endothelin-1 and Nephritis--Hereditary

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.

Topics: Actin Cytoskeleton; Animals; Basement Membrane; Cell Adhesion; Cell Movement; Cells, Cultured; Collagen Type IV; Disease Models, Animal; Endothelin-1; Female; Fluorescent Antibody Technique; Gene Expression Profiling; Male; Mice, 129 Strain; Microscopy, Confocal; Microscopy, Electron, Transmission; Nephritis, Hereditary; Pericytes; Receptor, Endothelin A; RNA-Seq; Signal Transduction; Stria Vascularis

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.

Topics: Animals; Biomechanical Phenomena; Disease Models, Animal; Endothelial Cells; Endothelin Receptor Antagonists; Endothelin-1; Fluorescent Antibody Technique; Gene Knockdown Techniques; Glomerular Basement Membrane; Hypertension; Isoxazoles; Laminin; Mesangial Cells; Mice; Mice, Inbred C57BL; Nephritis, Hereditary; Podocytes; Proteinuria; Pseudopodia; Receptor, Endothelin A; RNA Interference; RNA, Small Interfering; Signal Transduction; Thiophenes; Up-Regulation

Using a mouse model of Alport disease, Dufek et al. report that endothelial cell-derived endothelin-1 activates mesangial cells, which deposit abnormal laminin isoforms in the Alport glomerular basement membrane. This study extends findings obtained previously by this laboratory implicating mesangial cells in the early pathogenesis of Alport disease. Together with abnormalities in matrix receptor expression, cytoskeletal architecture, and proteinase secretion in podocytes, a clearer picture is emerging on the inception of proteinuria in Alport disease.

Topics: Animals; Endothelin-1; Glomerular Basement Membrane; Humans; Nephritis, Hereditary; Podocytes; Proteinuria

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 (ET

Topics: Animals; Basement Membrane; Body Temperature; Capillaries; Cell Line; Collagen Type IV; Disease Models, Animal; Endothelin-1; Extracellular Matrix; Gene Expression Regulation; Hypoxia; Isoxazoles; Laminin; Mice; Nephritis, Hereditary; Oxidative Stress; Phenotype; Stria Vascularis; Thiophenes