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

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

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

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

Podocyte loss in <i>Nphs2</i>^{<i>R140Q/-</i>} mice.

<p>(A) Number of podocytes reduced in induced mice with the course of the disease (columns represent 4–6 animals per group and time point). (B) Wt1 labelled podocytes in glomerulus in a healthy animal. Decreased Wt1 signal in <i>Nphs2</i>^{<i>R140Q/-</i>} animals 1 week (C), 2 weeks (D), and 4 weeks after induction (E). Wt1: green, nidogen: red, nucleus: blue. Magnification, X640.</p

Time course of clinical phenotype in different hereditary podocytopathy mouse models.

<p>Time course of clinical phenotype in different hereditary podocytopathy mouse models.</p

<i>Wt1</i> regulation in induced animals.

<p><i>Wt1</i> expression is significantly reduced in <i>Nphs2</i>^{<i>R140Q/-</i>} animals 4 weeks after disease induction (columns represent 5–7 animals per group and time point). **p = 0.0002.</p

Podocin loss leads to renal damage.

<p>(A) control, (B) week 2, (C) week 4, (D) week 12. (E) Percentage of total kidney area affected by fibrosis in <i>Nphs2</i>^{<i>R140Q/-</i>} mice increased with observation time (columns represent 4–10 animals per group and time point). (F) Proteinuria 2 weeks after induction is correlated with the tubulointerstitial fibrosis score at week 4 (n = 17; p = 0.01). SR staining; Magnification, X150. * p<0.05, ** p<0.001.</p

<i>Nphs2</i>^{<i>R140Q/-</i>} mice develop focal-segmental glomerulosclerosis (FSGS).

<p>(A) Glomerular sclerosis index (GSI) in healthy and <i>Nphs2</i>^{<i>R140Q/-</i>} mice. (B) Percentage of glomeruli affected by sclerosis in <i>Nphs2</i>^{<i>R140Q/-</i>} mice increased drastically over time (columns represent 4–10 animals per group and time point). (C-F) Evolution of glomerular lesions in induced animals. PAS staining; Magnification, X200.</p