Early atherosclerosis aggravates renal microvascular loss and fibrosis in swine renal artery stenosis

Abstract

Renal function in patients with atherosclerosis and renal artery stenosis (ARAS) deteriorates more frequently than in nonatherosclerotic RAS. We hypothesized that ARAS aggravates stenotic-kidney micro vascular loss compared to RAS. Domestic pigs were randomized to normal, RAS, and ARAS (RAS fed a high-cholesterol diet) groups (n = 7 each). Ten weeks later stenotic-kidney oxygenation, renal blood flow, and glomerular filtration rate (GFR) were evaluated in vivo, and micro vascular density by micro-computed tomography. Blood pressure in both RAS and ARAS was elevated; and stenotic-kidney renal blood flow and GFR similarly decreased. RAS decreased the density of small-size cortical microvessels (<200 μm), whereas ARAS extended the decrease to medium-sized microvessels (200-300 μm). Cortical hypoxia and interstitial fibrosis increased in both RAS and ARAS but correlated inversely with micro vascular density only in RAS. Atherosclerosis aggravates loss of stenotic-kidney microvessels, yet additional determinants likely contribute to cortical hypoxia and fibrosis in swine ARAS.

Keywords: Atherosclerosis; fibrosis; micro vascular injury; renal artery stenosis.

Figure 1

Renal microvasculature and oxygenation in Normal, renal artery stenosis (RAS), and atherosclerotic RAS (ARAS) pigs. A. Representative micro-CT images. B. In the renal cortex, RAS decreased the density of small-sized microvessels (<200 µm) throughout the whole cortex, while ARAS decreased the density of both small and medium-sized (200–300 µm) microvessels. C. Medullary microvessels decreased in both RAS and ARAS compared with Normal. D. Representative BOLD images. E. Basal renal cortical R2* increased in both RAS and ARAS compared with Normal, but medullary R2* were only elevated in ARAS. F. The medullary R2* response to furosemide was blunted in both RAS and ARAS. ^*P<0.05 vs. Normal; ^#P<0.05 vs. RAS.

Figure 2

Renal tissue remodeling. A. Representative renal trichrome, dihydroethidium (DHE), eNOS and p-eNOS immunofluorescence (all ×20). B. Renal cortical and medullary fibrosis increased in RAS compared with Normal, and aggravated in ARAS. C. Glomerular score increased in RAS and ARAS. D. Tubular injury score increased in RAS, and further aggravated in ARAS. E. DHE staining (normalized to DAPI-positive nuclei) increased in RAS compared with Normal, and in ARAS compared with Normal and RAS. F–G. eNOS expression decreased in RAS and ARAS compared with Normal, but p-eNOS only decreased in ARAS. ^*P<0.05 vs. Normal; ^#P<0.05 vs. RAS. Scale bar=50 µm.

Figure 3

Immunohistochemical staining. A. Representative ox-LDL, NF-κB and α-SMA staining (all ×20). B, C. ox-LDL and NF-κB immunoreactivity increased in ARAS compared with both Normal and RAS. D. Renal microvascular media-to-lumen ratio (α-SMA) increased in RAS compared with Normal, and further in ARAS compared with Normal and RAS. ^*P<0.05 vs. Normal; ^#P<0.05 vs. RAS. Scale bar=50 µm.

Figure 4

NF-κB and VEGF expression. A. Western blotting (representative 2 bands shown per group) in three groups. B, C. NF-κB increased in ARAS compared with Normal and RAS, but VEGF expression was not significantly different among the three groups. ^*P<0.05 vs. Normal; ^#P<0.05 vs. RAS.

Figure 5

Correlation of cortical medium-sized microvessels and medullary microvessels with RBF, GFR, cortical and medullary R2*, tubular injury score and fibrosis.