WO2014028059A1

WO2014028059A1 - Rac1 inhibitors for the treatment of alport glomerular disease

Info

Publication number: WO2014028059A1
Application number: PCT/US2013/032432
Authority: WO
Inventors: Dominic Cosgrove
Original assignee: Father Flanagans Boys Home
Current assignee: Father Flanagans Boys Home
Priority date: 2012-08-17
Filing date: 2013-03-15
Publication date: 2014-02-20
Anticipated expiration: 2015-02-17
Also published as: EP2884988A1; EP2884988A4

Description

RAC1 INHIBITORS FOR THE TREATMENT

OF ALPORT GLOMERULAR DISEASE

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Serial No.

61/684,566, filed August 17, 2012, and U.S. Provisional Application Serial No. 61/764,389, filed February 13, 2013, each of which is incorporated by reference herein in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. R01-DK55000 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

Alport syndrome (also referred to as hereditary nephritis) is a genetic disorder characterized by abnormalities in the basement membranes of the glomerulus (leading to hematuria, glomerulosclerosis, and end-stage kidney disease (ESRD)), cochlea (causing deafness), and eye (resulting in lenticonus and perimacular flecks). Alport syndrome is a primary basement membrane disorder caused by mutations in the collagen type IV COL4A3, COL4A4, or COL4A5 genes. Mutations in any of these genes prevent the proper production or assembly of the type IV collagen network, which is an important structural component of basement membranes in the kidney, inner ear, and eye. Basement membranes are thin, sheet-like structures that separate and support cells in many tissues. The abnormalities of type IV collagen in kidney basement membranes leads to irregular thickening and thinning and splitting of basement membranes, causing gradual scarring of the kidneys. Alport Syndrome causes progressive kidney damage. The glomeruli and other normal kidney structures such as tubules are gradually replaced by scar tissue, leading to kidney failure. Deafness and an abnormality in the shape of the lens called anterior lenticonus are other important features of Alport Syndrome. People with anterior lenticonus may have problems with their vision and may develop cataracts. The prevalence of Alport syndrome is estimated at approximately 1 in 5,000 births and it is estimated that the syndrome accounts for approximately 2.1 percent of pediatric patients with ESRD. Currently there is no specific treatment for Alport Syndrome; treatments are

symptomatic. Patients are advised on how to manage the complications of kidney failure and the proteinuria that develops is often treated with ACE inhibitors. Once kidney failure has developed, patients are given dialysis or can benefit from a kidney transplant, although this can cause problems. The body may reject the new kidney as it contains normal type IV collagen, which may be recognized as foreign by the immune system. Thus there is a need for improved therapeutic approaches for the treatment of Alport syndrome.

SUMMARY OF THE INVENTION

The present invention includes a method of treating Alport syndrome in a subject, the method including administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

The present invention includes a method of preventing glomerular disease progression in a subject diagnosed with Alport syndrome, the method including administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

The present invention includes a method of treating glomerulonephritis in a subject, the method including administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

The present invention includes a method of treating kidney injury due to biomechanical strain in Alport syndrome, the method including administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

The present invention includes a method of inhibiting deposition of laminin 211 in the glomerular basement membrane (GBM) in a subject, the method including administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

The present invention includes a method of inhibiting mesangial cell process invasion of the glomerular capillary loop in a kidney of a subject, the method including administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor. The present invention includes a method of inhibiting Alport glomerular pathogenesis in a subject; the method including: determining that the subject is at risk for developing Alport glomerular disease; and administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor to the subject. In some aspects, the determination that the subject is at risk for developing Alport glomerular disease is determined by family medical history, genetic testing, immunodiagnostic skin biopsy testing, and/or molecular diagnostic marker testing. In some aspects, the determination that the subject is at risk for developing Alport glomerular disease is made prior to the onset of proteinuria in the subject.

In some aspects of the methods of the present invention, the administration of an effective amount of a RACl inhibitor and/or a CDC42 inhibitor is initiated prior to the onset of proteinuria in the subject.

In some aspects of the methods of the present invention, the RACl inhibitor and/or a CDC42 inhibitor blocks CDC42 activation of the endothelin type I receptor and/or the endothelin type II receptor.

In some aspects of the methods of the present invention, the RACl inhibitor and/or a CDC42 inhibitor is an endothelin (ET) receptor antagonist. In some aspects, the endothelin (ET) receptor antagonist is a dual antagonist of both the ETA receptor and ET_B receptor.

In some aspects of the methods of the present invention, the RACl inhibitor and/or a CDC42 inhibitor is bosentan or a derivative thereof

In some aspects of the methods of the present invention, the RAC 1 inhibitor is

NSC23766 or a derivative thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A to IF. Laminin 211 localizes to the glomerular basement membrane (GBM) in Alport glomeruli. Dual immunofluorescence immunostaining was performed on wild type (Figs. 1A-1C) and Alport (Figs. ID- IF) glomeruli from 7 week 129 Sv mice. Glomerular basement membranes were labeled with labeled with anti-laminin a5 antibodies (Fig. 1 A and Fig. ID). Anti-laminin a2 immunostaining is shown in Fig. IB and Fig. IE. Note the irregular deposits of laminin 211 in the Alport GBM, especially in the thickened regions of the GBM (overlapping staining in Fig. ID and Fig. IE). Anti-laminin a2 immunostaining is not observed in the GBM of wild type mice (note the absence of overlapping immunostaining in Fig. 1 A and Fig. IB).

Figures 2A to 2L. Mesangial processes invade the capillary loops of Alport glomeruli where they co-localize with laminin 211 deposits. Dual immunofluorescence immunostaining was performed on wild type or Alport kidney sections from 7 week old 129 Sv mice. Figs. 2A- 2F show localization of laminin a2 and integrin a8 (a mesangial cell marker), and Figs. 2G-2L show localization of laminin a5 (a GBM marker) and integrin a8. Note circumferential co- localization of laminin a2 and integrin a8 in the Alport glomerulus in Figs. 2D-2F, and the co- localization of integrin a8 and laminin a5 in Figs. 2J-2L indicating invasion of the glomerular capillary tufts with mesangial processes.

Figures 3 A to 3C. Mesangial processes invade the capillary loops of human Alport glomeruli where they co-localize with laminin 511. Cryosections from human Alport kidneys were stained with antibodies specific for laminin a5 (Fig. 3A) and integrin a8 (Fig. 3B). The integrin a8-specific mesangial processes localize adjacent to the laminin 15-positive GBM, consistent with mesangial process invasion.

Figures 4 A to 4F. Hypertension exacerbates mesangial invasion of the glomerular capillary tufts in Alport mice. The X- linked Alport mouse model (on the C57 Bl/6 background) was made hypertensive by providing L-NAME salts in the drinking water from 5 weeks to 10 weeks of age. Control Alport mice were given normal drinking water. Glomeruli were analyzed by dual immunofluorescence immunostaining using antibodies against either laminin a2 or integrin a8. Extensive mesangial process invasion of the capillary tuft is observed in the glomeruli from the salt-treated mice relative to the mice given normal drinking water.

Figure 5. Extensive mesangial process invasion of the glomerular capillary tufts is observed in CD 151 knockout mice. Kidney cryosections from 8 week old wild type and CDC 151 KO mice (on the FVB background) were analyzed by dual immunofluorescence immunostaining using antibodies against either laminin a2 or integrin a8. Extensive mesangial process invasion of the capillary tuft is observed in the glomeruli from CD151 knockout mice relative to wild type mice. Note that the extent of mesangial process invasion in CD 151 knockout mice is much greater than that observed in Alport mice.

Figure 6. Biomechanical stretching of cultured primary mesangial cells induces expression of pro-migratory cytokines, CTGF and TGF-βΙ mRNA. Primary mesangial cell cultures from wild type mice were subjected to cyclic biomechanical stretching for 24 hours. R A from multiple replicates was analyzed by quantitative real time RT-PCR for CTGF and TGF-βΙ mRNA. Statistically significant increases in expression for both cytokines was observed (p<0.05).

Figure 7. l integrin deletion in Alport mice results in markedly reduced mesangial process invasion of the glomerular capillary tufts. Glomeruli from 7 week old integrin a 1 -null mice, Alport mice, and integrin a 1 -null Alport mice were analyzed by dual immunofluorescence immunostaining using antibodies against either laminin a2 or integrin a8. The degree of mesangial process invasion of the glomerular capillary tufts was greatly reduced in the integrin αΐ-null Alport mice relative to age/strain-matched Alport mice.

