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. 2011 Dec;22(12):2203-12.
doi: 10.1681/ASN.2011020202. Epub 2011 Nov 3.

Prorenin receptor is essential for normal podocyte structure and function

Yoichi Oshima  1 , Kenichiro Kinouchi, Atsuhiro Ichihara, Mariyo Sakoda, Asako Kurauchi-Mito, Kanako Bokuda, Tatsuya Narita, Hideaki Kurosawa, Ge-Hong Sun-Wada, Yoh Wada, Taketo Yamada, Minoru Takemoto, Moin A Saleem, Susan E Quaggin, Hiroshi Itoh
Affiliations

Prorenin receptor is essential for normal podocyte structure and function

Yoichi Oshima et al. J Am Soc Nephrol. 2011 Dec.

Abstract

The prorenin receptor is an accessory subunit of the vacuolar H(+)-ATPase, suggesting that it has fundamental functions beyond activation of the local renin-angiotensin system. Podocytes express the prorenin receptor, but its function in these cells is unknown. Here, podocyte-specific, conditional, prorenin receptor-knockout mice died of kidney failure and severe proteinuria within 4 weeks of birth. The podocytes of these mice exhibited foot process effacement with reduced and altered localization of the slit-diaphragm proteins nephrin and podocin. Furthermore, the podocytes contained numerous autophagic vacuoles, confirmed by enhanced accumulation of microtubule-associated protein 1 light chain 3-positive intracellular vesicles. Ablation of the prorenin receptor selectively suppressed expression of the V(0) c-subunit of the vacuolar H(+)-ATPase in podocytes, resulting in deacidification of intracellular vesicles. In conclusion, the prorenin receptor is important for the maintenance of normal podocyte structure and function.

