Experimental coronary artery stenosis accelerates kidney damage in renovascular hypertensive swine

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1 & International Society of Nephrology see commentary on page 676 Experimental coronary artery stenosis accelerates kidney damage in renovascular hypertensive swine Dong Sun,, Alfonso Eirin, Xiang-Yang Zhu, Xin Zhang, John A. Crane, John R. Woollard, Amir Lerman 3 and Lilach O. Lerman,3 Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota, USA; Department of Nephrology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, China and 3 Division Cardiovascular Disease, Mayo Clinic, Rochester, Minnesota, USA The impact of coronary artery stenosis () on renal injury is unknown. Here we tested whether the existence of, regardless of concurrent atherosclerosis, would induce kidney injury and magnify its susceptibility to damage from coexisting hypertension (). Pigs (seven each) were assigned to sham, left-circumflex, renovascular, and plus groups. Cardiac and nonstenotic kidney functions, circulating and renal inflammatory and oxidative markers, and renal and microvascular remodeling were assessed weeks later. Myocardial perfusion declined distal to. Systemic levels of PGF-a isoprostane, a marker of oxidative stress, increased in and plus, whereas single-kidney blood flow responses to acetylcholine were significantly blunted only in plus compared with sham,, and, indicating renovascular endothelial dysfunction. Tissue expression of inflammatory and oxidative markers were elevated in the pig kidney, and further magnified in plus, whereas angiogenic factor expression was decreased. Bendavia, a mitochondriatargeted peptide, decreased oxidative stress and improved renal function and structure in. Furthermore, and synergistically amplified glomerulosclerosis and renal fibrosis. Thus, mild myocardial ischemia, independent of systemic atherosclerosis, induced renal injury, possibly mediated by increased oxidative stress. Superimposed aggravates renal inflammation and endothelial dysfunction caused by, and synergistically promotes kidney fibrosis, providing impetus to preserve cardiac integrity in order to protect the kidney. Kidney International (5) 87, 79 77; doi:.38/ki..33; published online October KEYWORDS: coronary artery stenosis; renovascular hypertension; renal fibrosis Correspondence: Lilach O. Lerman, Division of Nephrology and Hypertension, Mayo Clinic, First Street SW, Rochester, Minnesota 5595, USA. lerman.lilach@mayo.edu Received October 3; revised 9 July ; accepted August ; published online October Atherosclerotic coronary artery disease is the most common cause of death in the United States, and it is often accompanied by diffuse vascular disease in other organs. An increase in systemic inflammation and oxidative stress may mediate some of the systemic manifestations of atherosclerosis, yet myocardial ischemia may impose adverse effects on remote organs and vascular territories. 3 For example, myocardial infarction and stroke augment inflammation, and in turn atherosclerosis, and they can trigger morphological and functional changes in the kidney. 5 However, the isolated effects on the kidney of myocardial ischemia dissociated from infarction and systemic atherosclerosis have been difficult to discern. Patients with coronary artery disease often also have hypertension (), an important cause of chronic kidney disease. 6 Previous studies have shown increased prevalence of renovascular disease in patients with coronary artery disease, 7 9 in whom renal insufficiency is the main clinical predictor of renal artery stenosis. Renal artery stenosis is an important cause of secondary in the elderly population, which may ultimately lead to end-stage renal disease and represents a sizeable fraction of new patients entering dialysis. Importantly, hypertensive patients at risk for renovascular disease often present with severe coronary artery disease, and renal function correlates with its presence. We have also recently shown that coexistence of coronary and renal artery diseases in human subjects magnifies renal injury., The preglomerular vasculature exposed to the develops progressive vascular pathology, 3 magnified by the loss of autoregulation with glomerular hypertrophy, hyperfiltration, and focal segmental glomerulosclerosis. Peritubular capillary loss and consequent hypoxia lead to tubular atrophy and interstitial fibrosis. We have also shown that renovascular blunts antioxidant defense mechanisms in the nonstenotic kidney. 5 These functional and structural alterations may render kidneys exposed to susceptible to other comorbidities, and amplify renal damage. However, whether nonatherosclerotic coronary artery stenosis () alone induces kidney injury or increases its vulnerability to adverse effects of coexisting remains unclear. The present study was therefore designed to test the hypothesis that isolated elicits kidney inflammation and Kidney International (5) 87,

