Super-Resolution Imaging of the Filtration Barrier Suggests a Role for Podocin R229Q in Genetic Predisposition to Glomerular Disease

Background: Diseases of the kidney's glomerular filtration barrier are a leading cause of end stage renal failure. Despite a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2, is enriched in cohorts of patients with FSGS. However, p.R229Q has been proposed to cause disease only when transassociated with specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results.

Methods: To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing (Pod^R231Q ). We assessed the phenotype using super-resolution microscopy and albuminuria measurements and evaluated the stability of the mutant protein in cell culture experiments.

Results: Heterozygous Pod^{R231Q/wild-type} mice did not present any overt kidney disease or proteinuria. However, homozygous Pod^R231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous Pod^{R231Q/wild-type} mice showed a more severe course of disease compared with Pod^{wild-type/wild-type} mice. Podocin protein levels were decreased in Pod^{R231Q/wild-type} and Pod^R231Q/R231Q mice as well as in human cultured podocytes expressing the podocin^R231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation.

Conclusions: Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients.