Sonic hedgehog connects podocyte injury to mesangial activation and glomerulosclerosis
Dong Zhou, Haiyan Fu, Yang Han, Lu Zhang, Shijia Liu, Lin Lin, Donna B. Stolz, Youhua Liu
Dong Zhou, Haiyan Fu, Yang Han, Lu Zhang, Shijia Liu, Lin Lin, Donna B. Stolz, Youhua Liu
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Research Article Nephrology

Sonic hedgehog connects podocyte injury to mesangial activation and glomerulosclerosis

Abstract

Glomerular disease is characterized by proteinuria and glomerulosclerosis, two pathologic features caused by podocyte injury and mesangial cell activation, respectively. However, whether these two events are linked remains elusive. Here, we report that sonic hedgehog (Shh) is the mediator that connects podocyte damage to mesangial activation and glomerulosclerosis. Shh was induced in glomerular podocytes in various models of proteinuric chronic kidney diseases (CKD). However, mesangial cells in the glomeruli, but not podocytes, responded to hedgehog ligand. In vitro, Shh was induced in podocytes after injury and selectively promoted mesangial cell activation and proliferation. In a miniorgan culture of isolated glomeruli, Shh promoted mesangial activation but did not affect the integrity of podocytes. Podocyte-specific ablation of Shh in vivo exhibited no effect on proteinuria after adriamycin injection but hampered mesangial activation and glomerulosclerosis. Consistently, pharmacologic blockade of Shh signaling decoupled proteinuria from glomerulosclerosis. In humans, Shh was upregulated in glomerular podocytes in patients with CKD and its circulating level was associated with glomerulosclerosis but not proteinuria. These studies demonstrate that Shh mechanistically links podocyte injury to mesangial activation in the pathogenesis of glomerular diseases. Our findings also illustrate a crucial role for podocyte-mesangial communication in connecting proteinuria to glomerulosclerosis.

Authors

Dong Zhou, Haiyan Fu, Yang Han, Lu Zhang, Shijia Liu, Lin Lin, Donna B. Stolz, Youhua Liu

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Figure 2

Shh selectively promotes mesangial cell activation and proliferation in vitro.

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Shh selectively promotes mesangial cell activation and proliferation in ...
(A) qPCR shows that Shh induced Gli1 and Gli2 expression in cultured mesangial cells in a dose-and time-dependent manner. *P < 0.05, **P < 0.01 versus controls (n = 3, Student-Newman-Keuls test). (B) Shh did not have a significant effect on Gli1 and Gli2 expression in cultured podocytes (n = 3, Student-Newman-Keuls test). (C) Representative micrographs show the phase-contrast images of glomerular mesangial cells after incubation with different doses of Shh for 48 hours. Scale bar: 10 μm. (D and E) Cell counting demonstrates that Shh promoted mesangial cell proliferation in a dose- and time-dependent manner. Glomerular mesangial cells were incubated with a fixed dose of Shh (100 ng/ml) for various periods of time (D) or with different concentrations of Shh for 3 days (E). Cell numbers (×104 per well) were counted and are presented. *P < 0.05, **P < 0.01 versus controls (n = 3, Student-Newman-Keuls test). (F and G) Colorimetric MTT assay shows that Shh promoted mesangial cell proliferation in a time- (F) and dose-dependent fashion (G). *P < 0.05, **P < 0.01 versus controls (n = 3, Student-Newman-Keuls test). (H and I) Shh did not affect podocyte proliferation, as assessed by MTT assay. (J) Representative micrographs show that Shh promoted mesangial cell DNA synthesis, as demonstrated by BrdU incorporation. Mesangial cells were incubated with 25 and 50 ng/ml Shh for 2 days, respectively. Cells were immunostained with mouse anti-BrdU antibody (red). SYTO-Green was used to visualize the nuclei. Arrows indicate BrdU+ cells. Scale bar: 20 μm. (K) Western blots show that Shh induced the expression of numerous proliferation-related genes in cultured mesangial cells. Cell lysates were subjected to Western blot analyses for PCNA, cyclin D1, c-fos, c-Myc, fibronectin (FN), PDGFR-β, α-SMA, Wnt1, Wnt2, β-catenin, and α-tubulin, respectively. (L) Representative micrographs show that Shh activated mesangial cells by inducing α-SMA expression after incubation for 2 days. Scale bar: 20 μm.