Dual inhibition of the endothelin and angiotensin receptor ameliorates renal and inner ear pathologies in Alport mice.

Alport syndrome (AS), a type IV collagen disorder, leads to glomerular disease and, in some patients, hearing loss. AS is treated with inhibitors of the renin-angiotensin system; however, a need exists for novel therapies, especially those addressing both major pathologies. Sparsentan is a single-molecule dual endothelin type-A and angiotensin II type 1 receptor antagonist (DEARA) under clinical development for focal segmental glomerulosclerosis and IgA nephropathy. We report the ability of sparsentan to ameliorate both renal and inner ear pathologies in an autosomal-recessive Alport mouse model. Sparsentan significantly delayed onset of glomerulosclerosis, interstitial fibrosis, proteinuria, and glomerular filtration rate decline. Sparsentan attenuated glomerular basement membrane defects, blunted mesangial filopodial invasion into the glomerular capillaries, increased lifespan more than losartan, and lessened changes in profibrotic/pro-inflammatory gene pathways in both the glomerular and the renal cortical compartments. Notably, treatment with sparsentan, but not losartan, prevented accumulation of extracellular matrix in the strial capillary basement membranes in the inner ear and reduced susceptibility to hearing loss. Improvements in lifespan and in renal and strial pathology were observed even when sparsentan was initiated after development of renal pathologies. These findings suggest that sparsentan may address both renal and hearing pathologies in Alport syndrome patients. © 2023 Travere Therapeutics, Inc and The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2ß1.

In Alport mice, activation of the endothelin A receptor (ETA R) in mesangial cells results in sub-endothelial invasion of glomerular capillaries by mesangial filopodia. Filopodia deposit mesangial matrix in the glomerular basement membrane (GBM), including laminin 211 which activates NF-κB, resulting in induction of inflammatory cytokines. Herein we show that collagen α1(III) is also deposited in the GBM. Collagen α1(III) localized to the mesangium in wild-type mice and was found in both the mesangium and the GBM in Alport mice. We show that collagen α1(III) activates discoidin domain receptor family, member 1 (DDR1) receptors both in vitro and in vivo. To elucidate whether collagen α1(III) might cause podocyte injury, cultured murine Alport podocytes were overlaid with recombinant collagen α1(III), or not, for 24 h and RNA was analyzed by RNA sequencing (RNA-seq). These same cells were subjected to siRNA knockdown for integrin α2 or DDR1 and the RNA was analyzed by RNA-seq. Results were validated in vivo using RNA-seq from RNA isolated from wild-type and Alport mouse glomeruli. Numerous genes associated with podocyte injury were up- or down-regulated in both Alport glomeruli and cultured podocytes treated with collagen α1(III), 18 of which have been associated previously with podocyte injury or glomerulonephritis. The data indicate α2ß1 integrin/DDR1 co-receptor signaling as the dominant regulatory mechanism. This may explain earlier studies where deletion of either DDR1 or α2ß1 integrin in Alport mice ameliorates renal pathology. © 2022 Boys Town National Research Hospital. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

RNA-seq analysis of gene expression profiles in isolated stria vascularis from wild-type and Alport mice reveals key pathways underling Alport strial pathogenesis.

Previous work demonstrates that the hearing loss in Alport mice is caused by defects in the stria vascularis. As the animals age, progressive thickening of strial capillary basement membranes (SCBMs) occurs associated with elevated levels of extracellular matrix expression and hypoxia-related gene and protein expression. These conditions render the animals susceptible to noise-induced hearing loss. In an effort to develop a more comprehensive understanding of how the underlying mutation in the COL4A3 gene influences homeostasis in the stria vascularis, we performed vascular permeability studies combined with RNA-seq analysis using isolated stria vascularis from 7-week old wild-type and Alport mice on the 129 Sv background. Alport SCBMs were found to be less permeable than wild-type littermates. RNA-seq and bioinformatics analysis revealed 68 genes were induced and 61 genes suppressed in the stria from Alport mice relative to wild-type using a cut-off of 2-fold. These included pathways involving transcription factors associated with the regulation of pro-inflammatory responses as well as cytokines, chemokines, and chemokine receptors that are up- or down-regulated. Canonical pathways included modulation of genes associated with glucose and glucose-1-PO4 degradation, NAD biosynthesis, histidine degradation, calcium signaling, and glutamate receptor signaling (among others). In all, the data point to the Alport stria being in an inflammatory state with disruption in numerous metabolic pathways indicative of metabolic stress, a likely cause for the susceptibility of Alport mice to noise-induced hearing loss under conditions that do not cause permanent hearing loss in age/strain-matched wild-type mice. The work lays the foundation for studies aimed at understanding the nature of strial pathology in Alport mice. The modulation of these genes under conditions of therapeutic intervention may provide important pre-clinical data to justify trials in humans afflicted with the disease.