Figures 8 A to 8D. Integrin αΐβΐ -dependent Racl/CDC42 activation mediates dynamic remodeling of the actin cytoskeleton and mesangial process invasion of the glomerular capillary tufts. Fig. 8A shows migration of primary cultured mesangial cells is significantly reduced under conditions of integrin l deletion, Integrin linked kinase inhibition, Racl inhibition, and CDC42 inhibition, but not AKT inhibition. In contrast, the migratory potential of cultured integrin al- null mesangial cells is unaffected by inhibition of either Racl or CDC42. Migration was measured by Boyden chamber assay in the presence or absence of ILK inhibitor, QLT-0267; Racl inhibitor, NSC 23766; CDC42 inhibitor, ML141; or the pan-AKT inhibitor GSK 690693. Multiple replicate experiments were performed on multiple independent derivations of mesangial cells and the data analyzed by Students-t-test. Asterisks denote statistically significant differences relative to 10% FCS (p<0.05). Fig. 8B shows treatment of cultured mesangial cells with LPS induced cytoskeletal rearrangement with numerous actin spikes (untreated cells, A; LPS treated cells, B), and these morphological changes are blocked by treatment of cells with either Racl inhibitors (C), or CDC42 inhibitors (D). Untreated integrin αΐ-null cells did not respond to LPS treatment. Fig. 8C shows treatment of cultured mesangial cells with LPS results in polarized localization of CDC42 and associated with filopodia (Figure 8C(b), insert, compared to Golgi and cytosolic localization of CCD42 in wild type cells (8C(a)). Pre-treatment of cells with the Racl inhibitor, NSC 23766, abolished LPS-activated polarized localization of CDC42 (8C(c)), indicating cross-talk between Racl and CDC42. Fig. 8D shows a GTP-Racl pull-pull down assay which confirms LPS-mediated activation of Racl in cultured mesangial cells, that was blocked by pre-treatment with Racl inhibitors, but not CDC42 inhibitor. Figures 9A to 9F. Treatment of Alport mice with Racl inhibitors partially ameliorates mesangial cell process invasion of the glomerular capillary tufts. Alport mice on the 129 Sv background were injected once daily with either saline or the Racl inhibitor NSC 23766 from 2 weeks to 6 weeks of age. Kidney cryosections were analyzed by dual immunofluorescence immunostaining using antibodies against either laminin a2 or integrin a8. The degree of mesangial process invasion of the glomerular capillary tufts was ameliorated in the Racl inhibitor-treated mice relative to mice injected with saline.

Figures 10A to IOC. Laminin 211 potentiates mesangial process invasion of the glomerular capillary loops in Alport mice, and promotes mesangial cell migration in vitro. In Fig. 10A laminin a2-deficient Alport mice show reduced mesangial process invasion of the glomerular capillary tufts. Cryosections of kidney tissue from 8 week old laminin a2-deficient Alport mice were analyzed by dual immunofluorescence immunostaining using antibodies against either laminin a5 or integrin a8. The degree of mesangial process invasion of the glomerular capillary tufts was greatly reduced in the laminin a2-null Alport mice relative to Alport mice (compare with Figs. 2J-2L). Fig. 10B shows wild type mesangial cells migrate more robustly on laminin 211 compared to laminin 521 (GBM laminin). Wound scratch assays were performed using wild type mesangial cells cultured on wither recombinant purified laminins or commercially available laminins extracted from either placenta (primarily laminin 511) or muscle (primarily laminin 211). Images shown are representative of multiple replicates. In Fig. IOC primary mesangial cells from laminin a2-deficient mice show impaired migratory potential relative to wild type mesangial cells. Boyden chamber assays were performed. Blinded cell counts from multiple replicates were analyzed. Asterisk denotes statistically significant differences (p<0.05).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

symptomatic. Patients are advised on how to manage the complications of kidney failure and the proteinuria that develops is often treated with ACE inhibitors. Once kidney failure has developed, patients are given dialysis or can benefit from a kidney transplant, although this can cause problems. The body may reject the new kidney as it contains normal type IV collagen, which may be recognized as foreign by the immune system. Thus there is a need for improved therapeutic agents for the treatment of individuals with Alport syndrome, especially for the treatment of presymptomatic individuals, before the onset of proteinuria.

Alport syndrome is characterized by delayed onset progressive glomerulonephritis associated with sensorineural hearing loss and retinal flecks (Kashtan and Michael, 1996, Kidney Int; 50(5): 1445-1463). The most common form (80%) is X-linked and caused by mutations in the type IV collagen COL4A5 gene (Barker et al, 1990, Science; 8; 248(4960): 1224-7). The two autosomal forms of the disease account for the remaining 20% of Alport patients, and result from mutations in the COL4A3 and COL4A4 genes (Mochizuki et al, 1994, Nat Genet; 8(1):77- 81). The a3(IV), a4(IV) and a5(IV) proteins form a heterotrimer and is assembled into a subepithelial network in the glomerular basement membrane that is physically and biochemically distinct from a subendothelial type IV collagen network comprised of l(IV) and a2(IV) heterotrimers (Kleppel et al., 1992, J Biol Chem; 267(6):4137-4142). Mutations in any one of the three type IV collagen genes that cause Alport syndrome results in the absence of all three proteins in the GBM due to an obligatory association to form functional heterotrimers (Kalluri and Cosgrove, 2000, J Biol Chem; 275(17): 12719-12724). Thus, the net result for all genetic forms of Alport syndrome is the absence of the a3(IV) a4(IV) a5(IV) subepithelial collagen network, resulting in a GBM type IV collagen network comprised only of l(IV) and a2(IV) heterotrimers.

This change in basement membrane composition does not result in immediate pathology. The GBM appears to function adequately for the first few years of life and sometimes past the first decade (Kashtan et al, 1998, Pediatr Nephrol; 12(4):269-27). This delayed onset predicts a triggering mechanism for glomerular disease initiation and a theoretical window for therapeutic intervention that may arrest or significantly ameliorate Alport renal disease in its earliest stages.

Alport syndrome results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. These mutations may be either autosomal recessive (mutations in either COL4A3 or COL4A4 genes (Mochizuki et al, 1994, Nat Genet; 8(1):77-81)) or X-linked (mutations in COL4A5 (Barker et al, 1990, Science; 8;248(4960): 1224-7)).

Mutations in any of these genes results in the absence of all three collagens (a3(IV), a4(IV), and a5(IV) in the GBM type IV collagen network due to an obligatory association to form heterotrimers. The result is a thinner and less crosslinked GBM collagen network resulting in delayed onset progressive glomerulonephritis. Until the observations of the present invention, the molecular trigger for disease onset is unknown.

Alport syndrome is also known as congenital hereditary hematuria, hematuria- nephropathy-deafness syndrome, hematuric hereditary nephritis, hemorrhagic familial nephritis, hemorrhagic hereditary nephritis, hereditary familial congenital hemorrhagic nephritis, hereditary hematuria syndrome, hereditary interstitial pyelonephritis, and hereditary nephritis.

The present invention includes methods of treating Alport syndrome in a subject by the administration of a RACl inhibitor and/or a CDC42 inhibitor. The administration of a RACl inhibitor and/or a CDC42 inhibitor may result in one or more of the following: inhibiting migration of mesangial cells, inhibiting irregular deposition of mesangial laminin 211 in the GBM, inhibiting accumulation of mesangial integrin α8β1 in the capillary loops, inhibiting invasion of the capillary loops by mesangial cell processes, inhibiting mesangial filopodial invasion of the glomerular capillary tuft, and/or preventing, or slowing the onset of proteinuria. The present invention includes methods of preventing, slowing, and/or managing glomerular disease progression in a subject diagnosed with Alport syndrome by the

administration of a RACl inhibitor and/or a CDC42 inhibitor.

The present invention includes methods of treating glomerulonephritis associated with Alport syndrome in a subject by administering a RACl inhibitor and/or a CDC42 inhibitor.

The present invention includes methods of treating kidney injury due to biomechanical strain in Alport syndrome by administering a RACl inhibitor and/or a CDC42 inhibitor.

The present invention includes methods of inhibiting deposition of laminin 211 in the glomerular basement membrane (GBM) by administering a RACl inhibitor and/or a CDC42 inhibitor. The laminins are major proteins in the basal lamina, a layer of the basement membrane, a protein network foundation for most cells and organs. Laminins are trimeric proteins that contain an a-chain, a β-chain, and a γ-chain, found in five, four, and three genetic variants, respectively. The laminin molecules are named according to their chain composition. Thus, laminin-511 contains a5, βΐ, and γΐ chains (Aumailley et al., 2005, Matrix Biol;

24(5):326-32). Fourteen other chain combinations have been identified in vivo. Laminin-211 (composed of α2, βΐ and γΐ chains (Ehrig et al, 1991, PNAS; 87:3264-3268) is the main laminin isoform in skeletal muscle (Leivo and Engvall, 1988, PNAS; 85: 1544-1588; and Patton, 1997, J Cell Biol; 139: 1507-1521) and identification of laminin a2 chain mutations in a severe form of congenital muscular dystrophy (merosin-deficient congenital muscular dystrophy; MDC1A) established the importance of laminin-211 for normal muscle function (Helbling-Leclerc et al., 1995, Nat Genet; 11 :216-218). The present invention demonstrates for the first time, the role of the deposition of laminin 211 in the glomerular basement membrane (GBM) in the pathogenesis of Alport syndrome.

The present invention includes methods of inhibiting mesangial cell process invasion of the glomerular capillary loop of the kidney by administering a RACl inhibitor and/or a CDC42 inhibitor. RACl (also referred to herein as Racl) is a member of the Rac subfamily (Racl-Rac4) of the Rho family of GTPases. Members of this superfamily appear to regulate a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases. Together with Rho (regulator of stress fibers) and Cdc42 (regulator of filopodia), Rac modulates the formation of focal adhesion (FA) complexes; membrane ruffles and lamellipodia that contribute to important cell functions related to cell attachment and movement.

