Abstract
Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV α3, α4 or α5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non‐invasive way. Here we isolated, immortalized, and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA‐sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte–glomerular endothelial cell co‐culture system, was established and AS podocyte co‐cultures showed a significantly higher permeability of albumin compared to control podocyte co‐cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..
Highlights
Alport syndrome (AS) is a genetic disorder that affects one in every 50 000 newborns
Increasing evidence indicates that AS podocytes possess an altered phenotype, beyond their altered collagen IV synthesis, which can be involved in the pathogenesis and progression of the disease
We isolated and characterized podocytes derived from the urine of three patients with AS, with genetic alterations of either COL4A3 or COL4A5, to assess common pathological features due to collagen IV mutations
Summary
Alport syndrome (AS) is a genetic disorder that affects one in every 50 000 newborns. Around 80% of AS families have X-linked inheritance caused by mutations in the COL4A5 gene, while the others have autosomal recessive, or less commonly autosomal dominant, disease, caused by mutations in COL4A3 or COL4A4 genes [1] These genes encode for three of the six different collagen IV α-chains (α1–α6), which are assembled in type IV collagen networks [2,3]. Podocytes express, assemble, and secrete collagen IV α3α4α5 proteins that replace the initial embryonic collagen IV α1α1α2 network in the glomerular basal membrane (GBM) [7]. After this developmental switch, the resulting mature GBM is a highly organized 300–400 nm thick collagen IV network [8]. In AS patients, alterations of the GBM, presenting as lamellation of the lamina densa, affect the function of podocytes, which appear to be the first altered cell type
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