.

[056] Selection of appropriate RNAi is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of RNAi that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.

[057] By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, 1988 J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.

[058] By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601 -608). This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art. [059] The term, "complementary" means that two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3'-end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence. Normally, the complementary sequence of the oligonucleotide has at least 80% or 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence. Preferably, alleles or variants thereof can be identified. A BLAST program also can be employed to assess such sequence identity.

[060] The term "complementary sequence" as it refers to a polynucleotide sequence, relates to the base sequence in another nucleic acid molecule by the base-pairing rules. More particularly, the term or like term refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 95% of the nucleotides of the other strand, usually at least about 98%, and more preferably from about 99% to about 100%. Complementary polynucleotide sequences can be identified by a variety of approaches including use of well-known computer algorithms and software, for example the BLAST program.

[061 ] The term "stability" in reference to duplex or triplex formation generally designates how tightly an antisense oligonucleotide binds to its intended target sequence; more particularly, "stability" designates the free energy of formation of the duplex or triplex under physiological conditions. Melting temperature under a standard set of conditions, e.g., as described below, is a convenient measure of duplex and/or triplex stability. Preferably, oligonucleotides of the invention are selected that have melting temperatures of at least 45 °C. when measured in 100 mM NaCl, 0.1 mM EDTA and 10 mM phosphate buffer aqueous solution, pH 7.0 at a strand concentration of both the oligonucleotide and the target nucleic acid of 1 .5 μΜ. Thus, when used under physiological conditions, duplex or triplex formation will be substantially favored over the state in which the antigen and its target are dissociated. It is understood that a stable duplex or triplex may in some embodiments include mismatches between base pairs and/or among base triplets in the case of triplexes. Preferably, modified oligonucleotides, e.g.

comprising LNA units, of the invention form perfectly matched duplexes and/or triplexes with their target nucleic acids.

[062] In accordance with the present invention, there may be employed conventional molecular biology, microbiology, recombinant DNA, immunology, cell biology and other related techniques within the skill of the art. See, e.g., Sambrook et al., (2001 ) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al., eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al., eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al., eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc. : Hoboken, NJ; Coico et al., eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.:

Hoboken, NJ; Coligan et al., eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc. : Hoboken, NJ; Enna et al., eds. (2005) Current Protocols in Pharmacology John Wiley and Sons, Inc.: Hoboken, NJ; Hames et al., eds. ( 1999) Protein Expression: A Practical Approach. Oxford University Press: Oxford; Freshney (2000) Culture of Animal Cells: A Manual of Basic Technique. 4th ed. Wiley-Liss; among others. The Current Protocols listed above are updated several times every year.

Compositions

[063] The compositions that embodied comprise agents which modulate cathepsin L or any molecules associated with the pathways leading to kidney disease. In general embodiments of the invention are directed to compositions modulating the expression, function or activity of cathepsin L (CatL). These compositions and methods are applicable to all cells or tissues in vivo whereby cathepsin L, dendrin, CD2AP etc. are found and as such are not to be construed or limited to the kidneys. In particular embodiments, these agents are effective as protective against kidney disease or disorders and in the treatment of patients with kidney disease or disorders. The agents can target various pathways or molecules involved in CatL expression, activity or function. These include agents which modulate CD2-associated protein (CD2AP), Transforming Growth Factor-beta 1 (TGF-βΙ ), dendrin and the like. For example, as shown in the examples section which follows, CD2-associated protein (CD2AP) leads to progressive renal failure in humans. CD2AP regulates the TGF-βΙ -dependent translocation of dendrin from the slit diaphragms (SDs) to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-βΙ . These examples, identify CD2AP as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.

[064] As further described in the examples section in detail, a cytosolic variant of cathepsin L (CatL) is involved in the pathogenesis of human glomerular diseases and the LPS mouse model of proteinuria. The data here also show that cytosolic CatL is also involved in models of chronic progressive glomerular disease such as CD2-associated protein (CD2AP) deficient or TGF-βΙ transgenic mice. Glomerular CatL expression in CD2AP^{" "} mice was normal by immunohistochemistry at the age of 1 week but increased at 3 weeks, i.e. the onset of glomerulosclerosis. Similar to the in vivo situation, conditionally immortalized podocytes from CD2AP^"7" mice initially expressed low cytosolic CatL levels and normal actin cytoskeleton, but with increasing passage number, these cells undergo a phenotypic change that is accompanied by the induction of high cytosolic CatL levels and cleavage of dynamin and synaptopodin along with rearrangement of the actin cytoskeleton. This phenotype could be rescued by treatment with CatL inhibitors or CatL shRNA. In both, in vivo and in vitro CD2AP^"'^" podocytes nuclear relocation of dendrin was observed. Dendrin was found to be a CD2AP-binding protein that promotes apoptosis upon nuclear import. Reporter assays in HE cells demonstrated strong upregulation of CatL but not cathepsin B (CatB) promoter activity upon co-transfection with wild type dendrin but not with a dendrin construct devoid of the nuclear import signal.

Knocking down of dendrin rescued the actin phenotype of late passage CD2AP^"A podocytes and restored dynamin and synaptopodin levels. CD2AP^{_ "} podocytes exhibited an increased susceptibility to TGF-βΙ induced apoptosis, which was also rescued by dendrin shRNA, CatL shRNA or CatL inhibitor treatment. In line with the finding that TGF-βΙ itself induces nuclear relocation of dendrin, CatL expression was also increased in TGF-β Ι transgenic mice where it correlated with glomerular disease progression. Dendrin is a transcriptional activator of CatL. CatL not only induces acute podocyte foot process (FP) effacement, but enhances podocyte susceptibility to apoptosis and is thus involved in glomerular disease progression.

[065] In one embodiment, a pharmaceutical composition comprises a therapeutically effective amount of an agent which modulates cathepsin L (CatL) transcription in vitro or in vivo. The inhibition or up-regulation of CatL can be measured by the levels of TGF-β Ι , dynamin, synaptopodin and/or RhoA expression in podocytes compared to a normal baseline control.

[066] In one embodiment, the agent modulates expression of CD2AP in vitro or in vivo. In another embodiment, the agent modulates CD2AP-mediated cellular signaling in vitro or in vivo. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. The results described in detail in the examples section which follow have shown that CD2AP regulates the TGF-pi-dependent translocation of dendrin from the slit diaphragm (SD) to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-βΙ . CD2AP acts as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression.

[067] In another preferred embodiment, the pharmaceutical composition comprises an agent which modulates expression of TGF-βΙ in vitro or in vivo.

[068] In another preferred embodiment, the agent modulates dendrin expression, function or activity and/or nuclear localization of dendrin in vitro or in vivo.

[069] In another preferred embodiment, the agent modulates binding of dendrin to a CatL promoter in vitro or in vivo. For example, the agent can be an antibody which prevents binding of dendrin to the promoter; the agent can be a mimetic, an interference RNA , and the like. In some aspects, the agent modulates binding of dendrin to cathepsin L, cathepsin L transcription factors, or cathepsin L transcription or transcription regulatory domains in vitro or in vivo. In another aspect, the agent binds to a CatL promoter and modulates transcription of CatL such that the agent affects cytosolic Cat L expression and/or cytosolic CatL localization.

[070] In preferred embodiments, the agent comprises: a small molecule, amino acid, oligonucleotide, polynucleotide, peptide, polypeptide, amino acid analogs, nucleic acid analogs, enzymes, antibodies, organic compound, inorganic compound, peptide nucleic acid, natural or synthetic compounds.

[071] In some embodiments, the agent is an antibody, an interference RNA or small molecule. The presence of the agent modulates the expression or function of the target molecule or associated molecules thereof.

[072] In a preferred embodiment, a method of treating kidney diseases or disorders in vivo comprises administering to a patient in need thereof, a therapeutically effective amount of one or more agents which modulate dendrin and/or cathepsin L (CatL) activity, expression or function. Preferably, a therapeutically effective amount inhibits CatL-mediated cleavage of dynamin, synaptopodin and/or RhoA expression in podocytes compared to a normal baseline control.

[073] In another embodiment, the agent inhibits expression or activity of TGF-βΙ in podocytes compared to a normal baseline control.

[074] In another embodiment, the agent modulates expression, function or activity of CD2AP, CD2AP-mediated cellular signaling. In some embodiments, an agent inhibits expression, function or activity of CD2AP, CD2AP-mediated cellular signaling.

[075] In another embodiment, the agent modulates dendrin nuclear localization of dendrin, binding of dendrin to a CatL promoter, binding of cathepsin L transcription factors, or cathepsin L transcription or transcription regulatory domains, binds to a CatL promoter and cytosolic Cat L expression and/or cytosolic CatL localization.

[076] In one embodiment, an agent modulates dendrin function, activity or expression. In some aspects the agent inhibits dendrin function, expression, or activity.

[077] In another embodiment, an agent modulates CatL expression, function or activity. [078] In some embodiments, the agent is a vector comprising an RNA interference molecule specific for dendrin or cathepsin L (CatL). The dendrin specific RNA interference molecule comprises a sequence set forth as SEQ ID NOS: 4 or 5, homologs or orthologs thereof.

[079] In another embodiment, the CatL specific RNA interference molecule comprises a sequence set forth as SEQ ID NOS: 1 , 2 or 3, homologs or orthologs thereof. Preferably the species is an animal, preferably, a human.

[080] In another preferred embodiment, the agent is an antibody specific for epitopes comprising SEQ ID NOS: 22 or 23, homologs or orthologs thereof. Preferably the sequences are specific for a human.

[081] In another embodiment, an oligonucleotide of a cathepsin L promoter comprising a sequence set forth as SEQ ID NOS: 15, 16, 17, 18, 19, 20 or 21 , homologs or orthologs thereof.

[082] In some embodiments, a method of treating a patient having a kidney disease or disorder comprises optionally administering to the patient as part of a therapeutic regimen one or more therapeutic compounds for treating kidney disease, disorders or symptoms thereof.

[083] In a preferred embodiment, a kidney disease or disorder comprises: proteinuria related disease such as: glomerular diseases, membranous glomerulonephritis, focal segmental glomerulonephritis, minimal change disease, nephrotic syndromes, pre-eclampsia, eclampsia, kidney lesions, collagen vascular diseases, stress, strenuous exercise, benign orthostatic

(postural) proteinuria, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, sarcoidosis, Alport's syndrome, diabetes mellitus, kidney damage due to drugs, Fabry's disease, infections, aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, Sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, cancer, Wegener's Granulomatosis or Glycogen Storage Disease Type 1 .

[084] In another preferred embodiment, an agent which modulates CatL is administered to patients suffering from or pre-disposed to developing a podocyte disease or disorder. Podocyte diseases or disorders include but are not limited to loss of podocytes (podocytopenia), podocyte mutation, an increase in foot process width, or a decrease in slit diaphragm length. In one aspect, the podocyte-related disease or disorder can be effacement or a diminution of podocyte density. In one aspect, the diminution of podocyte density could be due to a decrease in a podocyte number, for example, due to apoptosis, detachment, lack of proliferation, DNA damage or hypertrophy.

