Mitochondrial protection restores renal function in swine atherosclerotic renovascular disease

Abstract

Aims: The mechanisms responsible for renal injury in atherosclerotic renovascular disease (ARVD) are incompletely understood, and few therapeutic options are available to reverse it. We hypothesized that chronic renal damage involves mitochondrial injury, and that mitochondrial protection would reduce renal fibrosis and dysfunction in ARVD pigs.

Methods and results: Domestic pigs were studied after 10 weeks of ARVD or sham, treated for the last 4 weeks with daily subcutaneous injections (5 days/week) of vehicle or Bendavia (0.1 mg/kg), a tetrapeptide that preserves cardiolipin content in the mitochondrial inner membrane. Single-kidney haemodynamics and function were studied using fast-computer tomography, oxygenation using blood oxygen level-dependent magnetic resonance imaging, microvascular architecture, oxidative stress, and fibrosis ex vivo. Cardiolipin content was assessed using mass spectrometry and staining. Renal endothelial function was studied in vivo and ex vivo. In addition, swine renal artery endothelial cells incubated with tert-butyl hydroperoxide were also treated with Bendavia. Stenotic-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) decreased in ARVD + Vehicle compared with normal (318.8 ± 61.0 vs. 553.8 ± 82.8 mL/min and 48.0 ± 4.0 vs. 84.0 ± 3.8 mL/min, respectively) associated with loss of cardiolipin, intra-renal microvascular rarefaction, and hypoxia. Bendavia restored cardiolipin content in ARVD and improved vascular density, oxygenation, RBF (535.1 ± 24.9 mL/min), and GFR (86.6 ± 11.2 mL/min). Oxidative stress and fibrosis were ameliorated, and renovascular endothelial function normalized both in vivo and in vitro.

Conclusion: Preservation of mitochondrial cardiolipin attenuated swine stenotic-kidney microvascular loss and injury, and improved renal oxygenation, haemodynamics, and function. These observations implicate mitochondrial damage in renal deterioration in chronic experimental ARVD, and position the mitochondria as a central therapeutic target.

Keywords: Atherosclerosis; Hypertension; Mitochondria; Renal.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

Figures

Figure 1

Stenotic-kidney BOLD-MRIs showing hypoxic regions (red), and quantification of R2* in the cortex and medulla (n = 7/group).

Figure 2

(A) Stenotic-kidney cardiolipin content decreased in ARVD, but was restored in ARVD + Bendavia (n = 7/group). (B) Fluorescent staining for cardiolipin (red) and cytokeratin (green) confirmed decreased renal expression and staining intensity in ARVD, which was normalized in ARVD + Bendavia (n = 7/group). (C) Expression of Taz-1 mRNA was unaltered, whereas ALCAT-1 was down-regulated in ARVD + Bendavia (n = 7/group). (D) The numbers of TUNEL+ and caspase-3+ cells were elevated in ARVD, yet normalized in ARVD + Bendavia (n = 7/group). (E) Renal protein expression and ratio of Bax and Bcl-xl (n = 6/group).

Figure 3

(A) Renal production of superoxide anion (DHE) was increased in ARVD and attenuated by Bendavia (n = 7/group). (B) Renal expression of p47 was restored in Bendavia-treated ARVD (n = 6/group). Tubular injury score (C, H&E) and collagen IV immunostaining of (D) were elevated in both ARVD groups, yet improved in ARVD + Bendavia (n = 7/group).

Figure 4

(A) Tubulointerstitial fibrosis and glomerular score (trichrome) improved in ARVD + Bendavia (n = 7/group). (B) Renal expression of TGF-β remained up-regulated, but expression of PAI-1 and TIMP-1 was normalized in Bendavia-treated ARVD pigs (n = 6/group).

Figure 5

(A) Representative 3D micro-computed tomography images of the pig kidney and quantification of spatial density of size-specific microvessels in the inner and outer cortex (n = 7/group). (B) Tomographically isolated vessels and quantification of their tortuosity (n = 7/group). (C) Renal protein expression of VEGF and angiopoietin-1 was up-regulated in ARVD + Bendavia pigs (n = 6/group).

Figure 6

(A) Endothelial-dependent (left) and independent (right) relaxation responses of renal arterial segments from normal and ARVD pigs (chronically treated with Bendavia or Vehicle) to Ach and SNP (n = 7/group). (B) Quantification of cardiolipin expression (10N-nonyl acridine orange), apoptosis (TUNEL), DHE (n = 7/group), and eNOS immunoreactivity (n = 6/group) in stenotic-kidney artery sections. *P ≤ 0.05 vs. Normal + Vehicle, †P ≤ 0.05 vs. ARVD + Bendavia.

Figure 7

(A) Cardiolipin expression decreased in RAECs isolated from a normal animal and treated with tBHP, but was preserved in cells co-incubated with Bendavia, and the number of caspase-3-positive cells decreased DHE staining and eNOS immunoreactivity was normalized. (B) Expression of caspase-3, nitrotyrosine, and phosphorylated eNOS (peNOS) in untreated RAEC, RAEC + Bendavia, RAEC + tBHP, and RAEC + tBHP + Bendavia normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH).