Sex-specific effects of heme oxygenase-2 deficiency on renovascular hypertension

Abstract

Background: Heme oxygenase-2 (HO-2) is the main isoform responsible for the breakdown of heme and release of carbon monoxide in the vasculature. Vascular-derived carbon monoxide protects against excessive vasoconstriction due to agents such as angiotensin II (Ang II) and in states of deficiency of nitric oxide. The current study was designed to determine the role of HO-2 in the development of renovascular hypertension using HO-2 knockout mice.

Methods: Polyurethane cuffs were placed around the left renal artery of male and female HO-2 wild-type (WT), heterozygous (HET), and knockout (KO) mice between 16 and 24 weeks of age to induce renovascular hypertension. After 3 weeks, blood pressure was measured for 5 days, after which time both clipped and unclipped kidneys were harvested.

Results: No differences were observed in the blood pressure of sham mice between the different genotypes of both sexes. Cuffing of the left renal artery resulted in a significant increase in blood pressure in all genotypes of both sexes. In male mice, the increase in blood pressure was significantly greater in HET and KO mice as compared to WT mice (P < .05). This effect was not observed in female mice. Renovascular hypertension resulted in a significant increase (P < .05) in cardiac hypertrophy in male mice, which was not different between the genotypes. In female mice, HET and KO mice exhibited significantly greater (P < .05) cardiac hypertrophy as compared with WT mice.

Conclusion: These results demonstrate a sex-specific effect of HO-2 deficiency on the development of renovascular hypertension and its effects on the heart in response to the increase in blood pressure.

Keywords: Angiotensin II; bilirubin; blood pressure; carbon monoxide; renin.

Figure 1

Blood pressure response in A) male and B) female mice 3 weeks after sham surgery or clipping of the left renal artery, n=6/group. *= significant (P<0.05) difference as compared to corresponding value in sham operated. † = significant (P<0.05) difference as compared to corresponding value in WT mice.

Figure 2

Cardiac hypertrophy in A) male and B) female mice 3 weeks after sham surgery or clipping of the left renal artery, n=6/group. Degree of renal stenosis as determined by the ratio of the clipped kidney to the unclipped kidney weight in C) male and D) female mice 3 weeks after sham surgery or clipping of the left renal artery, n=6/group. *= significant (P<0.05) difference as compared to corresponding value in sham operated. † = significant (P<0.05) difference as compared to corresponding value in WT mice.

Figure 3

Quantitation of gene expression between the clipped and unclipped kidney in male mice via real time PCR. Expression of A) Renin, B) angiotensin converting enzyme (ACE), C) angiotensin type 1 receptor (AT1A), and D) endothelin B receptor (ETB). Each targeted gene was normalized to 18s rRNA in the clipped and unclipped kidney and this difference expressed as the −C_t difference. Thus, genes expressed at a higher level in the clipped kidney have a positive value and those expressed higher in the unclipped kidney have a negative value. *= significant (P<0.05) difference as compared to corresponding value in WT and HET mice. † = significant (P<0.05) difference as compared to corresponding value in WT and KO mice, n=4/group.

Figure 4

Renal inflammation in the unclipped kidney of male mice as determined by Western blot. A) Representative Western blots for interleukin 1β (IL-1β), transforming growth factor β1, (TGF-β1) active and pro and β-actin. B) Levels of IL-1β. C) Levels of pro- TGF-β1. D) levels of active- TGF-β1 *= significant (P<0.05) difference as compared to corresponding value in KO mice. . n=6 in WT and KO, n=4 in HET.

Figure 5

Renal inflammation and endoplasmic reticulum (ER) stress response gene expression in clipped and unclipped kidneys in male mice via real time PCR. A) Expression of tumor necrosis factor-α (TNF-α). B) Expression of glucose-regulated protein-78 (GRP78). C) Expression of X-box binding protein-1 (XBP1). † = significant (P<0.05) difference as compared to corresponding value in WT and KO mice, n=4/group.