In-vivo und in-vitro Charakterisierung von Podocinphosphomutanten

Täglich werden in den Nieren erwachsener Menschen etwa 180 L Primärharn gebildet. Die Bildung des Primärharns im Glomerulus erfolgt durch die Filtration von Blutplasma durch eine ladungs- und größenselektive glomeruläre Filtrationsbarriere hindurch, welche aus drei Schichten aufgebaut ist: dem fenestrierten Endothel der Kapillaren, der Basalmembran und den Podozyten. Eine Schädigung des Filters kann zur Entstehung eines nephrotischen Syndroms führen. Seltene, genetische Formen des nephrotischen Syndroms rückten in den letzten 20 Jahren in den Fokus der nephrologischen Forschung. Eine dieser genetischen Erkrankungen ist das Steroid-resistente nephrotische Syndrom, welches Mutationen verschiedener Gene als Ursache haben kann. Dazu gehört das Gen NPHS2, welches für das Schlitzmembranprotein Podocin kodiert. In dieser Arbeit wird der Einfluss von Phosphorylierungen des Proteins Podocin auf die Funktion des glomerulären Filters untersucht. Hierfür wurden mittels CRISPR-Cas9 Mauslinien generiert, die phosphoablating Podocinmutationen tragen (PodS382A bzw. PodT234A) und mit einer weiteren Mauslinie verkreuzt wurden, die ausschließlich das Analogon zur humanen kurzen Isoform von Podocin (PodΔExon5) exprimiert. In Heterozygotie entwickelten die Mäuse keinen Phänotypen. Die compound-heterozygoten Mäuse PodT234I/ΔExon5 und PodS382A/ΔExon5 entwickelten jedoch im Laufe des Untersuchungszeitraums eine Proteinurie sowie eine FSGS. Diese Ergebnisse sprechen dafür, dass die Phosphorylierung von Podocin an den entsprechenden Aminosäureresten für die intakte Funktion des glomerulären Filters essenziell ist und eine Fehlfunktion zur Entstehung glomerulärer Erkrankungen beiträgt. Es wird zudem mittels in-vitro Experimenten gezeigt, dass im Hinblick auf die Interkation mit anderen Schlitzmembranproteinen und auf die zelluläre Lokalisation keine signifikante Veränderung des Verhaltens der Podocin Phosphomutanten im Vergleich zu Podocin WT vorliegt. Dies weist darauf hin, dass die Krankheitsentstehung auf eine intrinsische Pathogenität der Phosphomutanten zurückzuführen ist, die sich offenbar erst in Kombination mit einer weiteren Mutation zeigt

In vivo characterization of a podocyte-expressed short podocin isoform

Abstract The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocin Δexon5 ) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes’ foot process morphology. Mice homozygous for podocin Δexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes’ slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocin Δexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels. The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex

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