The methods of the present invention may be used for the presymptomatic treatment of individuals, with the administration of a RACl inhibitor and/or a CDC42 inhibitor beginning after the determination or diagnosis of Alport syndrome, by prior to the onset of symptoms, such as for, example, proteinuria. The diagnosis of Alport syndrome in an individual may be made, for example, by family medical history, genetic testing, immunodiagnostic skin biopsy testing, and/or molecular diagnostic marker testing. Such methods may be combined with a step of obtaining a diagnosis of Alport syndrome by the use of one or more such diagnostic means.

A RACl inhibitor or a CDC42 inhibitor can block the activation of RAC1/CDC42 members of the rho family of small GTPases. Any of a wide variety of RACl inhibitors may be used with the methods described herein, including, but not limited to, NSC23766 and derivatives thereof (Gao et al, 2004, PNAS; 101 :7618-7623), EHT 1864 and derivatives thereof (Shutes et al, 2007, J Biol Chem; 282:35666-35678), W56 (Gao et al, 2001, J Biol Chem; 276:47530), F56 (Gao et al, 2001, J Biol Chem; 276:47530), and any of the RACl inhibitors described by Ferri et al. (J Med Chem 2009; 52(14):4087-90) and Hernandez et al. (P R Health &/ J2010; 29(4):348- 356). In some aspects of the methods described herein, a RACl inhibitor may be NSC23766 or a derivative thereof.

Human CDC42 is a small GTPase of the Rho-subfamily, which regulates signaling pathways that control diverse cellular functions including cell morphology, migration, endocytosis and cell cycle progression. Any of a wide variety of CDC42 inhibitors may be used with the methods described herein, including, but not limited to, secramine (Pelish et al, 2006, Nat Chem Biol; 2(l):39-46), ML141 (Surviladze et al, "A Potent and Selective Inhibitor of Cdc42 GTPase," Probe Reports from the NIH Molecular Libraries Program [Internet], Bethesda (MD): National Center for Biotechnology Information (US); 2010), or an endothelin receptor antagonist, such as, for example, bosentan, ambrisentan, or derivatives thereof.

Bosentan, an endothelin receptor antagonist, is indicated mainly for the treatment of pulmonary arterial hypertension (PAH) (see Rubin et al, 2002, N Engl J Med; 346(12): 896- 903). In 2007, bosentan was also approved in the European Union for reducing the number of new digital ulcers in patients with systemic sclerosis and ongoing digital ulcer disease. It is also known by the trade name TRACLEER, is designated chemically as 4-tert-butyl-N-[6-(2- hydroxy-ethoxy)-5-(2-methoxy-phenoxy)[2,2]-bipyrimidin-4-yl]- benzenesulfonamide monohydrate, has the chemical formula C27H₃iN₅0vS,and the CAS Registry number 157212-55- 0.

In some aspects of the methods described herein, a CDC42 inhibitor blocks CDC42 activation of endothelin type I and/or endothelin type II receptor. A CDC42 inhibitor may be an endothelin (ET) receptor antagonist (ERA) and may blocks endothelin receptors. Three main kinds of ERAs are know: selective ETA receptor antagonists (sitaxentan, ambrisentan

(LETAIRIS), atrasentan, BQ-123, zibotentan), which affect endothelin A receptors; dual antagonists (bosentan (TRACLEER), macitentan, tezosentan), which affect both endothelin A and B receptors; and selective ET_B receptor antagonists (BQ-788 and A192621).

In some aspects of the methods described herein, a CDC42 inhibitor is bosentan or a derivative thereof.

One or more additional therapeutic modalities may be administered along with one or more agents of the present disclosure. In some aspects of the present invention, the

administration of agents of the present disclosure may allow for the effectiveness of a lower dosage of other therapeutic modalities when compared to the administration of the other therapeutic modalities alone, providing relief from the toxicity observed with the administration of higher doses of the other modalities. One or more additional therapeutic agents may be administered before, after, and/or coincident to the administration of agents of the present disclosure. Agents of the present disclosure and additional therapeutic agents may be administered separately or as part of a mixture of cocktail. As used herein, an additional therapeutic agent may include, for example, an agent whose use for the treatment of Alport syndrome, kidney disease, kidney failure, and/or proteinuria is known to the skilled artisan. For example, an angiotensin-converting enzyme (ACE) inhibitor, such as ramipril or anapril, may be administered.

As used herein "treating" or "treatment" can include therapeutic and/or prophylactic treatments. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological

consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The agents of the present disclosure can be administered by any suitable means including, but not limited to, for example, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intravesical, or injection into or around the tumor. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intraperitoneal, and intratumoral administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for

administration will, in any event, determine the appropriate dose for the individual subject.

Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA. Such preparation may be pyrogen- free.

For enteral administration, the inhibitor may be administered in a tablet or capsule, which may be enteric coated, or in a formulation for controlled or sustained release. Many suitable formulations are known, including polymeric or protein microparticles encapsulating drug to be released, ointments, gels, or solutions which can be used topically or locally to administer drug, and even patches, which provide controlled release over a prolonged period of time. These can also take the form of implants.

The present invention includes compositions of one or more of the inhibitors described herein. A composition may also include, for example, buffering agents to help to maintain the pH in an acceptable range or preservatives to retard microbial growth. Such compositions may also include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler, diluents or

encapsulating substances which are suitable for administration to a human or other vertebrate animal. The compositions of the present disclosure are formulated in pharmaceutical

preparations in a variety of forms adapted to the chosen route of administration.

Therapeutically effective concentrations and amounts may be determined for each application herein empirically by testing the compounds in known in vitro and in vivo systems, such as those described herein, dosages for humans or other animals may then be extrapolated therefrom. With the methods of the present disclosure, the efficacy of the administration of one or more agents may be assessed by any of a variety of parameters known in the art.

It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods.

An agent of the present disclosure may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. For example, an agent of the present disclosure may be administered twice a day, three times a day, four times a day, or more. For example an agent of the present disclosure may be administered every other day, every third day, once a week, every two weeks, or once a month.at once, or may be divided into a number of smaller doses to be administered at intervals of time. In some applications, an agent of the present disclosure may be administered continuously, for example by a controlled release formulation or a pump.

It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the

compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods.

In some therapeutic embodiments, an "effective amount" of an agent is an amount that results in a reduction of at least one pathological parameter. Thus, for example, in some aspects of the present disclosure, an effective amount is an amount that is effective to achieve a reduction of at least about 10%, at least about 15%, at least about 20%, or at least about 25%, at least about 30%>, at least about 35%, at least about 40%>, at least about 45%, at least about 50%>, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%), at least about 80%>, at least about 85%, at least about 90%>, or at least about 95%, compared to the expected reduction in the parameter in an individual not treated with the agent.

As used herein, the term "subject" includes, but is not limited to, humans and non-human vertebrates. In preferred embodiments, a subject is a mammal, particularly a human. A subject may be an individual. A subject may be an "individual," "patient," or "host. In some aspects, a subject is an individual diagnosed with Alport syndrome. Diagnosis may be by any of a variety of means, including, but not limited to, family history, clinical presentation, pathological determination, and/or genetic testing. Such as subject may be a male or a female. Non-human vertebrates include livestock animals, companion animals, and laboratory animals. Non-human subjects also include non-human primates as well as rodents, such as, but not limited to, a rat or a mouse. Non-human subjects also include, without limitation, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits.

As used herein "in vitro" is in cell culture and "in vivo" is within the body of a subject.

As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.

As used herein, "isolated" refers to material that has been either removed from its natural environment (e.g., the natural environment if it is naturally occurring), produced using recombinant techniques, or chemically or enzymatically synthesized, and thus is altered "by the hand of man" from its natural state.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

The above discussion of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Example 1

Use of Rac 1/CDC42 inhibitors for the treatment of Alport glomerular disease

Alport glomerular disease is delayed onset and progressive, with onset generally occurring in the first decade of life. Abnormal laminins (laminin 211 and laminin 111) progressively accumulate in the glomerular basement membranes (GBM) of mice dogs and people with Alport syndrome. The present invention shows that laminin 211 activates focal adhesion kinase leading to downstream signaling through the NFkappaB transcription factor which results in maladaptive dysregulation of genes that drive the progression of Alport glomerular pathology. Thus the deposition of laminin 211 in the GBM is an important driver of glomerular pathogenesis in Alport syndrome. The present invention also shows that laminin 211 is being deposited by mesangial cell filopodia that are invading the interface between the glomerular endothelial cells and the GBM. This important observation indicates that the activation of filopodial invasion of the GBM is an early event in glomerular disease initiation in Alport syndrome. Agents that can attenuate or arrest the activation of mesangial filopodial invasion will have important therapeutic application. Cell culture studies show that filopodia formation involves dynamic actin microfilament remodeling that is regulated by the rho family of small GTPases. These structures emerge from lamellipodia, found at the leading edge of migrating cells. Lamellipodia formation is regulated by activation of the Ras related C3 botulinum toxin substrate 1 (RACl) small GTPase, and filopodia formation is regulated by the activation of cell division control 42 homologue (CDC42) small GTPase. Cross-talk between RAC 1 and CDC42 has been demonstrated.

