Experimental coronary artery stenosis accelerates kidney damage in renovascular hypertensive swine
2014; Elsevier BV; Volume: 87; Issue: 4 Linguagem: Inglês
10.1038/ki.2014.343
ISSN1523-1755
AutoresDong Sun, Alfonso Eirin, Xiang-Yang Zhu, Xin Zhang, John A. Crane, John R. Woollard, Amir Lerman, Lilach O. Lerman,
Tópico(s)Blood Pressure and Hypertension Studies
ResumoThe impact of coronary artery stenosis (CAS) on renal injury is unknown. Here we tested whether the existence of CAS, regardless of concurrent atherosclerosis, would induce kidney injury and magnify its susceptibility to damage from coexisting hypertension (HT). Pigs (seven each) were assigned to sham, left-circumflex CAS, renovascular HT, and CAS plus HT groups. Cardiac and nonstenotic kidney functions, circulating and renal inflammatory and oxidative markers, and renal and microvascular remodeling were assessed 10 weeks later. Myocardial perfusion declined distal to CAS. Systemic levels of PGF2-α isoprostane, a marker of oxidative stress, increased in CAS and CAS plus HT, whereas single-kidney blood flow responses to acetylcholine were significantly blunted only in CAS plus HT compared with sham, HT, and CAS, indicating renovascular endothelial dysfunction. Tissue expression of inflammatory and oxidative markers were elevated in the CAS pig kidney, and further magnified in CAS plus HT, whereas angiogenic factor expression was decreased. Bendavia, a mitochondria-targeted peptide, decreased oxidative stress and improved renal function and structure in CAS. Furthermore, CAS and HT synergistically amplified glomerulosclerosis and renal fibrosis. Thus, mild myocardial ischemia, independent of systemic atherosclerosis, induced renal injury, possibly mediated by increased oxidative stress. Superimposed HT aggravates renal inflammation and endothelial dysfunction caused by CAS, and synergistically promotes kidney fibrosis, providing impetus to preserve cardiac integrity in order to protect the kidney. The impact of coronary artery stenosis (CAS) on renal injury is unknown. Here we tested whether the existence of CAS, regardless of concurrent atherosclerosis, would induce kidney injury and magnify its susceptibility to damage from coexisting hypertension (HT). Pigs (seven each) were assigned to sham, left-circumflex CAS, renovascular HT, and CAS plus HT groups. Cardiac and nonstenotic kidney functions, circulating and renal inflammatory and oxidative markers, and renal and microvascular remodeling were assessed 10 weeks later. Myocardial perfusion declined distal to CAS. Systemic levels of PGF2-α isoprostane, a marker of oxidative stress, increased in CAS and CAS plus HT, whereas single-kidney blood flow responses to acetylcholine were significantly blunted only in CAS plus HT compared with sham, HT, and CAS, indicating renovascular endothelial dysfunction. Tissue expression of inflammatory and oxidative markers were elevated in the CAS pig kidney, and further magnified in CAS plus HT, whereas angiogenic factor expression was decreased. Bendavia, a mitochondria-targeted peptide, decreased oxidative stress and improved renal function and structure in CAS. Furthermore, CAS and HT synergistically amplified glomerulosclerosis and renal fibrosis. Thus, mild myocardial ischemia, independent of systemic atherosclerosis, induced renal injury, possibly mediated by increased oxidative stress. Superimposed HT aggravates renal inflammation and endothelial dysfunction caused by CAS, and synergistically promotes kidney fibrosis, providing impetus to preserve cardiac integrity in order to protect the kidney. Atherosclerotic coronary artery disease is the most common cause of death in the United States,1.Bitton A. Choudhry N.K. Matlin O.S. et al.The impact of medication adherence on coronary artery disease costs and outcomes: a systematic review.Am J Med. 2013; 126: e7-e27Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar 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,2.Hansson G.K. Inflammation, atherosclerosis, and coronary artery disease.N Engl J Med. 2005; 352: 1685-1695Crossref PubMed Scopus (6981) Google Scholar yet myocardial ischemia may impose adverse effects on remote organs and vascular territories.3.Kim E.J. Kim S. Kang D.O. et al.Metabolic activity of the spleen and bone marrow in patients with acute myocardial infarction evaluated by 18f-fluorodeoxyglucose positron emission tomograpic imaging.Circ Cardiovasc Imaging. 2014; 7: 454-460Crossref PubMed Scopus (87) Google Scholar For example, myocardial infarction and stroke augment inflammation, and in turn atherosclerosis,4.Dutta P. Courties G. Wei Y. et al.Myocardial infarction accelerates atherosclerosis.Nature. 