Attenuation of Angiotensin II-Induced Hypertension in BubR1 Low-Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

Abstract

Background Angiotensin II (Ang II) can cause hypertension and tissue impairment via AGTR1 (Ang II receptor type 1), particularly in renal proximal tubule cells, and can cause DNA damage in renal cells via nicotinamide adenine dinucleotide phosphate oxidase. BubR1 (budding uninhibited by benzimidazole-related 1) is a multifaceted kinase that functions as a mitotic checkpoint. BubR1 expression can be induced by Ang II in smooth muscle cells in vitro, but the relationship between systemic BubR1 expression and the Ang II response is unclear. Methods and Results Twenty 24-week-old male BubR1 low-expression mice (BubR1^L/L mice) and age-matched BubR1^+/+ mice were used in this study. We investigated how Ang II stimulation affects BubR1^L/L mice. The elevated systolic blood pressure caused by Ang II stimulation in BubR1^+/+ mice was significantly attenuated in BubR1^L/L mice. Additionally, an attenuated level of Ang II-induced perivascular fibrosis was observed in the kidneys of BubR1^L/L mice. Immunohistochemistry revealed that the overexpression of AGTR1 induced by Ang II stimulation was repressed in BubR1^L/L mice. We evaluated AGTR1 and Nox-4 (nicotinamide adenine dinucleotide phosphate oxidase-4) levels to determine the role of BubR1 in the Ang II response. Results from in vitro assays of renal proximal tubule cells suggest that treatment with small interfering RNA targeting BubR1 suppressed Ang II-induced overexpression of AGTR1. Similarly, the upregulation in Nox4 and Jun N-terminal kinase induced by Ang II administration was repressed by treatment with small interfering RNA targeting BubR1. Conclusions Ang II-induced hypertension is caused by AGTR1 overexpression in the kidneys via the upregulation of BubR1 and Nox4.

Keywords: angiotensin II type 1 receptor; budding uninhibited by benzimidazole‐related 1; hypertension; nicotinamide adenine dinucleotide phosphate oxidase‐4; renal proximal tubule cell.

Figure 1

Time‐dependent changes in systolic blood pressure (SBP) following angiotensin II (Ang II) infusion in mice. A, Time course of SBP levels in Ang II–stimulated BubR1 low‐expression (BubR1 ^L/L) mice and wild‐type (BubR1 ^+/+) mice from days 0 to 6 of Ang II infusion. n=10 BubR1 ^+/+ mice; n=11 BubR1 ^L/L mice. B, SBP levels at day 1 and day 6 of Ang II infusion. *P<0.01 vs BubR1 ^+/+ mice.

Figure 2

Histological analysis of tissues from BubR1 low‐expression (BubR1 ^L/L) mice and wild‐type (BubR1 ^+/+) mice after 1 week of angiotensin II (Ang II) infusion. A, Representative Sirius red‐stained kidney sections. Magnification, ×40; n=10 BubR1 ^+/+ mice; n=8 BubR1 ^L/L mice; scale bar, 100 μm. B, Perivascular fibrotic lesion area in the kidney, quantified using morphometry. *P<0.01 vs BubR1 ^+/+ mice. C, Representative Elastica van Gieson–stained sections of the thoracic aorta. Magnification, ×40; n=12 BubR1 ^+/+ mice; n=11 BubR1 ^L/L mice; scale bar, 100 μm. D, Area of aorta media, quantified using morphometry. *P<0.01 vs BubR1 ^+/+ mice. E, Representative Masson trichrome–stained sections of the heart. Magnification, ×40; n=12 BubR1 ^+/+ mice; n=11 BubR1 ^L/L mice; scale bar, 100 μm. F, Ejection fraction, evaluated by echocardiographs; n=12 control mice; n=11 BubR1 ^L/L mice. *P<0.01 vs BubR1 ^+/+ mice.

Figure 3

Immunohistochemistry of kidneys from BubR1 ^+/+ and BubR1 ^L/L mice before and after 1 week of angiotensin II (Ang II) infusion. A through C, Images of representative kidney sections that were immunostained for BubR1 (A), Ang II receptor type 1 (AGTR1) (B), or Ang II receptor type 2 (AGTR2) (C). Magnification, ×40; scale bar, 50 μm. BubR1, AGTR1, and AGTR2 expression levels in kidney with or without BubR1 reduction, with or without angiotensin II (Ang II) stimulation (n=5/group). *P<0.01. kpxls indicates kilo pixels; n.s., not significant.

Figure 4

In vitro assay of BubR1 reduction in human renal proximal tubule cells (RPTCs). A, BubR1 levels in RPTCs that were treated with siBubR1 or scrambled control siRNA (3 biological replicates each). *P<0.01. B, Angiotensin II (Ang II) receptor type 1 (AGTR1) expression levels in RPTCs with or without BubR1 reduction, with or without 10 μmol/L Ang II stimulation for 24 hours (n=3/group). *P<0.01. siBubR1 indicates short interfering RNA targeting BubR1.

Figure 5

Expression levels of Nox 4, JNK, and phospho‐JNK in human renal proximal tubule cells (RPTCs), with or without angiotensin II (Ang II) stimulation. A, Representative Western blot of Nox 4, JNK, and phospho‐JNK/JNK in siBubR1‐ or scramble siRNA control–treated RPTCs, with or without Ang II stimulation. B through D, Quantified expression levels of Nox 4 (B), JNK (C), and phospho‐JNK/JNK (D) from the blot in (A) (n=6/group). *P<0.01. JNK indicates Jun N‐terminal kinase; Nox4, nicotinamide adenine dinucleotide phosphate oxidase‐4; n.s., not significant; p‐JNK, phospho‐JNK; siBubR1, short interfering RNA targeting BubR1.

Figure 6

Immunohistochemistry of kidneys from BubR1 ^+/+ and BubR1 ^L/L mice before and after 1 week of angiotensin II (Ang II) infusion. Representative immunohistochemistry sections of Nox4 (A) and JNK (B) expression. Scale bar, 100 μm. Nox4 and SAPK/JNK expression levels in kidney with or without BubR1 reduction, with or without Ang II stimulation (n=5/group). Quantified expression levels of Nox 4 (C), and SAPK/JNK (D). *P<0.01. JNK indicates Jun N‐terminal kinase; Nox4, nicotinamide adenine dinucleotide phosphate oxidase‐4; n.s., not significant; SAPK, stress‐activated protein kinase.