This example determined that the mesangial process invasion into the capillary loops in Alport syndrome involves the activation of RACl and/or CDC42. The autosomal recessive Alport mouse model (a COL4A3 gene knockout mouse on the 129 Sv/J background) was treated with the small molecule inhibitor for RACl, NSC 23766 (commercial available) at a

concentration of 2 milligrams per kilogram, administered daily by intraperitoneal injection starting at 2 weeks of age (before proteinuria is observed). Kidneys were harvested at 6 weeks of age (at a stage when glomerular disease is well advanced and proteinuria is >300 mg/dl). One kidney was used for histology and the other for glomerular RNA isolation. The kidney cryosections were analyzed by dual immunofluorescence immunostaining for integrin a8 (a mesangial cell surface marker) and laminin a5 (a glomerular basement membrane marker). The results showed that the RAC 1 treated Alport mice demonstrated very little mesangial process invasion of the glomerular capillary loops, while saline injected controls had extensive mesangial process invasion of the capillary loops. Gene expression analysis was performed on the glomerular RNA for matrix metalloproteinase 10 and interleukin 6 (both transcripts are up- regulated by focal adhesion kinase-mediated signaling through NFkappaB) were significantly reduced in the samples from RACl inhibitor-treated Alport mice relative to saline-treated Alport mice.

Collectively, this example identifies novel therapeutic targets for the treatment of Alport syndrome, namely agents that can block the activation of RAC1/CDC42 members of the rho family of small GTPases and thus prevent invasion of the glomerular capillary tufts by mesangial lamellipodial/filopodial processes. By blocking mesangial process invasion, the deposition of laminin 211 in the GBM is abrogated, thus preventing the activation of maladaptive expression of proteins known to contribute to glomerular disease progression.

Example 2

Early Mechanism for Alport Glomerular Pathology

Alport syndrome results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. Mutations in any of these genes results in the absence of all 3 in the GBM type IV collagen network due to an obligatory association to form heterotrimers. The result is a thinner and less crosslinked GBM collagen network resulting in delayed onset progressive glomerulonephritis. The molecular trigger for disease onset is unknown. A comparative analysis of glomerular disease progression in Alport mice and CD151 knockout mice revealed a progressive irregular deposition of mesangial laminin 211 in the GBM. Co-localization studies showed that the mesangial integrin α8β1 also progressively accumulates in the capillary loops of both models, reflecting an invasion of the capillary loops by mesangial cell processes. L-NAME salt-induced hypertension accelerated mesangial filopodial invasion and laminin 211

accumulation, suggesting biomechanical strain plays a role in this mechanism. Mesangial cells showed reduced migratory potential when treated with either integrin linked kinase inhibitor, focal adhesion kinase inhibitor, or Racl inhibitors. Biomechanical stretching of cultured mesangial cells induced promigratory cytokines TGF-βΙ and CTGF. Treatment of Alport mice with Racl inhibitors reduced mesangial filopodial invasion of the glomerular capillary tuft. Laminin a2-deficient Alport mice show reduced GBM damage and triple the lifespan of Alport mice in spite of the hind limb paralysis, indicating a central role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings indicate

biomechanical insult results in the induction of mesangial filopodial invasion of the glomerular capillary tuft leading to the irregular deposition of mesangial laminin 211 and the initiation mechanism of Alport glomerular pathology.

Example 3

αΐβΐ integrin-mediated Racl/CDC42-dependent mesangial process invasion of glomerular capillary tufts in Alport syndrome

With this example, a comparative analysis of glomerular disease progression in Alport mice and CD151 knockout mice revealed a progressive irregular deposition of mesangial laminin 211 in the GBM. Co-localization studies showed that the mesangial integrin α8β1 also progressively accumulates in the capillary loops of both models as well as in human Alport glomeruli, indicating an invasion of the capillary loops by mesangial cell processes. L-NAME salt-induced hypertension accelerated mesangial cell process invasion and laminin 211 accumulation, suggesting biomechanical strain plays a role in this mechanism. Cultured mesangial cells showed reduced migratory potential when treated with either integrin linked kinase inhibitor, Racl inhibitors, CDC42 inhibitors, or by deletion of integrin al .

Biomechanical stretching of cultured mesangial cells induced promigratory cytokines TGF-βΙ and CTGF. Treatment of Alport mice with a Racl inhibitor reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin a2-deficient Alport mice show reduced mesangial process invasion, and laminin a2-null cells show reduced migratory potential, indicating a central role for mesangial laminins in progression of Alport glomerular

pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of mesangial cell process invasion of the glomerular capillary tuft leading to the irregular deposition of mesangial laminin 211 as an initiation mechanism of Alport glomerular pathology.

The activation of genes encoding GBM matrix molecules, matrix metalloproteinases (MMPs), and proinflammatory cytokines have all been linked to the progression of Alport glomerular disease. These, however, are events that occur after the onset of proteinuria therefore downstream of disease initiation events (Sayers et al, 1999, Kidney Int; 56(5): 1662-1673;

Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59; Rao et al, 2006, Am J Pathol; 169(1):32- 46; Zeisberg et al, 2006, PLoS Medicine; 3(4), elOO; and Cosgrove et al, 2008, Am J Pathol; 172(3):761-7737-l 1). Consistent with this notion, experiments aimed at blocking these pathways have offered only limited therapeutic benefit in mouse models for Alport syndrome (Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59; Rao et al, 2006, Am J Pathol; 169(1):32- 46; Zeisberg et al, 2006, PLoS Medicine; 3(4), elOO; and Koepke et al, 2007, Nephrol Dial Transplant; 22(4): 1062-9). One of the earliest events is the appearance of an irregular deposition of laminin 211 in the GBM of Alport mice (Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59), an observation confirmed in both Alport dogs and human patients with the disease (Kashtan et al., 2001, J Am Soc Nephrol; 12:252-60). This laminin is normally found only in the mesangium of the glomerulus, and is not expressed in the GBM at any stage of embryonic development (Miner et al, 1997, J Cell Biol; 137(3):685-701). Indeed several other mesangial matrix proteins appear in the GBM of Alport mice, including laminin 111 and fibronectin (Cosgrove et al, 1996, Genes Dev; 10(23): 2981-2992; and St John and Abrahamson, 2001 , Kidney Int; 60(3): 1037- 1046).

In the Alport glomerulus, the podocytes are exposed to GBM that has an embryonic type IV collagen composition (Kalluri et al, 1997, J Clin Invest; 99(10):2470-2478; and Harvey et al, 1998, Kidney Int; 54(6): 1857-1866). This could result in altered cell signaling that may trigger the onset of the disease. It has been proposed this type of mechanism may account for the reactivation of laminin 111 expression in podocytes (Abrahamson et al., 2003, Kidney Int;

63:826-34), a laminin found in the GBM during development (Miner et al., 1997, J Cell Biol; 137(3):685-701). Since the al(IV)/a2(IV) collagen network contains significantly fewer interchain disulfide crosslinks (Gunwar et al, 1998, J Biol Chem; 273(15):8767-75), and since the Alport GBM is thinner than normal (Kamenetsky et al., 2010, J Digital Imaging; 23:463- 474) the Alport GBM is likely to be more elastic, resulting in elevated biomechanical strain on the glomerular cells at their points of contact the GBM. Consistent with this, glomeruli from Alport mice have been shown to have elevated deformability relative to wild type glomeruli (Wyss et al, 2011, Am J Physiol Cell Physiol; 300:C397-C405), and salt-induced hypertension has been shown to accelerate glomerular disease progression in Alport mice (Meehan et al., 2009, Kidney Int; 76:968-976).

This example shows that deletion of laminin 211 in Alport mice ameliorates the mesangial process invasion of the glomerular capillary loops in Alport mice, demonstrating for the first time a functional role for GBM laminin 211 in Alport glomerular pathogenesis. The cellular origin of GBM laminin 211 has not been previously determined. This example shows that the source of GBM laminin 211 in Alport GBM is mesangial cell processes, which are invading the capillary tufts. Salt-mediated hypertension exacerbates this mesangial process invasion. A knockout mouse for the integrin α3β1 co-receptor CD 151, which results in reduced adhesion of podocytes pedicles to GBM laminin 521, also develops mesangial process invasion of the capillary loops with GBM deposition of laminin 211, demonstrating the same phenotype for a completely unrelated component of the capillary structural barrier. The CD 151 knockout mouse model also shows accelerated glomerular disease progression in response to hypertension (Sachs et al, 2012, J Clin Invest; 122(l):348-58). Mesangial cell culture studies show that biomechanical stretching induces promigratory cytokines TGF-βΙ and CTGF, both known to be induced in Alport glomeruli (Sayers et al, 1999, Kidney Int; 56(5): 1662-1673; and Koepke et al, 2007, Nephrol Dial Transplant; 22(4): 1062-9). Inhibitor studies indicate that migration is mediated through αΐβΐ integrin signaling through the Rho GTPases RACl and CDC42.

