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WO2012068344A2 - Régulation de cathepsine l par la dendrine de son facteur de transcription - Google Patents

Régulation de cathepsine l par la dendrine de son facteur de transcription Download PDF

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WO2012068344A2
WO2012068344A2 PCT/US2011/061142 US2011061142W WO2012068344A2 WO 2012068344 A2 WO2012068344 A2 WO 2012068344A2 US 2011061142 W US2011061142 W US 2011061142W WO 2012068344 A2 WO2012068344 A2 WO 2012068344A2
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catl
dendrin
expression
cd2ap
agent
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WO2012068344A3 (fr
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Jochen Reiser
Sanja Sever
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University of Miami
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University of Miami
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Definitions

  • 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.
  • CKD chronic kidney disease
  • 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.
  • 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.
  • FPs interdigitating foot processes
  • FPs interdigitating foot processes
  • 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.
  • FP effacement causes proteinuria, the first clinical sign in CKD.
  • FP effacement causes proteinuria, the first clinical sign in CKD.
  • 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- ⁇ ), dendrin and the like.
  • CD2AP CD2-associated protein
  • TGF- ⁇ Transforming Growth Factor-beta 1
  • dendrin dendrin
  • 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.
  • Figures 1 A- 1 H show that the high level of TGF- ⁇ ⁇ induces expression of CatL.
  • Figure 1 A is a graph showing the levels of Tgfbl, Ctsl, and control gene in glomeruli of TGF- ⁇ ⁇ 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- ⁇ ⁇ Tg mice. Kidneys of WT and TGF- ⁇ 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- ⁇ ⁇ Tg animals (arrows). Kidneys of WT and TGF- ⁇ 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
  • 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-73 ⁇ 4 3 ⁇ 47 Cd2ap ⁇ ' ⁇ cells. Note the loss of well-defined stress fibers and dramatic increase in number of transverse. arcs in high-7g/3 ⁇ 47 Cd2ap ⁇ / ⁇ cells. FAs and F-actin were visualized with anti-paxillin antibodies and rhodamine-phalloidin, respectively.
  • FIG. 1 Low-Tgfbl Cd2ap ⁇ / ⁇ podocytes could be transformed into high-Tgfbl Cd2ap ⁇ / ⁇ podocytes by high TGF- ⁇ levels.
  • WT and low-7g/3 ⁇ 47 Cd2ap ⁇ ' ⁇ passage cells were treated with 5 ng/ml TGF- ⁇ 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.
  • FIG. 2E is a blot showing the subcellular fractionation of low- and high-7g/3 ⁇ 4/ Cd2ap ⁇ ' ⁇ podocytes in isotonic sucrose.
  • S soluble
  • P particulate
  • Lamp-2 and mannosidase Manno
  • 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 ' ⁇ .
  • FIGS. 3A-3G show that cytosolic CatL activity regulates actin cytoskeleton in
  • FIG. 3A is a graph showing Ctsl mRNA levels, determined by RT-PCR, in high-7g/3 ⁇ 47 Cd2ap ⁇ ' ⁇ podocytes infected with different shRNA constructs (C2, C5, C6) downregulating endogenous CatL. Con, high-73 ⁇ 4/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-73 ⁇ 4/3 ⁇ 47 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/3 ⁇ 47 Cd2ap ⁇ / ⁇ podocytes and in high-7g/3 ⁇ 47 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/3 ⁇ 47 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 3 ⁇ 47 Cd2ap ⁇ ' ⁇ podocytes, and high- 7g/3 ⁇ 47 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.
  • Figures 4A and 4B show number of FAs within WT podocytes and high-7g 3 ⁇ 47
  • FIG. 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
  • 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.
  • 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-73 ⁇ 4 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.
  • FIG 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 ⁇ .
  • FIGS 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.
  • dNLS nuclear localization signal
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ .
  • 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.
  • 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- ⁇ for 24 hours.
  • Figure 9C is a graph showing that CatL mRNA levels are upregulated only in CD2AP " "(H,gh TGFp) podocytes.
  • 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.
  • FIG. 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
  • FIGS 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.
  • 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.
  • FIG. 10E 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.
  • 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.
  • CD2AP " ⁇ (H i gh TGF ) poc j OC ytes 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 ⁇ each) for 24 hours prior to starting the assay.
  • 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).
  • 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:
  • 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 13H are graphs showing the Effect of LPS on TGF- ⁇ ⁇ (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.
  • Embodiments of the present invention relate to compositions which regulate the cathepsin L (CatL) expression, function or activity.
  • 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- ⁇ , CD2-associated protein (CD2AP) or dendrin.
  • 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.
  • 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.
  • 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.
  • the genes or nucleic acid sequences are human.
  • determining means 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.
  • 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.
  • 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).
  • 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.
  • a patient in need thereof refers to any patient that is affected with a disorder characterized by proteinuria.
  • a patient in need thereof refers to any patient that may have, or is at risk of having a disorder characterized by proteinuria.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • other therapeutic agents e.g., radiation and/or chemotherapy.
  • 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.
  • a "therapeutically effective" amount of a compound 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • oligonucleotide 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.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • 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.
  • the oligonucleotide may be "chimeric,” that is, composed of different regions.
