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1.
J Pathol ; 262(3): 296-309, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38129319

RESUMO

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.


Assuntos
Nefrite Hereditária , Podócitos , Humanos , Camundongos , Animais , Integrina alfa1/genética , Integrina alfa1/metabolismo , Ramipril/farmacologia , Ramipril/metabolismo , Longevidade , Membrana Basal Glomerular/metabolismo , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/genética , Nefrite Hereditária/metabolismo , Podócitos/metabolismo , Laminina/genética , Laminina/metabolismo , Camundongos Knockout , Proteinúria/tratamento farmacológico , Proteinúria/genética , Proteinúria/metabolismo , Análise de Sequência de RNA
2.
J Pathol ; 260(3): 353-364, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37256677

RESUMO

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.


Assuntos
Orelha Interna , Nefrite Hereditária , Animais , Camundongos , Nefrite Hereditária/metabolismo , Receptores de Angiotensina/metabolismo , Receptores de Angiotensina/uso terapêutico , Membrana Basal Glomerular/metabolismo , Colágeno Tipo IV/genética , Orelha Interna/metabolismo , Orelha Interna/patologia , Endotelinas/metabolismo , Endotelinas/uso terapêutico
3.
Nat Commun ; 14(1): 972, 2023 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-36810733

RESUMO

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.


Assuntos
Proteínas da Matriz Extracelular , Proteínas de Membrana , Síndromes de Usher , Animais , Humanos , Camundongos , Proteínas da Matriz Extracelular/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Mutação , Síndromes de Usher/genética
4.
J Pathol ; 258(1): 26-37, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35607980

RESUMO

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.


Assuntos
Nefrite Hereditária , Podócitos , Animais , Membrana Basal/patologia , Colágeno Tipo III , Colágeno Tipo IV/genética , Receptor com Domínio Discoidina 1/genética , Membrana Basal Glomerular/patologia , Humanos , Integrina alfa2beta1 , Camundongos , Camundongos Knockout , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Podócitos/patologia , Pseudópodes/patologia , RNA
5.
Front Med (Lausanne) ; 9: 846152, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35223933

RESUMO

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.

6.
PLoS One ; 15(8): e0237907, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822386

RESUMO

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.


Assuntos
Regulação da Expressão Gênica/genética , Perda Auditiva Provocada por Ruído/metabolismo , Nefrite Hereditária/metabolismo , Estria Vascular/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Membrana Basal/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Matriz Extracelular/metabolismo , Feminino , Glucose/genética , Glucose/metabolismo , Perda Auditiva Provocada por Ruído/genética , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , RNA-Seq , Transdução de Sinais/genética , Estria Vascular/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Regulação para Cima
7.
Hear Res ; 390: 107935, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32234583

RESUMO

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.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Membrana Basal/ultraestrutura , Nefrite Hereditária/patologia , Pericitos/ultraestrutura , Estria Vascular/ultraestrutura , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Endotelina-1/farmacologia , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Masculino , Camundongos da Linhagem 129 , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Nefrite Hereditária/genética , Nefrite Hereditária/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , RNA-Seq , Receptor de Endotelina A/agonistas , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Transdução de Sinais , Estria Vascular/efeitos dos fármacos , Estria Vascular/metabolismo
9.
Kidney Int ; 94(2): 303-314, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29759420

RESUMO

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.


Assuntos
Aminoácido Oxirredutases/metabolismo , Inibidores Enzimáticos/uso terapêutico , Membrana Basal Glomerular/patologia , Mesângio Glomerular/patologia , Nefrite Hereditária/patologia , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/genética , Animais , Autoantígenos/genética , Colágeno Tipo IV/genética , Modelos Animais de Doenças , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Fibrose , Membrana Basal Glomerular/metabolismo , Mesângio Glomerular/metabolismo , Humanos , Laminina/metabolismo , Camundongos , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/genética , RNA Mensageiro/metabolismo , Regulação para Cima
10.
Matrix Biol ; 57-58: 45-54, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27576055

RESUMO

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.