Consistent with this, l integrin deletion in Alport mice was previously shown to ameliorate glomerular disease progression and slow the accumulation of laminin 211 in Alport GBM

(Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59). This example shows that mesangial process invasion of the capillary loops is ameliorated in al integrin null Alport mice. These data define a surprising role for biomechanical strain mediated-induction of mesangial cell process invasion as a key aspect of Alport glomerular disease initiation, and identify novel therapeutic targets to blocking this process.

Results

GBM laminin 211 in Alport mice is of mesangial origin. In the glomerulus, laminin 211 is normally found only in the mesangial matrix. Figures 1A to 1C demonstrate mesangial distribution of laminin 211 in wild type mice, which is distinct from the glomerular basement membrane (collagen a3(IV)). In Alport glomeruli, Figures ID to IF demonstrate the irregular distribution of laminin 211 in the GBM which appears to accumulate preferentially in irregularly thickened regions of the GBM (here the GBM is marked by immunostaining with antibodies specific for laminin a5). The cellular source of the GBM laminin 211 has never been

determined. Dual immunofluorescence labeling with antibodies against laminin a2 and integrin a8 show mesangial specific immunostaining in wild type glomeruli (Figs. 2A-2C), as reported previously (Hartner et al, 1999, Kidney Int; 56(4): 1468-80). In Alport glomeruli (at 7 weeks of age) immunostaining for both laminin a2 and integrin a8 appears to have spread into the capillary loops consistent with a mesangial cell process invasion of the capillary loops (Figs. 2D- 2F). Dual immunofluorescence immunostaining using the basement membrane marker laminin a5 with the mesangial marker integrin a8 confirms that integrin a8 immunostaining, while absent from the GBM in wild type mice (Figs. 2G-2I), is clearly present in most of the GBM of Alport mice (Figs. 2J-2L). Collectively these data indicate that GBM laminin 211 arises from a mesangial cell process invasion of the capillary loops, and thus is of mesangial cell origin.

To determine the relevance of this observation to human Alport syndrome cryosections from human Alport necropsy kidney sections were stained with antibodies specific for integrin a8 and laminin a5. The results in Fig. 3 show that mesangial processes are clearly present adjacent to the laminin a5-immunopisitive GBM in the human specimen.

Mesangial process invasion of the capillary loops is exacerbated by elevated

biomechanical strain. An earlier report demonstrated that hypertension exacerbated the progression of Alport glomerular disease (Meehan et al, 2009, Kidney Int; 76:968-976).

Hypertension accelerated several aspects of glomerular disease progression including proteinuria and induction of matrix metalloproteinases. The accumulation of GBM laminin 211 was also accelerated. Fig. 4 shows that salt-induced hypertension clearly accelerates the inundation of the glomerular capillary loops by mesangial processes as evidenced by the presence of integrin a8 immunopositivitiy in the GBM (Fig. 4D-4F).

It is likely the increased biomechanical stress on the glomerular capillary tuft in Alport glomeruli is due to the change in GBM type IV composition from dual networks of

al(IV)/a2(IV) and a3(IV)/a4(IV)/a5(IV) collagen to one comprised only of al(IV)/a2(IV) collagen. The latter is thinner and known to contain fewer interchain disulfide crosslinks (Gunwar et al, 1998, J Biol Chem; 273(15):8767-75) which would intuitively be expected to result in increasing the elasticity of the glomerular filtration barrier. In order to provide independent validation, a completely different model was examined that would also be expected to affect the elastic integrity of the glomerular filtration barrier, the CD151 knockout mouse. CD151 is a tetraspanin co-receptor for integrin α3β1 which functions to increase the affinity of integrin α3β1 for its GBM ligand, laminin a5 (Nishiuchi et al, 2005, Proc Natl Acad Sci USA; 102(6): 1939-44). Deletion of CD151 results in glomerular disease with morphological changes in the GBM strikingly similar to Alport syndrome (Baleato et al., 2008, Am J Pathol;

173(4):927-37). Recently it was shown that hypertension accelerates the progression of glomerular disease in the CD 151 knockout mouse, similar to our observations for the Alport mouse (Sachs et al, 2012, J Clin Invest; 122(l):348-58). Considering all of this, glomeruli from the CD151 knockout mouse were examined for mesangial process invasion and laminin 211 deposition in the GBM. The results in Fig. 5 are impressive, in that this mouse shows a near complete inundation of the glomerular capillary tufts with integrin a8 and laminin a2

immunopositivity, demonstrating mesangial process invasion and deposition of mesangial laminins in the GBM in this genetically unrelated model.

If biomechanical strain can induce the activation of mesangial process invasion of the capillary tuft, pro-migratory responses will be activated in vitro by mechanically stretching cultured primary mesangial cells. Primary cultured mesangial cells, derived from 129 Sv/J mice, were subjected to cyclic cell stretching using the Flexcell system for 24 hours. Expression of several pro-migratory cytokines was quantified by real time RT-PCR. The results in Fig. 6 demonstrate that expression of both TGF-βΙ and CTGF are significantly elevated in cells subjected to biomechanical stretching relative to cells cultured under identical conditions, but not subjected to stretch.

Mesangial cell migration (in vitro) and mesangial process invasion of the glomerular capillary loops (in vivo) are regulated by integrin αΐβΐ mediated Racl/CDC42 crosstalk. Earlier work demonstrated that deletion of al integrin markedly attenuated the progression of glomerular disease in Alport mice (Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59).

Although is highly likely that disease attenuation in integrin a 1 -null Alport mice emanates from the mesangial compartment where integrin αΐβΐ is highly expressed, the molecular mechanism underlying this effect has remained unclear. Fig. 7 shows that deletion of al integrin markedly reduces the dynamics of mesangial process invasion of the capillary tufts in Alport mice, consistent with the reduction in GBM laminin 211 deposition shown here and previously

(Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59).

Since it is well established that the formation of filopodia and lamellipodia require the concerted action of the small GTPases Racl and CDC42 (Vicente-Manzanares et al., 2009, J Cell Sci; 122(2): 199-206), cell migration assays were performed using the Boyden chamber approach to determine whether such a functional connection was evident in cultured wild type and integrin αΐ-null mesangial cells. The results in Fig. 8A show that integrin αΐ-null mesangial cells show a significant reduction in migratory potential relative to wild type mesangial cells. Migration of wild type cells was significantly reduced when cells were treated with either the integrin linked kinase inhibitor QLT-0267, the Racl inhibitor NSC 23766, or the CDC42 inhibitor ML 141. Cell migration of wild type cells were not affected by treatment with the pan AKT inhibitor GSK 690693. Integrin αΐ-null mesangial cell migration was significantly reduced when cells were treated with ILK inhibitors, but unaffected when treated with Racl inhibitors, demonstrating that deletion of al integrin abrogates Racl -dependent cell migration.

Treatment of cells with the bacterial endotoxin lipopolysaccharide (LPS) activates both Racl and CDC42 GTPases (Sanlioglu et al, 2001, J Biol Chem; 276(32):30188-98; and Fessler et al, 2004, J Biol Chem; 279(38):39989-98), and is known to induce the formation of both lamellipodia and filopodia in cultured mesangial cells (Bursten et al, 1991, Am J Pathol;

139(2):371-82). Cultured wild type mesangial cells were treated with LPS, the actin filaments stained with phalloidin, and the cultures examined for morphological changes. As shown in Fig. 8B, after 30 minutes, treated cells undergo a stark morphological change about half of the cells sprouting numerous filopodia (Fig. 8B(b), denoted by asterisks), that are easily discernable, blinded, in numerous replicate experiments. Cells treated with LPS in combination with either the Racl inhibitor NSC 23766 or the CDC42 inhibitor ML 141 could not be distinguished in blinded experiments form untreated wild type mesangial cells (Figs. 8B(c) and 8B(d), respectively.

Interestingly, treatment of integrin a 1 -null mesangial cells with LPS had no discernable effect on cell morphology. To further validate these findings, either wild type or a 1 -null mesangial cell cultures were stimulated with LPS in the presence or absence of either Racl or CDC42 inhibitors and performed immunofluorescence analysis for CDC42 localization and pull down assays for activated Racl . As shown in Fig. 8C, treatment of cells with LPS resulted in polarized localization of CDC42 associated with staining in adjacent filopodia (panel B insert), an established characteristic of CDC42 activation (Etienne-Manneville and Hall, 2001, Cell; 106:489-498; and Huang et al, 2011, J Cell Biochem; 112(6): 1572-1584). Treatment of these cells with Racl inhibitor abolished this polarized activation, indicating cross-talk between Racl and CDC42. Integrin αΐ-null mesangial cells did not respond to LPS activation with polarized CDC42 localization. Pull down assays demonstrate that LPS treatment does indeed activate RAcl, and that pre-treatment of cells with the Racl inhibitor abolishes its activation (Fig.

8C(d)). Interestingly, pre-treatment of cells with CDC42 inhibitors did not block LPS mediated Racl activation, suggesting that, while Racl inhibitors block LPS-CDC42 activation (Fig.

8C(c)), CDC42 inhibitors do not block Racl activation (Fig. 8C(d)).

