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.

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.

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.

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.

肾脏肾小球滤过屏障疾病是终末期肾衰竭的主要原因。尽管对儿童肾小球疾病相关基因的了解越来越多，但绝大多数成年患者缺乏明确的基因诊断。蛋白 podocin p.R229Q 由NPHS2中最常见的错义变异产生，在 FSGS 患者队列中富集。然而，p.R229Q 已被提议仅在与特定的额外遗传改变相关时才会引起疾病，并且基于人群的流行病学研究关于其与白蛋白尿的关联产生了模棱两可的结果。

为了测试 podocin p.R229Q 是否也可能导致成人复杂的疾病发病机制，我们使用基于 CRISPR/Cas9 的基因组编辑 ( Pod ^R231Q ) 在小鼠中引入了确切的遗传改变。我们使用超分辨率显微镜和白蛋白尿测量评估了表型，并评估了突变蛋白在细胞培养实验中的稳定性。

杂合子Pod ^{R231Q/野生型}小鼠未出现任何明显的肾脏疾病或蛋白尿。然而，纯合子Pod ^R231Q/R231Q小鼠随着年龄的增长出现白蛋白尿水平升高，超分辨率显微镜显示之前的超微结构形态学改变最近与疾病易感性有关。当注射肾毒性血清诱导肾小球损伤时，与Pod^{野生型/野生型}小鼠相比，杂合子Pod ^{R231Q/野生型}小鼠表现出更严重的病程。Pod ^{R231Q/野生型}和Pod ^R231Q/R231Q中的 Podocin 蛋白水平降低小鼠以及表达 podocin ^R231Q变体的人类培养的足细胞。我们的体外实验表明潜在的蛋白酶体降解增加。

我们的研究结果表明 podocin R231Q 自身发挥致病作用，支持 podocin R229Q 有助于成年患者遗传易感性的概念。