<i>TBC1D8B </i>Loss-of-Function Mutations Lead to X-Linked Nephrotic Syndrome via Defective Trafficking Pathways

International audienceSteroid-resistant nephrotic syndrome (SRNS) is characterized by high-range proteinuria and most often focal and segmental glomerulosclerosis (FSGS). Identification of mutations in genes causing SRNS has improved our understanding of disease mechanisms and highlighted defects in the podocyte, a highly specialized glomerular epithelial cell, as major factors in disease pathogenesis. By exome sequencing, we identified missense mutations in TBC1D8B in two families with an X-linked early-onset SRNS with FSGS. TBC1D8B is an uncharacterized Rab-GTPase-activating protein likely involved in endocytic and recycling pathways. Immunofluorescence studies revealed TBC1D8B presence in human glomeruli, and affected individual podocytes displayed architectural changes associated with migration defects commonly found in FSGS. In zebrafish we demonstrated that both knockdown and knockout of the unique TBC1D8B ortholog-induced proteinuria and that this phenotype was rescued by human TBC1D8B mRNA injection, but not by either of the two mutated mRNAs. We also showed an interaction between TBC1D8B and Rab11b, a key protein in vesicular recycling in cells. Interestingly, both internalization and recycling processes were dramatically decreased in affected individuals' podocytes and fibroblasts, confirming the crucial role of TBC1D8B in the cellular recycling processes, probably as a Rab11b GTPase-activating protein. Altogether, these results confirmed that pathogenic variations in TBC1D8B are involved in X-linked podocytopathy and points to alterations in recycling processes as a mechanism of SRNS

Cathepsin B increases ENaC activity leading to hypertension early in nephrotic syndrome

The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria‐dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full‐length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+/K+‐ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified

Adeno-associated virus gene therapy prevents progression of kidney disease in genetic models of nephrotic syndrome

Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2, encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03–mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9–mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Nephroprotective Efficacy of RAS Blockade in Mice Carrying R140Q Podocin Mutation

<p>INTRODUCTION:NPHS2 mutations cause hereditary nephrotic syndrome and progressive renal failure.We recently generated an inducible knock-in mouse model carrying R140Q,the analogue of the most common human mutation R138Q.These mice develop FSGS with nephrotic syndrome and progressive renal failure.Here we tested the efficacy of early and delayed RAS blockade in this model of hereditary podocyte disease.</p> <p>METHODS:In C57BL/6 mice with Nphs2Flox/R140Q/Cre+ genotype,hemizygosity for mutant podocin was induced by tamoxifen injection.Animals received combined high-dose ACE inhibition and AT1 receptor blockade(ramipril+candesartan 10mg/kg each,R+C)or remained untreated.Treatment was started either prophylactically(P)at time of induction or with a 4-week delay(D).Animals were either sacrificed after 5 wks(9 wks in D) or observed open-end.</p> <p>RESULTS:Induction of R140Q-podocin hemizygosity caused massive proteinuria peaking at 4 wks,followed by a gradual decrease as progressive renal failure developed.In the P animals,RAS blockade persistently inhibited proteinuria(13%of untreated animals at 4 wks,p<0.005).In the D group,proteinuria sharply decreased upon RAS blockade.Three-month survival was 31% in the untreated,67% in the D and 100% in the P group respectively(p<0.05).<br>Podocin protein abundance was almost totally lost in both untreated and treated animals,despite preserved or even increased mRNA expression.P group animals retained a higher number of podocytes per glomerulus and lower glomerulosclerosis and tubolointerstitial fibrosis indices than the untreated animals.In the D animals,histopathological lesions were slightly less marked at 9 wks than in the untreated animals at 5 wks.</p> <p>CONCLUSIONS:In mice carrying the most common human podocin mutation,RAS blockade attenatues proteinuria,podocyte loss and glomerulosclerosis despite persistently increased degradation of mutant podocin protein.Renal failure is delayed and survival prolonged.Treatment is more effective when applied prophylactically than when started in established nephropathy.Our findings indicate that early RAS blockade may provide effective nephroprotection in this hereditary podocytopathy.</p> <p> </p