[085] In one. embodiment, the podocyte-related disease or disorder can be due to a podocyte injury. In one aspect, the podocyte injury can be due to mechanical stress such as high blood pressure, hypertension, or ischemia, lack of oxygen supply, a toxic substance, an endocrinologic disorder, an infection, a contrast agent, a mechanical trauma, a cytotoxic agent (cis-platinum, adriamycin, puromycin), calcineurin inhibitors, an inflammation (e.g., due to an infection, a trauma, anoxia, obstruction, or ischemia), radiation, an infection (e.g., bacterial, fungal, or viral), a dysfunction of the immune system (e.g., an autoimmune disease, a systemic disease, or IgA nephropathy), a genetic disorder, a medication (e.g., anti -bacterial agent, anti-viral agent, antifungal agent, immunosuppressive agent, anti-inflammatory agent, analgesic or anticancer agent), an organ failure, an organ transplantation, or uropathy. In one aspect, ischemia can be sickle-cell anemia, thrombosis, transplantation, obstruction, shock or blood loss. In one aspect, the genetic disorders may include congenital nephritic syndrome of the Finnish type, the fetal membranous nephropathy or mutations in podocyte-specific proteins, such as a-actin-4, podocin and TRPC6.

[086] In one aspect, the podocyte-related disease or disorder can be an abnormal expression or function of slit diaphragm proteins such as podocin, nephrin, CD2AP, cell membrane proteins such as TRPC6, and proteins involved in organization of the cytoskeleton such as synaptopodin, actin binding proteins, lamb-families and collagens. In another aspect, the podocyte-related disease or disorder can be related to a disturbance of the GBM, to a disturbance of the mesangial cell function, and to deposition of antigen-antibody complexes and anti-podocyte antibodies. In another aspect, the podocyte-related disease or disorder can be tubular atrophy.

[087] A wide variety of agents can be used to target CatL and associated molecules. These agents may be designed to target signaling by having an in vivo activity which reduces the expression and/or activity of CatL and associated molecules and/or, for example, increases or decreases CDA2P expression, function or activity as compared to baseline controls. For example, the agents may regulate molecules based on the cDNA or regulatory regions, using for example, DNA-based agents, such as antisense inhibitors and ribozymes, can be utilized to target both the introns and exons of the target molecule genes as well as at the RNA level. [088] Alternatively, the agents may target the molecules based on the amino acid sequences including the three-dimensional protein structures of the target molecules. Protein-based agents, such as human antibody, non-human monoclonal antibody and humanized antibody, can be used to specifically target different epitopes on dendrin, CatL, TGF-βΙ molecules and the like.

Peptides or peptidomimetics can serve as high affinity inhibitors to specifically bind to the promoter site of CatL, inhibiting, for example, expression of cytosolic CatL; or in other aspects, increase CD2AP expression. The agents can be identified by a variety of means using the desired outcome to identify which would be suitable, for example, those that modulate CatL expression or function. An example of one agent is a small molecule.

[089] Small Molecules: In order to identify, small molecules as modulators of CatL, CD2AP, dendrin etc, small molecule test compounds can initially be members of an organic or inorganic chemical library. As used herein, "small molecules" refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. The small molecules can be natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular- Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the "split and pool" or "parallel" synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio., 1 :60 ( 1997). In addition, a number of small molecule libraries are commercially available.

[090| Small molecules may include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Also of interest as small molecules are structures found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such compounds may be screened to identify those of interest, where a variety of different screening protocols are known in the art.

[091] The small molecule may be derived from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the preparation of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known small molecules may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[092] As such, the small molecule may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e., a compound diversity combinatorial library. Combinatorial libraries, as well as methods for the production and screening, are known in the art.

[093] Chemical Libraries: Developments in combinatorial chemistry allow the rapid and economical synthesis of hundreds to thousands of discrete compounds. These compounds are typically arrayed in moderate-sized libraries of small molecules designed for efficient screening. Combinatorial methods can be used to generate unbiased libraries suitable for the identification of novel compounds. In addition, smaller, less diverse libraries can be generated that are descended from a single parent compound with a previously determined biological activity. In either case, the lack of efficient screening systems to specifically target therapeutically relevant biological molecules produced by combinational chemistry such as inhibitors of important enzymes hampers the optimal use of these resources.

[094] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks," such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, is formed by combining a set of chemical building blocks (amino acids) in a large number of combinations, and potentially in every possible way, for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. [095] A "library" may comprise from 2 to 50,000,000 diverse member compounds.

Preferably, a library comprises at least 48 diverse compounds, preferably 96 or more diverse compounds, more preferably 384 or more diverse compounds, more preferably, 10,000 or more diverse compounds, preferably more than 100,000 diverse members and most preferably more than 1 ,000,000 diverse member compounds. By "diverse" it is meant that greater than 50% of the compounds in a library have chemical structures that are not identical to any other member of the library. Preferably, greater than 75% of the compounds in a library have chemical structures that are not identical to any other member of the collection, more preferably greater than 90% and most preferably greater than about 99%.

[096] The preparation of combinatorial chemical libraries is well known to those of skill in the art. For reviews, see Thompson et al., Synthesis and application of small molecule libraries, Chem Rev 96:555-600, 1996; Kenan et al., Exploring molecular diversity with combinatorial shape libraries, Trends Biochem Sci 19:57-64, 1994; Janda, Tagged versus untagged libraries: methods for the generation and screening of combinatorial chemical libraries, Proc Natl Acad Sci USA. 91 : 10779-85, 1994; Lebl et al., One-bead-one-structure combinatorial libraries,

Biopolymers 37: 177-98, 1995; Eichler et al., Peptide, peptidomimetic, and organic synthetic combinatorial libraries, Med Res Rev. 15:481 -96, 1995; Chabala, Solid-phase combinatorial chemistry and novel tagging methods for identifying leads, Curr Opin Biotechnol. 6:632-9, 1995; Dolle, Discovery of enzyme inhibitors through combinatorial chemistry, Mol Divers.

2:223-36, 1997; Fauchere et al, Peptide and nonpeptide lead discovery using robotically synthesized soluble libraries, Can J. Physiol Pharmacol. 75:683-9, 1997; Eichler et al.,

Generation and utilization of synthetic combinatorial libraries, Mol Med Today 1 : 174-80, 1995; and Kay et al., Identification of enzyme inhibitors from phage-displayed combinatorial peptide libraries, Comb Chem High Throughput Screen 4:535-43, 2001 .

[097] Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to, peptoids (PCT Publication No. WO 91 /19735);

encoded peptides (PCT Publication WO 93/20242); random bio-oligomers (PCT Publication No. WO 92/00091 ); benzodiazepines (U.S. Pat. No. 5,288,514); diversomers, such as hydantoins, benzodiazepines and dipeptides (Hobbs, et al, Proc. Nat. Acad. Sci. USA, 90:6909-6913 ( 1993)); vinylogous polypeptides (Hagihara, et al., J. Amer. Chem. Soc. 1 14:6568 ( 1992));

nonpeptidal peptidomimetics with β-D-glucose scaffolding (Hirschmann, et al, J. Amer. Chem. Soc, 1 14:9217-9218 (1992)); analogous organic syntheses of small compound libraries (Chen, et al, J. Amer. Chem. Soc, 1 16:2661 ( 1994)); oligocarbamates (Cho, et al., Science, 261 : 1303 (1993)); and/or peptidyl phosphonates (Campbell, et al., J. Org. Chem. 59:658 ( 1994)); nucleic acid libraries (see, Ausubel, Berger and Sambrook, all supra); peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083); antibody libraries (see, e.g., Vaughn, et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287); carbohydrate libraries (see, e.g., Liang, et al, Science, 274: 1520- 1 522 (1996) and U.S. Pat. No. 5,593,853); small organic molecule libraries (see, e.g., benzodiazepines, Baum C&E News, January 18, page 33 ( 1993); isoprenoids (U.S. Pat. No. 5,569,588); thiazolidinones and metathiazanones (U.S. Pat. No. 5,549,974); pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519, 134); morpholino compounds (U.S. Pat. No. 5,506,337); benzodiazepines (U.S. Pat. No. 5,288,514); and the like.

[098] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem. Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd., Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Bio sciences, Columbia, Md., etc.).

[099] In addition to targeting CatL and associated molecules, agents may also be used which compete for binding sites or signaling sites.

[0100] In embodiments, an agent modulates the interactions of dendrin and CatL promoters. This data in the examples section describes the identification of dendrin as a transcription factor of CatL that specifically drives the expression of cathepsin L. Examples of agents, include without limitation: antibodies, aptamers, RNAi, small molecules, y high-affinity binding of specific synthetic or natural peptides that interfere with the assembly of the transcription factor- DNA binding and thus inhibit complex formation. In some aspects the agent inhibits dendrin expression (e.g. by siRNA) in order to inhibit its transcriptional activity in the nucleus for CatL.

[0101] One embodiment of the invention includes isolated antibodies, or fragments of those antibodies, that bind to, for example, dendrin, CatL promoter regions, or proteolytic sites, for example SEQ ID NOS: 23 or 24. As known in the art, the antibodies can be, for example, polyclonal, oligoclonal, monoclonal, chimeric, humanized, and/or fully human antibodies.

Embodiments of the invention described herein also provide cells for producing these antibodies.

[0102] Interference RNA: Detailed methods of producing the can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application 2002/0086356 of Tuschl et ah, the entire disclosure of which is herein incorporated by reference.

[0103] The ability of an RNAi containing a given target sequence to cause RNAi-mediated degradation of the target mRNA can be evaluated using standard techniques for measuring the levels of RNA or protein in cells. For example, RNA of the invention can be delivered to cultured cells, and the levels of target mRNA can be measured by Northern blot or dot blotting techniques, or by quantitative RT-PCR. RNAi-mediated degradation of target mRNA by an siRNA containing a given target sequence can also be evaluated with animal models, such as mouse models. RNAi-mediated degradation of the target mRNA can be detected by measuring levels of the target mRNA or protein in the cells of a subject, using standard techniques for isolating and quantifying mRNA or protein as described above.

[0104] In a preferred embodiment, siRNA molecules target overlapping regions of a desired sense/antisense locus, thereby modulating both the sense and antisense transcripts e.g. targeting dendrin. In another preferred embodiment, a composition comprises siRNA molecules, of either one or more, and/or, combinations of siRNAs, siRNAs that overlap a desired target locus, and/or target both sense and antisense (overlapping or otherwise). These molecules can be directed to any target that is desired for potential therapy of any disease or abnormality. Theoretically there is no limit as to which molecule is to be targeted. Furthermore, the technologies taught herein allow for tailoring therapies to each individual.

[0105] In preferred embodiments, the oligonucleotides can be tailored to individual therapy, for example, these oligonucleotides can be sequence specific for allelic variants in individuals, the up-regulation or inhibition of a target can be manipulated in varying degrees, such as for example, 10%, 20%, 40%, 100% expression relative to the control. That is, in some patients it may be effective to increase or decrease target gene expression by 10% versus 80% in another patient. [0106] Up-regulation or inhibition of gene expression may be quantified by measuring either the endogenous target RNA or the protein produced by translation of the target RNA.

