Angiotensin II stimulates in vitro branching morphogenesis of the isolated ureteric bud

Abstract

Mutations in the renin-angiotensin system (RAS) genes are associated with congenital anomalies of the kidney and urinary tract (CAKUT). As angiotensin (Ang) II, the principal effector peptide growth factor of the RAS, stimulates ureteric bud (UB) branching in whole intact embryonic (E) metanephroi, defects in UB morphogenesis may be causally linked to CAKUT observed under conditions of disrupted RAS. In the present study, using the isolated intact UB (iUB) assay, we tested the hypothesis that Ang II stimulates UB morphogenesis by directly acting on the UB, identified Ang II target genes in the iUB by microarray and examined the effect of Ang II on UB cell migration in vitro. We show that isolated E11.5 mouse iUBs express Ang II AT(1) and AT(2) receptor mRNA. Treatment of E11.5 iUBs grown in collagen matrix gels with Ang II (10(-5)M) increases the number of iUB tips after 48h of culture compared to control (4.8±0.4 vs. 2.4±0.2, p<0.01). A number of genes required for UB branching as well as novel genes whose role in UB development is currently unknown are targets of Ang II signaling in the iUB. In addition, Ang II increases UB cell migration (346±5.1 vs. 275±4.4, p<0.01) in vitro. In summary, Ang II stimulates UB cell migration and directly induces morphogenetic response in the iUB. We conclude that Ang II-regulated genes in the iUB may be important mediators of Ang II-induced UB branching. We hypothesize that Ang II-dependent cell movements play an important role in UB branching morphogenesis.

Figure 1

A: Isolated intact E11.5 ureteric buds (iUB) express Ret (300 bp), but not GDNF (291 bp), mRNA. E12.5 mouse kidneys (Kid) express both Ret and GDNF mRNA. B and C: iUB, immortalized UB cells (UBc) and E12.5 mouse kidneys express angiotensin (Ang) II AT₁R and AT₂R mRNA. D: Effect of Ang II on branching of iUBs. iUBs were dissected on E11.5 and grown in collagen matrix gels in the presence of GDNF, FGF1 and media (control) or GDNF, FGF1 and Ang II (10⁻⁵ M) for 48 hours. Numbers below each iUB image show number of UB tips (marked with arrows) at each time point. E: Bar graph shows the effect of treatments on the number of UB tips after 48 hours of culture. The number of iUB tips in higher in Ang II-treated iUBs compared with media control (Mean±SEM).

Figure 2

A: Bar graph shows that the number of ureteric bud-derived cells (UB cells) that migrated across the filter is higher in the presence of angiotensin (Ang) II compared to control (media) (Mean±SEM). B: Quantitative real-time reverse transcription-polymerase chain reaction confirming the changes in gene expression observed in the microarray analysis in isolated E11.5 ureteric buds treated with Ang II or media (control). Media values are normalized to 1 (Con- control) and data are presented as relative-fold difference. Du- Dusp8, Zfp- Zfp42, C20- Ccl20. C: Ang II upregulates Etv4 and Etv5 mRNA expression in E12.5 mouse metanephroi that were grown ex vivo for 24 h in the presence of Ang II (10⁻⁵ M) or media (control). After 24 h in culture, kidney explants were processed for whole-mount in situ hybridization. Representative images demonstrate that treatment with Ang II increases Etv4 (a) and Etv5 (c) mRNA expression in the UB compared to control (media, b, d).

Figure 3

Ingenuity pathway analysis of phosphatidylinositol 3-kinase (PI3K) signaling pathway affected by angiotensin II. Pink color indicates upregulated genes; green color indicates downregulated genes.