Pericyte abnormalities precede strial capillary basement membrane thickening in Alport mice.

In 129 Sv autosomal Alport mice, the strial capillary basement membranes (SCBMs) progressively thicken between 5 and 9 weeks of age resulting in a hypoxic microenvironment with metabolic stress and induction of pro-inflammatory cytokines and chemokines. These events occur concomitant with a drop in endocochlear potential and a susceptibility to noise-induced hearing loss under conditions that do not permanently affect age/strain-matched littermates. Here we aimed to gain an understanding of events that occur before the onset of SCBM thickening. Alport stria has normal thickness and shows levels of extracellular matrix (ECM) molecules in the SCBMs commensurate with wild-type mice. Hearing thresholds in the 3-week Alport mice do not differ from those of wild-type mice. We performed RNAseq analysis using RNA from stria vascularis isolated from 3-week Alport mice and wild type littermates. Data was processed using Ingenuity Pathway Analysis software and further distilled using manual procedures. RNAseq analysis revealed significant dysregulation of genes involved in cell adhesion, cell migration, formation of protrusions, and both actin and tubulin cytoskeletal dynamics. Overall, the data suggested changes in the cellular architecture of the stria might be apparent. To test this notion, we performed dual immunofluorescence analysis on whole mounts of the stria vascularis from these same animals stained with anti-isolectin gs-ib4 (endothelial cell marker) and anti-desmin (pericyte marker) antibodies. The results showed evidence of pericyte detachment and migration as well as the formation of membrane ruffling on pericytes in z-stacked confocal images from Alport mice compared to wild type littermates. This was confirmed by TEM analysis. Earlier work from our lab showed that endothelin A receptor blockade prevents SCBM thickening and ECM accumulation in the SCBMs. Treating cultured pericytes with endothelin-1 induced actin cytoskeletal rearrangement, increasing the ratio of filamentous to globular actin. Collectively, these findings suggest that the change in type IV collagen composition in the Alport SCBMs results in cellular insult to the pericyte compartment, activating detachment and altered cytoskeletal dynamics. These events precede SCBM thickening and hearing loss in Alport mice, and thus constitute the earliest event so far recognized in Alport strial pathology.

Lysyl oxidase like-2 contributes to renal fibrosis in Col4α3/Alport mice.

Lysyl oxidase like-2 (LOXL2) is an amine oxidase with both intracellular and extracellular functions. Extracellularly, LOXL2 promotes collagen and elastin crosslinking, whereas intracellularly, LOXL2 has been reported to modify histone H3, stabilize SNAIL, and reduce cell polarity. Although LOXL2 promotes liver and lung fibrosis, little is known regarding its role in renal fibrosis. Here we determine whether LOXL2 influences kidney disease in COL4A3 (-/-) Alport mice. These mice were treated with a small molecule inhibitor selective for LOXL2 or with vehicle and assessed for glomerular sclerosis and fibrosis, albuminuria, blood urea nitrogen, lifespan, pro-fibrotic gene expression and ultrastructure of the glomerular basement membrane. Laminin α2 deposition in the glomerular basement membrane and mesangial filopodial invasion of the glomerular capillaries were also assessed. LOXL2 inhibition significantly reduced interstitial fibrosis and mRNA expression of MMP-2, MMP-9, TGF-ß1, and TNF-α. LOXL2 inhibitor treatment also reduced glomerulosclerosis, expression of MMP-10, MMP-12, and MCP-1 mRNA in glomeruli, and decreased albuminuria and blood urea nitrogen. Mesangial filopodial invasion of the capillary tufts was blunted, as was laminin α2 deposition in the glomerular basement membrane, and glomerular basement membrane ultrastructure was normalized. There was no effect on lifespan. Thus, LOXL2 plays an important role in promoting both glomerular and interstitial pathogenesis associated with Alport syndrome in mice. Other etiologies of chronic kidney disease are implicated with our observations.

Endothelin-1 mediated induction of extracellular matrix genes in strial marginal cells underlies strial pathology in Alport mice.