Techniques for quantifying RNA and proteins are well known to one of ordinary skill in the art. In certain preferred embodiments, gene expression is inhibited by at least 10%, preferably by at least 33%, more preferably by at least 50%, and yet more preferably by at least 80%. In particularly preferred embodiments, of the invention gene expression is inhibited by at least 90%, more preferably by at least 95%, or by at least 99% up to 100% within cells in the organism. In certain preferred embodiments, gene expression is up-regulated by at least 10%, preferably by at least 33%, more preferably by at least 50%, and yet more preferably by at least 80%. In particularly preferred embodiments, of the invention gene expression is up-regulated by at least 90%, more preferably by at least 95%, or by at least 99% up to 100%) within cells in the organism.

[0107] Selection of appropriate RNAi is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of RNAi that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.

[0108] In a preferred embodiment, small interfering RNA (siRNA) either as RNA itself or as DNA, is delivered to a cell using aptamers or any other type of delivery vehicle known in the art. In certain embodiments, the nucleic acid molecules of the present disclosure can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore el al., Science 256:9923, 1992; Draper el al., PCT Publication No. WO 93/23569; Shabarova el al., Nucleic Acids Res. 19:4247, 1991 ; Bellon et al., Nucleosides & Nucleotides 16:95 1 , 1997; Bellon et al, Bioconjugate Chem. 8:204, 1997), or by hybridization following synthesis or deprotection.

[0109] In further embodiments, RNAi's can be made as single or multiple transcription products expressed by a polynucleotide vector encoding one or more siRNAs and directing their expression within host cells. An RNAi or analog thereof of this disclosure may be further comprised of a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the aptamers and RNAi's. In one embodiment, a nucleotide linker can be a linker of more than about 2 nucleotides length up to about 50 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By "aptamer" or "nucleic acid aptamer" as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule wherein the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold et al., Annu. Rev. Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2: 100, 2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical Chem. 45: 1628, 1999).

[0110] The invention may be used against protein coding gene products as well as nonprotein coding gene products. Examples of non-protein coding gene products include gene products that encode ribosomal RNAs, transfer RNAs, small nuclear RNAs, small cytoplasmic RNAs, telomerase RNA, RNA molecules involved in DNA replication, chromosomal rearrangement and the like.

[0111] In accordance with the invention, siRNA oligonucleotide therapies comprise administered siRNA oligonucleotide which contacts (interacts with) the targeted mRNA from the gene, whereby expression of the gene is modulated. Such modulation of expression suitably can be a difference of at least about 10% or 20% relative to a control, more preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% difference in expression relative to a control. It will be particularly preferred where interaction or contact with an siRNA oligonucleotide results in complete or essentially complete modulation of expression relative to a control, e.g., at least about a 95%, 97%, 98%, 99% or 100% inhibition of or increase in expression relative to control. A control sample for determination of such modulation can be comparable cells (in vitro or in vivo) that have not been contacted with the siRNA oligonucleotide.

[0112] In another preferred embodiment, the nucleobases in the siRNA may be modified to provided higher specificity and affinity for a target mRNA. For example nucleobases may be . substituted with LNA monomers, which can be in contiguous stretches or in different positions. The modified siRNA, preferably has a higher association constant (K_a) for the target sequences than the complementary sequence. Binding of the modified or non-modified siRNA's to target sequences can be determined in vitro under a variety of stringency conditions using hybridization assays and as described in the examples which follow.

[0113] Chimeric/modified RNAi's: In accordance with this invention, persons of ordinary skill in the art will understand that mRNA includes not only the coding region which carries the information to encode a protein using the three letter genetic code, including the translation start and stop codons, but also associated ribonucleotides which form a region known to such persons as the 5'-untranslated region, the 3 '-untranslated region, the 5' cap region, intron regions and intron/exon or splice junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated

ribonucleotides as well as to the coding ribonucleotides. In preferred embodiments, the

oligonucleotide is targeted to a translation initiation site (AUG codon) or sequences in the coding . region, 5' untranslated region or 3 '-untranslated region of an mRNA. The functions of messenger RNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing or maturation of the RNA and possibly even independent catalytic activity which may be engaged in by the RNA. The overall effect of such interference with the RNA function is to cause interference with protein expression.

[0114] Other agents: These can include any synthetic or natural peptides, glycoproteins, enzymes, modulators of signaling, inhibitors of assembly of transcription or translational factor complexes, organic or inorganic molecules and the like.

[0115] Other embodiments of the invention include isolated nucleic acid molecules encoding any of the targeted binding agents, antibodies or fragments thereof as described herein, vectors having isolated nucleic acid molecules or a host cell transformed with any of such nucleic acid molecules. It should be realized that embodiments of the invention also include any nucleic acid molecule which encodes an antibody or fragment of an antibody of the invention including nucleic acid sequences optimized for increasing yields of antibodies or fragments thereof when transfected into host cells for antibody production.

Administration of Compositions to Patients

[0116] The compositions or agents identified by the methods described herein may be administered to animals including human beings in any suitable formulation. For example, the compositions for modulating protein degradation may be formulated in pharmaceutically acceptable carriers or diluents such as physiological saline or a buffered salt solution. Suitable carriers and diluents can be selected on the basis of mode and route of administration and standard pharmaceutical practice. A description of exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington's Pharmaceutical Sciences, a standard text in this field, and in USP NF. Other substances may be added to the compositions to stabilize and/or preserve the compositions.

[0117] The compositions of the invention may be administered to animals by any conventional technique. The compositions may be administered directly to a target site by, for example, surgical delivery to an internal or external target site, or by catheter to a site accessible by a blood vessel. Other methods of delivery, e.g., liposomal delivery or diffusion from a device impregnated with the composition, are known in the art. The compositions may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously). For parenteral administration, the compositions are preferably formulated in a sterilized pyrogen-free form.

[0118] The compounds can be administered with one or more therapies. The

chemotherapeutic agents may be administered under a metronomic regimen. As used herein, "metronomic" therapy refers to the administration of continuous low-doses of a therapeutic agent. [0119] Dosage, toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for

determining the LDs₀ (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0120] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the

therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0121] As defined herein, a therapeutically effective amount of a compound (i.e., an effective dosage) means an amount sufficient to produce a therapeutically (e.g., clinically) desirable result. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compounds of the invention can include a single treatment or a series of treatments. [0122] Formulations: While it is possible for a composition to be administered alone, it is preferable to present it as a pharmaceutical formulation. The active ingredient may comprise, for topical administration, from 0.001 % to 10% w/w, e.g., from 1 % to 2% by weight of the formulation, although it may comprise as much as 10% w/w but preferably not in excess of 5% w/w and more preferably from 0.1 % to 1 % w/w of the formulation. The topical formulations of the present invention, comprise an active ingredient together with one or more acceptable carrier(s) therefor and optionally any other therapeutic ingredients(s). The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0123] Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear, or nose. Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified and sterilized by filtration and transferred to the container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are

phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01 %). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

[0124] Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops.

Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.

[0125] Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogels. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surface active such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

[0126] While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.

[0127] All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention. Embodiments of inventive compositions and methods are illustrated in the following examples.

EXAMPLES

[0128] The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

Example J : Regulation of cathepsin L by its transcription factor dendrin determines kidney disease [0129] Recent years have shed light on the molecular makeup of the slit diaphragm (SD) and podocyte FPs, mainly through human and mouse genetic studies. Clinically, acquired forms of glomerular dysfunction, such as those seen in diabetes mellitus, are far more common and follow similar patterns of injury. A key event in the development of podocyte FP effacement and proteinuria lies in the induction of a cytosolic form of the protease cathepsin L (CatL; encoded by Ctsl) in podocytes that triggers the disease process. Ctsl mRNA is found in all tissues, but is an enriched glomeruiar-specific transcript compared with other segments of the kidney. Via mechanisms of alternative translation of Ctsl mRNA, a cytosolic CatL protein that lacks the lysosomal targeting sequence can be produced in a few cell types, including podocytes.

Physiological functions of cytosolic CatL include processing of transcription factors as well as processing of histone H3 during embryonic stem cell differentiation in mice. In podocytes, cytosolic CatL proteolyzes the large GTPase dynamin and the actin-binding protein

synaptopodin. Both events result in disorganization of the podocyte actin cytoskeleton and FP effacement. FP effacement can be inhibited by blocking CatL activity or by protection of the target proteins from CatL cleavage.

[0130] An important genetic model to study the sequela of podocyte injury and glomerular disease is the Cd2ap^~/~ mouse model. CD2AP, a scaffolding protein, is essential for proper signaling at the SD. Thus, homozygous CD2AP mutation or haplo-insufficiency of the human CD2AP gene predisposes to renal disease, and mice lacking 1 copy of Cd2ap develop glomerular renal failure. Importantly, Tg mice expressing CD2AP only in podocytes prevented the development of proteinuria, which demonstrated that the renal failure is solely due to loss of CD2AP in podocytes.

[0131] Loss of CD2AP leads to increased expression of TGF-βΙ in podocytes and apoptosis, demonstrating that CD2AP somehow regulates podocyte survival by regulating expression of TGF-βΙ . Furthermore, high levels of TGF-βΙ have been associated with translocation of dendrin from the SD to the nucleus, which in turn increased podocyte apoptosis. Dendrin binds CD2AP and nephrin at the SD, which suggests that loss of CD2AP and subsequent increase in TGF-βΙ expression in podocytes might drive dendrin translocation in cells lacking CD2AP. Genetic deletion or proteolytic degradation of CD2AP allowed for an increase in podocyte TGF-β Ι signaling that in turn drove translocation of dendrin from the SD into the nucleus. Dendrin is a CatL transcription factor specifically promoting expression of cytosolic CatL. Cytosolic CatL in turn drives reorganization of the actin cytoskeleton by proteolytically processing dynamin and synaptopodin.

[0132] Alteration in the actin cytoskeleton rendered podocytes hypersensitive to proapoptotic signals. The prolonged presence of cytosolic CatL led to sustained loss of CD2AP, which in turn promoted dendrin translocation and drove sustained expression of cytosolic CatL, establishing progressive renal disease. In summary, these results demonstrated that CD2AP plays an unexpected role in the regulation of the expression of cytosolic CatL and linked SD injury with a proteolytic program that underlies progressive renal disease.

[0133] Methods

[0134] Cells and reagents. Synaptopodin knockdown (Asanuma K, et al. J Clin Invest. 2005;1 15: 1 188-198) and dynamin knockdown (Gu C, et al. EMBO J. 2010;29:3593-3606) podocytes were cultured as described previously. Staurosporine, angiotensin II and actinomycin D were all obtained from Sigma.

[0135] Animals. Cathepsin L knockout mice on a mixed C57B16/129J background was described previously (Nakagawa T, et al. Science. 1998; 280:450).

[0136] Cell culture and transient transfection. Mouse WT and Cd2ap^~'^~ (Schiffer M. et al. J Biol Chem. 2004; 279(35):37004-37012) podocytes were cultured as described previously (Mundel P, et al. Exp Cell Res. 1997; 236( 1 ):248-258). HEK 293 cells were maintained and transfected using Lipofectamine 2000 reagent (Invitrogen) as previously reported (Sever S, et al. J Clin Invest. 2007; 1 17(8):2095-210). Adenoviral infections of cultured podocytes were preformed as described previously (Sever S, et al, 2007).

