Downregulation of Spry-1, an inhibitor of GDNF/Ret, causes angiotensin II-induced ureteric bud branching

Abstract

Mutations of the renin-angiotensin system (RAS) genes are associated with congenital abnormalities of the kidney and urinary tract. We have shown that angiotensin (Ang) II stimulates ureteric bud (UB) branching in vitro. The present study tested the hypothesis that Ang II stimulates the GDNF/c-Ret/Wnt11 pathway. E12.5 mice metanephroi were grown for 24 hours in the presence or absence of Ang II or AT1R receptor antagonist candesartan and subjected to whole-mount ISH. c-Ret, a receptor tyrosine kinase for GDNF, and its downstream target Wnt11 were induced by Ang II in the UB tip cells. GDNF, a Wnt11-regulated gene expressed in the mesenchyme, was also upregulated by Ang II. In contrast, Ang II treatment downregulated Spry1, an endogenous inhibitor of Ret tyrosine kinase activity, in an AT1R-dependent manner. Quantitative RT-PCR analysis confirmed that Ang II decreases Spry1 mRNA levels in cultured UB cells. In vivo BrdU incorporation indicated that exogenous Ang II preferentially stimulates UB tip cell proliferation, while AT1R blockade increases TUNEL-positive apoptotic cells. These findings suggest a model in which AT1R-mediated inhibition of Spry1 gene expression releases c-Ret tyrosine kinase activity leading to upregulation of c-Ret and its downstream target gene, Wnt11. Enhanced Wnt11 expression induces GDNF in the adjacent mesenchyme. This causes focal bursts of UB tip cell proliferation, a decrease in UB tip cell apoptosis and branching.

Ontogeny of angiotensin-converting enzyme 2

“…Spatial distribution of ACE2 immunoreactivity during gestation shifts from diffuse staining in the UBs, nascent glomeruli, and the mesenchyme on E12.5 to a more localized expression in the tubular epithelia that resemble morphologically proximal tubules during E14.5-E18.5. We have recently reported that Ang II, when applied directly to whole intact E11.5 mouse metanephroi cultured in vitro, stimulates UB branching (18). The presence of ACE2 in the UB branches suggests a novel role for ACE2 in the regulation of UB morphogenesis.…”

“…Although all ACE2-mutant lines were generated with randomly mixed genetic backgrounds derived from C57BL/6 and 129 mice (11,15,16), the differences in cardiac structure and function, the dominant phenotype reported in ACE2-null adult mice (11), do not appear to be due to variability in genetic background (17). Because we have previously reported an important role for Ang II and its AT 1 R in ureteric bud (UB) morphogenesis during embryonic kidney development in CD1 mice (18), we chose to examine ACE2 ontogeny in CD1 mice.…”

Ontogeny of the (pro)renin receptor

“…Organs were homogenized in cold lysis buffer containing a cocktail of enzyme inhibitors (35). The samples were centrifuged, and the supernatants containing proteins (40 µg/lane) were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes.…”

“…SYBR Green quantitative real-time RT-PCR with (P)RR-specific primers (SABiosciences, Frederick, MD) was conducted as described previously (35). The quantity of (P)RR mRNA expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase mRNA expression.…”

“…Because β-actin cannot be used as a loading control for protein expression in the heart muscle (36), western blots for (P)RR expression in the heart tissue were reprobed with glyceraldehyde 3-phosphate dehydrogenase antibody. Immunoreactive bands were visualized using the enhanced chemiluminescence detection system (ECL; Amersham, Piscataway, NJ) as previously described (35).…”