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Figures

Figure 1.
Figure 1.
Podocyte-specific ablation of Atp6ap2 inevitably caused renal failure. (A) Kaplan-Meier survival curves showing lethality within 4 weeks of birth in the conditional prorenin receptor-knockout (CKO) group. (B) Biochemical analysis of urine and plasma showing renal failure and severe renal protein wasting in CKO mice on postnatal day (P) 21. Cr, creatinine; TP, total protein; TC, total cholesterol; ns, not significant. The data are the means ± SD (n = 10). **P < 0.001 versus wild-type (WT) control.
Figure 2.
Figure 2.
Podocyte-specific deletion of the prorenin receptor caused glomerular sclerosis. Light microscopic examination of kidneys was performed from wild-type (WT) and conditional prorenin receptor-knockout (CKO) mice on postnatal day (P) 1, P7, P14, and P21 using periodic acid-Schiff and periodic acid-silver methenamine staining. No differences were evident on P1. On P7, focal glomerular changes, consisting of the proliferation of mesangial cells, appeared in kidneys from CKO mice. On P14, diffuse glomerular changes, such as proliferation of the mesangial matrix, segmental sclerosis, hypertrophic changes of parietal epithelial cells without crescent formations, and mild tubulointerstitial changes, such as tubular casts and dilation, were observed in kidneys from CKO mice. On P21, kidney glomeruli from CKO mice revealed diffuse and global mesangial sclerosis. Tubulointerstitial changes, including tubular dilation, tubular atrophy with disappearance of the brush border, and extensive tubular casts were prominent. Scale bars, 100 μm (left panel), 20 μm (center and right panels). Arrowheads indicate tubular casts. The asterisks indicate mesangial expansion and sclerosis.
Figure 3.
Figure 3.
The prorenin receptor-deleted podocytes developed foot process effacement and intracellular vacuoles. Electron microscopic examination of kidneys was performed from wild-type (WT) and conditional prorenin receptor-knockout (CKO) mice on postnatal day (P) 1, P7, P14, and P21. On P1, there were no obvious differences between WT and CKO mice. As early as P7, kidneys from CKO mice were found to contain prominently enlarged podocytes with extensive foot process effacement and actin filament aggregation. Numerous electron-dense autophagic vacuoles containing partially digested cellular components were observed in the cytoplasm of these podocytes. Mesangial matrix expansion was also noted in CKO glomeruli. Ultimately, on P21, the capillary lumen of kidneys from CKO mice had collapsed because of the presence of highly vacuolated podocytes and mesangial expansion. Basement membranes were intact without any electron-dense deposits in any of the glomeruli. Scale bars, 10 μm (left panels), 5 μm (left center panels), 2 μm (right center panels), and 1 μm (right panels). C, glomerular capillary; E, endothelial cells; P, podocytes; V, vesicular structure. Arrows indicate foot processes. White arrowheads indicate hypertrophic change of parietal epithelial cells. Black arrowheads indicate glomerular basement membrane. Asterisks indicate mesangial expansion and sclerosis.
Figure 4.
Figure 4.
Reduced expression and altered localization of slit-diaphragm proteins nephrin and podocin in the podocyte-specific prorenin receptor deleted kidney. (A and B) Immunofluorescent staining of nephrin (A) and podocin (B) was performed on postnatal day (P) 1, P7, and P14 in wild-type (WT) and conditional prorenin receptor-knockout (CKO) mice. Over the course of development, the localization of nephrin and podocin shifted from the glomerular basement membrane to podocyte cell bodies in glomeruli from CKO mice. (C) Nephrin and podocin expression were significantly decreased in kidneys from CKO mice on P1, P7, and P14. Scale bars, 20 μm.
Figure 5.
Figure 5.
The significant podocyte loss by the prorenin receptor deletion. Immunofluorescent staining of kidneys was performed from wild-type (WT) and conditional prorenin receptor-knockout (CKO) mice on postnatal day (P) 14 and P21 for the podocyte nuclear marker WT1. The number of WT1-positive podocytes in glomeruli from CKO mice was similar to that in WT mice on P14 but decreased significantly in glomeruli from CKO mice on P21, probably reflecting increased cell death or detachment from glomerular basement membrane of podocytes. Scale bars, 50 μm (left panels), 20 μm (right panels). Graphed data show the means ± SD. *P < 0.05 compared with WT control.
Figure 6.
Figure 6.
Autophagosomes in the prorenin receptor-null podocytes. Immunofluorescent staining of kidneys was performed from wild-type (WT) and conditional prorenin receptor-knockout (CKO) mice on postnatal day (P) 14 for the late endosome and lysosome marker RAB7, lysosomal-associated membrane protein 2 (LAMP2), and the autophagosome marker light chain 3 (LC3). Glomeruli from CKO mice showed increased expression for RAB7, LAMP2, and LC3. Note that these markers are colocalized with nephrin, meaning that these markers correspond with vacuoles of podocytes. Scale bars, 50 μm (left panels), 20 μm (center panels), and 10 μm (right panels).
Figure 7.
Figure 7.
ATP6AP2 is indispensable for the assembly of the vacuolar H+-ATPase (V-ATPase). (A) In human cultured podocytes, the expression of ATP6AP2 mRNA is reduced after transfection of ATP6AP2 short interference RNA (siATP6AP2). Graphed data show the means ± SD. *P < 0.05 compared with vehicle control. (B) Protein levels of ATP6AP2 and the c-subunit of the V0 segment of V-ATPase are significantly decreased in cultured podocytes after transfection of ATP6AP2 siRNA. Transfection of ATP6AP2 siRNA had no effect on the E2-subunit of V1. Note that nephrin and podocin expressions were reduced in podocytes after ATP6AP2 siRNA treatment. (C) Defective acidification in podocytes after knockdown of the ATP6AP2 gene and treatment with bafilomycin and chloroquine. The nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI; blue). (D and E) RAB7-positive vacuoles (D) and light chain 3 (LC3)-positive autophagic vacuoles (E) after treatment with ATP6AP2 siRNA, bafilomycin, and chloroquine. Scale bars, 20 μm. (F) Quantitative analysis of LysoTracker, RAB7, and LC3-positive vacuoles after treatment with vehicle, ATP6AP2 siRNA, bafilomycin, and chloroquine. Graphed data show the means ± SD. *P < 0.05 compared with vehicle control.
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