Angiotensin II AT2 receptor regulates ureteric bud morphogenesis

Abstract

ANG II AT2 receptor (AT2R)-deficient mice exhibit abnormal ureteric bud (UB) budding, increased incidence of double ureters, and vesicoureteral reflux. However, the role of the AT2R during UB morphogenesis and the mechanisms by which aberrant AT2R signaling disrupts renal collecting system development have not been fully defined. In this study, we mapped the expression of the AT2R during mouse metanephric development, examined the impact of disrupted AT2R signaling on UB branching, cell proliferation, and survival, and investigated the cross talk of the AT2R with the glial-derived neurotrophic factor (GDNF)/c-Ret/Wnt11 signaling pathway. Embryonic mouse kidneys express AT2R in the branching UB and the mesenchyme. Treatment of embryonic day 12.5 (E12.5) metanephroi with the AT2R antagonist PD123319 or genetic inactivation of the AT2R in mice inhibits UB branching, decreasing the number of UB tips compared with control (34 +/- 1.0 vs. 43 +/- 0.6, P < 0.01; 36 +/- 1.8 vs. 48 +/- 1.3, P < 0.01, respectively). In contrast, treatment of metanephroi with the AT2R agonist CGP42112 increases the number of UB tips compared with control (48 +/- 1.8 vs. 39 +/- 12.3, P < 0.05). Using real-time quantitative RT-PCR and whole mount in situ hybridization, we demonstrate that PD123319 downregulates the expression of GDNF, c-Ret, Wnt11, and Spry1 mRNA levels in E12.5 metanephroi grown ex vivo. AT(2)R blockade or genetic inactivation of AT2R stimulates apoptosis and inhibits proliferation of the UB cells in vivo. We conclude that AT2R performs essential functions during UB branching morphogenesis via control of the GDNF/c-Ret/Wnt11 signaling pathway, UB cell proliferation, and survival.

Fig. 1.

Immunolocalization of ANG II A₂ receptor (AT₂R) protein in the fetal kidney on embryonic (E) day E11.5 and E12. A and B: on E11.5, AT₂R is detectable using antibody concentrations of 1/100. In a T-stage ureteric bud (UB), AT₂R immunostaining (brown) is present in both luminal and basolateral aspects of the UB followed by the induced mesenchyme (arrows). C: on E12, AT₂R is detectable using antibody concentrations of 1/200. In the E12 metanephros, distinct but modest staining is present in the stromal mesenchyme, vesicles, and UB tips. D: the adrenal medulla exhibits the strongest immunostaining. E and F: control consecutive sections demonstrate marked attenuation in AT₂R immunostaining after preadsorbtion of the primary antibody with its immunogen. Ad, adrenal gland.

Fig. 2.

Immunolocalization of AT₂R protein in the fetal kidney on E14 and E16. A and B: AT₂R is detectable using antibody concentrations of 1/400. On E14, AT₂R immunoreactivity is present in both luminal and basolateral membranes of UB branches followed by nephron progenitors and medullary stroma. C: on E16, AT₂R is expressed diffusely in inner cortical tubules, which resemble morphologically proximal tubules, followed by glomeruli and medullary stroma. D: control section where the addition of the primary antibody was omitted demonstrates no staining. UB, ureteric bud; G, glomerulus; S, stroma; T, tubules.

Fig. 3.

Effect of ANG II and PD123319 on UB cell branching. Top: RT-PCR and Western blot analysis of AT₂R. A: ethidium bromide-stained gel showing RT-PCR analysis of AT₂R mRNA expression. B: Western blot (0.02 mg total protein/lane) showing AT₂R protein expression. KRNK cells (positive control); P1, postnatal day 1 mouse kidney. C–F: phase-contrast micrographs of UB cells grown for 48 h in collagen gels in the presence of ANG II (10⁻⁵ M) alone or combined with PD123319 (10⁻⁶ M). G: bar graph shows the effect of treatments on the number of UB cells with processes after 48 h of culture.

Fig. 4.