2012; 487: 325-329Crossref PubMed Scopus (753) Google Scholar and they can trigger morphological and functional changes in the kidney.5.Lu J. Wang X. Wang W. et al.Abrogation of lectin-like oxidized LDL receptor-1 attenuates acute myocardial ischemia-induced renal dysfunction by modulating systemic and local inflammation.Kidney Int. 2012; 82: 436-444Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar 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 (HT), an important cause of chronic kidney disease.6.Appel L.J. Wright Jr, J.T. Greene T. et al.Intensive blood-pressure control in hypertensive chronic kidney disease.N Engl J Med. 2010; 363: 918-929Crossref PubMed Scopus (438) Google Scholar Previous studies have shown increased prevalence of renovascular disease in patients with coronary artery disease,7.Weber-Mzell D. Kotanko P. Schumacher M. et al.Coronary anatomy predicts presence or absence of renal artery stenosis. A prospective study in patients undergoing cardiac catheterization for suspected coronary artery disease.Eur Heart J. 2002; 23: 1684-1691Crossref PubMed Google Scholar, 8.Liang F. Hu D.Y. Wu M.Y. et al.The incidence of renal artery stenosis in the patients referred for coronary artery bypass grafting.Indian J Nephrol. 2012; 22: 13-17Crossref PubMed Scopus (5) Google Scholar, 9.Ollivier R. Boulmier D. Veillard D. et al.Frequency and predictors of renal artery stenosis in patients with coronary artery disease.Cardiovasc Revasc Med. 2009; 10: 23-29Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar in whom renal insufficiency is the main clinical predictor of renal artery stenosis. Renal artery stenosis is an important cause of secondary HT 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.10.Macedo T.A. Pedrosa R.P. Costa-Hong V. et al.Renal artery stenosis predicts coronary artery disease in patients with hypertension.PLoS One. 2013; 8: e58635Crossref PubMed Scopus (4) Google Scholar We have also recently shown that coexistence of coronary and renal artery diseases in human subjects magnifies renal injury.11.Khangura K.K. Eirin A. Kane G.C. et al.Cardiac function in renovascular hypertensive patients with and without renal dysfunction.Am J Hypertens. 2014; 27: 445-453Crossref PubMed Scopus (20) Google Scholar,12.Khangura K.K. Eirin A. Kane G.C. et al.Extrarenal atherosclerotic disease blunts renal recovery in patients with renovascular hypertension.J Hypertens. 2014; 32: 1300-1306Crossref PubMed Scopus (11) Google Scholar The preglomerular vasculature exposed to the HT develops progressive vascular pathology,13.Bidani A.K. Polichnowski A.J. Loutzenhiser R. et al.Renal microvascular dysfunction, hypertension and CKD progression.Curr Opin Nephrol Hypertens. 2013; 22: 1-9Crossref PubMed Scopus (109) Google Scholar 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.14.Hill G.S. Hypertensive nephrosclerosis.Curr Opin Nephrol Hypertens. 2008; 17: 266-270Crossref PubMed Scopus (137) Google Scholar We have also shown that renovascular HT blunts antioxidant defense mechanisms in the nonstenotic kidney.15.Rodriguez-Porcel M. Krier J.D. Lerman A. et al.Combination of hypercholesterolemia and hypertension augments renal function abnormalities.Hypertension. 2001; 37: 774-780Crossref PubMed Google Scholar These functional and structural alterations may render kidneys exposed to HT susceptible to other comorbidities, and amplify renal damage. However, whether nonatherosclerotic coronary artery stenosis (CAS) alone induces kidney injury or increases its vulnerability to adverse effects of coexisting HT remains unclear. The present study was therefore designed to test the hypothesis that isolated CAS elicits kidney inflammation and oxidative stress, which are exacerbated by HT and impair renal function. Renal artery stenosis increased mean arterial pressure (MAP) in both HT and CAS+HT compared with sham (Table 1, P=0.03 for both). Body weight and plasma renin activity were not significantly different among the groups, but serum creatinine was elevated in CAS, HT, and CAS+HT (Table 1, P<0.04 each), affected by both CAS and HT. Urinary protein, affected by HT, increased significantly only in CAS+HT compared with sham and CAS (Table 1, P=0.006 and P=0.045, respectively). Circulating PGF2-α isoprostane levels increased in CAS and CAS+HT compared with sham and HT (Figure 1a, P 0.05 for all).Table 1Systemic characteristics and cardiac function in the four groups (mean±s.e.m., n=7 each)P-value for two-way ANOVAShamHTCASCAS+HTHTCASHT × CASBody weight, kg48.0±2.848.3±5.251.6±4.646.1±5.0MAP, mmHg96.7±2.8116.5±7.8aP<0.05 vs. sham.101.3±5.6112.5±7.0aP<0.05 vs. sham.0.0280.9610.519PRA, pg/ml/min0.17±0.100.25±0.160.22±0.070.24±0.150.5050.7860.648Creatinine, mg/dl1.17±0.311.58±0.17aP<0.05 vs. sham.1.62±0.32aP<0.05 vs. sham.1.80±0.16aP<0.05 vs. sham.0.0160.0080.326Urine protein, μg/ml16.9±2.522.1±5.918.4±6.133.4±4.7aP<0.05 vs. sham.,bP<0.05 vs. CAS.0.0470.1990.319Norepinephrine, ng/ml0.08±0.020.10±0.020.04±0.01cP<0.05 vs. HT.0.05±0.010.2900.010.583Degree of CAS, %0077.3±8.9aP<0.05 vs. sham.,cP<0.05 vs. HT.74.0±10.3aP<0.05 vs. sham.,cP<0.05 vs. HT.Stroke volume, ml48±250±348±537±3aP<0.05 vs. sham.,cP<0.05 vs. HT.0.2620.0450.078Ejection fraction, %50±357±445±545±30.4210.0460.426E/A1.07±0.061.02±0.151.20±0.151.14±0.120.6520.3130.946Abbreviations: ANOVA, analysis of variance; CAS, coronary artery stenosis; E/A, early-to-late left ventricular filling velocities; HT, hypertension; MAP, mean arterial pressure; PRA, plasma renin activity.a P<0.05 vs. sham.b P<0.05 vs. CAS.c P<0.05 vs. HT. Open table in a new tab Download .doc (.04 MB) Help with doc files Supplementary Table 1 Abbreviations: ANOVA, analysis of variance; CAS, coronary artery stenosis; E/A, early-to-late left ventricular filling velocities; HT, hypertension; MAP, mean arterial pressure; PRA, plasma renin activity. Ten weeks after induction, the pigs developed significant and similar degree of CAS (Table 1, Figure 1d), but stroke volume and cardiac output decreased owing to CAS only in CAS+HT compared with sham and HT (Table 1, Figure 1e, P≤0.03 for each). The ejection fraction (albeit slightly affected by CAS) and early and late left ventricular (LV) filling velocities (E/A) were not significantly different among the four groups (Table 1). Myocardial perfusion distal to CAS and CAS+HT was lower than in sham (Figure 1f, P=0.02 and P=0.01, respectively), whereas LV muscle mass (LVMM) was increased in HT and CAS+HT (Figure 1c and g, P≤0.03 for both). The degree of renal artery stenosis was not different between HT and CAS+HT, but renal blood flow (RBF) and glomerular filtration rate (GFR) of the nonstenotic kidney were elevated only in HT; RBF was increased by HT, suppressed by CAS, and showed a significant interaction between the two (Table 2). GFR also tended to be blunted by the interaction HT × CAS (Figure 1h). RBF response to Ach was attenuated by CAS only in CAS+HT (P=0.08 vs. baseline, Figure 1i), and became lower than sham (P=0.004), by a significant interaction CAS × HT (Table 2).Table 2Nonstenotic kidney hemodynamics and function in sham, CAS, HT, and CAS+HT pigs (mean±s.e.m.,n=7 each)P-value for two-way ANOVAShamHTCASCAS+HTHTCASHT × CASDegree of renal artery stenosis, %081.7±7.5aP<0.05 vs. sham.,bP<0.05 vs. CAS.080.7±5.7aP<0.05 vs. sham.,bP<0.05 vs. CAS.RBF, ml/min Basal469.0±63.1779.4±81.1aP<0.05 vs. sham.422.3±82.5cP<0.05 vs. HT.428.5±51.0cP<0.05 vs. HT.0.0310.0080.037 Ach677.4±32.5dP<0.05 vs. baseline.969.4±83.3aP<0.05 vs. sham.,dP<0.05 vs. baseline.590.6±111.0cP<0.05 vs. HT.,dP<0.05 vs. baseline.482.0±48.5aP<0.05 vs. sham.,cP<0.05 vs. HT.0.1920.00030.007Perfusion, ml/min/ml Cortex: baseline4.10±0.475.03±0.753.80±0.46cP<0.05 vs. HT.4.03±0.30cP<0.05 vs. HT.0.1410.1030.371 Ach6.21±0.29dP<0.05 vs. baseline.6.30±0.59dP<0.05 vs. baseline.4.94±0.60aP<0.05 vs. sham.,cP<0.05 vs. HT.,dP<0.05 vs. baseline.5.12±0.40aP<0.05 vs. sham.,cP<0.05 vs. HT.,dP<0.05 vs. baseline.0.7810.0170.920 Medulla: baseline2.84±0.442.19±0.101.98±0.352.00±0.350.3700.1460.342 Ach3.77±0.55dP<0.05 vs. baseline.3.47±0.43dP<0.05 vs. baseline.3.82±0.47dP<0.05 vs. baseline.2.48±0.39aP<0.05 vs. sham.,bP<0.05 vs. CAS.0.0950.3280.281RVR, mmHg/min/ml0.18±0.020.16±0.020.27±0.04aP<0.05 vs. sham.cP<0.05 vs. HT.0.29±0.04aP<0.05 vs. sham.,cP<0.05 vs. HT.0.5110.0120.174Abbreviations: Ach, acetylcholine; ANOVA, analysis of variance; CAS, coronary artery stenosis; HT, hypertension; RBF, renal blood flow; RVR, renal vascular resistance.a P<0.05 vs. sham.b P<0.05 vs. CAS.c P<0.05 vs. HT.d P<0.05 vs. baseline. Open table in a new tab Abbreviations: Ach, acetylcholine; ANOVA, analysis of variance; CAS, coronary artery stenosis; HT, hypertension; RBF, renal blood flow; RVR, renal vascular resistance. Basal cortical perfusion in CAS and CAS+HT was lower than in HT, and after Ach infusion it was also lower than in sham (Table 2, P<0.05 for each). Basal medullary perfusion was not significantly different among the four groups, but its response to Ach was blunted only in CAS+HT. Renal vascular resistance was elevated in CAS and CAS+HT compared with sham and HT (Table 