  • "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.
  • 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.
  • 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.
  • 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.
  • nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 - ethanocytosin, N 6 ,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.
  • 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.
  • nucleoside includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Romberg and Baker, DNA Replication, 2nd Ed.
  • nucleosides 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
  • Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
  • the term "gene” means the gene and all currently known variants thereof and any further variants which may be elucidated.
  • 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).
  • variants 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.
  • SNPs single nucleotide polymorphisms
  • 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.
  • mRNA means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which may be elucidated.
  • RNA molecules 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.
  • 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).
  • 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)).
  • the mediators of RNA-dependent gene silencing are 21 -25 nucleotide "small interfering" RNA duplexes (siRNAs).
  • 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).
  • RISC RNA Induced Silencing Complex
  • 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).
  • nt nucleotides
  • 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.
  • RNAi 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.
  • 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.
  • enzymatic RNA 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.
  • 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.
  • 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.
  • TAR HIV trans-activation response
  • TAR HIV trans-activation response
  • 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.
  • 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.
  • the complementary sequence of the oligonucleotide has at least 80% or 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence.
  • alleles or variants thereof can be identified.
  • a BLAST program also can be employed to assess such sequence identity.
  • 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.
  • 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.
  • duplex or triplex formation 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 ⁇ .
  • a stable duplex or triplex may in some embodiments include mismatches between base pairs and/or among base triplets in the case of triplexes.
  • modified oligonucleotides e.g.
  • LNA units comprising LNA units, of the invention form perfectly matched duplexes and/or triplexes with their target nucleic acids.
  • 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.:
  • compositions that embodied comprise agents which modulate cathepsin L or any molecules associated with the pathways leading to kidney disease.
  • agents which modulate cathepsin L or any molecules associated with the pathways leading to kidney disease are directed to compositions modulating the expression, function or activity of cathepsin L (CatL).
  • CatL cathepsin L
  • 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.
  • 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.
  • CD2-associated protein CD2-associated protein
  • TGF- ⁇ Transforming Growth Factor-beta 1
  • dendrin agents which modulate CD2-associated protein
  • CD2AP CD2-associated protein
  • SDs slit diaphragms
  • 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- ⁇ .
  • 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- ⁇ transgenic mice.
  • CD2AP CD2-associated protein
  • 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.
  • 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.
  • CatB cathepsin B
  • CD2AP _ podocytes exhibited an increased susceptibility to TGF- ⁇ induced apoptosis, which was also rescued by dendrin shRNA, CatL shRNA or CatL inhibitor treatment.
  • TGF- ⁇ itself induces nuclear relocation of dendrin
  • CatL expression was also increased in TGF- ⁇ ⁇ 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.
  • a pharmaceutical composition comprises a therapeutically effective amount of an agent which modulates cathepsin L (CatL) transcription in vitro or in vivo.
  • CatL cathepsin L
  • the inhibition or up-regulation of CatL can be measured by the levels of TGF- ⁇ ⁇ , dynamin, synaptopodin and/or RhoA expression in podocytes compared to a normal baseline control.
  • 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.
  • 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).
  • 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- ⁇ .
  • CD2AP acts as the gatekeeper of the podocyte TGF- ⁇ response through its regulation of CatL expression.
  • the pharmaceutical composition comprises an agent which modulates expression of TGF- ⁇ in vitro or in vivo.
  • the agent modulates dendrin expression, function or activity and/or nuclear localization of dendrin in vitro or in vivo.
  • the agent modulates binding of dendrin to a CatL promoter in vitro or in vivo.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CatL dendrin and/or cathepsin L
  • a therapeutically effective amount inhibits CatL-mediated cleavage of dynamin, synaptopodin and/or RhoA expression in podocytes compared to a normal baseline control.
  • the agent inhibits expression or activity of TGF- ⁇ in podocytes compared to a normal baseline control.
  • 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.
  • 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.
  • an agent modulates dendrin function, activity or expression. In some aspects the agent inhibits dendrin function, expression, or activity.
  • an agent modulates CatL expression, function or activity.
  • 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.
  • the CatL specific RNA interference molecule comprises a sequence set forth as SEQ ID NOS: 1 , 2 or 3, homologs or orthologs thereof.
  • the species is an animal, preferably, a human.
  • the agent is an antibody specific for epitopes comprising SEQ ID NOS: 22 or 23, homologs or orthologs thereof.
  • the sequences are specific for a human.
  • 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.
  • 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.
  • 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
  • FSGS focal segmental glomerulosclerosis
  • 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 .