Effect of media, PD123319, CGP42112, or genetic inactivation of the AT₂R on UB branching in mouse metanephroi. A and B: after 48 h in culture, kidney explants were costained with anti-pancytokeratin antibody to label the UB (red) and Lotus tetragonolobus agglutinin (LTA) to label proximal tubule epithelia (green). C and D: UB is visualized with anti-pancytokeratin antibody (green). The number of UB tips was compared between the treatment groups. A–D: representative images of branching UBs in paired metanephroi. A: left kidney, media; B: right kidney, PD123319 (PD). C: left kidney, media. D: right kidney, CGP42112 (10⁻⁹ M; CGP). E: bar graph showing the effect of media, PD123319, or CGP42112 on the number of UB tips. F–H: effect of genetic inactivation of the AT₂R on UB branching on E13.5. Metanephroi were costained with anti-pancytokeratin antibody to label the UB (green) and anti-WT1 antibody to label the mesenchyme and podocytes (red). F: AT₂R^+/+ kidney. G: AT₂R^−/− kidney. H: bar graph showing the effect of AT₂R genotype on the number of UB tips. Numbers on A–D and F and G indicate the number of UB tips.

Fig. 5.

AT₂R mRNA expression in the mouse metanephros on E11.5–E13.5 as determined by in situ hybridization (ISH). A–D: whole mount ISH. E and F: ISH on kidney sections. A: on E11.5, AT₂R mRNA is expressed predominantly in the UB stalk and mesenchyme (M) adjacent to the T-stage UB tip (blue staining). AT₂R mRNA is not present in the UB tips (outlined by dotted lines). C: on E12.5, AT₂R mRNA is expressed in the ureter, UB branches, and mesenchyme. E: on E13.5, AT₂R mRNA is present in the UB, mesenchyme, and maturing glomeruli (G). B, D, and F: control kidneys hybridized with the sense AT₂R probe show remarkably attenuated staining.

Fig. 6.

AT₂R blockade downregulates Spry1, glial-derived neurotrophic factor (GDNF), c-Ret, and Wnt11 mRNA expression in E12.5 mouse metanephroi that were grown ex vivo for 24 h. After 24 h in culture, kidney explants were processed for whole mount in situ hybridization or total RNA was extracted and Spry1, GDNF, c-Ret, and Wnt11 mRNA levels were analyzed by real-time quantitative RT-PCR. A: representative images demonstrate that treatment with PD123319 decreases the expression of c-Ret and Wnt11 in the UB tips, of GDNF in the mesenchyme, and of Spry1 in the UB tips and mesenchyme. Note that GDNF staining in the UB is nonspecific. B: bar graph showing that treatment with PD123319 decreases Spry1, GDNF, c-Ret, and Wnt11 mRNA levels as determined by RT-PCR.

Fig. 7.

Effect of AT₂R antagonist PD123319 on UB cell apoptosis and proliferation. A–D: apoptotic cells were identified by terminal uridine triphosphate (UTP) nick-end labeling (TUNEL; brown staining). A: media. B and C: PD123319 (10⁻⁶ M). D: kidney treated with TACS-nuclease to generate DNA breaks in every cell (positive control). E: bar graph shows the effect of media or PD123319 on the number of TUNEL-positive cells in the UB tip and stalk cells. F–I: proliferating cells are identified by anti-phospho-histone H3 (pH3) antibody (red staining). UB epithelia are visualized with anti-cytokeratin antibody (green). F and G: media. H and I: PD123319 (10⁻⁶ M). PD123319-treated metanephroi have less pH3 staining in UB (arrows) compared with control (media; marked by rectangles in G and I). J: bar graph shows the effect of media or PD123319 on cell proliferation index in the UB tip and stalk cells.

Fig. 8.

Effect of AT₂R genotype on cell apoptosis and proliferation in E13.5 mouse metanephroi. A–D: apoptotic cells are identified by anti-caspase 3 antibody staining (green). B and D: merged images show cell nuclei visualized with 4′-6-diamidino-2-phenylindole (DAPI; blue) and UBs visualized with anti-cytokeratin antibody (red). E: bar graph shows the effect of AT₂R genotype on the number of caspase 3-positive cells in the UB and mesenchyme. F–I: proliferating cells are identified by anti-pH3 antibody (green). Merged images show cell nuclei visualized with DAPI (blue) and UBs visualized with anti-cytokeratin antibody (red). J: bar graph shows the effect of AT₂R genotype on the number of pH3-positive cells in the UB and mesenchyme. Magnification ×20.