Alport syndrome, a type IV collagen disorder, manifests as glomerular disease associated with hearing loss with thickening of the glomerular and strial capillary basement membranes (SCBMs). We have identified a role for endothelin-1 (ET-1) activation of endothelin A receptors (ETARs) in glomerular pathogenesis. Here we explore whether ET-1 plays a role in strial pathology. Wild type (WT) and Alport mice were treated with the ETAR antagonist, sitaxentan. The stria vascularis was analyzed for SCBM thickness and for extracellular matrix (ECM) proteins. Additional WT and Alport mice were exposed to noise or hypoxia and the stria analyzed for hypoxia-related and ECM genes. A strial marginal cell line cultured under hypoxic conditions, or stimulated with ET-1 was analyzed for expression of hypoxia-related and ECM transcripts. Noise exposure resulted in significantly elevated ABR thresholds in Alport mice relative to wild type littermates. Alport stria showed elevated expression of collagen α1(IV), laminin α2, and laminin α5 proteins relative to WT. SCBM thickening and elevated ECM protein expression was ameliorated by ETAR blockade. Stria from normoxic Alport mice and hypoxic WT mice showed upregulation of hypoxia-related, ECM, and ET-1 transcripts. Both ET-1 stimulation and hypoxia up-regulated ECM transcripts in cultured marginal cells. We conclude that ET-1 mediated activation of ETARs on strial marginal cells results in elevated expression of ECM genes and thickening of the SCBMs in Alport mice. SCBM thickening results in hypoxic stress further elevating ECM and ET-1 gene expression, exacerbating strial pathology.

Endothelin A receptor activation on mesangial cells initiates Alport glomerular disease.

Recent work demonstrates that Alport glomerular disease is mediated through a biomechanical strain-sensitive activation of mesangial actin dynamics. This occurs through a Rac1/CDC42 cross-talk mechanism that results in the invasion of the subcapillary spaces by mesangial filopodia. The filopodia deposit mesangial matrix proteins in the glomerular basement membrane, including laminin 211, which activates focal adhesion kinase in podocytes culminating in the up-regulation of proinflammatory cytokines and metalloproteinases. These events drive the progression of glomerulonephritis. Here we test whether endothelial cell-derived endothelin-1 is up-regulated in Alport glomeruli and further elevated by hypertension. Treatment of cultured mesangial cells with endothelin-1 activates the formation of drebrin-positive actin microspikes. These microspikes do not form when cells are treated with the endothelin A receptor antagonist sitaxentan or under conditions of small, interfering RNA knockdown of endothelin A receptor mRNA. Treatment of Alport mice with sitaxentan results in delayed onset of proteinuria, normalized glomerular basement membrane morphology, inhibition of mesangial filopodial invasion of the glomerular capillaries, normalization of glomerular expression of metalloproteinases and proinflammatory cytokines, increased life span, and prevention of glomerulosclerosis and interstitial fibrosis. Thus endothelin A receptor activation on mesangial cells is a key event in initiation of Alport glomerular disease in this model.

Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

EIAV-based retinal gene therapy in the shaker1 mouse model for usher syndrome type 1B: development of UshStat.

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.

Photoreceptors in whirler mice show defective transducin translocation and are susceptible to short-term light/dark changes-induced degeneration.

Usher syndrome combines congenital hearing loss and retinitis pigmentosa (RP). Mutations in the whirlin gene (DFNB31/WHRN) cause a subtype of Usher syndrome (USH2D). Whirler mice have a defective whirlin gene. They have inner ear defects but usually do not develop retinal degeneration. Here we report that, in whirler mouse photoreceptors, the light-activated rod transducin translocation is delayed and its activation threshold is shifted to a higher level. Rhodopsin mis-localization is observed in rod inner segments. Continuous moderate light exposure can induce significant rod photoreceptor degeneration. Whirler mice reared under a 1500 lux light/dark cycle also develop severe photoreceptor degeneration. Previously, we have reported that shaker1 mice, a USH1B model, show moderate light-induced photoreceptor degeneration with delayed transducin translocation. Here, we further show that, in both whirler and shaker1 mice, short-term moderate light/dark changes can induce rod degeneration as severe as that induced by continuous light exposure. The results from shaker1 and whirler mice suggest that defective transducin translocation may be functionally related to light-induced degeneration, and these two symptoms may be caused by defects in Usher protein function in rods. Furthermore, these results indicate that both Usher syndrome mouse models possess a light-induced retinal phenotype and may share a closely related pathobiological mechanism.