[0137] Lentiviral infection. Lentiviral shRNA plasmids for CatL were obtained from Open Biosystems and were used to generate lentiviral transduction particles in HEK 293T cells. A target set of 3 clones was used with pLK0.1 <-puro as the parental vector (see Table 1 for sequences(SEQ ID NOS: 1 -5)). Lentiviral shRNA plasmids for dendrin were obtained from Sigma Aldrich and were used to generate lentiviral transduction particles in HEK 293T cells. A target set of 2 clones was used with pLK0.1 <-puro as a parental vector (Table 1 ). Lentiviral knockdown of mouse CatL and dendrin were performed in differentiated mouse and high-7g ¾7 Cd2ap^~/~ podocytes according to the protocol from the RNAi Consortium. Cells were harvested to assay for knockdown efficiency using.quantitative PCR and Western blot. In addition, changes in phenotype were monitored using immunofluorescence.

[0138] Antibodies. The following primary antibodies were used: mouse anti-actin (Sigma- Aldrich), rhodamine- and FITC-phalloidin (Invitrogen), mouse anti-dynamin, mouse anti- paxillin (Millipore), rabbit anti-WTl , rabbit anti-RhoA, goat anti-synaptopodin (Santa Cruz Biotechnology), mouse anti-GAPDH, rabbit anti-mannosidase II (Abeam), rabbit anti-CD2AP (Dustin ML, et al. Cell. 1998;94(5):667-677), mouse anti-synaptopodin (Mundel P., et al. J Cell Biol. 1997; 139( 1 ) : 193-204), rabbit anti-dendrin (Asanuma K, et al. Proc Natl Acad Sci USA. 2007; 104(24): 10134-10139), rabbit anti-CatL (Ishidoh K, Kominami E. FEBS Lett.

1994;352(3):281-284), rabbit anti-a-actinin-4 (Kaplan JM, et al. Nat Genet. 2000;24(3):251- 256), rabbit anti-nephrin (Holzman LB, et al. Kidney Int. 1999;56(4): 1481— 1491 ), and rat anti- Lamp-2 (Developmental Studies Hybridoma Bank). CatL epitope was generated in house, and cytosolic CatL antibody production was outsourced to Picono Rabbit Farm and Laboratory.

[0139] Animals and treatments. TGF-βΙ Tg mice (Kopp JB, et al. Lab Invest. 1996;

74(6):991-1003) and Cd2ap^~/~ mice on a mixed C57BL6/129J background (Shih NY, et al. Science. 1999; 286(5438):312—315) were described previously. Dendrin knockout mice were generated by replacing the entire coding region of 2 exons with EGFP lox-Ubl -EM7-Neolox cassette (Regeneron Pharmaceuticals). The elimination of dendrin in knockout mice was confirmed by immunohistochemistry. The mouse model of LPS-induced proteinuria was as previously described (Reiser J, et al. J Biol Chew. 2004; 279(33):34827-34832). The rat puromycin aminonucleoside-induced nephrosis model was as previously described (Kim SW, et al. Am J Physiol Renal Physiol. 2004; 286(5):F922-F935). Urine microalbumin was assessed by densitometric analysis of Bis-Tris gels (Invitrogen) loaded by standard BSA (Bio-Rad

Laboratories) and urine samples. The urine creatinine measurement was carried out using a colorimetric endpoint assay with a commercial kit (Cayman Chemical). Animal protocols and procedures were reviewed for ethical and humane standards and approved by the institutional Animal Care Committees of Massachusetts General Hospital and University of Miami.

[0140] Isolation and processing of glomeruli. Glomeruli were isolated from kidneys of 8- to 12-week-old LPS- and PBS-treated (control) mice using a sequential sieve technique with mesh sizes of 180, 100, and 71 μιη. The fraction collected from the 71 -μιη sieve was maintained for soup/pellet fractionation as previously described (Sever S, et al. J Clin Invest. 2007; 1 17(8):2095-2104). Isolated glomeruli were homogenized in CHAPS buffer containing 20 mM Tris (pH 7.5), 500 mM NaCl, 0.5 % (w/v) CHAPS, and protease inhibitors (Roche) using Dounce homogenizer. Subsequently, the extract was centrifuged for 10 minutes at 1 5,000 g.

[01411 Isolation of nuclear fraction. Podocytes were detached, precipitated, and washed with PBS. Cell pellets were immediately resuspended in 400 μΐ chilled buffer A (10 mM HEPES, pH 7.9; 10 mM C1; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol [DTT]; and 0.5 mM PMSF). This resuspension mixture was incubated on ice for 15 minutes without vortexing, and then 25 μΐ of 10% NP-40 was added followed by vigorous vortexing for 10 seconds at 4°C. The sample was centrifuged at 1 ,500 g for 1 minute at 4°C. The resulting supernatant was collected and stored as the cytosolic fraction. The remaining nuclear pellet was resuspended in 50 μΐ ice- cold buffer B (20 mM HEPES, pH 7.9; 400 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; and 0.5 nM PMSF), rocked vigorously at 4°C for 2 hours, and then centrifuged at maximum speed for 10 minutes at 4°C. The nuclear and cytosolic fractions were separated by 12% SDS- PAGE, transferred on a PVDF membrane (Bio-Rad Laboratories), and analyzed by Western blotting.

[0142] Immunohistochemistry, immunofluorescence, and immunoblotting. Mouse and rat kidney tissue was harvested and either immersion-fixed in formaldehyde (Electron Microscopy Sciences) for paraffin embedding or embedded in OCT compound (Sakura Finetek) for frozen sections. Sections of paraffin-processed kidney were stained with H&E. The protocol to study kidney biopsy samples from FSGS and MCD patients was reviewed and approved with a consent waiver by the University of Miami Institutional Review Board. Kidney biopsies were stained with N- and C-terminal CD2AP antibodies according to standard protocols (Wei C, et al. Nat Med. 2008; 14(1 ):55-63). Immunofluorescence of cultured podocytes and Western blotting were performed as described previously (Sever S, et al. J Clin Invest. 2007; 1 17(8):2095-2104).

Images were captured using a LSM 5 PASCAL laser scanning microscope (Zeiss) and a *40 objective. Quantification of stress fibers, FAs, CatL, and hematoxylin immunostain intensities were performed using ImageJ software.

[0143] Coimmunoprecipilalion studies. The recombinant mouse FLAG-dendrin and GFP- tagged CD2AP variants (full-length CD2AP, CD2AP-NH₂, and CD2AP-COOH) were expressed in HEK 293 cells. FLAG fusion proteins were immunoprecipitated from cell lysates using anti- FLAG-M2 beads (Sigma-Aldrich), and eluates were analyzed by immunoblotting using antibodies to FLAG (Sigma-Aldrich) or GFP (Invitrogen).

[0144] Endopeptidase cleavage site score. To assess the susceptibility of CD2AP for cleavage by CatL in silico, the prediction of endopeptidase substrates (PEPS) bioinformatics tool was used (Lohmuller T, et al. Biol Chem. 2003;384(6):899-909). A score above the threshold of 0.01 estimates protein sequences to be within 100 peptide motifs (of 10,000).

[0145] Purification ofCDIAP and dendrin. FLAG-CD2AP and FLAG-dendrin were expressed in HEK 293 cells, immobilized on anti-FLAG M2 agarose (Sigma- Aldrich), and eluted with FLAG peptide (Sigma-Aldrich).

[0146] Proteolytic processing of CD2AP by CatL. CD2AP was diluted in buffer containing 200 mM NaCl, 10 mM HEPES (pH 7.0), 2 mM EGTA, 1 mM MgCl₂, and 1 mM DTT. When indicated, 20 μΜ CatL inhibitor I (Calbiochem) was added. The reaction was initiated by addition of 0.5 μΐ purified CatL enzyme (Sigma-Aldrich), and samples were placed at 37°C in a water bath for 10-30 minutes. Total assay volume was 20 μΐ. The reaction was terminated with addition of ^χ4 sample buffer (Invitrogen).

[0147] Deletion of CD2AP cleavage site LSAAE. Deletion of the CatL cleavage site LSAAE from the CD2AP amino acid sequence was done using the Quick-Change II Site Directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions.

[0148] Quantitative PCR. Cells were treated with TRlzol reagent (Invitrogen) to allow complete cell lysis, followed by RNA extraction per the manufacturer's protocol. RNA was quantitated and cDNA synthesis was performed using the Protoscript First strand cDNA synthesis kit (New England Biolabs). Quantitative PCR was performed using Brilliant SYBR Green Master Mix (Stratagene) and specific primers for CatL, dendrin, dynamin, RhoA, and synaptopodin (Table 2; SEQ ID NOS: 6-14)) in MX3000P QPCR System (Agilent

Technologies). Normalization across samples was performed using the average of the constitutive gene Gapdh. Fold expression changes were determined using the comparative CT method for relative quantification with the calculation 2^{" CT}, and data were graphed using either Excel (Microsoft) or Prism (Graph-Pad) software.

[0149| Kidney total RNA isolation and quantitative RT-PCR. Harvested mouse kidneys were homogenized in TRlzol reagent (Invitrogen) for 40 seconds using POWERGEN 125 (Fisher Scientific) at maximum speed. Total RNA was isolated according to the manufacturer's protocol. Quality and quantity of total RNA was checked by Bio-analyzer (Agilent Technologies). 1 μg kidney total RNA was reversely transcribed into single-strand cDNA. Quantitative RT-PCR was performed as described previously (Ju W, et al. Mol Cell Biol.

2006;26(2):654-667). Expression of GAPDHand β-actin was used to normalize the sample amount.

[0150] CatL activity assays. Prior to enzyme assays, cytosolic fraction containing CatL was isolated by subcellular fraction as described previously (Damke H, et al. J Cell Biol. 1994; 127(4):915-934). Activity assays were performed using the CatL and CatB fluorescent substrate Z-Phe-Arg-7-amido-4methylcoumarin hydrochloride (Sigma-Aldrich) at different pHs. 5 μΙ supernatant (fixed protein concentration) from each sample was added in triplicate to a 96-well, all-black flat-bottomed plate, followed by addition of 175 μΙ freshly made assay buffer (340 mM sodium acetate, 60 mM acetic acid, 4 mM disodium EDTA, 8 mM dithiothreitol). The CatB- specific inhibitor CA074 (Enzo Life Sciences) was added to the wells to cancel out the contribution of CatB. The cysteine protease inhibitor E64 (Biomol) was added to the wells designated for negative control. The mixture was incubated at room temperature for 1 minute to activate the enzyme, immediately followed by the addition of 25 μΙ of 20 μΜ fluorescent substrate. Fluorescence of free aminomethyl coumarin was determined as a kinetic interval assay, with readings taken every 5 minutes for 3 hours at 30°C by excitation at 370 nm and emission at 460 nm using a SpectraMax M2E (Molecular Devices). Data were collected every 5 minutes.