An inducible mouse model of podocin-mutation-related nephrotic syndrome

Mutations in the NPHS2 gene, encoding podocin, cause hereditary nephrotic syndrome. The most common podocin mutation, R138Q, is associated with early disease onset and rapid progression to end-stage renal disease. Knock-in mice carrying a R140Q mutation, the mouse analogue of human R138Q, show developmental arrest of podocytes and lethal renal failure at neonatal age. Here we created a conditional podocin knock-in model named NPHS2 R140Q/-, using a tamoxifen-inducible Cre recombinase, which permits to study the effects of the mutation in postnatal life. Within the first week of R140Q hemizygosity induction the animals developed proteinuria, which peaked after 4-5 weeks. Subsequently the animals developed progressive renal failure, with a median survival time of 12 (95% CI: 11-13) weeks. Foot process fusion was observed within one week, progressing to severe and global effacement in the course of the disease. The number of podocytes per glomerulus gradually diminished to 18% compared to healthy controls 12-16 weeks after induction. The fraction of segmentally sclerosed glomeruli was 25%, 85% and 97% at 2, 4 and 8 weeks, respectively. Severe tubulointerstitial fibrosis was present at later disease stage and was correlated quantitatively with the level of proteinuria at early disease stages. While R140Q podocin mRNA expression was elevated, protein abundance was reduced by more than 50% within one week following induction. Whereas miRNA21 expression persistently increased during the first 4 weeks, miRNA-193a expression peaked 2 weeks after induction. In conclusion, the inducible R140Q-podocin mouse model is an auspicious model of the most common genetic cause of human nephrotic syndrome, with a spontaneous disease course strongly reminiscent of the human disorder. This model constitutes a valuable tool to test the efficacy of novel pharmacological interventions aimed to improve podocyte function and viability and attenuate proteinuria, glomerulosclerosis and progressive renal failure

Whereas mRNA expression of mutant <i>Nphs2</i> is elevated, podocin protein abundance is diminished.

<p>(A) <i>Nphs2</i>^{<i>R140Q/-</i>} animals showed an elevated expression level of mutated podocin mRNA during the first four weeks following induction (analysis is based on 4–6 animals per group and time point) WT: wild type. (B) Western blot analysis of total kidney extracts showing partial podocin protein loss during first two weeks, subtotal loss after 4–6 weeks and complete loss at attainment of end-stage renal disease (week 12–16) (analysis is based on 4–6 animals per group and time point; p<0.05).</p

Progressive loss of glomerular podocin abundance in the course of disease.

<p>Podocin (green), nidogen (red) and nucleus (blue) staining of glomeruli of healthy and <i>Nphs2</i>^{<i>R140Q/-</i>} animals. (A) Normally expressed podocin in glomerulus of a healthy animal. (B) Partial podocin loss one week after the induction. (C) Immense podocin loss in <i>Nphs2</i>^{<i>R140Q/-</i>} animals four weeks after the induction. (D) Subtotal to total podocin loss at the end stage disease. Magnification, X640.</p

Podocyte foot process effacement in <i>Nphs2</i>^{<i>R140Q/-</i>} mice.

<p>(A) Ultrastructural studies showed regular foot processes (FP) in healthy control animals on the opposite side of endothelial cells (En) lining the capillary lumen (L). (B) Irregularly shaped or fused FPs in <i>Nphs2</i>^{<i>R140Q/-</i>} mice one week after induction (arrow). (C and D) Progression of focal changes to global fusion of FPs in <i>Nphs2</i>^{<i>R140Q/-</i>} animals over time (Magnification, X10000). 3D modelling of glomerular structure showed no GBM denudation in <i>Nphs2</i>^{<i>R140Q/-</i>} animal (F) compared to controls (E). Blue colour represents GBM, pink and green represent FPs of adjacent podocytes. Severely affected FP number and organization in <i>Nphs2</i>^{<i>R140Q/-</i>} animals (G). GBM thickening in <i>Nphs2</i>^{<i>R140Q/-</i>} animals (H) (G and H: analysis is based on 3 animals per group). ** p<0.01, **** p<0.0001.</p