To examine the effect of Racl inhibitors on Alport glomerular disease progression, either wild type or Alport mice were treated with inhibitors by IP injection from 2 weeks to 6 weeks of age. Glomeruli were examined for mesangial process invasion of the capillary tufts by dual immunofluorescence microscopy using antibodies specific for either integrin a8 or the GBM marker laminin a5. The results in Fig. 9 demonstrate that saline-injected mice show significant co-localization of integrin a8 and laminin a5 throughout many of the glomerular capillary tufts, while mice injected with the Racl inhibitor showed very little mesangial process invasion.

Combined, the data in Fig. 7, Fig. 8, and Fig. 9 confirm that mesangial process invasion of the glomerular capillaries is a Racl -dependent process, and that Racl activation is attenuated by integrin al deletion both in vitro and in vivo. Furthermore, LPS activation of filopodia in wild type mesangial cells (but not in a 1 -null mesangial cells) involves both Racl and CDC42 activation, suggesting αΐβΐ integrin-dependent cross talk between the two small GTPases in the signaling complex.

Laminin 211 enhances mesangial cell migration and mesangial process invasion of the capillary loops. In a related study to determine whether GBM laminin 211 contributed mechanistically to the progression of Alport glomerular disease, a laminin a2-deficient mouse was crossed with the Alport mouse to produce a double knockout. One effect of laminin o2 deficiency was a marked reduction of mesangial process invasion of the capillary loops (Fig. 10A). This indicates that laminin 211 might facilitate mesangial process invasion of the capillary loops. Thus, cell migration assays were performed on either laminin 211 or laminin 521 (GBM laminin). Two different laminin preparations were used. One was extracted laminin from either placenta (primarily laminin 511) or muscle (primarily laminin 211); the other commercially available purified recombinant laminin heterotrimers. A scratch wound assay was used as opposed to the Boyden chamber, to determine the role of specific extracellular matrix in potentiating mesangial cell migration. As shown in Fig. 10B, wild type mesangial cells migrate much more efficiently on laminin 211 compared to laminin 521. While the effect was more pronounced on the muscle laminin preparation relative to the placental laminin preparation, it is also clear on the pure recombinant laminin substrates. To more directly confirm the role of laminin a2 in migratory potential, the relative migration of wild type mesangial cells to mesangial cells derived from laminin a2-deficient mice was measured, this time using the Boyden chamber approach. The results in Fig. IOC represent multiple derivations of both cell types, and demonstrate a statistically significant reduction in the migratory potential of laminin a2-deficient mesangial cells relative to wild type mesangial cells. Collectively the data in Fig. 10 indicate that laminin 211 deposition by the mesangial processes functionally contributes to the process invasion of the capillary tuft in Alport and CD 151 -knockout glomeruli.

Discussion

Earlier studies of Alport mouse, dog, and humans reported the presence of "abnormal" laminins in the GBM, including laminin 211 and laminin 1 11 (Cosgrove et al., 2000, Am J Pathol; 157(5): 1649-59; Kashtan et al, 2001, J Am Soc Nephrol; 12:252-60; and Abrahamson et al., 2003, Kidney Int; 63:826-34). These laminins tend to accumulate in areas of irregular thickening of the GBM, and these thickened areas have been shown to be more permeable to ferritin, suggesting that they are comprised of loosely assembled or partially degraded extracellular matrix (Abrahamson et al., 2007, J Am Soc Nephrol; 18:2465-72). In addition to the "abnormal" laminins, fibronectin and heparin sulfate proteoglycans have also been reported to accumulate in the GBM of Alport mice (Cosgrove et al, 1996, Genes Dev; 10(23): 2981- 2992). What all of these ECM components have in common is that they are predominantly found in the mesangial matrix (Schlondorff and Banas, 2009, J Am Soc Nephrol; 20: 1179-87).

This example determined that these abnormal GBM matrix molecules that progressively accumulate in the Alport GBM are of mesangial cell origin. Integrin a8 was used as a specific mesangial cell surface marker to demonstrate that mesangial processes invade the capillary tufts and co-localize with laminin 211, a mesangial laminin. Integrin a8 is expressed in mesangial cells, but not in other glomerular cell types (Hartner et al, 1999, Kidney Int; 56(4): 1468-80), and its expression is generally restricted to smooth muscle cells and neuronal cell types (Bossy et al, 1991, EMBO J; 10(9):2375-2385; and Schnapp et al, 1995, J Cell Sci; 108:537-544). Mesangial process invasion of the glomerular capillary tufts was exacerbated by hypertension, indicating that the mechanism triggering this event was mediated by biomechanical stress, likely at the interface between the mesangial processes and the sub-endothelial interface with the glomerular capillaries, an area known to provide important structural support for the capillary loops

(Schlondorff and Banas, 2009, J Am Soc Nephrol; 20: 1179-87). The Alport mutations, which can be either autosomal recessive (mutations in either COL4A3 or COL4A4 genes (Mochizuki et al, 1994, Nat Genet; 8(1):77-81)) or X-linked (mutations in COL4A5 (Barker et al, 1990, Science; 8;248(4960): 1224-7)) result in the absence of the collagen a3(IV)/a4(IV)/a5(IV) network from the GBM. The consequence is a thinner GBM comprised of only l(IV) and a2(IV) collagens, which have been shown to contain fewer interchain disulfide crosslinks (Gunwar et al, 1998, J Biol Chem; 273(15):8767-75). This structural change alters the biomechanical properties of the capillary tuft, resulting in stresses on the cells comprising the tuft even under normal glomerular blood pressures.

A second model was examined, the CD151 knockout mouse, which would also show enhanced strain on the capillary tufts. In this model, enhanced strain arises as a result of reduced adhesion of the podocyte pedicles to the GBM due to reduced affinity for the podocyte integrin α3β1 for its GBM ligand laminin 521 (Nishiuchi et al, 2005, Proc Natl Acad Sci USA;

102(6): 1939-44). Mesangial process invasion of the glomerular capillary tufts in the CD 151 mouse was even more robust than that for the Alport model. Like the Alport model (Meehan et al, 2009, Kidney Int; 76:968-976), glomerular pathology in the CD151mouse model, which shows ultrastructural lesions in the GBM strikingly similar to Alport syndrome (Baleato et al., 2008, Am J Pathol; 173(4):927-37; and Sachs et al, 2006, J Cell Biol; 175(l):33-9) is significantly exacerbated under hypertensive conditions (Sachs et al, 2012, J Clin Invest;

122(l):348-58). Collectively this evidence supports the notion that mutations affecting structural integrity of the glomerular capillary tuft result in unnatural stresses on the cells comprising the tuft. In the mesangial cell compartment this results in mesangial cell invasion of the tuft and deposition of matrix proteins in the GBM that are of mesangial cell origin.

Earlier work showed that deletion of the mesangial integrin αΐβΐ in Alport mice resulted in a marked attenuation in the progression of the glomerular pathology, with reduced proteinuria and a near doubling of lifespan (Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59). The mechanism underlying the influence of mesangial αΐβΐ integrin on Alport renal disease progression has, until the present exmaple, remained unclear. This example shows that mesangial process invasion is markedly attenuated in integrin a 1 -null Alport mice relative to strain/age matched Alport mice. This indicates that the signaling pathway that activates actin cytoskeletal rearrangements is perturbed in the absence of αΐβΐ integrin. Further, decreased migratory potential was observed for primary cultures of a 1 -null mesangial cells relative to wild type mesangial cells from strain/age matched mice (Fig. 8A).

Lipopolysaccharide, which activates both Racl and CDC42 in wild type mesangial cells, failed to activate Racl or CDC42 (Fig. 8B), and failed to activate actin cytoskeletal

rearrangements in cultured a 1 -null mesangial cells. Collectively these data explain why deletion of l-integrin results in attenuation of Alport glomerular pathogenesis and indicate that αΐβΐ integrin is a key sensor of biomechanical strain at the glomerular capillary tuft and participates in the adhesive signaling mechanism that links to the Rho GTPases Racl and CDC42 which activate actin polymerization dynamics required to process invasion of the glomerular capillary tufts.

Classically, Racl activation is associated with lamellipodia formation and CDC42 activation is associated with filopodia formation (Nobes and Hall, 1995, Cell; 81(l):53-62). Recently, evidence for crosstalk between the two Rho GTPases has emerged (Zamudio-Meza et al, 2009, J Gen Virol; 90(Pt 12):2902-11). This phenomenon is likely regulated through the guanine nucleotide exchange factor βΐρίχ, which contains binding sites for both CDC42 and Racl (Chahdi et al, 2004, Biochem Biophys Res Commun; 317(2):384-9; and Chahdi et al, 2005, J Biol Chem; 280(l):578-84). This example provides evidence for cross-talk between Racl and CDC42 in cultured mesangial cells regulating actin cytoskeletal rearrangement including: showing that treatment of mesangial cells with LPS, known to activate rapid actin cytoskeletal rearrangement (Bursten et al., 1991, Am J Pathol; 139(2):371-82), activates Racl in wild type mesangial cells (Fig. 8C(d)); showing that membrane localization of CDC42, a known prerequisite for its activation, is blocked by addition of RAC1 inhibitors coincident with LPS stimulation (Fig. 8C(a-c)); and showing that inclusion of either Racl inhibitors or CDC42 inhibitors upon stimulation of mesangial cell cultures with LPS blocks actin cytoskeletal rearrangements (Fig. 8B).