[0151] SEAP reporter assay. HE 293 cells were triple-transfected using Lipofectamine 2000 reagent (Invitrogen) with the following 3 plasmids: (a) pSEAP2-Basic, containing either the full-length rat CatL promoter (construct A; Figure 6C), 1 of 2 partial deletion constructs (constructs B and C; Figure 6C), or the full-length CatB promoter (Liu G, et al. J Biol Chem. 2006;281 (5 1 ):39681-39692); (b) WT rat dendrin, rat dendrin with a mutated nuclear localization signal (Asanuma , et al. Proc Natl Acad Sci U SA. 2007; 104(24): 10134-10139), or the empty vector; and (c) pMetLuc-Control for normalization (Clontech). All transfections were performed in triplicate. Medium was analyzed for SEAP and luciferase activity according to the manufacturer's instructions in a GloMax-96 Microplate Luminometer (Promega).

[0152] EMSA. 4 overlapping fragments (fragments D-G; Figure 6C) of the rat CatL promoter portion between -1 ,215 and -339 (Liu G, et al. J Biol Chem. 2006;281 (51 ):39681 - 39692) were incubated with purified FLAG-dendrin for EMSA using the Electrophoretic Mobility Shift Assay kit (Molecular Probes) and visualized with SYBR green. Promoter fragment E, which bound to dendrin, was further divided into 4 overlapping 60-bp

oligonucleotides (see Table 3 for sequences (SEQ ID NOS: 15-21 )); oligonucleotide 4, which exhibited dendrin binding, was divided again into 3 overlapping 24-bp oligonucleotides (Table 3), which were biotinylated at the 5' end and used for EMSA using the LIGHTSH1FT

Chemiluminescent EMSA kit (Thermo Scientific). Corresponding unlabeled WT

oligonucleotides were used for competition in 200-fold excess. Finally, oligonucleotide 4-1 , which exhibited dendrin binding, was used to design 3 different mutant oligonucleotides (see Figure 6C for sequences) that were used for competition assays in 200-fold excess.

[0153] Apoptosis Assays. Wild type mouse (Control) and CD2AP^"HH'^{8h TGFp)} podocytes were grown in 24 well dishes and allowed to differentiate for 10 days. Upon differentiation, cells were infected with 30 μΐ of lentiviruses to knock down dendrin or CatL for 24 hours in presence of 8 g l polybrene. After 24 hours, the medium was replaced with serum-starved RPMi (0.2% FBS, 1% Penicillin/Streptomycin, all from Invitrogen). Twenty-four hours later, cells were treated with apoptotic inducers, e.g., 1 , 2 and 5 ng/ml TGF-βΙ , 10 ng/ml actinomycin D and 100 nM angiotensin II for an additional 24 hours or 1 μΜ staurosporine for 1 h. When indicated, the cells were treated with 20 μΜ E-64 in serum-starved medium for 24 hours. Apoptosis assays were performed using the Cell Death Detection ELISA PLUS kit (Roche) as per the

manufacturers protocol.

[0154] Statistics. Statistical analysis was performed by 2-tailed Student's / test. A P value less than 0.05 was considered significant. Unless otherwise indicated, data are reported as mean ± SEM.

[0155] Results:

[0156] Increased TGF-β Ι signaling induces CatL expression. Most forms of glomerular diseases, such as diabetic nephropathy, are associated with increases in TGF-βΙ signaling (Bottinger EP, Bitzer M. J Am Soc Nephrol. 2002; 13( 10):2600-2610). Ctsl mRNA and CatL protein levels are upregulated in glomeruli of patients with diabetic nephropathy (Sever S, et al. J Clin Invest. 2007; 1 17(8):2095-2104). It was examined whether mice Tg for TGF-βΙ exhibit elevated CatL expression. These animals, which express TGF-βΙ in the liver controlled by an albumin promoter, display elevated plasma concentrations of TGF-β Ι , exhibit podocyte apoptosis at an early stage of disease, and develop variable degrees of C D and proteinuria (Schiffer M, et al. J Clin Invest. 2001 ; 108(6):807-816; Ju W, et al. Am J Pathol.

2009;174(6):2073-2085). Because all animals have high levels of circulatory TGF-βΙ , which in itself is not sufficient to explain the variability in the kidney phenotype (glomerulosclerosis), real-time PCR (RT-PCR) was used to examine mRNA levels of Tgfbl in isolated kidneys of TGF-βΙ Tg animals. There was an increase in mRNA levels for Tgfbl, which correlated well with severity of the kidney phenotype (Figures 1 A-IH). A similar correlation was detected with respect to Ctsl mRNA levels in glomeruli. Together, these data showed that there was a strong association between Tgfbl and Ctsl mRNA levels and severity of glomerulosclerosis (r = 0.96 and r = 0.97, respectively). The increase in Ctsl mRNA levels in glomeruli translated into an increase in CatL protein levels in podocytes, as revealed by immunohistochemical staining for CatL (Figures 1 B and 1 C). Together, these data showed that increased exogenous levels of TGF-βΙ can increase both endogenous kidney TGF-βΙ levels and CatL expression.

[0157] High levels of TGF-βΙ induce translocation of dendrin from the plasma membrane to the nucleus of podocytes in culture. The data herein showed increased nuclear dendrin staining in TGF-βΙ Tg mice (Figures I D and I E). To explore the identified connection between increased TGF-βΙ and CatL levels in the glomerulus and TGF-βΙ -dependent nuclear localization of dendrin in podocytes, the Cd2ap^~/~ mouse model of progressive kidney disease was used. . Cd2ap^~/~ mice are born normal, but develop heavy proteinuria and renal failure approximately 4 weeks after birth and die usually at 6 weeks of age (Shih NY, et al. Science.

1999;286(5438):312-315, Schiffer M, et al. J Clin Invest. 2001 ; 108(6):807-816). As glomerular disease progresses in these mice, podocyte apoptosis increases, driven in part by an increase in intraglomerular TGF-βΙ signaling. Given the increased TGF-β Ι levels in podocytes lacking CD2AP at the time of proteinuria onset, these mice represent an ideal model to examine the connection between TGF-βΙ signaling and CatL expression in podocytes. It was first examined whether increased CatL levels coincide with proteinuria onset in Cd2ap^~/~ animals. Whereas glomeruli of both WT and Cd2ap^~/~ mice exhibited low-level staining for CatL at 1 week, staining for CatL increased in glomeruli of 3-week-old Cd2ap^{~ ~~} mice (Figures I F and 1 G). The staining indicated that a majority of CatL was present within the podocytes (Figure 1 G, arrows). Next, it was examined whether dendrin could be found in the nucleus in Cd2ap^~/~ animals. The classical plasma membrane staining was observed for dendrin in Cd2ap^{~ ~} mice at 1 week of age, when mice were still healthy (Figure 1 H). Importantly, dendrin exhibited a nuclear staining pattern in 4-week-old Cd2ap^~/~ mice (Figure 1 H), which demonstrated that dendrin translocated into the nucleus of podocytes during the early stages of podocyte injury. In contrast, dendrin staining in the glomeruli of 1 - or 4-week-old WT mice exhibited a characteristic membrane pattern (Figure 1 H), in agreement with its SD localization. Together, these data identified a link between loss of the SD adaptor protein CD2AP, increase in autocrine levels of TGF-βΙ , dendrin translocation into the nucleus, and increased levels of CatL in podocytes.

[0158] Loss of CD2AP leads to expression of cytosolic CatL in podocytes. To elucidate the molecular mechanism that regulates expression of CatL in a TGF-βΙ -dependent manner, podocytes in culture were used. The results showed that the conditionally immortalized podocytes derived from Cd2ap^"/~ mice were indistinguishable from the WT podocytes, with well-defined focal adhesions (FAs) and stress fibers (Figure 2A, left panels). Similar to the course of pathology in Cd2ap^"/~ mice, Cd2ap^^~ podocytes cultured for more than 6 weeks developed distinct alteration in the actin cytoskeleton, loss of mature FAs and increased number of focal complexes, and loss of well-defined stress fibers and dramatically increased number of transverse arcs (Figure 2A). The alteration in the actin cytoskeleton was similar to that observed in cells treated with LPS and was consistent with FP effacement at the onset of proteinuria in Cd2ap^{_ "} mice. Because increased glomerular levels of TGF-βΙ that can act in a

paracrine/autocrine fashion has been shown to occur at the onset of proteinuria Tgfbl mRNA levels in Cd2ap^{~ ~~} podocytes that contained WT organization of the actin cytoskeleton (early passage) were compared with those with altered actin cytoskeleton (late passage). Indeed, late- passage Cd2ap^~/~ podocytes exhibited higher Tgfbl mRNA levels than did early-passage Cd2ap^~ '^~ podocytes (Figure 2C). To further explore the connection between high Tgfbl mRNA levels and actin cytoskeleton alterations, it was examined whether addition of high levels of recombinant TGF-βΙ in the media could induce alteration of the podocyte actin cytoskeleton. Culturing of WT podocytes with 5 ng/ml TGF-βΙ did not induce alteration in their actin cytoskeleton (Figure 2B). In contrast, identical treatment of early-passage Cd2ap^{~ ~} podocytes induced dramatic alteration of their actin cytoskeleton (Figure 2B). This TGF^ l-dependent alteration of the actin cytoskeleton coincided with increased dendrin staining in the nucleus in Cd2ap^~/~ cells (Figure 2B). Notably, dendrin was detected in the nucleus to some degree even in WT cells (Figure 2B; and Figure 9A). [0159] Even at the basal state, Cd2ap cells exhibited higher Tgfbl mRNA levels than did WT cells, and this level was further increased upon culturing cells in TGF-βΙ rich media (Figure 2C). Thus, podocytes lacking CD2AP were susceptible to increased levels of TGF-βΙ in media, which in turn drove reorganization of their actin cytoskeleton. Consequently, Cd2ap^~~/~

podocytes with low Tgfbl mRNA levels exhibited WT organization of the actin cytoskeleton, and those with high Tgfbl mRNA levels exhibited altered organization of the actin cytoskeleton (Figure 2, A and B). Experimentally, the switch from low to high Tgfbl was achieved by prolonged culturing of Cd2ap^{~ ~} podocytes to allow for endogenous autocrine TGF-βΙ signaling (Figure 2A and Figure 9A) as well as by adding high levels of recombinant TGF-β Ι into the media to rapidly transduce the Tgfbl signal (Figures 2B and 2C). In both instances, the levels of endogenous Tgfbl mRNA correlated with nuclear dendrin staining (Figure 2B and Figure 9A).

[0160] Importantly, the increase in TGF-βΙ induced an increase in Ctsl mRNA levels in Cd2ap^~'^~ cells. In contrast, expression of Ctsl mRNA in WT cells was resistant to high levels of TGF-βΙ (Figure 2D). However, even in WT cells, the higher the TGF-β level in the media, the higher the level of Ctsl mRNA (Figure 9B), which evidences that prolonged high levels of exogenous TGF-β may trigger an increase in Ctsl mRNA. Similar observations were made when Ctsl mRNA levels were compared among WT cells, hig -Tgfbl Cd2ap^~/" cells, and cells treated with LPS (Figure 2E). Increased Ctsl mRNA was not observed in podocytes expressing a gain- of-function mutation in the a-actinin-4 gene (Actn4; Figure 2E) or in podocytes in which dynamin or synaptopodin were downregulated (Figure 9C). These data, consistent with the findings in animals (Figures 1 A and I B), evidencing that increased TGF-β Ι signaling has a specific effect on CatL expression.