2) owing to CAS. CAS also decreased proximal and distal tubular intratubular concentration in CAS+HT (Figure 1j), suggesting impaired tubular fluid reabsorption. Trichrome staining showed increased renal fibrosis in HT and CAS compared with sham pigs (P=0.003 and P=0.001, respectively). Compared with other groups, renal fibrosis and glomerular score in CAS+HT increased significantly (Figures 2a–c, P<0.01 each), showing interactions between CAS and HT in both fibrosis (Figure 2b, P=0.0008) and glomerulosclerosis (Figure 2c, P=0.04). Dihydroethidium (DHE) staining was elevated in CAS (P=0.03 vs. sham) and further exacerbated in CAS+HT (Figure 2a-DHE and d, P=0.006 vs. sham and P=0.03 vs. HT). Compared with sham, renal capillary density decreased in HT (Figure 2, P=0.02), yet in CAS+HT it decreased further compared with all groups (P<0.04 each). Microvascular wall thickening (media-to-lumen ratio) increased in CAS (P=0.0003 vs. sham), but further in HT and CAS+HT (P<0.001 each). Tubular injury observed in HT and CAS was further aggravated in CAS+HT (P<0.001). None showed CAS × HT interactions. Compared with sham, renal protein expression of TNFα increased in CAS (Figure 3a and Supplementary Figure S1 online, P=0.03) and further in CAS+HT (P=0.004 and P=0.002, respectively). Monocyte chemoattractant protein (MCP)-1 and GP91-phox expressions were all higher in CAS and CAS+HT than in sham and HT (Figure 3a and Supplementary Figure S1 online), affected by CAS alone (P<0.003 each), whereas nitrotyrosine was not. Hypoxia-inducible factor (HIF)-1α expression increased owing to CAS only in CAS+HT kidneys compared with sham and HT (Figure 3b and Supplementary Figure S2 online), suggesting tissue hypoxia. Vascular endothelial growth factor (VEGF) expression decreased in HT and further decreased in CAS+HT, owing to both CAS and HT. Renal TGF-β1 levels increased in HT compared with sham, but markedly increased in CAS and CAS+HT (Figure 3b and d and Supplementary Figure S2 online). Tissue inhibitor of metalloproteinases (TIMP)-1 was elevated by HT in CAS+HT compared with the other groups (P 0.05 vs. sham for all). Download .doc (.04 MB) Help with doc files Supplementary Table 2 Download .jpg (.18 MB) Help with files Supplementary Figure 3 In additional animals chronically treated with the mitochondria-targeted antioxidant bendavia, myocardial perfusion was not different from that in untreated CAS, yet serum creatinine that was elevated in CAS decreased in CAS+Bendavia, and MAP and renal hemodynamics were unchanged compared with CAS (Supplementary Table S3 online). Kidney fibrosis, DHE, microvascular media/lumen ratio, and tubular injury in CAS+Bendavia all decreased compared with untreated CAS (Supplementary Figure S4 online), and TNF-α, P67, GP91, and TGF-β1 were downregulated, whereas eNOS expression remained unchanged (Supplementary Figure S5 online). Download .doc (.04 MB) Help with doc files Supplementary Table 3 Download .jpg (.23 MB) Help with files Supplementary Figure 4 Download .jpg (.09 MB) Help with files Supplementary Figure 5 This study demonstrates that a hemodynamically significant but nonatherosclerotic CAS leads to renal dysfunction, oxidative stress, inflammation, endothelial dysfunction, and fibrosis, which are associated with systemic oxidative stress. Furthermore, coexisting experimental HT synergistically accelerates CAS-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 CAS and HT. 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.2.Hansson G.K. Inflammation, atherosclerosis, and coronary artery disease.N Engl J Med. 2005; 352: 1685-1695Crossref PubMed Scopus (6981) Google Scholar Nevertheless, myocardial infarction per se can also trigger systemic inflammation that can accelerate atherosclerosis4.Dutta P. Courties G. Wei Y. et al.Myocardial infarction accelerates atherosclerosis.Nature. 2012; 487: 325-329Crossref PubMed Scopus (753) Google Scholar or renal inflammation in mice.16.Ruparelia N. Digby J.E. Jefferson A. et al.Myocardial infarction causes inflammation and leukocyte recruitment at remote sites in the myocardium and in the renal glomerulus.Inflamm Res. 2013; 62: 515-525Crossref PubMed Scopus (52) Google Scholar 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,17.Ronco C. McCullough P. Anker S.D. et al.Cardio-renal syndromes: report from the consensus conference of the acute dialysis quality initiative.Eur Heart J. 2010; 31: 703-711Crossref PubMed Scopus (685) Google Scholar particularly in association with heart failure.18.Heywood J.T. Fonarow G.C. Costanzo M.R. et al.High prevalence of renal dysfunction