Mesangial cell cultures subjected to cyclic biomechanical strain expressed elevated levels of the pro-migratory cytokines CTGF and TGF-βΙ, providing further evidence that

biomechanical strain activates actin cytoskeletal dynamics required for mesangial process invasion. Both CTGF and TGF-βΙ signaling have been shown to activate CDC42 (Edlund Set al, 2002, Mol Bio Cell; 13:902-14; and Crean et al, 2004, FASEB J; 18(13): 1541-3), and both cytokines have been shown to be induced in Alport glomeruli (Sayers et al, 1999, Kidney Int; 56(5): 1662-1673; and Kashtan et al, 2001, J Am Soc Nephrol; 12:252-60) indicating that activation of these signaling pathways might be an important underlying mechanism for the activation of mesangial process invasion of glomerular capillary tufts in Alport syndrome.

Indeed, earlier work showed that inhibition of TGF-βΙ in the Alport mouse resulted in abrogation of GBM thickening, in support of this notion (Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59). And, when TGF-βΙ was inhibited in al integrin-null Alport mice, a synergistic improvement in glomerular disease was observed suggesting that TGF-βΙ and integrin l are working through distinct pathways (Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59). Based on the current study, these pathways may converge on strain-mediated activation of

Racl/CDC42 in the mesangial cell compartment.

While the deposition of laminin 211 in the GBM of Alport mice was described more than 10 years ago (Cosgrove et al, 2000, Am J Pathol; 157(5): 1649-59; and Kashtan et al, 2001, J Am Soc Nephrol; 12:252-60), a functional role for this laminin in Alport glomerular pathology has not been described. This example shows reduced mesangial process invasion of the glomerular capillary loops in Alport mice that are also lacking laminin a2, indicating that laminin 211 itself promotes the migration of processes into the glomerular capillary loops (Fig. 9A). Consistent with this, the example shows that wild type mesangial cells migrate more robustly when cultured on laminin 211 compared to laminin 521, and that primary mesangial cells from laminin a2-deficient mice show impaired migration relative primary wild type mesangial cells from age/strain matched mice (Figs. 9B and 9C). While modulation of mesangial cell migration by ECM has been described previously (Person et al, 1988, Am J Pathol; 133(3):609-14), this example shows that the strain-mediated mesangial process invasion of the capillary loops is enhanced by mesangial cell secreted laminin 211, which may explain why laminin 211 accumulates in the patchy irregularly thickened regions of the Alport GBM (see Fig. 1).

This example shows that the changes in the biophysical properties of the Alport glomerular capillary tuft results in biomechanical stresses that result in the induction of pathologic processes. Parallel observations in Alport and CD151 mouse models, including mesangial process invasion of the glomerular capillary tufts and deposition of laminin 211 provide additional support, since the two mouse models arise from mutations that would be expected to relax the structural integrity of the glomerular capillary tufts, but are otherwise mechanistically unrelated to each other. Recent studies of the biophysical properties of Alport glomeruli from pre-proteinuric mice reported increased deformability and suggested the glomeruli were "squishy" (Wyss et al, 2011, Am J Physiol Cell Physiol; 300:C397-C405). Collectively, this example supports a model where biomechanical stresses on the glomerular capillary tufts activate a promigratory signaling cascade in mesangial cells involving integrin αΐβΐ -mediated activation of Racl/CDC42 crosstalk. This activation culminates in the invasion of the capillary loops by mesangial processes. These processes clearly deposit laminin 211, which further exacerbates the mesangial process invasion. In addition to laminin 211, other mesangial matrix molecules are likely deposited in the GBM, and local action of mesangial cytokines (TGF-βΙ and CTGF, for example) and MMPs might also contribute to the structural and functional properties of the Alport GBM (irregular thickening, splitting, permeability, etc). In addition, all of these events are very likely to influence podocyte cell health. Thus, mesangial process invasion of the GBM is an important early event that precipitates glomerulosclerosis in Alport syndrome. The observation of mesangial process invasion of glomerular capillary loops in human Alport glomeruli provides relevance for these observations to the human disease. A better understanding of the activation process might reveal novel targets capable of preventing this event and arresting the Alport glomerular pathogenesis in its pre-initiated state.

Methods

Mice. All mice used in these studies were on pure genetic backgrounds. Autosomal recessive Alport mice were on the 129 Sv background. X-linked Alport mice were on the C57 Bl/6 background, laminin a2-deficient mice were on the 129 Sv background, integrin a 1 -null mice were on the 129 Sv background (Gardner et al, 1996, Dev Biol; 175(2):301-13), and CD151 knockout mice were on the FVB background (Takeda et al, 2007, Blood; 109(4): 1524- 32). All experiments were performed using strain/age-matched control mice. All animal studies were conducted in accordance to USDA approved standards and under the approval of the institutional IACUC. Every effort was made to minimize pain and discomfort.

Immunofluorescence microscopy. Fresh frozen kidneys were sectioned at 8 μιη and acetone fixed. Sections were incubated overnight at 4°C with 0.3% PBST (Triton X-100), 5% Fetal Bovine Serum, and with two of the following antibodies: rat anti-mouse Laminin-2 antibody (Sigma) at 1 :200, goat anti-mouse Integrin a8 antibody (R & D Systems) at 1 : 100, rabbit anti-mouse Laminin-5 antibody at 1 :200, rabbit anti-human Laminin-5 antibody (GeneTex) at 1 :500, rabbit anti-mouse CDC42 antibody (ProteinTech) at 1 :50, and goat anti- mouse a-actinin-4 antibody (Santa Cruz) at 1 :50. Affinity purified rabbit anti-collagen a3(IV) antibodies were as previously described. Slides were rinsed with IX PBS and incubated with the appropriate Alexa Fluor donkey secondary antibodies at 1 :300 for 1 hour at room temperature. They were then rinsed again with IX PBS and mounted with Vectashield Mounting Medium with Dapi (Vector).

MES Migration (insert). Transwell cell migration assays were performed as described by Daniel et al. {Lab Invest. 2012 Jun;92(6):812-26) with some modifications. 8 micron, 24-well plate control inserts (BD Bioscience, Bedford, MA) were coated overnight at 4°C with 100 μΐ of 0.1% gelatin/PBS then washed IX with PBS. MES cultures were incubated in 1% FCS overnight, then 0.05% BSA-containing media for at least 8 hours, washed IX with PBS and carefully tryspinized to ensure a single cell suspension and limited "clumping" of cells. After serum-neutralization and subsequent centrifugation, ~ 100,000 cells were resuspended in 1.5 mis of 0.05 % BSA media containing activators/inhibitors. The wells of a 24-well plate were filled with 0.75 mis of 10%> FCS-containing media plus activators/inhibitors (excluding 0.05 % BSA control well). 0.5 ml of cell-suspension was loaded into the gelatin-coated insert and the insert placed in a well. Wells were visually inspected for bubbles beneath insert and equal distribution of cell-suspension. Cells were allowed to migrate overnight (-18 hrs). Using a moistened cotton swab, non-migrated cells were librated from the apical-side of the insert by gentle but firm rubbing. A second swab repeated the removal and was followed by a single wash with PBS. Inserts were fixed, stained and washed (2X) in companion 24 well plate(s) containing 0.5 mis MEOH, 0.5 mis 1% Toluidine Blue in 1% Borax and 0.5 mis distilled H20, respectively. Inserts were air dried and counted at 100 X magnification. Five fields were counted on each insert including one center and four periphery areas. Data was expressed as relative to 10 % FCS control well (set equal to one).

Scratch wound migration assay. For Basal Lamina studies Superfrost™ Plus (VWR) microscope slides were coated with the following: 100 ng/ml Merosin™ (Millipore), 100 ng/ml human placental laminin (Sigma-Aldrich), 20 ng/ml human rlaminin-21 1 (BioLamina), or 20 ng/ml human rlaminin-521 (BioLamina) per manufacturer's suggestion. Slide(s) were placed in a tissue culture dish and an 8 x 8 mm cloning ring (Bellco Glass) placed on the coated area. A 100 μΐ of cell suspension (-30,000 cells) in 1% FBS-containing media was added to the cloning ring and the cells were allowed to attach for ~8 hours, PBS was placed in the dish and the ring removed. A -0.3-0.5 mm swath of cells was removed was by running a serological pipette at a ~ 45° angle through the monolayer. After capturing images of removed cells, slides were incubated for 24 hours in 1% FBS containing media, washed with PBS, fixed in methanol for 5 minutes, air dried and stained for 30 minutes with modified Giemsa Stain (Sigma- Aldrich). Images of previously photographed fields were captured using a Leica MZ10F Microscope fitted with a DFC310FX camera.

Biomechanical stretching of cultured mesangial cells. Low passage, sub confluent, primary mesangial cells were trypsized and seeded onto Bioflex™ 6-well plates (Flexcell International Corp) coated with Rat tail type I collagen (BD Biosciences). Cells were plated in 5% FCS containing media at densities that resulted in 20-40 % confluence. 0.5% FCS media was placed on the cells the next day. 48 hours later the media was changed and the cultures exposed to a regimen of 60 cycles of stretch and relaxation per minute with amplitude of 10 % radial surface elongation. The Flexercell Strain Unit FX4000 (Flexcell International Corp., Hillsborough, NC.) was used to induce stretch/relaxation for 18 hours according to

manufacturer's directions. Cells grown identically, but not exposed to stretch, served as controls.

