Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

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 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.

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.

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.

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.

The usherin mutation c.2299delG leads to its mislocalization and disrupts interactions with whirlin and VLGR1.

Usher syndrome (USH) is the leading cause of combined deafness-blindness with type 2 A (USH2A) being the most common form. Knockout models of USH proteins, like the Ush2a-/- model that develops a late-onset retinal phenotype, failed to mimic the retinal phenotype observed in patients. Since patient's mutations result in the expression of a mutant protein and to determine the mechanism of USH2A, we generated and evaluated an usherin (USH2A) knock-in mouse expressing the common human disease-mutation, c.2299delG. This mouse exhibits retinal degeneration and expresses a truncated, glycosylated protein which is mislocalized to the photoreceptor inner segment. The degeneration is associated with a decline in retinal function, structural abnormalities in connecting cilium and outer segment and mislocaliztion of the usherin interactors very long G-protein receptor 1 and whirlin. The onset of symptoms is significantly earlier compared to Ush2a-/-, proving expression of mutated protein is required to recapitulate the patients' retinal phenotype.

Glomerular pathology in Alport syndrome: a molecular perspective.

We have known for some time that mutations in the genes encoding 3 of the 6 type IV collagen chains are the underlying defect responsible for both X-linked (where the COL4A5 gene is involved) and autosomal (where either COL4A3 or COL4A4 genes are involved) Alport syndrome. The result of these mutations is the absence of the sub-epithelial network of all three chains in the glomerular basement membrane (GBM), resulting, at maturity, in a type IV collagen GBM network comprising only α1(IV) and α2(IV) chains. The altered GBM functions adequately in early life. Eventually, there is onset of proteinuria associated with the classic and progressive irregular thickening, thinning, and splitting of the GBM, which culminates in end-stage renal failure. We have learned much about the molecular events associated with disease onset and progression through the study of animal models for Alport syndrome, and have identified some potential therapeutic approaches that may serve to delay the onset or slow the progression of the disease. This review focuses on where we are in our understanding of the disease, where we need to go to understand the molecular triggers that set the process in motion, and what emergent therapeutic approaches show promise for ameliorating disease progression in the clinic.

Molecular and Cellular Mechanisms Underlying the Initiation and Progression of Alport Glomerular Pathology.

Alport syndrome results from a myriad of variants in the COL4A3, COL4A4, or COL4A5 genes that encode type IV (basement membrane) collagens. Unlike type IV collagen α1(IV)2α2(IV)1 heterotrimers, which are ubiquitous in basement membranes, α3/α4/α5 have a limited tissue distribution. The absence of these basement membrane networks causes pathologies in some, but not all these tissues. Primarily the kidney glomerulus, the stria vascularis of the inner ear, the lens, and the retina as well as a rare link with aortic aneurisms. Defects in the glomerular basement membranes results in delayed onset and progressive focal segmental glomerulosclerosis ultimately requiring the patient to undergo dialysis and if accessible, kidney transplant. The lifespan of patients with Alport syndrome is ultimately significantly shortened. This review addresses the consequences of the altered glomerular basement membrane composition in Alport syndrome with specific emphasis on the mechanisms underlying initiation and progression of glomerular pathology.

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.

Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells.

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

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.

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.

Human alpha1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by alpha1beta1 integrin.

Human noncollagenous domain 1 of the alpha1 chain of type IV collagen [alpha1(IV)NC1], or arresten, is derived from the carboxy terminal of type IV collagen. It was shown to inhibit angiogenesis and tumor growth in vivo; however, the mechanisms involved are not known. In the present study we demonstrate that human alpha1(IV)NC1 binds to alpha1beta1 integrin, competes with type IV collagen binding to alpha1beta1 integrin, and inhibits migration, proliferation, and tube formation by ECs. Also, alpha1(IV)NC1 pretreatment inhibited FAK/c-Raf/MEK/ERK1/2/p38 MAPK activation in ECs but had no effect on the PI3K/Akt pathway. In contrast, alpha1(IV)NC1 did not affect proliferation, migration, or the activation of FAK/c-Raf/MEK1/2/p38/ERK1 MAPK pathway in alpha1 integrin receptor knockout ECs. Consistent with this, alpha1(IV)NC1 elicited significant antiangiogenic effects and tumor growth inhibition in vivo but failed to do the same in alpha1 integrin receptor knockout mice. This suggests a highly specific, alpha1beta1 integrin-dependent antiangiogenic activity of alpha1(IV)NC1. In addition, alpha1(IV)NC1 inhibited hypoxia-induced expression of hypoxia-inducible factor 1alpha and VEGF in ECs cultured on type IV collagen by inhibiting ERK1/2 and p38 activation. This unravels a hitherto unknown function of human alpha1(IV)NC1 and suggests a critical role for integrins in hypoxia and hypoxia-induced angiogenesis. Collectively, the above data indicate that alpha1(IV)NC1 is a potential therapeutic candidate for targeting tumor angiogenesis.