and its impact on outcome in 118,465 patients hospitalized with acute decompensated heart failure: a report from the ADHERE database.J Card Fail. 2007; 13: 422-430Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar However, it remained unclear whether myocardial ischemia due to CAS alone, without either atherosclerosis or myocardial infarction, aggravates renal injury. In our study, the slight but significant decrease in myocardial perfusion distal to the CAS indicates the hemodynamic significance of the stenoses. Our chronic CAS model was nonatherosclerotic, noninfarcted, and exhibited preserved cardiac function. Nevertheless, CAS increased kidney oxidative stress (DHE and GP91), inflammation (TNFα and MCP-1), 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 CAS,19.Urbieta-Caceres V.H. Lavi R. Zhu X.Y. et al.Early atherosclerosis aggravates the effect of renal artery stenosis on the swine kidney.Am J Physiol Renal Physiol. 2010; 299: F135-F140Crossref PubMed Scopus (32) Google Scholar supported by the downregulation of eNOS. This impact on the kidney might have been mediated by factors released from the heart, eliciting an increase in systemic oxidative stress (PGF2-α isoprostanes, Supplementary Figure S6 online) observed in CAS, whereas systemic levels of inflammatory markers remained unaltered. Indeed, the drop in serum creatinine, renal fibrosis, and remodeling after chronic antioxidant treatment of CAS pigs implicates oxidative stress in the mechanisms by which CAS adversely affected the kidney. These observations position CAS as a risk factor for kidney damage independent of, but likely aggravated by, atherosclerosis or infarction. Download .jpg (.08 MB) Help with files Supplementary Figure 6 HT is an important cause for renal disease.6.Appel L.J. Wright Jr, J.T. Greene T. et al.Intensive blood-pressure control in hypertensive chronic kidney disease.N Engl J Med. 2010; 363: 918-929Crossref PubMed Scopus (438) Google Scholar,20.Bidani A.K. Griffin K.A. Pathophysiology of hypertensive renal damage: implications for therapy.Hypertension. 2004; 44: 595-601Crossref PubMed Scopus (286) Google Scholar Importantly, when HT coexisted with CAS, cardiac function and renal hemodynamics further deteriorated. Basal RBF and cortical perfusion and responses to Ach were impaired in CAS+HT, indicating exacerbated endothelial dysfunction. Mild capillary loss observed in HT was exacerbated in CAS+HT, evoking tissue hypoxia, suggested by the upregulation of HIF-1α, accompanied by microvascular remodeling.21.Zhu X.Y. Daghini E. Chade A.R. et al.Role of oxidative stress in remodeling of the myocardial microcirculation in hypertension.Arterioscler Thromb Vasc Biol. 2006; 26: 1746-1752Crossref PubMed Scopus (39) Google Scholar Interestingly, intratubular concentration, an index of tubular integrity and function, decreased in the CAS+HT proximal and distal tubules, implying a defect in urinary concentration mechanisms. Capillary injury may reduce blood supply to the vulnerable renal tubules.22.Sun D. Bu L. Liu C. et al.Therapeutic effects of human amniotic fluid-derived stem cells on renal interstitial fibrosis in a murine model of unilateral ureteral obstruction.PLoS One. 2013; 8: e65042Crossref PubMed Scopus (48) Google Scholar Thus, coexisting experimental CAS and HT 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 HT aggravates the effects of CAS on the kidney may be multifactorial. Impaired systolic function may partly account for the lack of an increase in RBF and GFR, observed in HT, despite a similar increase in renal perfusion pressure. Furthermore, systemic oxidative stress was elevated similarly in CAS and CAS+HT, likely originating from the ischemic myocardium. The decrease in serum creatinine after antioxidant treatment implicates oxidative stress in renal dysfunction. Notably, CAS+HT additionally showed elevated circulating TGF-β1, possibly derived from the stenotic kidney.23.Urbieta-Caceres V.H. Zhu X.Y. Jordan K.L. et al.Selective improvement in renal function preserved remote myocardial microvascular integrity and architecture in experimental renovascular disease.Atherosclerosis. 2012; 221: 350-358Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar The elevated renal TGFβ1 expression in HT, amplified in CAS and CAS+HT, was likely produced locally in response to injury. TIMP-1 also increased further in CAS+HT, which likely exacerbated renal fibrosis. Hence, several parallel injury pathways might converge to amplify kidney injury when CAS+HT coexist. Yet, unaltered circulating levels of multiple inflammatory mediators argue against systemic inflammation underpinning the effects of CAS or its interaction with HT. In addition, prolonged and severe tissue inflammation and oxidative stress interfere with the upregulation of angiogenic factors,24.Chade A.R. Zhu X. Lavi R. et al.Endothelial progenitor cells restore renal function in chronic experimental renovascular disease.Circulation. 