[0161] Upregulation of Ctsl mRNA resulted in a dramatic increase in the amount of cytosolic CatL (also referred to as short-form CatL; Figure 2F, lane 3; Figure 9D). Indeed, cytosolic CatL was also found in the nucleus (Figure 9E. Cytosolic CatL downregulates protein levels of dynamin, synaptopodin, and RhoA. High-Tgfbl Cd2ap^~'^~ cells exhibited lower levels of dynamin, synaptopodin, and RhoA without affecting the level of a-actinin-4 (Figure 9F). The decreased protein levels were not caused by downregulation of mRNA for those proteins (Figures 9G and 91), which evidences that decreased levels were caused by proteolytic processing of these proteins by cytosolic CatL. Together, these data evidence that Cd2ap^~'^~ podocytes in culture phenocopy alterations that occur in podocytes in Cd2ap^~/~ animals at the onset of proteinuria: increased expression of endogenous TGF-βΙ , dendrin translocation into the nucleus (Figure 1 H), and increased expression of CatL (Figure 1 G).

[0162] Cytosolic CatL alters the aclin cytoskeleton in Cd2ap^~/~ cells. This study also identified the presence of cytosolic CatL in high-7g/Z>7 Cdlap ^~ podocytes. To further explore the hypothesis that the presence of cytosolic CatL drives alterations of the actin cytoskeleton in Cd2ap^~/~ cells by proteolytically processing a subset of proteins, CatL activity was inhibited by lentivirus-based shRNAs and by using the small-molecule cysteine protease inhibitor E64, which blocks CatL. Downregulation of CatL by RNAi was confirmed using RT-PCR and Western blot analysis (Figures 3A and 3B). 3 different shRNAs downregulated CatL protein levels 70%-90% and significantly reduced CatL activity in the cytoplasm of ig -Tgftl Cd2ap^~'^~ cells (Figure 3B, and 3C). In agreement with the fact that the enzymatic assay measures activities of both CatL and CatB, addition of CA074, a specific CatB inhibitor, resulted in further lowering of the measured activity to the basal level (Figure 3C), thus defining the specificity of the assay.

Therefore, the CatB inhibitor CA074 was included in all subsequent assays. As shown in Figure 3D, downregulation of CatL in high-7¾ ¾7 Cd2ap^~/~ cells by shRNA-C6 resulted in CatL activity similar to that observed in WT cells. Immunofluorescence and Western blot analysis

demonstrated that CatL was active in the cytoplasm of high-7 ¾7 Cd2ap^~'^~ cells (Figure 2F). If loss of dynamin and synaptopodin was indeed the result of identified activity of cytosolic CatL, then treatment of cells with E64 or downregulation of CatL expression by shRNA should exhibit a protective effect on the loss of these proteins. As expected, both treatments resulted in increased levels of dynamin, synaptopodin, and RhoA (Figures 3E-3G). In agreement with increased levels of these proteins in treated high-7g Z>/ Cd2ap^'/~ cells, CatL inhibition partially restored the number of podocyte FAs as well as stress fibers (Figures 4A and 4B). Together, these data demonstrated an increase in cytosolic CatL in high-TgfiJ Cd2ap^~'^~ podocytes, which in turn drives reorganization of the actin cytoskeleton through proteolytic processing of a subset of proteins that regulates the actin cytoskeleton.

[0163] Interestingly, downregulation of CatL in WT podocytes, either by siRNA or by addition of E64 (Figures 10A-10D), resulted in an increase in FA size (Figures 10E-10G).

Thus, although the number of FAs per cell was similar, there was a shift toward more mature FAs. This could be explained by the increase in levels of dynamin, synaptopodin, and - to some extent - RhoA (Figure 10H). Together, these data evidence that endogenous levels of cytosolic CatL regulate turnover of the FAs in cultured podocytes.

[0164] Nuclear dendrin regulates expression of cytosolic CatL. The data herein, identified a link between dendrin localization in the nucleus and expression of cytosolic CatL. To test whether nuclear dendrin (encoded by Ddn) directly induces expression of cytosolic CatL, dendrin was downregulated in high-7 /¾7 Cd2ap^~/~ cells using shR As (Figures 5A and 5B). This led to partial restoration of FAs and stress fibers within the cell body, demonstrating that loss of dendrin had a direct consequence on actin organization in ig -Tgfbl Cd2ap^~/~ cells, that was also indicated by FAs formed within the cell body that connected short actin filaments (Figures 5C and 5D). Strikingly, downregulation of dendrin resulted in downregulation of Ctsl mRNA and CatL protein (Figures 5E and 5F). Furthermore, downregulation of dendrin also partially lowered CatL activity in the cytoplasm of high-Tgfbl Cd2ap^~'^~ cells (Figure 5G). In agreement with the decrease in CatL activity, downregulation of dendrin also increased the levels of dynamin and synaptopodin in high-7g/Z>7 Cd2ap^~/~ cells (Figure 5F).

[0165] igh-TgfbJ Cd2ap^~'^~ cells were hypersensitive to proapoptotic signals such as high levels of TGF-βΙ , staurosporine, actinomycin D, or angiotensin II (Figure 5H). It was examined whether downregulation of dendrin and/or CatL has functional consequences on Cd2ap^~/~ podocyte survival, importantly, downregulation of dendrin in high-Tgfbl Cd2ap^"'^~ cells resulted in partial protection from TGF-βΙ -induced apoptosis (Figure 51). This partial rescue may be due to partial downregulation of cytosolic CatL activity (Figures 5F and 5G), and thus only partial rescue of the actin cytoskeleton, in high-Tgfbl Cd2ap^{~ ~} cells (Figures 5C-5F), or possible additional roles of nuclear dendrin in addition to regulation of CatL expression. In support of the first hypothesis, downregulation of CatL or addition of E64 resulted in complete protection from TGF-pi-induced apoptosis of high-7g ¾7 Cd2ap^'/" cells (Figure 51). Downregulation of dendrin or CatL protected high-Tgfbl Cd2ap^~/~ cells only from apoptosis induced by TGF-β Ι , not from other apoptotic signals (Figures 1 1 A— 1 1 C). Taken together, these results demonstrated that the specific proapoptotic effect of TGF-β Ι on high-Tgfbl Cd2ap^{~ ~} cells is the result of dendrin translocation into the nucleus, where it regulates expression of cytosolic CatL.

[0166] Dendrin is a CatL transcription factor. These results thus far identified a correlation between the presence of dendrin in the nucleus of podocytes and the expression of cytosolic CatL. It was therefore examined whether dendrin can directly regulate the expression of CatL by acting as its transcription factor. First, it was examined whether heterologous expression of dendrin in HEK 293 cells can enhance transcriptional activity of the CatL promoter. Using triple transfection experiments in HEK 293 cells, a plasmid encoding for secreted alkaline phosphatase (SEAP) was expressed under the control of the rat CatL promoter, dendrin, and Metridia luciferase under the control of a constitutively active promoter to normalize for cell number and transfection efficiency. Compared with the empty vector, dendrin induced normalized SEAP activity 5-fold (Figures 6A and 6B). The transcriptional activity of dendrin was completely abolished by mutation of its nuclear localization signal (Figures 6A and 6B), demonstrating that nuclear translocation of dendrin is essential for mediating its effect on CatL transcription. In contrast, CatB promoter activity was lower at baseline and was not inducible by dendrin (Figures 6A and 6B), demonstrating specificity of dendrin for CatL. Without wishing to be bound by theory, it was speculated that dendrin might directly bind to the CatL promoter to act as a transcription factor. To identify the dendrin-binding site on the CatL promoter, the effect of dendrin was studied, on SEAP transcription from partial deletion constructs of the CatL promoter, containing either bp -1 ,224 to -49 or bp -^09 to -49 (Figure 6C). Whereas the former construct did not significantly differ from the full-length promoter, the latter lost baseline activity as well as its inducibility by dendrin (Figure 6B), thus mapping the putative dendrin binding site to a region between -1 ,224 and -409 of the CatL promoter. Using EMSA with DNA fragments of decreasing length, direct binding of dendrin to the CatL promoter was detected and fine- mapped the dendrin binding site to a 24-bp region (Figures 6D and 6E). Introduction of a 3-bp mutation in the 5'-terminal part of this region, but not in the middle or the 3'-terminal part, completely abolished dendrin binding (Figure 6C). Together, these analyses demonstrated that nuclear dendrin directly binds to a specific region of the CatL promoter and acts as a

transcription factor.

[0167] Cytosolic CatL proteolytically processes CD2AP. Destruction of the SD is a common signature event that occurs literally in all proteinuric glomerular diseases. Given the

observations downstream of CD2AP deletion, it was sought to determine whether loss of CD2AP could be the starting point in many renal diseases at the time the SD is affected. Without wishing to be bound by theory, it was hypothesized that one method of CD2AP destruction could be through proteolysis, possibly by CatL, closing the regulatory loop. It was tested whether CatL, in addition to dynamin and synaptopodin, might target CD2AP that contains CatL cleavage sites within its AA sequence.

[0168] To examine whether CatL can proteolyze CD2AP, N-terminal GFP-tagged and C- terminal FLAG-tagged recombinant CD2AP constructs were generated. Incubation of recombinant CD2AP with purified CatL at pH 4.5 and pH 5.5 led to a complete digestion of the protein (Figure 7A), in accordance with unspecific proteolytic activity of CatL at lysosomal pH. However, at neutral pH 7.0, an approximately 55-kDa N-terminal fragment was detected using anti-GFP or N-terminal anti-CD2AP antibodies (Figure 7A and Figure 12). Although the prediction of endopeptidase substrates algorithm (Lohmuller T, et al. Biol Chem.

2003;384(6):899-909) predicted 1 1 putative CatL cleavage sites within the CD2AP protein, data from the cleavage assay suggested that CatL recognizes the QPLGS sequence situated between the second and third domains of src homology domain 3 (Figure 7B). To further test the full spectrum of CD2AP cleavage by CatL, additional proteolytic experiments were performed. A 32-kDa C-terminal CD2AP fragment (termed p32) was also detected by C-terminal anti-CD2AP antiserum (Figure 7C, lanes 2-4, and Figure 12), suggestive of cleavage through the site LSAAE (Figure 7D). To better define the relevance of these CD2AP cleavage fragments, the generation of CD2AP cleavage fragments was tested in cells. For these experiments, WT Ctsl mRNA (which generates both lysosomal and cytosolic CatL protein) or a CatL construct that contains a deletion of the first AUG site and thus encodes selectively for cytosolic CatL were used.

Cytosolic CatL alone was sufficient to yield p32, which could not be detected using anti-FLAG antibody (Figure 7E). Generation of p32 was prevented by coincubation of transfected HEK 293 cells with E64 (Figure 7E). To determine which cleavage sites are required for p32 generation, a C-terminal FLAG-tagged CD2AP fragment (71 kDa) as well as a C-terminal FLAG-tagged CD2AP mutant with deleted LSAAE site (Figure 7, C and D) were expressed in HEK 293 cells and immobilized on FLAG beads before digestion with purified CatL enzyme. Whereas WT CD2AP was proteolyzed to p32, deletion of LSAAE protected it from CatL cleavage (Figure 7F). Of note, the deletion of QPLGS did not prevent generation of p32, which evidences that LSAAE is the more critical site within CD2AP that is being targeted by cytosolic CatL.

