Super-Resolution imaging of the Filtration Barrier Suggests a Role for podocin R229O in Genetic Predisposition to Glomerular disease

Significance Statement Podocin R229Q results from the most frequent missense variant in NPHS2, and its association with FSGS when podocin R229Q is transassociated with a second mutation in NPHS2 is well recognized. However, because results from observational studies are ambiguous and appropriate animal studies are lacking, its isolated pathogenic potency is not entirely clear. In this study, the authors introduced this genetic alteration in mice and assessed the phenotype using super-resolution microscopy and albuminuria measurements. They demonstrated a deleterious effect of the variant on podocyte morphology and on the integrity of the glomerular filtration barrier under basal conditions and after external glomerular injury. Because this finding suggests that this mutation confers a genetic predisposition to glomerular disease, it has implications for a large number of carriers worldwide. 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.