2009; 119: 547-557Crossref PubMed Scopus (194) Google Scholar,25.Chade A.R. Zhu X. Mushin O.P. et al.Simvastatin promotes angiogenesis and prevents microvascular remodeling in chronic renal ischemia.FASEB J. 2006; 20: 1706-1708Crossref PubMed Scopus (117) Google Scholar and HT is characterized by capillary loss.14.Hill G.S. Hypertensive nephrosclerosis.Curr Opin Nephrol Hypertens. 2008; 17: 266-270Crossref PubMed Scopus (137) Google Scholar In the present study, CAS+HT showed a small additive effect on renal inflammation (e.g., TNFα) and oxidative stress, which downregulated protein expression of VEGF and eNOS compared with HT, suppressing capillary density. Downregulation of eNOS may also underlie endothelial dysfunction and unaltered nitrotyrosine expression. Thus, coexistence of CAS and HT may promote microvascular regression through amplified inflammation and oxidative stress. Limitations: In our models, CAS and HT developed over 10 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 CAS+HT has significant pathophysiological implications for evolution of kidney fibrosis. The unchanged renal function and structure during femoral artery stenosis underscore the specific effects of CAS and mild myocardial ischemia on the kidney. As typical for the renovascular HT, systemic plasma renin activity levels were not increased in chronic HT and CAS+HT, yet local angiotensin-II activity cannot be excluded. In conclusion, this study demonstrates that nonatherosclerotic CAS alone augments renal inflammation, increases systemic and renal oxidative stress, and elicits renal injury and dysfunction. Coexistence of CAS and HT 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 CAS and HT 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 CAS and HT, regardless of the atherosclerosis burden. Further studies are also needed to examine the ability of percutaneous coronary intervention to preserve renal function. Twenty-eight female domestic pigs (initially weighing 25–35kg) were studied after approval of the Institutional Animal Care and Use Committee and randomized to four groups (n=7 each): control, CAS only, HT only, and CAS with HT. Animals were anesthetized with intramuscular Telazol (Fort Dodge Animal Health, New York, NY) (5mg/kg) and xylazine (2mg/kg), intubated, and anesthesia was maintained with intravenous ketamine (0.2mg/kg/min) and xylazine (0.03mg/kg/min).26.Eirin A. Ebrahimi B. Zhang X. et al.Changes in glomerular filtration rate after renal revascularization correlate with microvascular hemodynamics and inflammation in swine renal artery stenosis.Circ Cardiovasc Interv. 2012; 5: 720-728Crossref PubMed Scopus (56) Google Scholar The femoral and carotid arteries were catheterized, followed by a heparin bolus (5000IU). 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.21.Zhu X.Y. Daghini E. Chade A.R. et al.Role of oxidative stress in remodeling of the myocardial microcirculation in hypertension.Arterioscler Thromb Vasc Biol. 2006; 26: 1746-1752Crossref PubMed Scopus (39) Google Scholar,27.Urbieta Caceres V.H. Lin J. Zhu X.Y. et al.Early experimental hypertension preserves the myocardial microvasculature but aggravates cardiac injury distal to chronic coronary artery obstruction.Am J Physiol Heart Circ Physiol. 2011; 300: H693-H701Crossref PubMed Scopus (20) Google Scholar,28.Favreau F. Zhu X.Y. Krier J.D. et al.Revascularization of swine renal artery stenosis improves renal function but not the changes in vascular structure.Kidney Int. 2010; 78: 1110-1118Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar 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. Ten weeks later, all pigs underwent renal and cardiac angiography, for which they were similarly anesthetized, intubated, and mechanically ventilated with room air.24.Chade A.R. Zhu X. Lavi R. et al.Endothelial progenitor cells restore renal function in chronic experimental renovascular disease.Circulation. 2009; 119: 547-557Crossref PubMed Scopus (194) Google Scholar,29.Chade A.R. Rodriguez-Porcel M. Herrmann J. et al.Antioxidant intervention blunts renal injury in experimental renovascular disease.J Am Soc Nephrol. 