2 D Sun et al.: Coronary artery stenosis accelerates kidney damage oxidative stress, which are exacerbated by and impair renal function. RESULTS Renal artery stenosis increased mean arterial pressure (MAP) in both and þ compared with sham (Table, P ¼.3 for both). Body weight and plasma renin activity were not significantly different among the groups, but serum creatinine was elevated in,, and þ (Table, Po. each), affected by both and. Urinary protein, affected by, increased significantly only in þ compared with sham and (Table, P ¼.6 and P ¼.5, respectively). Circulating PGF -a isoprostane levels increased in and þ compared with sham and (Figure a, Po.5 each), whereas systemic transforming growth factor (TGF)-b levels increased by in and þ (Figure b, P ¼. and P ¼.3 vs. sham, respectively). Norepinephrine levels were slightly reduced in only compared with. Systemic levels of granulocyte macrophage colony-stimulating factor, interleukin (IL)-a, IL-b, IL-ra, IL-, IL-, IL-6, IL-8, IL, IL, IL8, and tumor necrosis factor (TNF)-a were not significantly different among the four groups (Supplementary Table S online, P.5 for all). Cardiac function Ten weeks after induction, the pigs developed significant and similar degree of (Table, Figure d), but stroke volume and cardiac output decreased owing to only in þ compared with sham and (Table, Figure e, Pp.3 for each). The ejection fraction (albeit slightly affected by ) and early and late left ventricular (LV) filling velocities (E/A) were not significantly different among the four groups (Table ). Myocardial perfusion distal to and þ was lower than in sham (Figure f, P ¼. and P ¼., respectively), whereas LV muscle mass (LVMM) was increased in and þ (Figure c and g, Pp.3 for both). Renal hemodynamics and function The degree of renal artery stenosis was not different between and þ, but renal blood flow (RBF) and glomerular filtration rate (GFR) of the nonstenotic kidney were elevated only in ; RBF was increased by, suppressed by, and showed a significant interaction between the two (Table ). GFR also tended to be blunted by the interaction (Figure h). RBF response to Ach was attenuated by only in þ (P ¼.8 vs. baseline, Figure i), and became lower than sham (P ¼.), by a significant interaction (Table ). Basal cortical perfusion in and þ was lower than in, and after Ach infusion it was also lower than in sham (Table, Po.5 for each). Basal medullary perfusion was not significantly different among the four groups, but its response to Ach was blunted only in þ. Renal vascular resistance was elevated in and þ compared with sham and (Table ) owing to. also decreased proximal and distal tubular intratubular concentration in þ (Figure j), suggesting impaired tubular fluid reabsorption. Renal histology Trichrome staining showed increased renal fibrosis in and compared with sham pigs (P ¼.3 and P ¼., respectively). Compared with other groups, renal fibrosis and glomerular score in þ increased significantly (Figures a c, Po. each), showing interactions between and in both fibrosis (Figure b, P ¼.8) and glomerulosclerosis (Figure c, P ¼.). Dihydroethidium (DHE) staining was elevated in (P ¼.3 vs. sham) and further exacerbated in þ (Figure a-dhe and d, P ¼.6 vs. sham and P ¼.3 vs. ). Compared with sham, renal capillary density decreased in (Figure, P ¼.), yet in þ it decreased further compared with all groups (Po. each). Microvascular wall thickening (media-to-lumen ratio) increased in (P ¼.3 vs. sham), but further in and þ Table Systemic characteristics and cardiac function in the four groups (mean±s.e.m., n ¼ 7 each) P-value for two-way ANOVA þ Body weight, kg 8.±.8 8.3±5. 5.6±.6 6.±5. MAP, mm Hg 96.7±.8 6.5±7.8 a.3±5.6.5±7. a PRA, pg/ml/min.7±..5±.6.±.7.± Creatinine, mg/dl.7±.3.58±.7 a.6±.3 a.8±.6 a Urine protein, mg/ml 6.9±.5.±5.9 8.±6. 33.±.7 a,b Norepinephrine, ng/ml.8±..±..±. c.5± Degree of, % 77.3±8.9 a,c 7.±.3 a,c Stroke volume, ml 8± 5±3 8±5 37±3 a,c Ejection fraction, % 5±3 57± 5±5 5± E/A.7±.6.±.5.±.5.± Abbreviations: ANOVA, analysis of variance;, coronary artery stenosis; E/A, early-to-late left ventricular filling velocities;, hypertension; MAP, mean arterial pressure; PRA, plasma renin activity. a Po.5 vs. sham. b Po.5 vs.. c Po.5 vs.. 7 Kidney International (5) 87, 79 77

