Cyclooxygenase-2-dependent phosphorylation of the pro-apoptotic protein Bad inhibits tonicity-induced apoptosis in renal medullary cells

During antidiuresis, cell survival in the renal medulla requires cyclooxygenase-2 (COX-2) activity. We have recently found that prostaglandin E2 (PGE2) promotes cell survival by phosphorylation and, hence, inactivation of the pro-apoptotic protein Bad during hypertonic stress in Madin-Darby canine kidney (MDCK) cells in vitro. Here we determine the role of COX-2-derived PGE(2) on phosphorylation of Bad and medullary Apoptosis in vivo using COX-2-deficient mice. Both wild-type and COX-2-knockout mice constitutively expressed Bad in tubular epithelial cells of the renal medulla. Dehydration caused a robust increase in papillary COX-2 expression, PGE2 excretion, and Bad phosphorylation in wild-type, but not in the knockout mice. The abundance of cleaved Caspase-3, a marker of Apoptosis, was significantly higher in papillary homogenates, especially in tubular epithelial cells of the knockout mice. Knockdown of Bad in MDCK cells decreased tonicity-induced Caspase-3 activation. Furthermore, the addition of PGE2 to cells with knockdown of Bad had no effect on Caspase-3 activation; however, PGE2 caused phosphorylation of Bad and substantially improved cell survival in mock-transfected cells. Thus, tonicity-induced COX-2 expression and PGE2 synthesis in the renal medulla entails phosphorylation and inactivation of the pro-apoptotic protein Bad, thereby counteracting Apoptosis in renal medullary epithelial cells.