α1ß1 integrin/Rac1-dependent mesangial invasion of glomerular capillaries in Alport syndrome.

Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. L-NAME salt-induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2-deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2-null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1ß1-dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology.

Light-induced translocation of RGS9-1 and Gß5L in mouse rod photoreceptors.

The transducin GTPase-accelerating protein complex, which determines the photoresponse duration of photoreceptors, is composed of RGS9-1, Gß5L and R9AP. Here we report that RGS9-1 and Gß5L change their distribution in rods during light/dark adaptation. Upon prolonged dark adaptation, RGS9-1 and Gß5L are primarily located in rod inner segments. But very dim-light exposure quickly translocates them to the outer segments. In contrast, their anchor protein R9AP remains in the outer segment at all times. In the dark, Gß5L's interaction with R9AP decreases significantly and RGS9-1 is phosphorylated at S(475) to a significant degree. Dim light exposure leads to quick de-phosphorylation of RGS9-1. Furthermore, after prolonged dark adaptation, RGS9-1 and transducin Gα are located in different cellular compartments. These results suggest a previously unappreciated mechanism by which prolonged dark adaptation leads to increased light sensitivity in rods by dissociating RGS9-1 from R9AP and redistributing it to rod inner segments.

Regulated vesicular trafficking of specific PCDH15 and VLGR1 variants in auditory hair cells.

Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia, and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal colocalization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular subpools. The apically trafficked pool colocalized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associated with membrane microdomains and SNAP25. Moreover, coimmunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance, and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions.

Role for a novel Usher protein complex in hair cell synaptic maturation.

The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23), protocadherin-15 (PCDH15) and the very large G-protein coupled receptor 1 (VLGR1) have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1-/- mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzer(av3J) mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well.

Moderate light-induced degeneration of rod photoreceptors with delayed transducin translocation in shaker1 mice.

PURPOSE. Usher syndrome is characterized by congenital deafness associated with retinitis pigmentosa (RP). Mutations in the myosin VIIa gene (MYO7A) cause a common and severe subtype of Usher syndrome (USH1B). Shaker1 mice have mutant MYO7A. They are deaf and have vestibular dysfunction but do not develop photoreceptor degeneration. The goal of this study was to investigate abnormalities of photoreceptors in shaker1 mice. METHODS. Immunocytochemistry and hydroethidine-based detection of intracellular superoxide production were used. Photoreceptor cell densities under various conditions of light/dark exposures were evaluated. RESULTS. In shaker1 mice, the rod transducin translocation is delayed because of a shift of its light activation threshold to a higher level. Even moderate light exposure can induce oxidative damage and significant rod degeneration in shaker1 mice. Shaker1 mice reared under a moderate light/dark cycle develop severe retinal degeneration in less than 6 months. CONCLUSIONS. These findings show that, contrary to earlier studies, shaker1 mice possess a robust retinal phenotype that may link to defective rod protein translocation. Importantly, USH1B animal models are likely vulnerable to light-induced photoreceptor damage, even under moderate light.

Collagen XIII induced in vascular endothelium mediates alpha1beta1 integrin-dependent transmigration of monocytes in renal fibrosis.

Alport syndrome is a common hereditary basement membrane disorder caused by mutations in the collagen IV α3, α4, or α5 genes that results in progressive glomerular and interstitial renal disease. Interstitial monocytes that accumulate in the renal cortex from Alport mice are immunopositive for integrin α1ß1, while only a small fraction of circulating monocytes are immunopositive for this integrin. We surmised that such a disparity might be due to the selective recruitment of α1ß1-positive monocytes. In this study, we report the identification of collagen XIII as a ligand that facilitates this selective recruitment of α1ß1 integrin-positive monocytes. Collagen XIII is absent in the vascular endothelium from normal renal cortex and abundant in Alport renal cortex. Neutralizing antibodies against the binding site in collagen XIII for α1ß1 integrin selectively block VLA1-positive monocyte migration in transwell assays. Injection of these antibodies into Alport mice slows monocyte recruitment and protects against renal fibrosis. Thus, the induction of collagen XIII in endothelial cells of Alport kidneys mediates the selective recruitment of α1ß1 integrin-positive monocytes and may potentially serve as a therapeutic target for inflammatory diseases in which lymphocyte/monocyte recruitment involves the interaction with α1ß1 integrin.

Biomechanical strain causes maladaptive gene regulation, contributing to Alport glomerular disease.