Collagen IV diseases: A focus on the glomerular basement membrane in Alport syndrome.

Alport syndrome is the result of mutations in any of three type IV collagen genes, COL4A3, COL4A4, or COL4A5. Because the three collagen chains form heterotrimers, there is an absence of all three proteins in the basement membranes where they are expressed. In the glomerulus, the mature glomerular basement membrane type IV collagen network, normally comprised of two separate networks, α3(IV)/α4(IV)/α5(IV) and α1(IV)/α2(IV), is comprised entirely of collagen α1(IV)/α2. This review addresses the current state of our knowledge regarding the consequence of this change in basement membrane composition, including both the direct, via collagen receptor binding, and indirect, regarding influences on glomerular biomechanics. The state of our current understanding regarding mechanisms of glomerular disease initiation and progression will be examined, as will the current state of the art regarding emergent therapeutic approaches to slow or arrest glomerular disease in Alport patients.

Stage-specific action of matrix metalloproteinases influences progressive hereditary kidney disease.

BACKGROUND: Glomerular basement membrane (GBM), a key component of the blood-filtration apparatus in the in the kidney, is formed through assembly of type IV collagen with laminins, nidogen, and sulfated proteoglycans. Mutations or deletions involving alpha3(IV), alpha4(IV), or alpha5(IV) chains of type IV collagen in the GBM have been identified as the cause for Alport syndrome in humans, a progressive hereditary kidney disease associated with deafness. The pathological mechanisms by which such mutations lead to eventual kidney failure are not completely understood. METHODS AND FINDINGS: We showed that increased susceptibility of defective human Alport GBM to proteolytic degradation is mediated by three different matrix metalloproteinases (MMPs)--MMP-2, MMP-3, and MMP-9--which influence the progression of renal dysfunction in alpha3(IV)-/- mice, a model for human Alport syndrome. Genetic ablation of either MMP-2 or MMP-9, or both MMP-2 and MMP-9, led to compensatory up-regulation of other MMPs in the kidney glomerulus. Pharmacological ablation of enzymatic activity associated with multiple GBM-degrading MMPs, before the onset of proteinuria or GBM structural defects in the alpha3(IV)-/- mice, led to significant attenuation in disease progression associated with delayed proteinuria and marked extension in survival. In contrast, inhibition of MMPs after induction of proteinuria led to acceleration of disease associated with extensive interstitial fibrosis and early death of alpha3(IV)-/- mice. CONCLUSIONS: These results suggest that preserving GBM/extracellular matrix integrity before the onset of proteinuria leads to significant disease protection, but if this window of opportunity is lost, MMP-inhibition at the later stages of Alport disease leads to accelerated glomerular and interstitial fibrosis. Our findings identify a crucial dual role for MMPs in the progression of Alport disease in alpha3(IV)-/- mice, with an early pathogenic function and a later protective action. Hence, we propose possible use of MMP-inhibitors as disease-preventive drugs for patients with Alport syndrome with identified genetic defects, before the onset of proteinuria.

X-Linked Alport Dogs Demonstrate Mesangial Filopodial Invasion of the Capillary Tuft as an Early Event in Glomerular Damage.

BACKGROUND: X-linked Alport syndrome (XLAS), caused by mutations in the type IV collagen COL4A5 gene, accounts for approximately 80% of human Alport syndrome. Dogs with XLAS have a similar clinical progression. Prior studies in autosomal recessive Alport mice demonstrated early mesangial cell invasion as the source of laminin 211 in the glomerular basement membrane (GBM), leading to proinflammatory signaling. The objective of this study was to verify this process in XLAS dogs. METHODS: XLAS dogs and WT littermates were monitored with serial clinicopathologic data and kidney biopsies. Biopsies were obtained at set milestones defined by the onset of microalbuminuria (MA), overt proteinuria, onset of azotemia, moderate azotemia, and euthanasia. Kidney biopsies were analyzed by histopathology, immunohistochemistry, and electron microscopy. RESULTS: XLAS dogs showed progressive decrease in renal function and progressive increase in interstitial fibrosis and glomerulosclerosis (based on light microscopy and immunostaining for fibronectin). The only identifiable structural abnormality at the time of microalbuminuria was ultrastructural evidence of mild segmental GBM multilamination, which was more extensive when overt proteinuria developed. Co-localization studies showed that mesangial laminin 211 and integrin α8ß1 accumulated in the GBM at the onset of overt proteinuria and coincided with ultrastructural evidence of mild cellular interpositioning, consistent with invasion of the capillary loops by mesangial cell processes. CONCLUSION: In a large animal model, the induction of mesangial filopodial invasion of the glomerular capillary loop leading to the irregular deposition of laminin 211 is an early initiating event in Alport glomerular pathology.