2004; 15: 958-966Crossref PubMed Scopus (112) Google Scholar After angiography, nonstenotic regional kidney perfusion, RBF, and GFR were evaluated using multidetector CT (MDCT, SOMATOM Definition-64; Siemens, Forchheim, Germany), and again after a 10-minute suprarenal arterial infusion of acetylcholine (Ach, 5μg/kg/min). Thirty minutes later, CT studies were conducted to assess cardiac structure and systolic and diastolic function in vivo.21.Zhu X.Y. Daghini E. Chade A.R. et al.Role of oxidative stress in remodeling of the myocardial microcirculation in hypertension.Arterioscler Thromb Vasc Biol. 2006; 26: 1746-1752Crossref PubMed Scopus (39) Google Scholar,27.Urbieta Caceres V.H. Lin J. Zhu X.Y. et al.Early experimental hypertension preserves the myocardial microvasculature but aggravates cardiac injury distal to chronic coronary artery obstruction.Am J Physiol Heart Circ Physiol. 2011; 300: H693-H701Crossref PubMed Scopus (20) Google Scholar,30.Daghini E. Primak A.N. Chade A.R. et al.Assessment of renal hemodynamics and function in pigs with 64-section multidetector CT: comparison with electron-beam CT.Radiology. 2007; 243: 405-412Crossref PubMed Scopus (102) Google Scholar 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 cross-sectional images from the aorta, renal cortex, and medulla, and time-attenuation curves were generated.30.Daghini E. Primak A.N. Chade A.R. et al.Assessment of renal hemodynamics and function in pigs with 64-section multidetector CT: comparison with electron-beam CT.Radiology. 2007; 243: 405-412Crossref PubMed Scopus (102) Google Scholar 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.30.Daghini E. Primak A.N. Chade A.R. et al.Assessment of renal hemodynamics and function in pigs with 64-section multidetector CT: comparison with electron-beam CT.Radiology. 2007; 243: 405-412Crossref PubMed Scopus (102) Google Scholar 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.30.Daghini E. Primak A.N. Chade A.R. et al.Assessment of renal hemodynamics and function in pigs with 64-section multidetector CT: comparison with electron-beam CT.Radiology. 2007; 243: 405-412Crossref PubMed Scopus (102) Google Scholar For myocardial perfusion, regions of interest were traced at the lateral LV wall distal to the CAS, and time-attenuation curves were analyzed as we have shown before.21.Zhu X.Y. Daghini E. Chade A.R. et al.Role of oxidative stress in remodeling of the myocardial microcirculation in hypertension.Arterioscler Thromb Vasc Biol. 2006; 26: 1746-1752Crossref PubMed Scopus (39) Google Scholar,27.Urbieta Caceres V.H. Lin J. Zhu X.Y. et al.Early experimental hypertension preserves the myocardial microvasculature but aggravates cardiac injury distal to chronic coronary artery obstruction.Am J Physiol Heart Circ Physiol. 2011; 300: H693-H701Crossref PubMed Scopus (20) Google Scholar,30.Daghini E. Primak A.N. Chade A.R. et al.Assessment of renal hemodynamics and function in pigs with 64-section multidetector CT: comparison with electron-beam CT.Radiology. 2007; 243: 405-412Crossref PubMed Scopus (102) Google Scholar 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,24.Chade A.R. Zhu X. Lavi R. et al.Endothelial progenitor cells restore renal function in chronic experimental renovascular disease.Circulation. 2009; 119: 547-557Crossref PubMed Scopus (194) Google Scholar and assessed as the decrease in arterial luminal diameter and area at the most stenotic point compared with a stenosis-free segment. Blood samples were collected from the inferior vena cava for measurement of SCr (Arbor Assays, Ann Arbor, MI), PGF2-α isoprostane (ELISA, Cayman, Ann Arbor, MI), plasma renin activity (radioimmunoassay, DiaSorin, Stillwater, MN), norepinephrine (Abnova GmbH, Heidelberg, Germany), and TGF-β1 (R&D, Minneapolis, MN). granulocyte–macrophage colony-stimulating factor, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, and TNFα 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 (100mg/kg; Sleepaway, Fort Dodge Laboratories, Fort Dodge, IA). Kidneys were removed and fresh-frozen or preserved in formalin. Trichrome stainings were performed in 5-μm paraffin midhilar renal cross-sections to assess fibrosis by a computer-aided image analysis program (AxioVision 4.8.2, Carl Zeiss Microscopy, Thornwood, NY). In each slide, trichrome staining was semiautomatically quantified in 6–10 fields, expressed as a fraction of kidney surface area, and the results from all fields were averaged. Glomerular score (% of sclerotic out of 100 glomeruli) was assessed as described.24.Chade A.R. Zhu X. Lavi R. et al.Endothelial progenitor cells restore renal function in chronic experimental renovascular disease.Circulation. 