Histone deacetylases are critical regulators of the renin-angiotensin system during ureteric bud branching morphogenesis

Abstract

Mutations in the genes encoding components of the renin-angiotensin system (RAS) in mice or humans cause congenital abnormalities of the kidney and urinary tract. We hypothesized that absence of angiotensin (Ang) II in angiotensinogen (AGT)-deficient mice leads to defects in ureteric bud (UB) branching and that RAS genes are critically dependent on histone deacetylase (HDAC) activity. The number of UB tips was lower in AGT-/- compared with AGT+/+ embryonic (E) day E13.5 metanephroi (24+/-1.5 versus 36+/-3.7, p<0.05). Real-time RT-PCR demonstrated that pharmacological inhibition of HDAC activity with Scriptaid increases AGT, renin, angiotensin-converting enzyme (ACE), and AT1 receptor (AT1R) mRNA levels in E12.5 mouse metanephroi and early mesenchymal (MK3) cells. Furthermore, Scriptaid enhanced Ang II induced decrease in Sprouty (Spry) 1 gene expression in cultured UB cells. Treatment of intact E12.5 mouse metanephroi grown ex vivo with Ang II (10(-5) M, 24 h) increased HDAC-1 and decreased total acetylated histone H3 protein levels. These findings indicate that lack of endogenous Ang II in AGT-deficient mice inhibits UB branching. We conclude that intact RAS is critical in structural integrity of the renal collecting system and that UB morphogenetic program genes, such as AGT, renin, ACE, AT1R, or Spry1, are epigenetically controlled via HDACs.

Figure 1

Effect of angiotensinogen (AGT) genotype on UB branching in double transgenic Hoxb7-GFP/AGT embryos on E13.5. (A–D): UB images. Numbers represent tip number. BP, branch point. Scale bar: 200 μm. Magnification ×10. (E, F): Bar graphs showing the effect of AGT genotype on the number of UB tips and branch points. *p < 0.05.

Figure 2

Effect of HDAC inhibitor Scriptaid (S) or its inactive analogue Nullscript (N) on angiotensinogen (AGT), renin, angiotensin-convertin enzyme (ACE), and Ang II AT₁ receptor (AT₁R) mRNA levels in cultured MK3 cells and embryonic metanephroi. (A): Ethidium bromide-stained gel shows semi-quantitative RT-PCR analysis of the RAS mRNA in MK3 cells. (B, C): Bar graphs showing the effect of Nullscript and Scriptaid on RAS mRNA expression in MK3 cells (B) and E12.5 metanephroi (C) as determined by quantitative RT-PCR. *p < 0.05 vs. Nullscript (control = 100%), **p < 0.001 vs. Nullscript. (D): Bar graph showing the effect of HDAC-1 inhibition with morpholino in MK4 cells. *p < 0.05, **p < 0.01 vs. control (= 100%).

Figure 3

Effect of media (control) or Ang II on HDAC-1 and histone H3 protein expression. (A, E): E12.5 mouse kidneys were grown ex vivo in the presence of media or Ang II (10⁻⁵ M) for 24 h. Whole kidney lysates were subjected to immunoblotting with anti-HDAC-1 (A) or anti-acetylated or total histone H3 (E) antibodies. After stripping, top membrane in “E” was reprobed with anti-total histone H3 antibody. (C): Quiescent UB cells were treated with media or Ang II (10⁻⁵ M) for 24 h and cell lysates were subjected to immunoblotting with anti-HDAC-1 antibody. (B, D, F): Bar graphs showing the effect of media or Ang II on target protein levels. Bars represent ratio of HDAC-1/β-actin (B, D) or acetylated/total histone H3 (F). *p < 0.05.

Figure 4

Effect of HDAC inhibition on Spry1 gene expression. (A): Bar graph showing the effect of media, HDAC inhibitor Scriptaid, Ang II combined with Scriptaid, or inactive Scriptaid analogue Nullscript on Spry1 mRNA levels in cultured UB cells. HDAC inhibition with Scriptaid decreases Spry1 mRNA levels. Scriptaid further down-regulates Ang II-induced decrease in Spry1 mRNA levels. *p < 0.05 vs. Media, **p < 0.05 vs. Scriptaid or Ang II + Nullscript. (B): Bar graph showing the effect of HDAC-1 knockdown in MK4 cells on Spry1 mRNA. *p < 0.01 vs. control (= 100%).

Figure 5

A proposed model for HDAC-RAS cross-talk in ureteric bud branching. Inhibition of HDAC activity induces RAS gene expression leading to AT₁R-dependent repression of Spry1. This causes activation of c-Ret tyrosine kinase activity and induction of UB branching. In addition, enhanced AT₁R signaling induces HDAC-1 expression leading to deacetylation of histone H3. This exerts a negative feedback on the RAS activity.