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Feasibility of repairing glomerular basement membrane defects in Alport syndrome.

ABSTRACT: Alport syndrome is a hereditary glomerular disease that leads to kidney failure. It is caused by mutations affecting one of three chains of the collagen ?3?4?5(IV) heterotrimer, which forms the major collagen IV network of the glomerular basement membrane (GBM). In the absence of the ?3?4?5(IV) network, the ?1?1?2(IV) network substitutes, but it is insufficient to maintain normal kidney function. Inhibition of angiotensin-converting enzyme slows progression to kidney failure in patients with Alport syndrome but is not a cure. Restoration of the normal collagen ?3?4?5(IV) network in the GBM, by either cell- or gene-based therapy, is an attractive and logical approach toward a cure, but whether or not the abnormal GBM can be repaired once it has formed and is functioning is unknown. Using a mouse model of Alport syndrome and an inducible transgene system, we found that secretion of ?3?4?5(IV) heterotrimers by podocytes into a preformed, abnormal, filtering Alport GBM is effective at restoring the missing collagen IV network, slowing kidney disease progression, and extending life span. This proof-of-principle study demonstrates the plasticity of the mature GBM and validates the pursuit of therapeutic approaches aimed at normalizing the GBM to prolong kidney function.

SUBMITTER: Lin X

PROVIDER: S-EPMC3968506 | biostudies-other | 2014 Apr

REPOSITORIES: biostudies-other

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Feasibility of repairing glomerular basement membrane defects in Alport syndrome.

Lin Xiaobo X Suh Jung Hee JH Go Gloriosa G Miner Jeffrey H JH

Journal of the American Society of Nephrology : JASN 20131121 4

Alport syndrome is a hereditary glomerular disease that leads to kidney failure. It is caused by mutations affecting one of three chains of the collagen α3α4α5(IV) heterotrimer, which forms the major collagen IV network of the glomerular basement membrane (GBM). In the absence of the α3α4α5(IV) network, the α1α1α2(IV) network substitutes, but it is insufficient to maintain normal kidney function. Inhibition of angiotensin-converting enzyme slows progression to kidney failure in patients with Alp ...[more]

PMID: 24262794

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Project description:BackgroundCD151 is a cell-surface molecule of the tetraspanin family. Its lateral interaction with laminin-binding integrin ɑ3β1 is important for podocyte adhesion to the glomerular basement membrane (GBM). Deletion of Cd151 in mice induces glomerular dysfunction, with proteinuria and associated focal glomerulosclerosis, disorganisation of GBM and tubular cystic dilation. Despite this, CD151 is not routinely screened for in patients with nephrotic-range proteinuria. We aimed to better understand the relevance of CD151 in human kidney disease.MethodsNext-generation sequencing (NGS) was used to detect the variant in CD151. Electron and light microscopy were used to visualise the filtration barrier in the patient kidney biopsy, and immunoreactivity of patient red blood cells to anti-CD151/MER2 antibodies was performed. Further validation of the CD151 variant as disease-causing was performed in zebrafish using CRISPR-Cas9.ResultsWe report a young child with nail dystrophy and persistent urinary tract infections who was incidentally found to have nephrotic-range proteinuria. Through targeted NGS, a novel, homozygous truncating variant was identified in CD151, a gene rarely reported in patients with nephrotic syndrome. Electron microscopy imaging of patient kidney tissue showed thickening of GBM and podocyte effacement. Immunofluorescence of patient kidney tissue demonstrated that CD151 was significantly reduced, and we did not detect immunoreactivity to CD151/MER2 on patient red blood cells. CRISPR-Cas9 depletion of cd151 in zebrafish caused proteinuria, which was rescued by injection of wild-type CD151 mRNA, but not CD151 mRNA containing the variant sequence.ConclusionsOur results indicate that a novel variant in CD151 is associated with nephrotic-range proteinuria and microscopic haematuria and provides further evidence for a role of CD151 in glomerular disease. Our work highlights a functional testing pipeline for future analysis of patient genetic variants. A higher resolution version of the Graphical abstract is available as Supplementary information.

Project description: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.

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Feasibility of repairing glomerular basement membrane defects in Alport syndrome.

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Feasibility of repairing glomerular basement membrane defects in Alport syndrome.

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