The cyclin-dependent kinase inhibitor p21 is required for TGF-β1-induced podocyte apoptosis

Abstract

Background. Reduced podocyte number is a critical determinant in the development of glomerulosclerosis. Transforming growth factor-β1 (TGF-β1) induces podocyte apoptosis, but the cell cycle events are not known. The cyclin-dependent kinase (CDK) inhibitor p21 increases in podocytes in diseases where TGF-β increases. Accordingly, we studied the role of p21 in podocyte apoptosis. Methods. Immortalized and primary p21+/+ and p21-/-mouse podocytes were used. Apoptosis was measured by Hoechst 33342 staining and caspase-3 activity following the exposure to TGF-β1 or puromycin aminonucleoside. p21 and specific Bcl-2-related family proteins levels were measured by Western blot analysis. To prove a role for p21, we reconstituted p21 expression in p21-/- podocytes utilizing an adenovirus vector. Results. TGF-β1 increased the protein levels of p21 in p21+/+ podocytes, and this coincided with apoptosis. In contrast, TGF-β1 did not induce apoptosis in p21-/- podocytes. Restoring p21 expression increased apoptosis in p21-/- podocytes following exposure to TGF-β1. TGF-β1 increased the protein levels of an antiapoptotic Bcl-2 in p21-/- podocytes, but not in p21+/+ podocytes. Moreover, TGF-β1 did not increase Bcl-2 expression in p21-/- podocytes in which p21 expression was restored. Finally, puromycin aminonucleoside also induced apoptosis in p21+/+ podocytes, but not in p21-/- podocytes. Conclusion. Podocyte apoptosis induced by TGF-β1 and puromycin aminonucleoside requires p21, and Bcl-2 plays a crucial role downstream of p21 in mediating this effect. These results suggest that p21 may play a critical role in the decrease in podocyte number in disease status accompanied by increased TGF-β1. © 2005 by the International Society of Nephrology.