Patients with Alport's syndrome develop a number of pro-inflammatory cytokine and matrix metalloproteinase (MMP) abnormalities that contribute to progressive renal failure. Changes in the composition and structure of the glomerular basement membranes likely alter the biomechanics of cell adhesion and signaling in these patients. To test if enhanced strain on the capillary tuft due to these structural changes contributes to altered gene regulation, we subjected cultured podocytes to cyclic biomechanical strain. There was robust induction of interleukin (IL)-6, along with MMP-3, -9, -10, and -14, but not MMP-2 or -12 by increased strain. Neutralizing antibodies against IL-6 attenuated the strain-mediated induction of MMP-3 and -10. Alport mice treated with a general inhibitor of nitric oxide synthase (L-NAME) developed significant hypertension and increased IL-6 and MMP-3 and -10 in their glomeruli relative to those of normotensive Alport mice. These hypertensive Alport mice also had elevated proteinuria along with more advanced histological and ultrastructural glomerular basement membrane damage. We suggest that MMP and cytokine dysregulation may constitute a maladaptive response to biomechanical strain in the podocytes of Alport patients, thus contributing to glomerular disease initiation and progression.

Localization and expression of clarin-1, the Clrn1 gene product, in auditory hair cells and photoreceptors.

The Usher syndrome 3A (CLRN1) gene encodes clarin-1, which is a member of the tetraspanin family of transmembrane proteins. Although identified more than 6 years ago, little is known about its localization or function in the eye and ear. We developed a polyclonal antibody that react with all clarin-1 isoforms and used it to characterize protein expression in cochlea and retina. In the cochlea, we observe clarin-1expression in the stereocilia of P0 mice, and in synaptic terminals present at the base of the auditory hair cells from E18 to P6. In the retina, clarin-1 localizes to the connecting cilia, inner segment of photoreceptors and to the ribbon synapses. RT-PCR from P0 cochlea and P28 retina show mRNAs encoding only isoforms 2 and 3. Western blots show that only isoform 2 is present in protein extracts from these same tissues. We examined clarin-1 expression in the immortomouse-derived hair cell line UB/OC-1. Only isoform 2 is expressed in UB/OC-1 at both mRNA and protein levels, suggesting this isoform is biologically relevant to hair cell function. The protein co-localizes with microtubules and post-transgolgi vesicles. The subcellular localization of clarin-1 in hair cells and photoreceptors suggests it functions at both the basal and apical poles of neurosensoriepithelia.

Progressive morphological and functional defects in retinas from alpha1 integrin-null mice.

PURPOSE: The role of integrin/cell matrix interactions between the RPE and the basement membrane in retinal maintenance and function is not well characterized. In this study the functional importance of alpha1beta1 integrin for retinal pigment epithelial cell homeostasis and retinal health was assessed by comparing alpha1 integrin knockout mice with strain- and age-matched wild-type mice. METHODS: Immunolocalization and Western blot analysis of retinas and ARPE19 cells were performed to examine the expression of alpha1beta1 integrin in the RPE. Retinal abnormality was assessed by funduscopy, histology, and transmission electron microscopy. Progressive retinal damage was quantified by direct counting of rod photoreceptors. Light-induced translocation of arrestin and alpha-transducin was documented by immunohistochemical analysis of retinal cryosections. RESULTS: Integrin alpha1beta1 localizes to the basal aspect of retinal pigment epithelial cells colocalizing with the basal lamina of the RPE. Integrin alpha1-null mice have delayed-onset progressive retinal degeneration associated with thickening of the basement membrane, dysmorphology of basal processes, synaptic malformations, and funduscopic abnormalities. Integrin alpha1-null mice display marked delays in transducin translocation compared with dark-adapted wild-type mice after exposure to light. CONCLUSIONS: Collectively, these data suggest an essential role for alpha1beta1 integrin/basement membrane interactions in the RPE in basement membrane metabolism and translocation of transducin in photoreceptors. This is the first report describing evidence supporting an essential role for integrin/basement membrane interaction in the RPE. Further, this report demonstrates a direct link between integrin alpha1beta1 function in retinal pigment epithelial and molecular defects in photoreceptor cell function before retinal abnormality is apparent.

Integrin alpha1beta1 regulates matrix metalloproteinases via P38 mitogen-activated protein kinase in mesangial cells: implications for Alport syndrome.

Previous work has shown that integrin alpha1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin alpha1-null mice and integrin alpha1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin alpha1. Similarly, cultured mesangial cells from alpha1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin alpha1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin alpha1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in alpha1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.