Assuntos
Autoantígenos/genética , Membrana Basal/metabolismo , Colágeno Tipo IV/genética , Glomérulos Renais/metabolismo , Nefrite Hereditária/genética , Autoantígenos/metabolismo , Membrana Basal/patologia , Colágeno Tipo IV/metabolismo , Regulação da Expressão Gênica , Humanos , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Glomérulos Renais/patologia , Mutação , Nefrite Hereditária/metabolismo , Nefrite Hereditária/patologia , Ligação Proteica , Multimerização Proteica , Receptores de Colágeno/genética , Receptores de Colágeno/metabolismo , Transdução de Sinais
11.
PLoS One ; 11(12): e0168343, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27959966

RESUMO

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.


Assuntos
Mesângio Glomerular/metabolismo , Nefrite Hereditária/genética , Nefrite Hereditária/fisiopatologia , Pseudópodes/metabolismo , Albuminas/química , Animais , Biópsia , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Cães , Membrana Basal Glomerular/patologia , Humanos , Imuno-Histoquímica , Laminina/genética , Masculino , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Mutação , Proteinúria/metabolismo
12.
Hear Res ; 341: 100-108, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27553900

RESUMO

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.


Assuntos
Endotelina-1/metabolismo , Matriz Extracelular/genética , Nefrite Hereditária/fisiopatologia , Estria Vascular/citologia , Animais , Membrana Basal/patologia , Temperatura Corporal , Capilares/patologia , Linhagem Celular , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hipóxia/patologia , Isoxazóis/química , Laminina/metabolismo , Camundongos , Estresse Oxidativo , Fenótipo , Estria Vascular/metabolismo , Tiofenos/química
13.
Kidney Int ; 90(2): 300-310, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27165837

RESUMO

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.


Assuntos
Endotelina-1/metabolismo , Células Mesangiais/metabolismo , Nefrite Hereditária/metabolismo , Podócitos/metabolismo , Receptor de Endotelina A/metabolismo , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Antagonistas dos Receptores de Endotelina/farmacologia , Antagonistas dos Receptores de Endotelina/uso terapêutico , Imunofluorescência , Técnicas de Silenciamento de Genes , Membrana Basal Glomerular/metabolismo , Hipertensão/metabolismo , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Laminina/metabolismo , Células Mesangiais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Nefrite Hereditária/genética , Proteinúria/tratamento farmacológico , Pseudópodes/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor de Endotelina A/genética , Transdução de Sinais , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Regulação para Cima
14.
PLoS One ; 9(6): e99083, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24915008

RESUMO

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.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Laminina/metabolismo , Nefrite Hereditária/enzimologia , Nefrite Hereditária/etiologia , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Membrana Basal Glomerular/efeitos dos fármacos , Membrana Basal Glomerular/enzimologia , Membrana Basal Glomerular/patologia , Membrana Basal Glomerular/ultraestrutura , Proteínas I-kappa B/metabolismo , Interleucina-6/metabolismo , Cinética , Metaloproteinases da Matriz/metabolismo , Camundongos Knockout , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Inibidor de NF-kappaB alfa , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/patologia , Podócitos/enzimologia , Podócitos/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/metabolismo , Tetraspanina 24/metabolismo
15.
PLoS One ; 9(4): e94272, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24705452

RESUMO

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.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Vírus da Anemia Infecciosa Equina/genética , Síndromes de Usher/genética , Síndromes de Usher/terapia , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Ordem dos Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/metabolismo , Humanos , Macaca , Masculino , Camundongos , Camundongos Knockout , Miosina VIIa , Miosinas/genética , Fenótipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Transporte Proteico , Retina/metabolismo , Retina/patologia , Transducina/metabolismo
16.
Exp Eye Res ; 118: 145-53, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24211856

RESUMO

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.