2009; 119: 547-557Crossref PubMed Scopus (194) Google Scholar CD31 (1:50; AbD Serotec, Oxford, UK) immunofluorescence was used to investigate capillary density.31.Muczynski K.A. Cotner T. Anderson S.K. Unusual expression of human lymphocyte antigen class II in normal renal microvascular endothelium.Kidney Int. 2001; 59: 488-497Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Microvascular remodeling was assessed by wall-to-lumen ratio using α-smooth muscle actin (α-SMA) staining (1:50; Dako, Glostrup, Denmark).32.Chade A.R. Mushin O.P. Zhu X. et al.Pathways of renal fibrosis and modulation of matrix turnover in experimental hypercholesterolemia.Hypertension. 2005; 46: 772-779Crossref PubMed Scopus (61) Google Scholar Tubular injury was scored as described previously.33.Nangaku M. Alpers C.E. Pippin J. et al.CD59 protects glomerular endothelial cells from immune-mediated thrombotic microangiopathy in rats.J Am Soc Nephrol. 1998; 9: 590-597PubMed Google Scholar Oxidative stress indicated by in situ production of superoxide anion was quantified in 30-μm DHE-stained slides.34.Ebrahimi B. Eirin A. Li Z. et al.Mesenchymal stem cells improve medullary inflammation and fibrosis after revascularization of swine atherosclerotic renal artery stenosis.PLoS One. 2013; 8: e67474Crossref PubMed Scopus (84) Google Scholar Standard western blotting protocols were followed as described,35.Eirin A. Zhu X.Y. Urbieta-Caceres V.H. et al.Persistent kidney dysfunction in swine renal artery stenosis correlates with outer cortical microvascular remodeling.Am J Physiol Renal Physiol. 2011; 300: F1394-F1401Crossref PubMed Scopus (68) Google Scholar,36.Chade A.R. Rodriguez-Porcel M. Grande J.P. et al.Mechanisms of renal structural alterations in combined hypercholesterolemia and renal artery stenosis.Arterioscler Thromb Vasc Biol. 2003; 23: 1295-1301Crossref PubMed Scopus (142) Google Scholar using specific antibodies against TGF-β1 (1:200, Santa Cruz, Dallas, TX), MCP-1, HIF-1α (both 1:1000, Abcam, Cambridge, UK), TNFα (1:100, Santa Cruz), GP91-phox, p67-phox (both 1:200, Santa Cruz), nitrotyrosine (1:200, Cayman), TIMP-1, VEGF (both 1:200, Santa Cruz), and eNOS (1:1000, BD BioSciences, San Jose, CA). Protein expression was determined in one sham, CAS, or nonstenotic HT and CAS+HT kidney in each animal, and the intensities of the protein bands were quantified and normalized for a GAPDH (1:5000, Abcam) loading control. To exclude the possible nonspecific effects of an intravascular local-irritant coil on the remote kidney, coils were implanted endovascularly in the femoral arteries of two additional pigs, and studies were conducted 10 weeks later (n=4 kidneys). To further explore the role of oxidative stress in mediating the effect of CAS on the kidney, additional animals with CAS received chronic daily mitochondria-targeted antioxidant Bendavia (0.1mg/kg/day, subcutaneous) for 10 weeks.37.Eirin A. Li Z. Zhang X. et al.A mitochondrial permeability transition pore inhibitor improves renal outcomes after revascularization in experimental atherosclerotic renal artery stenosis.Hypertension. 2012; 60: 1242-1249Crossref PubMed Scopus (106) Google Scholar After 10 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=4 kidneys). Results are expressed as mean±s.e.m. Two-way analysis of variance was used to test for the effect of CAS and HT; their interaction was analyzed with Tukey's multiple comparison adjustment for the least-squares 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 JMP10 and significance was accepted for P≤0.05. This study was partly supported by NIH grant numbers DK73608, HL77131, HL121561, DK100081, C06-RR018898, HL-92954, and AG-31750. The authors thank Stealth Peptides Incorporated for providing Bendavia for the studies. Table S1. Circulating inflammatory mediators in the experimental groups (mean±s.e.m., n=7 each). Table S2. 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 (CAS), and CAS+Bendavia pigs (mean±s.e.m.). Figure S1. Full blots showing renal expression of inflammatory and oxidative factors. Figure S2. Full blots showing renal expression of growth factors. Figure S3. Evaluation of pigs with femoral artery stenosis (FAS). Figure S4. Renal tissue remodeling in pigs with coronary artery stenosis (CAS) treated by the mitochondria-targeted peptide Bendavia. Figure S5. Renal expression of inflammatory, oxidative, and growth factors in CAS+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 athttp://www.nature.com/ki Download .doc (.03 MB) Help with doc files Supplementary Figure Legends
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