3 D Sun et al.: Coronary artery stenosis accelerates kidney damage PGF-α isoprostane, pg/ml TGF-β, pg/ml + + A A A A R R R R + Coronary artery stent location Cardiac output, I/min Myocardial perfusion, ml/min/g LVMM, g/kg body wt 3 GFR, ml/min ΔRBF, ml/min & + ITC, arbitrary units 8 6 Proximal Henle s Distal + Figure Systemic, cardiac, and renal characteristics in sham, hypertension (), coronary artery stenosis (), and þ pigs. (a) Circulating PGF-a isoprostane. (b) Systemic transforming growth factor (TGF)-b. (c-d) Representative cardiac CT images obtained at the middle left-ventricle (LV). In hypertensive animals ( and þ ), the LV wall is thickened. The arrow in C shows myocardial lateral wall thinning distal to a high-grade (illustrated in d). (e) Cardiac output. (f) Myocardial perfusion. (g) LV muscle mass (LVMM). (h) Glomerular filtration rate (GFR). (i) Change in renal blood flow (DRBF) in response to acetylcholine (Ach). (j) Intratubular concentration (ITC) in the different nephron segments. Po.5 vs. sham; Po.5 vs. ; & Po.5 vs.. (Po. each). Tubular injury observed in and was further aggravated in þ (Po.). None showed interactions. Compared with sham, renal protein expression of TNFa increased in (Figure 3a and Supplementary Figure S online, P ¼.3) and further in þ (P ¼. and P ¼., respectively). Monocyte chemoattractant protein (MCP)- and GP9-phox expressions were all higher in and þ than in sham and (Figure 3a and Supplementary Figure S online), affected by alone (Po.3 each), whereas nitrotyrosine was not. Hypoxiainducible factor (HIF)-a expression increased owing to only in þ kidneys compared with sham and (Figure 3b and Supplementary Figure S online), suggesting Kidney International (5) 87,

4 D Sun et al.: Coronary artery stenosis accelerates kidney damage Table Nonstenotic kidney hemodynamics and function in sham,,, and þ pigs (mean±s.e.m., n ¼ 7 each) P-value for two-way ANOVA þ Degree of renal artery stenosis, % 8.7±7.5 a,b 8.7±5.7 a,b RBF, ml/min Basal 69.± ±8. a.3±8.5 c 8.5±5. c Ach 677.±3.5 d 969.±83.3 a,d 59.6±. c,d 8.±8.5 a,c Perfusion, ml/min/ml Cortex: baseline.±.7 5.3± ±.6 c.3±.3 c Ach 6.±.9 d 6.3±.59 d.9±.6 a,c,d 5.±. a,c,d Medulla: baseline.8±..9±..98±.35.± Ach 3.77±.55 d 3.7±.3 d 3.8±.7 d.8±.39 a,b RVR, mm Hg/min/ml.8±..6±..7±. ac.9±. a,c.5..7 Abbreviations: Ach, acetylcholine; ANOVA, analysis of variance;, coronary artery stenosis;, hypertension; RBF, renal blood flow; RVR, renal vascular resistance. a Po.5 vs. sham. b Po.5 vs.. c Po.5 vs.. d Po.5 vs. baseline. tissue hypoxia. Vascular endothelial growth factor (VEGF) expression decreased in and further decreased in þ, owing to both and. Renal TGF-b levels increased in compared with sham, but markedly increased in and þ (Figure 3b and d and Supplementary Figure S online). Tissue inhibitor of metalloproteinases (TIMP)- was elevated by in þ compared with the other groups (Po. for each). Protein expression of endothelial nitric-oxide synthase (enos) was downregulated in, and further in and þ (Pp. each), showing a significant interaction x (P ¼.3). Additional mechanistic exploration Induction of femoral artery stenosis instead of in additional pigs, using the same local-irritant coil, had no effect on renal function or endothelial function (Supplementary Table S online), oxidative stress, or fibrosis (Supplementary Figure S3 online) (P.5 vs. sham for all). In additional animals chronically treated with the mitochondria-targeted antioxidant bendavia, myocardial perfusion was not different from that in untreated, yet serum creatinine that was elevated in decreased in þ Bendavia, and MAP and renal hemodynamics were unchanged compared with (Supplementary Table S3 online). Kidney fibrosis, DHE, microvascular media/lumen ratio, and tubular injury in þ Bendavia all decreased compared with untreated (Supplementary Figure S online), and TNF-a, P67, GP9, and TGF-b were downregulated, whereas enos expression remained unchanged (Supplementary Figure S5 