Angiotensin II-induced activation of c-Ret signaling is critical in ureteric bud branching morphogenesis

The renin-angiotensin system (RAS) plays a critical role in ureteric bud (UB) and kidney morphogenesis. Mutations in the genes encoding components of the RAS cause a spectrum of congenital abnormalities of the kidney and urinary tract (CAKUT). However, the mechanisms by which aberrations in the RAS result in CAKUT are poorly understood. Given that c-Ret receptor tyrosine kinase (RTK) is a major inducer of UB branching, the present study tested the hypothesis that angiotensin (Ang) II-induced activation of c-Ret plays a critical role in UB branching morphogenesis. E12.5 mice metanephroi were grown for 24h in the presence or absence of Ang II, Ang II AT(1) receptor (AT(1)R) antagonist candesartan, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or ERK1/2 inhibitor PD98059. Ang II increased the number of UB tips (61+/-2.4 vs. 45+/-4.3, p<0.05) compared with control. Quantitative RT-PCR analysis demonstrated that Ang II increased c-Ret mRNA levels in the kidney (1.35+/-0.05 vs. 1.0+/-0, p<0.01) and in the UB cells (1.28+/-0.04 vs. 1.0+/-0, p<0.01) compared to control. This was accompanied by increased Tyr(1062)Ret phosphorylation by Ang II (5.5+/-0.9 vs. 1.8+/-0.4 relative units, p<0.05). In addition, treatment of UB cells with Ang II (10(-5)M) increased phosphorylation of Akt compared to control (213+/-16 vs. 100+/-20%, p<0.05). In contrast, treatment of metanephroi or UB cells with candesartan decreased c-Ret mRNA levels (0.72+/-0.06 vs. 1.0+/-0, p<0.01; 0.68+/-0.07 vs. 1.0+/-0, p<0.05, respectively) compared with control. Ang II-induced UB branching was abrogated by LY294002 (24+/-2.6 vs. 37+/-3.0, p<0.05) or PD98059 (33+/-2.0 vs. 48+/-2.2, p<0.01). These data demonstrate that Ang II-induced UB branching depends on activation of Akt and ERK1/2. We conclude that cross-talk between the RAS and c-Ret signaling plays an important role in the development of the renal collecting system.