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. 2013 Apr;24(5):713-21.
doi: 10.1681/ASN.2012080844. Epub 2013 Mar 7.

N-wasp Is Required for Stabilization of Podocyte Foot Processes

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Free PMC article

N-wasp Is Required for Stabilization of Podocyte Foot Processes

Christoph Schell et al. J Am Soc Nephrol. .
Free PMC article

Abstract

Alteration of cortical actin structures is the common final pathway leading to podocyte foot process effacement and proteinuria. The molecular mechanisms that safeguard podocyte foot process architecture and maintain the three-dimensional actin network remain elusive. Here, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin nucleation, is required to stabilize podocyte foot processes. Mice lacking N-WASP specifically in podocytes were born with normal kidney function but developed significant proteinuria 3 weeks after birth, suggesting an important role for N-WASP in maintaining foot processes. In addition, inducing deletion of N-WASP in adult mice resulted in severe proteinuria and kidney failure. Electron microscopy showed an accumulation of electron-dense patches of actin and strikingly altered morphology of podocyte foot processes. Although basic actin-based processes such as cell migration were not affected, primary cultures of N-WASP-deficient podocytes revealed significant impairment of dynamic actin reorganization events, including the formation of circular dorsal ruffles. Taken together, our findings suggest that N-WASP-mediated actin nucleation of branched microfilament networks is specifically required for the maintenance of foot processes, presumably sustaining the mechanical resistance of the filtration barrier.

Figures

Figure 1.
Figure 1.
The actin nucleation machinery is highly enriched in podocyte FPs and conditional deletion of N-WASP in podocytes results in severe proteinuria and early lethality. (A`–A````) Confocal images of human glomeruli stained with specific antibodies against N-WASP and Nephrin; cytoplasmic accumulation is indicated by white arrow, and colocalization of Nephrin and N-WASP in close proximity to the glomerular capillary is marked by a white asterisk. (B) Immunogold labeling with an antibody against N-WASP reveals presence of N-WASP in primary as well as secondary FPs (black arrowheads). (C and D) N-WASP–positive gold particles are present in cortical actin bundles (arrowheads) and also in actin patches in primary FPs (asterisks). (E) Schematic representation of generation of N-WASPfl/fl*Nphs2Cre mice. (F) Efficiency of N-WASP deletion is confirmed by Western blotting on glomerular lysates from wild-type and N-WASPfl/fl*Nphs2Cre mice aged 3 weeks. (G) Follow-up on body weight gain in respective WT, heterozygote, and homozygous N-WASPfl/fl*Nphs2Cre animals. (H) Proteinuria is present at 3 weeks after birth. (I) Increased mortality is observed in respective knockout animals. **P <0.01; ***P<0.001. PP, primary foot process; FP, secondary foot process; GBM, glomerular basement membrane; E, endothelium; WT, wild-type; a, no difference.
Figure 2.
Figure 2.
Actin cluster accumulation accompanies N-WASP deletion in podocytes. (A–D) Knockout and wild-type kidneys are analyzed by scanning EM and transmission EM. Podocyte morphology and FP development exhibit no major differences at 1 week in podocytes from N-WASP fl/fl*Nphs2Cre mice compared with respective controls (slit diaphragms are indicated by white arrows in A and B). (C and D) At 3 weeks of age, significant ultrastructural alterations are detectable, including numerous microvillus extensions on the surface of podocytes in N-WASP KO animals (scanning EM shown in D; black arrows); furthermore electron-dense, belt like structures in close proximity to the GBM are present in effaced FPs of KO animals (white asterisks in D`` and D```). At higher magnifications, these electron-dense patches are identified as clumped actin accumulations (D``` and D````). (E) Further detailed views display almost detached podocytes, scarcely connected via thinned primary processes to the GBM. (G) Regular actin filaments extend from condensed actin patches in effaced secondary processes (black arrows indicate actin filaments, white asterisks mark actin patches). (H and I) Cryo-EM reveals actin condensations in respective KO mice (black arrows mark slit diaphragm region and white asterisks indicate actin condensations) already at 2 weeks of age.
Figure 3.
Figure 3.
CDR formation is impaired in primary N-WASP KO podocytes. (A) Live cell imaging of N-WASP KO induced and wild-type podocytes. Upon application of EGF (20 ng/ml), wild-type and KO cells form lamellipodial protrusions (black arrows). (B) Staining with phalloidin detects CDRs after stimulation with EGF (white arrows indicate CDRs). Quantification of CDR-positive cells after stimulation with EGF shows that N-WASP KO podocytes (Nphs2*Cre and Nphs2*rtTA*TetOCre induced cells) exhibit a diminished capacity to form CDRs compared with wild-type control cells. (C) Pretreatment with the N-WASP inhibitor wiskostatin also results in impaired dorsal ruffle formation in primary podocytes (white arrows indicate CDR formation). (D) Macropinocytosis assessed by dextran uptake shows no major difference between wild-type and KO cells (white arrows indicate dextran-positive macropinosomes). Assessment of macropinosome number per cell shows no difference between wild-type and mutant podocytes. In addition, size distribution of respective vesicles indicates a clear response in wild-type and KO podocytes upon EGF stimulation. **P<0.01; ***P<0.001.
Figure 4.
Figure 4.
N-WASP is required for the maintenance of podocyte FPs. (A) Schematic illustration of generation of N-WASPfl/fl*Nphs2rtTA*TetOCre mice. Timeline illustrates the induction protocol: 4 weeks after birth, doxycycline is applied for a 2-week period and urine is collected every week. (B and C) Five-week postinduction periodic acid–Schiff staining shows signs of proteinuria in N-WASPfl/fl*Nphs2rtTA*TetOCre mice (black arrows in C` indicate proteinaceous casts). A subset of glomeruli shows first signs of segmental scarring (white asterisk in C```). (D and E) Ultrastructural analysis detects FP effacement (black arrows) and actin condensations in effaced podocyte FPs (white asterisks) of N-WASPfl/fl*Nphs2rtTA*TetOCre animals (5 weeks after induction). (F) Measurement of proteinuria after completion of doxycycline induction shows first detectable increase after 1 week and further increase over the observational period. *P<0.05; **P<0.001.

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