[0169] To learn about the functional relevance of partially degraded CD2AP, the known interactions that CD2AP undergoes with synaptopodin (Huber TB, et al. J Clin Invest. 2006; 1 16(5): 1337-1345), the SD protein nephrin (Huber TB, et al. Mol Cell Biol. 2003; 23(14):4917- 4928), and dendrin (Asanuma , et al. Proc Natl Acad Sci U S A. 2007; 104(24): 10134-10139) were analyzed. Co-immunoprecipitation studies were performed with GFP- and FLAG-tagged protein combinations expressed in HE 293 cells. Both the N-terminal fragment and p32 were still able to bind synaptopodin and nephrin, but only N-terminal CD2AP bound dendrin (Figure 7G). These data evidence that loss of CD2AP as a result of proteolysis would trigger a cellular cascade similar to that in Cd2ap^~/" podocytes, which is characterized by the dendrin translocation into the nucleus. In line with the biochemical data, loss of protein levels were detected for CD2AP in podocytes treated with LPS (Figure 13A), which increases cytosolic CatL activity (Sever S, et al. J Clin Invest. 2007; 1 17(8):2095-2104). Downregulation of CD2AP was also observed in the soluble fraction of isolated glomeruli from mice treated with LPS (Figure 13B). There was a decrease in staining for CD2AP in glomeruli of LPS-treated mice (Figures 13C and 13D). Finally, in agreement with the data in cells (Figures 3C and 3D), there was an increase in CatL activity in soluble extracts generated from isolated glomeruli of LPS-treated mice (Figure 13E). Together, these data provide further evidence that activity of cytosolic CatL underlies podocyte injury through degradation of CD2AP. The LPS model, in contrast to the Cd2ap^~/~ model (Figure 1 G), was reversible, as CatL induction was transient and without significant podocyte TGF-βΙ induction. Thus, time-limited LPS-ind ced CatL expression and subsequent downregulation of CD2AP did not lead to significant dendrin translocation into the nucleus (Figure 13F), which could explain the reversibility of the process. This is in line with dendrin knockout mice developing proteinuria after LPS treatment, as the initial CatL increase was dendrin independent (Figures 13G and 13H).

[0170] Having analyzed the role of CD2AP as gatekeeper of the TGF-β Ι response in podocytes, CD2AP expression was also analyzed in the human progressive glomerular disease focal segmental glomerulosclerosis (FSGS) and compared it with the nonprogressive minimal change disease (MCD; Figures 7H and 71). No reduction in CD2AP staining was detected in glomeruli of patients with MCD (Figure 71), which is caused by angiopoietin-like 4 protein (Clement LC, et al. Nat Med. 201 1 ; 17( 1 ): 1 1 7- 122) or by increased c-mip expression (Zhang SY, et al. Sci Signal. 2010;3(147):ra39). In contrast, glomeruli in human progressive FSGS, which have increased CatL expression (Sever S, et al. J Clin Invest. 2007; 1 17(8):2095-2 I 04), showed a prominent loss of N-terminal CD2AP (Figure 71) when using the anti-N-terminal CD2AP antisera, but not when using the anti-C-terminal CD2AP antisera (Figure 12). Together, these data evidence that loss of CD2AP (genetic and proteolytic) is a key upstream event in the development of progressive glomerular diseases such as FSGS.

[0171] Discussion

[0172] Here a proteolytic program under control of a functional SD was identified.

According to this regulatory system, the intact SD requires full-length CD2AP that keeps transcription factors, such as dendrin, at the plasma membrane (Figures 8A, 8B). Injury to the SD by mutations in CD2AP, genetic deletion, or enzymatic destruction allow for translocation of dendrin from the plasma membrane to the nucleus (Figures 8A, 8B). Nuclear dendrin binds the CatL promoter and turns on sustained expression of cytosolic CatL. CatL-mediated podocyte injury has 2 components: (a) reorganization of the podocyte actin cytoskeleton owed to the proteolytic downregulation of dynamin, synaptopodin, and RhoA (e.g., loss of FAs and stress fibers), which underlies FP effacement, and (b) decreased podocyte survival caused by decreased TGF-β threshold. Thus, the injured podocytes become hypersensitive to TGF-β proapoptotic signals, and TGF-p^-driven podocyte death promotes progression of kidney diseases. The above data also showed that cytosolic CatL was capable of regulating sustained expression of itself by degrading CD2AP, which explains why restoration of podocyte structure and function in the clinic is often time-limited with more damage to occur as the injury persists. Because podocyte injury is directly linked to proteinuria, it is generally assumed that proteinuria is the cause for increasing kidney damage. The data herein, however, argue that a main reason for progressive renal disease lies in the cellular injury mechanism of podocytes, rather than in proteinuria.

[0173] Role for TGF-βΙ signaling in regulating cytosolic CatL expression in podocytes. TGF-β Ι is a pleotropic cytokine that has been previously implicated in pathogenesis of renal fibrosis and, ultimately, end-stage kidney diseases. The TGF-β isoforms (TGF^ 1-TGF-P3) are widely expressed and act on virtually every cell type in mammals by engaging a ubiquitous intracellular signaling cascade of Smad family proteins through ligand-induced activation of heteromeric transmembrane TGF-β receptor kinases. In addition, TGF-β receptors can activate Smad-independent signaling mechanisms, including MAP s and PI3K (Derynck R, et al.

Nature. 2003; 425(6958):577-584). However, molecular mechanisms of activation of Smad- independent pathways by TGF-β receptors are still not fully defined. It has been shown that loss of the adaptor protein CD2AP leads to increased expression of TGF-βΙ in podocytes, where its autocrine effects lead to activation of proapoptotic p38MAP as well as inhibition of the antiapoptotic PI3 /AKT pathway. Here, progression to end-stage kidney disease and, ultimately, death in mice lacking CD2AP were attributed to increased TGF-βΙ -mediated podocyte death.

[0174] The results herein, showed that loss of CD2AP in podocytes, and the subsequent increase in TGF-βΙ expression, led to translocation of the SD protein dendrin from the plasma membrane into the nucleus. Nuclear dendrin then acted as transcription factor for cytosolic CatL. Thus, this study identified yet another role for TGF-βΙ signaling in podocytes: regulation of CatL expression. These results evidence that loss of direct interactions between CD2AP and dendrin at the SD facilitated dendrin translocation in a TGF-βΙ -dependent manner (Figures ID and 1 H). In agreement with this rationale, high levels of TGF-β Ι were not sufficient to induce significant dendrin translocation into the nucleus in WT podocytes (Figure 2B), nor did TGF-βΙ induce high levels of Ctsl mRNA expression (Figure 2D). These data also showed that the presence of CD2AP protected podocytes from the effects of high levels of TGF-βΙ signaling with respect to dendrin translocation and, thus, cytosolic CatL expression. Together, these data suggest that CD2AP plays the role of a gatekeeper with respect to effects of TGF-βΙ signaling on podocytes. This is particularly interesting given that dose-dependent effects of TGF-βΙ determine podocyte function under various conditions, and losing a controlled cellular intake ultimately results in podocyte death (Schiffer M, et al. J Clin Invest. 2001 ; 108(6):807-816). In line with the observation in podocytes that link Smad-independent prosurvival signaling of TGF-βΙ with CD2AP, the data evidence that loss of CD2AP switches the cellular balance from prosurvival to proapoptotic via induction of cytosolic CatL. The gatekeeping effect on podocyte TGF-βΙ by CD2AP was compromised in the face of high intrarenal TGF-βΙ production (Figure 1 A), as this was sufficient to translocate dendrin to the nucleus and increase expression of CatL. The data therefore identified a direct link between TGF-βΙ signaling in podocytes and cytosolic CatL levels. In addition, the findings herein, identified a parallel signaling pathway from the SD to the actin cytoskeleton, which was mediated by interactions between CD2AP and dendrin.

[0175] Role of cytosolic CatL for podocyte apoptosis. Cytosolic CatL degrades dynamin and synaptopodin (Sever S, et al. J Clin Invest. 2007; 1 17(8):2095-2104; Faul C, et al. Nat Med. 2008; 14(9):931-938). Loss of synaptopodin has been associated with downregulation of RhoA. Downregulation of dynamin and RhoA signaling results in loss of stress fibers and FAs, the hallmark of effaced podocytes. Dynamin can regulate actin cytoskeleton in podocytes independently and in parallel to RhoA signaling. Thus, it seems as if cytosolic CatL specifically targets both pathways involved in regulating turnover of FAs and stress fibers. The role of cytosolic CatL in podocyte injury was discovered using the LPS injury model (Sever S, et al. J Clin Invest. 2007; 1 1 7(8):2095— 2104). Importantly, LPS-induced proteinuria is reversible, and does not lead to progressive glomerular injury (Reiser J, et al. J Clin Invest. 2004; 1 13(10): 1390- 1397). Based on the LPS model, FP effacement and proteinuria in itself do not necessarily lead to progressive kidney injury.

[0176] The present findings data evidence that down regulation of dynamin, synaptopodin, RhoA, and CD2AP drives the major reorganization of the actin cytoskeleton, FP effacement; and proteinuria, but not podocyte death. Indeed, high-Tgfbl Cd2ap^~/~ podocytes did not exhibit significantly elevated basal level of apoptpsis compared with WT podocytes (Figure 51).

However, these cells did exhibit hypersensitivity to TGF- i-induced apoptosis. Together, these data evidence that cytosolic CatL-induced FP effacement and proteinuria occur before podocyte death and might represent a repair stage, in which cell structure is given up in order to survive. However, the repair process is timed, because as shown here, effacement was associated with a heightened level of susceptibility to TGF-pi-mediated apoptosis. Thus, the sustained presence of cytosolic CatL, coupled with increased levels of TGF-βΙ in podocytes, drives podocyte apoptosis and progression to end-stage kidney diseases, as observed in TGF-βΙ Tg and Cd2ap^~'^~ mice (Figures 1 B-1 H). In contrast, if injury signal does not lead to increase in TGF-βΙ signaling in podocytes, as in the case of reversible LPS-induced proteinuria, podocytes have the capability to switch off CatL expression and restore cellular structure and function. This model was corroborated with the observation of high levels of Ctsl mRNA and CatL protein, as well as degraded CD2AP (Figures 7H and 71), in patients with diverse progressive kidney diseases such as FSGS, diabetic nephropathy, and membranous nephropathy, but not in those with MCD. The studies herein, evidence that progression to C D is most likely driven by the sustained presence of cytosolic CatL.

[0177] Role of dendrin in progressive kidney diseases. Although the presence of dendrin in the nucleus has been associated with a proapoptotic phenotype, its exact role in this process has not, prior to the studies herein, been identified. The results herein, argue for a direct correlation between the presence of nuclear dendrin and the expression of cytosolic CatL. Expression of lysosomal and extracellular CatL is regulated by transcription factors such as ZHX proteins. The results herein, identified dendrin as a transcription factor specifically driving expression of cytosolic CatL. The dendrin-dependent increase in Ctsl mRNA in podocytes translated to a dramatic increase in cytosolic CatL expression, without increased levels of lysosomal CatL (Figures 2E and 2F). The molecular mechanism that couples increased transcription by dendrin with initiated translation from the downstream AUG sites is clearly operational in podocytes. What mechanism switches off CatL expression, as in a case of LPS-induced CatL expression, remains an open question that needs to be pursued in the future.

