Podocyte-Specific Deletion of Yes-Associated Protein Causes FSGS and Progressive Renal Failure - PubMed (original) (raw)

Podocyte-Specific Deletion of Yes-Associated Protein Causes FSGS and Progressive Renal Failure

Monica Schwartzman et al. J Am Soc Nephrol. 2016 Jan.

Abstract

FSGS is the most common primary glomerular disease underlying ESRD in the United States and is increasing in incidence globally. FSGS results from podocyte injury, yet the mechanistic details of disease pathogenesis remain unclear. This has resulted in an unmet clinical need for cell-specific therapy in the treatment of FSGS and other proteinuric kidney diseases. We previously identified Yes-associated protein (YAP) as a prosurvival signaling molecule, the in vitro silencing of which increases podocyte susceptibility to apoptotic stimulus. YAP is a potent oncogene that is a prominent target for chemotherapeutic drug development. In this study, we tested the hypothesis that podocyte-specific deletion of Yap leads to proteinuric kidney disease through increased podocyte apoptosis. Yap was selectively silenced in podocytes using Cre-mediated recombination controlled by the podocin promoter. Yap silencing in podocytes resulted in podocyte apoptosis, podocyte depletion, proteinuria, and an increase in serum creatinine. Histologically, features characteristic of FSGS, including mesangial sclerosis, podocyte foot process effacement, tubular atrophy, interstitial fibrosis, and casts, were observed. In human primary FSGS, we noted reduced glomerular expression of YAP. Taken together, these results suggest a role for YAP as a physiologic antagonist of podocyte apoptosis, the signaling of which is essential for maintaining the integrity of the glomerular filtration barrier. These data suggest potential nephrotoxicity with strategies directed toward inhibition of YAP function. Further studies should evaluate the role of YAP in proteinuric glomerular disease pathogenesis and its potential utility as a therapeutic target.

Keywords: focal segmental glomerulosclerosis; glomerular disease; podocyte.

Copyright © 2016 by the American Society of Nephrology.

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Figures

Figure 1.

Figure 1.

Immunofluorescence labeling of YAP in glomeruli. Upper panels: YAP (red) colocalizes with the podocyte-specific marker synaptopodin (green) with additional nuclear expression colocalizing with DAPI (blue) detected. Lower panels: confirmation of reduction in YAP protein expression with podocyte-specific gene silencing. Scale bar, 50 _µ_m.

Figure 2.

Figure 2.

Kidney appearance and function with Yap gene silencing. (A) _Yap_-KO kidneys are of comparable size to controls (Ctrl), but exhibit a corrugated capsule at 12 weeks of age. (B) SDS-PAGE Coomassie-blue staining of standard BSA (numbers indicate _μ_g/_μ_l) and urine from _Yap_-KO and control mice. Proteinuria first appears between 5 and 6 weeks of age in _Yap_-KO mice. In each respective lane, 5 _μ_l of urine and 1 _μ_l of BSA standard were loaded. (C) Albumin-creatinine ratios demonstrating weekly progression of proteinuria in _Yap_-KO and control mice. At week 12, P<0.01. (D) Serum creatinine measurements showing a decrease in kidney function in _Yap_-KO mice. Ctrl, Pod-Cre-Yapflox/flox.

Figure 3.

Figure 3.

PAS stainings of _Yap_-KO and control (Ctrl) mice at 12 weeks. (A,B) Ctrl kidney sections at low and high power. (C) _Yap_-KO mice with focal glomerulosclerosis and tubular dilatation and casts. (D) _Yap_-KO mouse with a lesion of focal segmental glomerulosclerosis. (E) Quantification of glomerular and tubular injury in 12-week-old mice. Scale bars, (A) and (C) 70 _µ_m; (B) and (D) 50 _µ_m.

Figure 4.

Figure 4.

Severe kidney disease in aged _Yap_-KO mice. (A) 32-week-old _Yap_-KO mouse PAS section with segmentally and globally sclerosed glomeruli. (B) 32-week-old _Yap_-KO mouse PAS section with diffuse tubular dilatation and proteinaceous casts. Scale bars, 50 _µ_m.

Figure 5.

Figure 5.

Electron microscopy images of _Yap_-KO mice. (A) Control with normal-appearing mesangial matrix. (B) Control with intact podocyte foot processes. (C) _Yap_-KO with moderate mesangial matrix accumulation (*). (D) _Yap_-KO with patchy foot process effacement. (E) _Yap_-KO with extensive FSGS with severe mesangial matrix expansion (**). (F) _Yap_-KO with diffuse podocyte foot process effacement. Scale bars, 3 _µ_m.

Figure 6.

Figure 6.

Podocyte apoptosis in _Yap_-KO mice. (A) PAS staining with apoptotic cell nucleus (arrow) in _Yap_-KO mouse at 12 weeks. (B) Terminal deoxynucleotidyl transferase–mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling–positive nuclei in _Yap_-KO but not control glomeruli at 12 weeks. (C) Anti-cleaved poly adenosine diphosphate ribose polymerase (PARP) immunofluorescence staining in control and _Yap_-KO mice at 12 weeks. (D) Cleaved caspase 3 staining in control and _Yap_-KO mice at 7.5 weeks. Scale bars, (A) 20 _µ_m; (B-D) 50 _µ_m.

Figure 7.

Figure 7.

Morphometric measurements. (A) Toluidine-blue staining showing quantification of podocyte number in adjacent epon sections 1-_µ_m thick. Only podocyte nuclei (asterisk) present in the sample section but not present in the look-up section (red arrows) were counted. Scale bar: 20 _µ_m. (B) Podocyte number was significantly reduced in _Yap_-KO mice at 12 weeks compared with the control. (C) Glomerular volumes were not significantly increased in _Yap_-KO compared with control (_P_=NS). (D) Podocyte density was reduced in _Yap_-KO mice compared with controls (_n_=3 mice, 30 total glomeruli per group).

Figure 8.

Figure 8.

Expression of YAP in human glomeruli. (A) YAP expression in normal glomeruli compared with a patient with FSGS. Scale bar, 50 _µ_m. (B) Quantification of fluorescence intensity.

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