Angiotensin II Type 1 Receptor–EGF Receptor Cross-Talk Regulates Ureteric Bud Branching Morphogenesis

Abstract

Angiotensinogen-, angiotensin-converting enzyme–, and angiotensin II (Ang II) type 1 receptor (AT₁R)–deficient mice exhibit a dilated renal pelvis (hydronephrosis) and a small papilla. These abnormalities have been attributed to impaired development of the ureteral and pelvic smooth muscle. Defects in the growth and branching of the ureteric bud (UB), which gives rise to the collecting system, have not been examined carefully. This study tested the hypothesis that Ang II stimulates UB growth and branching in the intact metanephros. Immunohistochemistry demonstrated that embryonic mouse kidneys express AT₁R in the UB and its branches. Embryonic day 11.5 metanephroi were microdissected from Hoxb7–green fluorescence protein mice and grown for 48 h in serum-free medium in the presence or absence of Ang II. The number of green fluorescence protein–positive UB branch points (BP) and tips was monitored in each explant at 24 and 48 h. Ang II increased the number of UB tips and BP at 24 h (tips: 24.3 ± 1.1 versus 18.3 ± 0.7, P < 0.01; BP: 14.4 ± 0.6 versus 11.7 ± 0.6, P < 0.01) and 48 h (tips: 30.2 ± 1.3 versus 22.9 ± 0.8, P < 0.01; BP: 21.3 ± 0.9 versus 15.7 ± 0.6, P < 0.01) compared with control. In contrast, treatment of metanephroi with the AT₁R antagonist candesartan inhibited UB branching, decreasing the number of UB tips and BP. Similarly, inhibition of EGF receptor (EGFR) tyrosine kinase activity abrogated Ang II–stimulated UB branching. A cross-talk between the renin-angiotensin system and EGFR signaling was elicited at the cellular level by the ability of Ang II to induce tyrosine phosphorylation of EGFR in UB cells and through abrogation of Ang II–induced UB cell branching using an EGFR tyrosine kinase inhibitor. These data demonstrate that Ang II, acting via the AT₁R, stimulates UB branching morphogenesis. This process depends on tyrosine phosphorylation of the EGFR. Cooperation of AT₁R and EGFR signaling therefore is important in the development of the renal collecting system.