[0178] Regardless of the exact mechanism by which dendrin drives expression of cytosolic CatL, the surprising discovery that dendrin was a transcription factor for CatL uncovered the physiological connection between the SD (i.e., CD2AP) and the regulation of podocyte cytoskeleton as well as cellular survival. This functional proteolytic system ties the structure of podocytes with their survival properties in which the input level of TGF-βΙ acts as a modifier. Together, the above data establish the mechanistic base for progressive podocyte disease and provide a rationale as to why proteinuric kidney diseases are generally more prone to progression and podocyte depletion. Since TGF-βΙ not only has deleterious effects on podocytes, but is also part of physiological responses, one could assume that at normal levels, it allows some dendrin to activate the CatL promoter to produce small amount of cytosolic CatL that is present to help regulate the podocyte physiological dynamic of the FP cytoskeleton (Figures 10A-10H).

However, persistent high TGF-βΙ input into podocytes likely drives aggravating glomerular injury through dendrin translocation. This study also showed that downregulation of dendrin or CatL was sufficient to overcome TGF-β Ι -mediated susceptibility to apoptosis. In agreement with these findings, stabilizing the phenotype in Cd2ap^~'^~ mice can be achieved by crossing them with mice deficient in dendrin, resulting in delayed proteinuria onset and improved survival, which suggests that the events of FP effacement and podocyte apoptosis can be separated by specific interventions. The results of this study allows for the conceptualization of strategies for renal protection that work in concert with antiproteinuric modalities by aiming at rescuing podocyte survival. Combining both approaches would result in improved human health.

[0179] Tables 1 through 3 contain sequences set forth as SEQ ID NOS: 1 through 21. Table S1. Lenaviral shRNA plasmids.

Table S2. Primers for quantitative PCR.

Table S3. Sixty and 24 bp oligonucleotides on CatL promoter.

Claims

What is claimed is:

1. A pharmaceutical composition comprising a therapeutically effective amount of an agent which modulates cathepsin L (CatL) transcription in vitro or in vivo, as measured by TGF-βΙ, dynamin, synaptopodin and/or R oA expression in ceils or tissues as compared to a normal baseline control.

2. The pharmaceutical composition of claim 1, wherein the agent modulates cathepsin L expression, function, activity or cytosolic translocation in a cell in vitro or in vivo.

3. The pharmaceutical composition of claim 2, wherein the agent modulates expression of CD2AP in vitro or in vivo,

4. The pharmaceutical composition of claim 3, wherem the agent modulates CD2AP- mediated cellular signaling in vitro or in vivo.

5. The pharmaceutical composition of claim 1, wherein the agent modulates expression of TGF-βΙ in vitro or in vivo.

6. The pharmaceutical composition of claim 1 , wherein the agent modulates dendrin expression, function or activity and/or nuclear localization of dendrin in vitro or in vivo.

7. The pharmaceutical composition of claim 6, wherem the agent modulates binding of dendrin to a CatL promoter in vitro or in vivo.

8. The pharmaceutical composition of claim 1 , wherem the agent modulates binding of dendrin to cathepsin L, cathepsin L transcription factors, or cathepsin L transcription or transcri tion regulatory domains in vitro or in vivo.

9. The pharmaceutical composition of claim 1, wherein the agent binds to a CatL promoter and modulates transcription of CatL.

10. The pharmaceutical composition of claim 9, wherein the agent modulates cytosolic Cat L expression and/or cytosolic CatL localization.

11. The pharmaceutical composition of clai 1 , wherein the CatL molecule cleaves dynamin, synaptopodin and RhoA proteins and decreases the expression thereof as compared to normal baseline controls.

12. The pharmaceutical composition of claim 1, wherein the agent comprises: a small molecule, amino acid, oligonucleotide, polynucleotide, peptide, polypeptide, amino acid analogs, nucleic acid analogs, enzymes, antibodies, organic compound, inorganic compound, peptide nucleic acid, natural or synthetic compounds.

13. The pharmaceutical composition of claim 1, wherein the agent is an antibody, an aptamer, an interference RNA, synthetic peptides, natural peptides, or small molecules.

14. A method of treating kidney diseases or disorders in vivo compri sing administering to a patient in need thereof, a therapeutically effective amount of one or more agents which modulate dendrin and/or cathepsin L (CatL) activity, expression or function; and,

treating kidney diseases or disorders.

15. The method of claim 14, wherein a therapeutically effective amount inhibits CatL-mediated cleavage of dynamin, synaptopodin and/or RhoA expression in podoeytes compared to a normal baseline control.

16. The method of claim 14, wherein the agent inhibits expression or activity of TGF-βΙ in podoeytes compared to a normal baseline control.

17. The method of claim 14, wherein the agent modulates expression of CD2AP in a patient.

18. The method of claim 17, wherein the agent modulates CD2A -mediated cellular signaling in a patient.

19. The method of claim 14, wherein the agent modulates dendrin nuclear localization of dendrin in a patient.

20. The method of claim 14, wherein the agent modulates binding of dendrin to a CatL promoter in a patient.

21. The method of claim 20, wherein the agent modulates binding of cathepsin L transcription factors, or cathepsin L transcription or transcription regulatory domains in a patient.

22. The method of claim 14, wherein the agent binds to a CatL promoter and modulates transcription of CatL.

23. The method of claim 14, wherein the agent modulates cytosolic Cat L expression and/or cytosolic CatL localization.

24. The method of claim 14, wherein the agent comprises: a small molecule, amino acid, oligonucleotide, polynucleotide, peptide, polypeptide, amino acid analogs, nucleic acid analogs, enzymes, antibodies, organic compound, inorganic compound, peptide nucleic acid, natural or synthetic compounds.

25. The pharmaceutical composition of claim 14, wherein the agent is an antibody, an interference RNA or small molecule.

26. The method of claim 14, wherein one or more agents are optional ly administered to a patient as part of a therapeutic regimen comprising one or more therapeutic compounds for treating kidney disease, disorders or symptoms thereof.

27. The method of claim 14, wherein the kidney disease or disorder comprises: podocyte diseases or disorders, proteinuria, glomerular diseases, progressive glomerular disease membranous glomerulonephritis, focal segmental glomerulonephritis, minimal change disease, nephrotic syndromes, pre-eelampsia, eclampsia, kidney lesions, collagen vascular diseases, stress, strenuous exercise, benign orthostatic (postural) proteinuria, focal segmental

glomerulosclerosis (FSGS), IgA nephropathy, IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, sarcoidosis, Alport's syndrome, diabetes mellitus, kidney damage due to drugs, Fabry's disease, infections, aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, Sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, diabetic nephropathy (D^'N), lupus nephritis, Wegener's

Granulomatosis or Glycogen Storage Disease Type 1.

28. A vector comprising an RNA interference molecule specific for dendrin or cathepsin L (CatL),

29. The vector of claim 28, wherein the dendrin specific RNA interference molecule comprises a sequence set forth as SEQ ID NOS: 4 on.

30. The vector of claim 28, wherein the CatL specific RNA interference molecule comprises a sequence set forth as SEQ ID NOS: 1, 2 or 3.

31. An oligonucleotide of a cathepsin L promoter comprising a sequence set forth as SEQ ID NOS: 15, 16, 17, 18, 19, 20 or 21.

32. An antibody specific for epitopes comprising SEQ ID NOS: 22 or 23.

33. A method of protecting a ceil from TGF-βΙ, comprising contacting a cell with a

therapeutically effective amount of an agent which modulates cathepsin L (CatL) transcription in vitro or in vivo, as measured by TGF-βΙ, dynamin, synaptopodm and/or RhoA expression in podocytes compared to a normal baseline control.

34. A method of modulating cathepsm L in a patient in vitro or in vivo, comprising

administering to a patient a therapeutical ly effective amount of an agent which modulates cathepsin L (CatL) transcription in vitro or in vivo, as measured by TGF-βΙ , dynamin, synaptopodin and/or RhoA. expression in cells or tissues compared to a nonnal baseline control.

35. The method of claim 34, wherein the agent modulates cathepsin L expression, function, activity or cytosolic translocation in a ceil in vitro or in vivo.

36. The method of claim 34, wherein the agent modulates expression, function or acti vity of CD2AP in vitro or in vivo.

37. The method of claim 34, wherein the agent modulates expression of TGF-βΙ in vitro or in vivo.

38. The method of claim 34, wherein the agent modulates dendrin expression, function or acti vity and/or nuclear localization of dendrin in vitro or in vivo.

39. The method of claim 34, wherein the agent modulates binding or interaction of dendrin to a CatL promoter and/or inhibits assembly of transcription factors associated with CatL

transcription in vitro or in vivo.

40. The method of claim 34, wherein the agent modulates binding of dendrin to cathepsin L, cathepsin L transcription factors, or cathepsin L transcription or transcription regulatory domains in vitro or in vivo.

41. The method of claim 34, wherein the agent binds to a CatL promoter and modulates transcription of CatL.

42. The method of claim 34, wherein the agent modulates cytosolic Cat L expression and/or cytosolic CatL localization.

43. The method of claim 34, wherein the agent compri ses: a small molecule, amino acid, oligonucleotide, polynucleotide, peptide, polypeptide, amino acid analogs, nucleic acid analogs, enzymes, antibodies, organic compound, inorganic compound, peptide nucleic acid, natural or synthetic compounds.

44. The method of claim 34, wherein the agent is an antibody, an aptamer, an interference RNA, synthetic peptides, natural peptides, or small molecules.

45. A pharmaceutical composition comprising a therapeutically effective amount of an agent which modulates dendrin activity, expression or function in vitro or in vivo, as measured by cathepsm L (CatL.) transcription in a cell as compared to a normal baseline control.

46. The pharmaceutical composition of claim 45, wherein the agent modulates dendrin expression, function or activity and/or nuclear localization of dendrin in vitro or in vivo.

47. The pharmaceutical composition of claim 45, wherein the agent modulates binding of dendrin to a CatL promoter in vitro or in vivo.

48. The pharmaceutical composition of claim 45, wherein the agent modulates binding of dendrin to cathepsm L, cathepsm L transcription factors, or cathepsm I. transcription or transcription regulatory domains in vitro or in vivo.

49. The pharmaceutical composition of claim 45, wherein the agent modulates binding or interaction of dendrin to a CatL promoter and/or inhibits assembly of transcription factors associated with CatL transcription in vitro or in vivo.

50. The pharmaceutical composition of claim 45, wherein the agent comprises: a small molecule, amino acid, oligonucleotide, polynucleotide, peptide, polypeptide, amino acid analogs, nucleic acid analogs, enzymes, antibodies, organic compound, inorganic compound, peptide nucleic acid, natural or synthetic compounds.

51 . The pharmaceutical composition of claim 45, wherein the agent is an antibody, an aptamer, an interference RNA, synthetic peptides, natural peptides, or small molecules.