Cardiovascular manifestations of renovascular hypertension in diabetic mice

Abstract

Purpose. Type 2 diabetes is the leading cause of end stage renal disease in the United States. Atherosclerotic renal artery stenosis is commonly observed in diabetic patients and impacts the rate of renal and cardiovascular disease progression. We sought to test the hypothesis that renovascular hypertension, induced by unilateral renal artery stenosis, exacerbates cardiac remodeling in leptin-deficient (db/db) mice, which serves as a model of human type II diabetes. Methods. We employed a murine model of renovascular hypertension through placement of a polytetrafluoroethylene cuff on the right renal artery in db/db mice. We studied 109 wild-type (non-diabetic, WT) and 95 db/db mice subjected to renal artery stenosis (RAS) or sham surgery studied at 1, 2, 4, and 6+ weeks following surgery. Cardiac remodeling was assessed by quantitative analysis of the percent of myocardial surface area occupied by interstitial fibrosis tissue, as delineated by trichrome stained slides. Aortic pathology was assessed by histologic sampling of grossly apparent structural abnormalities or by section of ascending aorta of vessels without apparent abnormalities. Results. We noted an increased mortality in db/db mice subjected to RAS. The mortality rate of db/db RAS mice was about 23.5%, whereas the mortality rate of WT RAS mice was only 1.5%. Over 60% of mortality in the db/db mice occurred in the first two weeks following RAS surgery. Necropsy showed massive intrathoracic hemorrhage associated with aortic dissection, predominantly in the ascending aorta and proximal descending aorta. Aortas from db/db RAS mice showed more smooth muscle dropout, loss of alpha smooth muscle actin expression, medial disruption, and hemorrhage than aortas from WT mice with RAS. Cardiac tissue from db/db RAS mice had more fibrosis than did cardiac tissue from WT RAS mice. Conclusions. db/db mice subjected to RAS are prone to develop fatal aortic dissection, which is not observed in WT mice with RAS. The db/db RAS model provides the basis for future studies directed towards defining basic mechanisms underlying the interaction of hypertension and diabetes on the development of aortic lesions.

Keywords: Cardiovascular disease; Diabetes; Hypertension; Renal artery stenosis.

Figure 1. Photograph of aorta dissection showing the severe rupture just distal to aortic root.

Representative photograph of aortic rupture. In the study, most aortic ruptures were found in the ascending aorta.

Figure 2. db/db mice showed higher mean pathology score at both early and late time points.

Representative images of aorta illustrating semiquantitative histologic assessment scores. (A) Shows normal aorta (score of 0). (B) Focal myocyte dropout (Score of 1). (C) Multifocal myocyte dropout (Score of 2). (D) Medial disruption and hemorrhage (Score of 3). Mean pathology score of db/db and WT early and late time points animals subjected to RAS surgery. ^∗p = 0.007, ^∗∗p = 0.0034 in comparison to WT group.

Figure 3. db/db mice showed more α smooth muscle actin loss in aorta at both early and late time points compared to WT.

Representative images of aorta illustrating semiquantitative assessment of α smooth muscle actin (α SMA) loss at 400× using α SMA staining. (A) shows no loss of α SMA stain (score of 0). (B) Focal loss of α SMA stain (Score of 1). (C) Multifocal loss of α SMA stain (Score of 2). D. extensive loss of α SMA stain (Score of 3). (E) Mean α SMA stain loss score of db/db and WT early and late time points animals. ^∗p = 0.0152, ^∗∗p = 0.002, ^#p = 0.0001 in comparison to respective sham groups. ^$p = 0.0001.

Figure 4. Abnormal aorta showed increased overall wall thickness.

Mean aortic medial thickness was greater in aortas with histopathological scores of 1, 2, or 3 versus a score of 0 in both WT and db/db mice (^∗p = 0.002, ^∗∗p = 0.0001).

Figure 5. Increased cardiac fibrosis in db/db RAS mice compared to WT at later time points.

Myocardial fibrosis was assessed by quantitative image analysis of trichrome stained sections at 200× magnification obtained from (A) WT sham, (B) db/db sham, (C) WT RAS and (D) db/db RAS mice (E). The mean percentage of fibrosis in db/db and WT mice at early and late time points. Both WT and db/db showed increase % fibrosis following RAS compared to their respective sham at both early and late time points. db/db RAS mice had significantly more fibrosis compared to WT RAS at late time points. ^∗p = 0.02, ^∗∗p = 0.009, ^#p = 0.001, ^$p = 0.0001 compared to their respective shams and ^&p = 0.03.