Real time qRT-PCR. Total RNA was reverse transcribed using Superscript® III

(Invitrogen) with 01igo(dT)₂o Primer (Invitrogen). The real time PCR was carried out using TaqMan® Gene Expression Master Mix (Applied Biosystems), and quantified using ABI Prism® 7000 sequence detection system (Applied Biosystems). Samples were normalized to Mouse GAPDH Endogenous Control VIC® Probe (Applied Biosystems catalogue #4352339E) which was run alongside the CTGF and TGFP-l TaqMan® Gene Expression Assay Probes (Applied Biosystems catalogue #4331182). Each of the samples were run in triplicate with a final reaction volume of 50μ1 with the following cycling parameters: 50°C for 2min, 95°C for lOmin, followed by 40 cycles of a two-step PCR consisting of 95°C for 15s and 60°C for lmin. Relative changes in gene expression were determined by calculating the fold change using the comparative C_T method of 2^~ΔΔσΓ.

Activation of mesangial cell cultures by treatment with LPS. Sub-confluent mesangial cells were tryspinized; plated at low density on Rat tail type 1 collagen (BD Biosciences) coated cytology slides (VWR) and incubated overnight in 1% FCS-containing media. One hour after the addition of serum-free media, ΙμΜ CDC42 Inhibitor (KSC-23-233) and 10 μΜ Rac-1 Inhibitor, NSC-23766 (Tocris) were added to individual slides and allowed to incubate for an additional hour. lOng/ml Lipopolysaccharides (Sigma- Aldrich) was added to cells, incubated 1 hour, fixed in ice cold acetone for 5 minutes and allowed to air dry ~ 2 hours. Cells were stained with a 1 : 100 dilution of antibodies specific for CDC42 (10155-1-AP, Proteintech™), and phalloidin (Molecular Probes) imaged. Untreated, LPS alone and LPS plus inhibitors treatments were repeated on two different derivations of primary mesangial cells with qualitatively consistent results.

Pull down assay. Pull down experiments for Racl in mesangial cells were done using the Racl Activation Assay Bicochem Kit (BK035, Cytoskeleton Inc, CO) and according to manufacturer instructions with minor modifications. Briefly, 500-800 μg of protein lysates were incubated with 20 μΐ of PAK-PBD beads for 1 hour at 4°C. Pull down samples and total protein lysates (30-50 μg of protein) were run in a 12% SDS-PAGE gel, transferred to PVDF

membranes and blocked in 5% milk for 30 minutes at room temperature. Rac-1 antibody incubation was done overnight at 4°C with rocking. After secondary antibody incubation and several washes membranes were developed using the ECL Plus kit (32134, Pierce, IL) pull-down experiments or the SuperSignal West Femto kit (34094, Pierce, IL) for total lysates. Films were exposed for 40 min and 5 min respectively and developed using a film processor (Biomedical Imaging Systems, Model SRX-101A).

Confocal microscopy. Slides were coverslipped using Vectashield mounting medium containing DAPI to counter-stain the nuclei (Vector Lab, CA) and confocal images captured using a Zeiss AxioPlan 2IF MOT microscope interfaced with a LSM510 META confocal imaging system, using a 63X NA: 1.4 oil objective. Final figures were assembled using Adobe Photoshop and Illustrator software (Adobe Systems, CA).

Example 4

Treatment of 129 Sv Alport Mice with Bosentan

129 Sv mice, as described in the previous examples, five animals per group, are being dosed daily with bosentan at 10 mg/kg by oral gavage using a carboxymethylcellulose vehicle from 2 to 7 weeks of age. The groups are: WT vehicle; Alport vehicle; WT Bosentan; and Alport Bosentan. Proteinuria and blood urea nitrogen (BUN) will be measured. Dual immunofluorescence immunostaining will be performed with antibodies for integrin alpha 8 and laminin alpha5, as well as integrin laminin alpha 2 and pFAK397. Glomerulosllerosis and fibrosis scores will be determined using histochemical stain (Masson's trichrome). With this example, treated mice will show reduced proteinuria and BUN measures and reduced fibrosis and glomerulosclerosis scores. It is expected that integrin alpha 8 immunostaining in the glomerular capillary loops will be markedly reduced if not absent, and that laminin alpha 2 in the GBM will be markedly reduced with concomitant reduction of PFAK397 immunostaining in the podocytes (which may be absent). A reduction in mesangial process invasion, which will be ascertained by the dual immunostaining experiments, will indicate that the endothelin blockade directly effects the activation of this process, and link this effect to improved renal health. The numbers will allow statistical analysis of the data.

Example 5

Treatment of C57 Bl/6 X-linked Alport Mice with Bosentan

Experimental Design Strategy. C57 Bl/6 X-linked Alport mice will be used. Animals will be in groups of 5 each, wild type and Alport given either vehicle or Bosentan at 100 mg/kg via oral gavage (once daily) and/or ramipril in the drinking water (10 mg/kg based on established water consumption measures) from 5 weeks of age. Two cohorts each consisting of 5 X-linked Alport mice and 5 wild type mice; no treatment, Bosentan alone, ramipril alone, and Bosentan plus ramipril. Cohort one. Urine and blood will be collected every two weeks from initiation of the study until end stage (determined as the date where animals lose >15% of their body weight, which is a humane end point caused by wasting associated with uriemia). Urine will be normalized to urinary creatinine levels by colorimetric microplate assay, and normalized urine samples will be assayed for albumin using a microplate ELISA assay. Blood urea nitrogen levels will be measured in plasma using a colorimetric microplate assay.

Cohort two. These animals will be harvested at 20 weeks of age. This is an age where advanced renal disease is uniformly evident in untreated mice, but not end stage (which is typically 23-26 weeks). Animals will be anesthetized and then transcardially perfused first with PBS, then one kidney clamped off at the renal artery and the other transcardially perfused with Dynabeads for magnetic recovery of glomeruli and isolation of glomerular RNA.

The data of the previous examples (showing elevated levels of endothelin 1 in Alport glomerular endothelium as well as induction of endothelin 1 in Alport glomeruli in mice made hypertensive by treatment with L-NAME salts, combined with the existence of a large amount of published work showing that endothelin 1 activates Racl and Cdc42 in glomerular mesangial cells leading to activation of actin cytoskeleton remodeling in those cells) indicate that endothelin receptor activation on mesangial cells by endothelin 1 is at least partly, in not wholly responsible for Racl/Cdc42 mediated mesangial process invasion of glomerular capillary tufts. Thus blockade of endothelin receptors by treatment of mice with Bosentan is expected to block this process and prevent/reduce deposition of laminin 211 in the GBM, potentially arresting the progression of the disease. It is possible that multiple pathways converge to fully activate these Rho GTPases, in which case a partial, but not complete, amelioration of the glomerular pathology will be observed.

The combination therapy with ramipril will be of interest, since these two therapies would be expected to provide synergistic benefit, given that they work through distinct mechanisms. The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

What is claimed is:

1. A method of treating Alport syndrome in a subject, the method comprising administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

2. A method of preventing glomerular disease progression in a subject diagnosed with Alport syndrome, the method comprising administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

3. A method of treating glomerulonephritis in a subject, the method comprising administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

4. A method of treating kidney injury due to biomechanical strain in Alport syndrome, the method comprising administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

5. A method of inhibiting deposition of laminin 211 in the glomerular basement membrane (GBM) in a subject, the method comprising administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

6. A method of inhibiting mesangial cell process invasion of the glomerular capillary loop in a kidney of a subject, the method comprising administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor.

7. A method of inhibiting Alport glomerular pathogenesis in a subject; the method comprising: determining that the subject is at risk for developing Alport glomerular disease; and administering an effective amount of a RACl inhibitor and/or a CDC42 inhibitor to the subject.

8. The method of claim 7, wherein the determination that the subject is at risk for developing Alport glomerular disease is determined by family medical history, genetic testing,

immunodiagnostic skin biopsy testing, and/or molecular diagnostic marker testing.

9. The method of claim 7, wherein the determination that the subject is at risk for developing Alport glomerular disease is made prior to the onset of proteinuria in the subject.

10. The method of any one of claim 1 to 9, wherein the administration of an effective amount of a RACl inhibitor and/or a CDC42 inhibitor is initiated prior to the onset of proteinuria in the subject.

11. The method of any one of claim 1 to 10, wherein the RACl inhibitor and/or a CDC42 inhibitor blocks CDC42 activation of the endothelin type I receptor and/or the endothelin type II receptor.

12. The method of any one of claim 1 to 11, wherein the RACl inhibitor and/or a CDC42 inhibitor comprises an endothelin (ET) receptor antagonist.

13. The method of claim 12, wherein the endothelin (ET) receptor antagonist comprises a dual antagonist of both the ETA receptor and ET_B receptor.

14. The method of any one of claim 1 to 13, wherein the RACl inhibitor and/or a CDC42 inhibitor comprises bosentan or a derivative thereof

15. The method of any one of claim 1 to 14, wherein the RACl inhibitor comprises NSC23766 or a derivative thereof.