online). DISCUSSION This study demonstrates that a hemodynamically significant but nonatherosclerotic leads to renal dysfunction, oxidative stress, inflammation, endothelial dysfunction, and fibrosis, which are associated with systemic oxidative stress. Furthermore, coexisting experimental synergistically accelerates -induced kidney fibrosis and glomerulosclerosis. These were accompanied by renovascular endothelial dysfunction and pronounced systemic and kidney tissue oxidative stress, microvascular injury, and hypoxia, which might contribute to progression of renal injury that characterizes coexisting and. The current studies therefore demonstrate an interaction between cardiac and renal function beyond systemic atherosclerosis. Coronary artery disease remains the main cause of death in the Western society. Its chief etiology is atherosclerosis, in which cytokines instigate the release of inflammatory and cytotoxic molecules. Nevertheless, myocardial infarction per se can also trigger systemic inflammation that can accelerate atherosclerosis or renal inflammation in mice. 6 However, the contribution of chronic low-level myocardial ischemia to renal disease is unknown. In recent years, deleterious interactions along the cardiac renal axis have been increasingly recognized, 7 particularly in association with heart failure. 8 However, it remained unclear whether myocardial ischemia due to alone, without either atherosclerosis or myocardial infarction, aggravates renal injury. In our study, the slight but significant decrease in myocardial perfusion distal to the indicates the hemodynamic significance of the stenoses. Our chronic model was nonatherosclerotic, noninfarcted, and exhibited preserved cardiac function. Nevertheless, increased kidney oxidative stress (DHE and GP9), inflammation (TNFa and MCP-), fibrosis, tubular injury, and microvascular remodeling. Moreover, renal vascular resistance was elevated and cortical perfusion responses to Ach were blunted, indicating endothelial dysfunction and decreased nitric oxide bioavailability in, 9 supported by the downregulation of enos. This impact on the kidney might have been mediated by factors released from the heart, 7 Kidney International (5) 87, 79 77

5 D Sun et al.: Coronary artery stenosis accelerates kidney damage + PAS Kidney fibrosis α-sma CD3 DHE Trichrome 8 6 &Δ + Glomerulosclerosis &Δ DHE/DAPI + Capillary count (/36 μm ) 8 6 & + Media/lumen.5.5 & + Tubular injury score 3 & + Figure Renal tissue remodeling. (a) Representative renal trichrome (), dihydroethidium (DHE) (), CD3 immunofluorescence (), a-sma (), and PAS staining (). Kidney fibrosis increased in hypertension () and coronary artery stenosis () compared with sham; kidney fibrosis and glomerular score increased synergistically in þ compared with the other groups (b, c). DHE staining (normalized to DAPI-positive nuclei) increased in compared with sham, and in þ compared with sham and (d). Capillary density (CD3 immunofluorescence) in decreased compared with sham, but in þ it decreased lower than all other groups (e). Renal microvascular media-to-lumen ratio (a-sma) increased in compared with sham, and further in and þ compared with sham and (f). Tubular injury score (PAS staining) increased in and, and further aggravated in þ (g). Po.5 vs. sham; Po.5 vs. ; & Po.5 vs. ; D Po.5 for synergistic interaction. Scale bar ¼ 5 mm. eliciting an increase in systemic oxidative stress (PGF -a isoprostanes, Supplementary Figure S6 online) observed in, whereas systemic levels of inflammatory markers remained unaltered. Indeed, the drop in serum creatinine, renal fibrosis, and remodeling after chronic antioxidant treatment of pigs implicates oxidative stress in the mechanisms by which adversely affected the kidney. These observations position as a risk factor for kidney damage independent of, but likely aggravated by, atherosclerosis or infarction. Kidney International (5) 87,