Assuntos
Adaptação Ocular/fisiologia , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transducina/genética , Translocação Genética , Animais , Western Blotting , Contagem de Células , Imuno-Histoquímica , Masculino , Camundongos , Fotoperíodo , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Transducina/metabolismo
17.
Int J Biochem Cell Biol ; 46: 80-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24239741

RESUMO

The 10 different genes associated with the deaf/blind disorder, Usher syndrome, encode a number of structurally and functionally distinct proteins, most expressed as multiple isoforms/protein variants. Functional characterization of these proteins suggests a role in stereocilia development in cochlear hair cells, likely owing to adhesive interactions in hair bundles. In mature hair cells, homodimers of the Usher cadherins, cadherin 23 and protocadherin 15, interact to form a structural fiber, the tip link, and the linkages that anchor the taller stereocilia's actin cytoskeleton core to the shorter adjacent stereocilia and the elusive mechanotransduction channels, explaining the deafness phenotype when these molecular interactions are perturbed. The conundrum is that photoreceptors lack a synonymous mechanotransduction apparatus, and so a common theory for Usher protein function in the two neurosensory cell types affected in Usher syndrome is lacking. Recent evidence linking photoreceptor cell dysfunction in the shaker 1 mouse model for Usher syndrome to light-induced protein translocation defects, combined with localization of an Usher protein interactome at the periciliary region of the photoreceptors suggests Usher proteins might regulate protein trafficking between the inner and outer segments of photoreceptors. A distinct Usher protein complex is trafficked to the ribbon synapses of hair cells, and synaptic defects have been reported in Usher mutants in both hair cells and photoreceptors. This review aims to clarify what is known about Usher protein function at the synaptic and apical poles of hair cells and photoreceptors and the prospects for identifying a unifying pathobiological mechanism to explain deaf/blindness in Usher syndrome.


Assuntos
Células Ciliadas Auditivas/patologia , Células Fotorreceptoras/patologia , Síndromes de Usher/genética , Síndromes de Usher/patologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Células Ciliadas Auditivas/metabolismo , Humanos , Células Fotorreceptoras/metabolismo , Síndromes de Usher/metabolismo
18.
Am J Pathol ; 183(4): 1269-1280, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23911822

RESUMO

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.


Assuntos
Capilares/patologia , Mesângio Glomerular/irrigação sanguínea , Mesângio Glomerular/patologia , Integrina alfa1beta1/metabolismo , Nefrite Hereditária/metabolismo , Nefrite Hereditária/patologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Capilares/efeitos dos fármacos , Capilares/metabolismo , Capilares/fisiopatologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/patologia , Membrana Basal Glomerular/fisiopatologia , Membrana Basal Glomerular/ultraestrutura , Mesângio Glomerular/fisiopatologia , Mesângio Glomerular/ultraestrutura , Humanos , Hipertensão/complicações , Hipertensão/metabolismo , Hipertensão/patologia , Hipertensão/fisiopatologia , Laminina/metabolismo , Camundongos , Camundongos Knockout , Nefrite Hereditária/complicações , Nefrite Hereditária/fisiopatologia , Transporte Proteico/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
19.
PLoS One ; 8(3): e58832, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555598

RESUMO

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.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Luz , Proteínas de Membrana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Adaptação à Escuridão/fisiologia , Feminino , Masculino , Proteínas de Membrana/química , Camundongos , Fosforilação , Ligação Proteica , Transporte Proteico/efeitos da radiação , Segmento Interno das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Serina/metabolismo
20.
J Neurosci ; 32(40): 13841-59, 2012 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-23035094

RESUMO

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.


Assuntos
Caderinas/metabolismo , Polaridade Celular/fisiologia , Células Ciliadas Auditivas/ultraestrutura , Precursores de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Vesículas Transportadoras/fisiologia , Fator 1 de Ribosilação do ADP/análise , Animais , Química Encefálica , Proteínas Relacionadas a Caderinas , Caderinas/biossíntese , Caderinas/genética , Compartimento Celular , Diferenciação Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Células Ciliadas Auditivas/metabolismo , Imunoprecipitação , Camundongos , Camundongos Mutantes Neurológicos , Mutação , Órgão Espiral/química , Órgão Espiral/ultraestrutura , Mapeamento de Interação de Proteínas , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Relação Estrutura-Atividade , Proteína 25 Associada a Sinaptossoma/análise , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Vesículas Transportadoras/química , Síndromes de Usher/metabolismo , Proteínas rab5 de Ligação ao GTP/análise
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