  • 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.
  • the podocyte-related disease or disorder can be effacement or a diminution of podocyte density.
  • 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.
  • the podocyte-related disease or disorder can be due to a podocyte injury.
  • 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
  • a medication e.g.
  • ischemia can be sickle-cell anemia, thrombosis, transplantation, obstruction, shock or blood loss.
  • 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.
  • 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.
  • 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.
  • the podocyte-related disease or disorder can be tubular atrophy.
  • 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.
  • 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.
  • 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- ⁇ 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.
  • small molecules test compounds can initially be members of an organic or inorganic chemical library.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a linear combinatorial chemical library such as a polypeptide library
  • a “library” may comprise from 2 to 50,000,000 diverse member compounds.
  • 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.
  • “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.
  • 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%.
  • 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);
  • 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.
  • agents may also be used which compete for binding sites or signaling sites.
  • 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.
  • 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.
  • the agent inhibits dendrin expression (e.g. by siRNA) in order to inhibit its transcriptional activity in the nucleus for CatL.
  • 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.
  • 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.
  • Interference RNA Detailed methods of producing the can be obtained using a number of techniques known to those of skill in the art.
  • 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.
  • RNAi containing a given target sequence can be evaluated using standard techniques for measuring the levels of RNA or protein in cells.
  • 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.
  • siRNA molecules target overlapping regions of a desired sense/antisense locus, thereby modulating both the sense and antisense transcripts e.g. targeting dendrin.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • small interfering RNA 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.
  • 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.
  • 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.
  • a nucleotide linker can be a linker of more than about 2 nucleotides length up to about 50 nucleotides in length.
  • the nucleotide linker can be a nucleic acid aptamer.
  • 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.
  • 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.
  • 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.
  • the invention may be used against protein coding gene products as well as nonprotein coding gene products.
  • 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.
  • siRNA oligonucleotide therapies comprise administered siRNA oligonucleotide which contacts (interacts with) the targeted mRNA from the gene, whereby expression of the gene is modulated.
  • 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.
  • the nucleobases in the siRNA may be modified to provided higher specificity and affinity for a target mRNA.
  • 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.
  • RNAi's include 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.
  • oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated
  • 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.
  • agents 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.
  • inventions 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.
  • compositions or agents identified by the methods described herein may be administered to animals including human beings in any suitable formulation.
  • 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.
  • 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).
  • the compositions are preferably formulated in a sterilized pyrogen-free form.
  • the compounds can be administered with one or more therapies.
  • the compounds can be administered with one or more therapies.
  • chemotherapeutic agents may be administered under a metronomic regimen.
  • “metronomic” therapy refers to the administration of continuous low-doses of a therapeutic agent.
  • LDs 0 the dose lethal to 50% of the population
  • 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.
  • 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.
  • the route of administration utilized for any compound used in the method of the invention.
  • 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.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of a compound 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.
  • 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.
  • compositions 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.
  • 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
  • Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
  • 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.
  • an agent to hasten drying and to cool the skin such as an alcohol or acetone
  • a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
  • 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.
  • Example J Regulation of cathepsin L by its transcription factor dendrin determines kidney disease [0129]
  • SD slit diaphragm
  • podocyte FPs mainly through human and mouse genetic studies.
  • 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.
  • CatL protease cathepsin L
  • 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.
  • cytosolic CatL 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
  • FP effacement can be inhibited by blocking CatL activity or by protection of the target proteins from CatL cleavage.
  • CD2AP a scaffolding protein
  • CD2AP a scaffolding protein
  • 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.
  • 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.
  • 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 3 ⁇ 47 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.
  • 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.
  • TGF- ⁇ Tg mice Kopp JB, et al. Lab Invest. 1996;
  • 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
  • the resulting supernatant was collected and stored as the cytosolic fraction.