6 D Sun et al.: Coronary artery stenosis accelerates kidney damage a TNFα 6 KDa MCP- KDa GP9-phox 65 KDa NT 7 KDa + c Protein expression (vs. GAPDH) GAPDH 36 KDa TNFα MCP- GP9-phox NT b HIF-α 6 KDa VEGF KDa TGF-β.5 KDa TIMP- KDa enos KDa GAPDH 36 KDa + d Protein expression (vs. GAPDH) & + & Δ HIF-α VEGF TGF-β TIMP- enos Figure 3 Renal expression of inflammatory, oxidative, and growth factors. Representative ( bands shown per group) immunoblotting of (a) tumor necrosis factor (TNF)-a, monocyte chemoattractant protein (MCP)-, GP9-phox, and nitrotyrosine (NT); (b) hypoxia-inducible factor (HIF)-a, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-b, tissue inhibitor of metalloproteinase (TIMP)-, and endothelial nitric oxide synthase (enos). Inflammatory and oxidative markers were elevated in coronary artery stenosis (), and further augmented in þ hypertension (). (c) Hypoxia and fibrotic markers increased in þ, whereas enos decreased; VEGF expression decreased in and further decreased in þ (d). Po.5 vs. sham; Po.5 vs. ; & Po.5 vs. ; D Po.5 for synergistic interaction. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. is an important cause for renal disease. 6, Importantly, when coexisted with, cardiac function and renal hemodynamics further deteriorated. Basal RBF and cortical perfusion and responses to Ach were impaired in þ, indicating exacerbated endothelial dysfunction. Mild capillary loss observed in was exacerbated in þ, evoking tissue hypoxia, suggested by the upregulation of HIF-a, accompanied by microvascular remodeling. Interestingly, intratubular concentration, an index of tubular integrity and function, decreased in the þ proximal and distal tubules, implying a defect in urinary concentration mechanisms. Capillary injury may reduce blood supply to the vulnerable renal tubules. Thus, coexisting experimental and exacerbate impairment in cardiac and renal function, and induce renal capillary injury, thereby magnifying tubular injury, interstitial fibrosis, and ultimately glomerulosclerosis (Supplementary Figure S6 online). The mechanism by which aggravates the effects of on the kidney may be multifactorial. Impaired systolic function may partly account for the lack of an increase in RBF and GFR, observed in, despite a similar increase in renal perfusion pressure. Furthermore, systemic oxidative stress was elevated similarly in and þ, likely originating from the ischemic myocardium. The decrease in serum creatinine after antioxidant treatment implicates oxidative stress in renal dysfunction. Notably, þ additionally showed elevated circulating TGF-b, possibly derived from the stenotic kidney. 3 The elevated renal TGFb expression in, amplified in and þ, was likely produced locally in response to injury. TIMP- also increased further in þ, which likely exacerbated renal fibrosis. Hence, several parallel injury pathways might converge to amplify kidney injury when þ coexist. Yet, unaltered circulating levels of multiple inflammatory mediators argue against systemic inflammation underpinning the effects of or its interaction with. In addition, prolonged and severe tissue inflammation and oxidative stress interfere with the upregulation of angiogenic factors,,5 and is characterized by capillary loss. In the present study, þ showed a small additive effect on renal inflammation (e.g., TNFa) and oxidative stress, which downregulated protein expression of VEGF and enos compared with, suppressing capillary density. Downregulation of enos may also underlie endothelial dysfunction and unaltered nitrotyrosine expression. Thus, coexistence of and may promote microvascular regression through amplified inflammation and oxidative stress. 7 Kidney International (5) 87, 79 77

7 D Sun et al.: Coronary artery stenosis accelerates kidney damage Limitations: In our models, and developed over weeks, which is shorter than the usual disease durations in humans, who often also have other comorbidities. This might limit the clinical translation power of our observations. Nevertheless, this exposure sufficed to illustrate that þ has significant pathophysiological implications for evolution of kidney fibrosis. The unchanged renal function and structure during femoral artery stenosis underscore the specific effects of and mild myocardial ischemia on the kidney. As typical for the renovascular, systemic plasma renin activity levels were not increased in chronic and þ, yet local angiotensin-ii activity cannot be excluded. In conclusion, this study demonstrates that nonatherosclerotic alone augments renal inflammation, increases systemic and renal oxidative stress, and elicits renal injury and dysfunction. Coexistence of and aggravates renal microvascular injury and consequently tissue hypoxia, it synergistically magnifies kidney fibrosis, and it may thereby contribute to increased incidence of renal failure seen when and coexist. These observations underscore the cross talk between the myocardium and the kidney and the need for careful screening in order to assess the relative risk and to ensure adequacy of management in patients with concurrent and, regardless of the atherosclerosis burden. Further studies are also needed to examine the ability of percutaneous coronary intervention to preserve renal function. MATERIALS AND METHODS Experimental design Twenty-eight female domestic pigs (initially weighing 5 35 kg) were studied after approval of the Institutional Animal Care and Use Committee and randomized to four groups (n ¼ 7 each): control, only, only, and with. Induction of and Animals were anesthetized with intramuscular Telazol (Fort Dodge Animal Health, New York, NY) (5 mg/kg) and xylazine ( mg/kg), intubated, and anesthesia was maintained with intravenous ketamine (. mg/kg/min) and xylazine (.3 mg/kg/min). 6 The femoral and carotid arteries were catheterized, followed by a heparin bolus (5 IU). Under fluoroscopic guidance, local-irritant coils were implanted in the left-circumflex coronary artery and/or the proximal-middle right renal artery, as previously described.,7,8 MAP was subsequently measured by a PhysioTel telemetry system (Data Sciences International, St Paul, MN) implanted at baseline in the left femoral artery. Controls underwent sham renal and/or coronary angiography. Measurement of kidney and cardiac function Ten weeks later, all pigs underwent renal and cardiac angiography, for which they were similarly anesthetized, intubated, and mechanically ventilated with room air.,9 After angiography, nonstenotic regional kidney perfusion, RBF, and GFR were evaluated using multidetector CT (MDCT, SOMATOM Definition-6; Siemens, Forchheim, Germany), and again after a -minute suprarenal arterial infusion of acetylcholine (Ach, 5 mg/kg/min). Thirty minutes later, CT studies were conducted to assess cardiac structure and systolic and diastolic function in vivo.,7,3 All images were analyzed with the ANALYZE software package (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN). For renal function, regions of interest were selected in the crosssectional images from the aorta, renal cortex, and medulla, and time-attenuation curves were generated. 3 The parameters obtained from the vascular curve in each region of the kidney were used to calculate cortical and medullary perfusion. Intratubular concentration was calculated for each nephron segment as the ratio of the area under each tubular curve to that of the cortical vascular curve, divided by normalized GFR. Renal volume was measured using planimetry, and RBF was calculated as the sum of cortical and medullary blood flows (product of cortical and medullary perfusion and volumes) and GFR from the cortical proximal tubular curve. 3 Renal vascular resistance was calculated as MAP/RBF. For cardiac function, LV stroke volume, cardiac output, ejection fraction, E/A, and LVMM were calculated as previously described. 3 For myocardial perfusion, regions of interest were traced at the lateral LV wall distal to the, and time-attenuation curves were analyzed as we have shown before.,7,3 Angiography A guide catheter was positioned under fluoroscopic guidance in the right renal artery and left main coronary artery for selective injections of contrast media. The degree of stenosis was measured by quantitative angiography, as described previously, and assessed as the decrease in arterial luminal diameter and area at the most stenotic point compared with a stenosis-free segment. Sample collection Blood samples were collected from the inferior vena cava for measurement of SCr (Arbor Assays, Ann Arbor, MI), PGF -a isoprostane (ELISA, Cayman, Ann Arbor, MI), plasma renin activity (radioimmunoassay, DiaSorin, Stillwater, MN), norepinephrine (Abnova GmbH, Heidelberg, Germany), and TGF-b (R&D, Minneapolis, MN). granulocyte macrophage colony-stimulating factor, IL-a, IL-b, IL-ra, IL-, IL-, IL-6, IL-, IL-, IL-8, and TNFa were measured by Luminex (Millipore, Billerica, MA). Urine samples were collected for urinary protein (Coomassie Plus assay, Thermo Scientific, Waltham, MA). After completion of all studies, the pigs were euthanized with a lethal intravenous dose of sodium pentobarbital ( mg/kg; Sleepaway, Fort Dodge Laboratories, Fort Dodge, IA). Kidneys were removed and fresh-frozen or preserved in formalin. Renal histology and immunohistochemistry Trichrome stainings were performed in 5-mm paraffin midhilar renal cross-sections to assess fibrosis by a computer-aided image analysis program (AxioVision.8., Carl Zeiss Microscopy, Thornwood, NY). In each slide, trichrome staining was semiautomatically quantified in 6 fields, expressed as a fraction of kidney surface area, and the results from all fields were averaged. Glomerular score (% of sclerotic out of glomeruli) was assessed as described. CD3 (:5; AbD Serotec, Oxford, UK) immunofluorescence was used to investigate capillary density. 