  • the remaining nuclear pellet was resuspended in 50 ⁇ 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.
  • 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).
  • PEPS endopeptidase substrates
  • 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).
  • CD2AP 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 MgCl 2 , and 1 mM DTT.
  • 20 ⁇ CatL inhibitor I (Calbiochem) was added.
  • the reaction was initiated by addition of 0.5 ⁇ 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 ⁇ .
  • the reaction was terminated with addition of ⁇ 4 sample buffer (Invitrogen).
  • 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
  • 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 ⁇ g 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.
  • 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.
  • 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.
  • 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.
  • 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
  • oligonucleotides were used for competition in 200-fold excess.
  • 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.
  • 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 ⁇ 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).
  • apoptotic inducers e.g., 1 , 2 and 5 ng/ml TGF- ⁇ , 10 ng/ml actinomycin D and 100 nM angiotensin II for an additional 24 hours or 1 ⁇ staurosporine for 1 h.
  • the cells were treated with 20 ⁇ 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
  • TGF- ⁇ ⁇ signaling induces CatL expression.
  • 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- ⁇ exhibit elevated CatL expression.
  • RT-PCR real-time PCR
  • mice As glomerular disease progresses in these mice, podocyte apoptosis increases, driven in part by an increase in intraglomerular TGF- ⁇ signaling. Given the increased TGF- ⁇ ⁇ levels in podocytes lacking CD2AP at the time of proteinuria onset, these mice represent an ideal model to examine the connection between TGF- ⁇ signaling and CatL expression in podocytes. It was first examined whether increased CatL levels coincide with proteinuria onset in Cd2ap ⁇ / ⁇ animals.
  • 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.
  • 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.
  • 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- ⁇ that can act in a
  • 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).
  • 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).
  • CatB inhibitor CA074 was included in all subsequent assays. As shown in Figure 3D, downregulation of CatL in high-73 ⁇ 4 3 ⁇ 47 Cd2ap ⁇ / ⁇ cells by shRNA-C6 resulted in CatL activity similar to that observed in WT cells. Immunofluorescence and Western blot analysis
  • igh-TgfbJ Cd2ap ⁇ ' ⁇ cells were hypersensitive to proapoptotic signals such as high levels of TGF- ⁇ , 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- ⁇ -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.
  • downregulation of CatL or addition of E64 resulted in complete protection from TGF-pi-induced apoptosis of high-7g 3 ⁇ 47 Cd2ap '/" cells ( Figure 51).
  • 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.
  • SEAP secreted alkaline phosphatase
  • 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.
  • CatB promoter activity was lower at baseline and was not inducible by dendrin ( Figures 6A and 6B), demonstrating specificity of dendrin for CatL.
  • dendrin might directly bind to the CatL promoter to act as a transcription factor.
  • 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.
  • CD2AP cleavage fragments were tested in cells.
  • WT Ctsl mRNA which generates both lysosomal and cytosolic CatL protein
  • a CatL construct that contains a deletion of the first AUG site and thus encodes selectively for cytosolic CatL were used.
  • 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).
  • FSGS human progressive glomerular disease focal segmental glomerulosclerosis
  • MCD nonprogressive minimal change disease
  • 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.
  • 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- ⁇ threshold.
  • the injured podocytes become hypersensitive to TGF- ⁇ proapoptotic signals, and TGF-p ⁇ -driven podocyte death promotes progression of kidney diseases.
  • 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.
  • TGF- ⁇ ⁇ is a pleotropic cytokine that has been previously implicated in pathogenesis of renal fibrosis and, ultimately, end-stage kidney diseases.
  • the TGF- ⁇ 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- ⁇ receptor kinases.
  • TGF- ⁇ receptors can activate Smad-independent signaling mechanisms, including MAP s and PI3K (Derynck R, et al.
  • the data therefore identified a direct link between TGF- ⁇ signaling in podocytes and cytosolic CatL levels.
  • the findings herein identified a parallel signaling pathway from the SD to the actin cytoskeleton, which was mediated by interactions between CD2AP and dendrin.
  • 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.
  • 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).
  • 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).
  • FP effacement and proteinuria in itself do not necessarily lead to progressive kidney injury.
  • podocytes In contrast, if injury signal does not lead to increase in TGF- ⁇ 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.
  • TGF- ⁇ 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).
  • Tables 1 through 3 contain sequences set forth as SEQ ID NOS: 1 through 21.

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Abstract

La présente invention concerne des compositions modulant l'expression, la fonction ou l'activité de la cathepsine L. (CatL.) qui sont efficaces en tant qu'agents de protection contre la maladie ou des troubles des reins et dans le traitement de patients atteints de la maladie ou de troubles des reins. Les agents peuvent cibler diverses voies ou molécules impliquées dans l'expression, l'activité ou la fonction de CatL.
PCT/US2011/061142 2010-11-17 2011-11-17 Régulation de cathepsine l par la dendrine de son facteur de transcription Ceased WO2012068344A2 (fr)

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CN107245502A (zh) * 2017-06-14 2017-10-13 中国科学院武汉病毒研究所 Cd2结合蛋白（cd2ap）和其相互作用蛋白

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US9144594B2 (en) * 2005-11-08 2015-09-29 University Of Miami Cathepsin L mediated diseases and associated methods and products

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107245502A (zh) * 2017-06-14 2017-10-13 中国科学院武汉病毒研究所 Cd2结合蛋白（cd2ap）和其相互作用蛋白
CN107245502B (zh) * 2017-06-14 2020-11-03 中国科学院武汉病毒研究所 Cd2结合蛋白(cd2ap)和其相互作用蛋白

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