3 Microvascular remodeling was assessed by wall-to-lumen ratio using a-smooth muscle actin (a-sma) staining (:5; Dako, Glostrup, Denmark). 3 Tubular injury was scored as described previously. 33 Oxidative stress Kidney International (5) 87,

8 D Sun et al.: Coronary artery stenosis accelerates kidney damage indicated by in situ production of superoxide anion was quantified in 3-mm DHE-stained slides. 3 Western blotting Standard western blotting protocols were followed as described, 35,36 using specific antibodies against TGF-b (:, Santa Cruz, Dallas, TX), MCP-, HIF-a (both :, Abcam, Cambridge, UK), TNFa (:, Santa Cruz), GP9-phox, p67-phox (both :, Santa Cruz), nitrotyrosine (:, Cayman), TIMP-, VEGF (both :, Santa Cruz), and enos (:, BD BioSciences, San Jose, CA). Protein expression was determined in one sham,, or nonstenotic and þ kidney in each animal, and the intensities of the protein bands were quantified and normalized for a GAPDH (:5, Abcam) loading control. Mechanistic exploration To exclude the possible nonspecific effects of an intravascular localirritant coil on the remote kidney, coils were implanted endovascularly in the femoral arteries of two additional pigs, and studies were conducted weeks later (n ¼ kidneys). To further explore the role of oxidative stress in mediating the effect of on the kidney, additional animals with received chronic daily mitochondria-targeted antioxidant Bendavia (. mg/kg/day, subcutaneous) for weeks. 37 After weeks of diet and intervention, studies were conducted in vivo using MDCT to measure myocardial and renal perfusion, and ex vivo for representative tissue studies (n ¼ kidneys). Statistical analysis Results are expressed as mean±s.e.m. Two-way analysis of variance was used to test for the effect of and ; their interaction was analyzed with Tukey s multiple comparison adjustment for the leastsquares means, and post hoc comparisons among groups were performed using unpaired Student s t-test. Paired Student s t-test was performed for comparisons within groups (baseline vs. Ach). For data not normally distributed, comparisons were done using nonparametric tests. All analyses were performed in JMP and significance was accepted for Pp.5. DISCLOSURE Dr Lilach O. Lerman received research funding from Stealth Peptides Incorporated. The remaining authors declare no conflict of interest. ACKNOWLEDGMENTS This study was partly supported by NIH grant numbers DK7368, HL773, HL56, DK8, C6-RR8898, HL-995, and AG The authors thank Stealth Peptides Incorporated for providing Bendavia for the studies. SUPPLEMENTARY MATERIAL Table S. Circulating inflammatory mediators in the experimental groups (mean±s.e.m., n ¼ 7 each). Table S. Systemic characteristics, kidney hemodynamics and function in sham and femoral artery stenosis (FAS) pigs (mean±s.e.m.). Table S3. Systemic characteristics, kidney hemodynamics and function in sham, coronary artery stenosis (), and þ Bendavia pigs (mean±s.e.m.). Figure S. Full blots showing renal expression of inflammatory and oxidative factors. Figure S. Full blots showing renal expression of growth factors. Figure S3. Evaluation of pigs with femoral artery stenosis (FAS). Figure S. Renal tissue remodeling in pigs with coronary artery stenosis () treated by the mitochondria-targeted peptide Bendavia. Figure S5. Renal expression of inflammatory, oxidative, and growth factors in þ Bendavia pigs. Figure S6. Proposed mechanisms of the cardio-renal interaction in the study. Supplementary material is linked to the online version of the paper at REFERENCES. Bitton A, Choudhry NK, Matlin OS et al. The impact of medication adherence on coronary artery disease costs and outcomes: a systematic review. Am J Med 3; 6: e7 e7.. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 5; 35: Kim EJ, Kim S, Kang DO et al. 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