Textor, Stephen C.

Background— Atherosclerotic renal artery stenosis (ARAS) is known to reduce renal blood flow, glomerular filtration rate (GFR) and amplify kidney hypoxia, but the relationships between these factors and tubulointerstitial injury in the poststenotic kidney are poorly understood. The purpose of this study was to examine the effect of renal revascularization in ARAS on renal tissue hypoxia and renal injury. Methods and Results— Inpatient studies were performed in patients with ARAS (n=17; >60% occlusion) before and 3 months after stent revascularization, or in patients with essential hypertension (n=32), during fixed Na + intake and angiotensin converting enzyme/angiotensin receptors blockers Rx. Single kidney cortical, medullary perfusion, and renal blood flow were measured using multidetector computed tomography, and GFR by iothalamate clearance. Tissue deoxyhemoglobin levels ( R 2 *) were measured by blood oxygen level–dependent MRI at 3T, as was fractional kidney hypoxia (percentage of axial area with R 2 *>30/s). In addition, we measured renal vein levels of neutrophil gelatinase–associated lipocalin, monocyte chemoattractant protein-1, and tumor necrosis factor-α. Pre-stent single kidney renal blood flow, perfusion, and GFR were reduced in the poststenotic kidney. Renal vein neutrophil gelatinase–associated lipocalin, tumor necrosis factor-α, monocyte chemoattractant protein-1, and fractional hypoxia were higher in untreated ARAS than in essential hypertension. After stent revascularization, fractional hypoxia fell ( P <0.002) with increased cortical perfusion and blood flow, whereas GFR and neutrophil gelatinase–associated lipocalin, monocyte chemoattractant protein-1, and tumor necrosis factor-α remained unchanged. Conclusions— These data demonstrate that despite reversal of renal hypoxia and partial restoration of renal blood flow after revascularization, inflammatory cytokines and injury biomarkers remained elevated and GFR failed to recover in ARAS. Restoration of vessel patency alone failed to reverse tubulointerstitial damage and partly explains the limited clinical benefit of renal stenting. These results identify potential therapeutic targets for recovery of kidney function in renovascular disease.

Morbidity and mortality attributable to hypertension are higher in black essential hypertensive (EH) compared with white EH patients, possibly related to differential effects on vascular injury and repair. Although circulating endothelial progenitor cells (EPCs) preserve endothelial integrity, inflammatory endothelial cells (IECs) detach from sites of injury and represent markers of vascular damage. We hypothesized that blood levels of IECs and inflammatory markers would be higher in black EH compared with white EH patients. Inferior vena cava and renal vein levels of CD34+/KDR+ (EPC) and VAP-1+ (IEC) cells were measured by fluorescence-activated cell sorting in white EH and black EH patients under fixed sodium intake and blockade of the renin–angiotensin system, and compared with systemic levels in normotensive control subjects (n=19 each). Renal vein and inferior vena cava levels of inflammatory cytokines and EPC homing factors were measured by Luminex. Blood pressure, serum creatinine, lipids, and antihypertensive medications did not differ between white and black EH patients, and EPC levels were decreased in both. Circulating IEC levels were elevated in black EH patients, and inversely correlated with EPC levels ( R 2 =0.58; P =0.0001). Systemic levels of inflammatory cytokines and EPC homing factors were higher in black EH compared with white EH patients, and correlated directly with IECs. Renal vein inflammatory cytokines, EPCs, and IECs did not differ from their circulating levels. Most IECs expressed endothelial markers, fewer expressed progenitor cell markers, but none showed lymphocyte or phagocytic cell markers. Thus, increased release of cytokines and IECs in black EH patients may impair EPC reparative capacity and aggravate vascular damage, and accelerate hypertension-related complications.

Obesity-metabolic disorders (ObM) often accompany renal artery stenosis (RAS). We hypothesized that the coexistence of ObM and RAS magnifies inflammation and microvascular remodeling in the stenotic kidney (STK) and aggravates renal scarring. Twenty-eight obesity-prone Ossabaw pigs were studied after 16 wk of a high-fat/high-fructose diet or standard chow including ObM-sham, ObM-RAS, Lean-RAS, or Lean-sham (normal control) groups. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed by multidetector computed tomography (CT), renal oxygenation and tubular transport capability by blood-oxygen-level-dependent MRI, and microcirculation by micro-CT for vessel density, and Western blotting for protein expressions of angiogenic factors (VEGF/FLK-1). Renal vein and inferior vena cava levels of inflammatory cytokines were measured to evaluate systemic and kidney inflammation. Macrophage (MØ) infiltration and subpopulations, fat deposition in the kidney, and inflammation in perirenal and abdominal fat were also examined. GFR and RBF were decreased in Lean-STK but relatively preserved in ObM-STK. However, ObM-STK showed impaired tubular transport function, suppressed microcirculation, and stimulated glomerulosclerosis. ObM diet interacted with RAS to blunt angiogenesis in the STK, facilitated the release of inflammatory cytokines, and led to greater oxidative stress than Lean-STK. The ObM diet also induced fat deposition in the kidney and infiltration of proinflammatory M1-MØ, as also in perirenal and abdominal fat. Coexistence of ObM and RAS amplifies renal inflammation, aggravates microvascular remodeling, and accelerates glomerulosclerosis. Increased adiposity and MØ-accentuated inflammation induced by an ObM diet may contribute to structural injury in the post-STK kidney.

Objective— Endothelial outgrowth cells (EOC) decrease inflammation and improve endothelial repair. Inflammation aggravates kidney injury in renal artery stenosis (RAS), and may account for its persistence upon revascularization. We hypothesized that EOC would decrease inflammatory (M1) macrophages and improve renal recovery in RAS. Approach and Results— Pigs with 10 weeks of RAS were studied 4 weeks after percutaneous transluminal renal angioplasty (PTRA+stenting) or sham, with or without adjunct intrarenal delivery of autologous EOC (10×10 6 ), and compared with similarly treated normal controls (n=7 each). Single-kidney function, microvascular and tissue remodeling, inflammation, oxidative stress, and fibrosis were evaluated. Four weeks after PTRA, EOC were engrafted in injected RAS-kidneys. Stenotic-kidney glomerular filtration rate was restored in RAS+EOC, RAS+PTRA, and RAS+PTRA+EOC pigs, whereas stenotic-kidney blood flow and angiogenesis were improved and fibrosis attenuated only in EOC-treated pigs. Furthermore, EOC increased cell proliferation and decreased the ratio of M1 (inflammatory)/M2 (reparative) macrophages, as well as circulating levels and stenotic-kidney release of inflammatory cytokines. Cultured-EOC released microvesicles in vitro and induced phenotypic switch (M1-to-M2) in cultured monocytes, which was inhibited by vascular endothelial growth factor blockade. Finally, a single intrarenal injection of rh-vascular endothelial growth factor (0.05 μg/kg) in 7 additional RAS pigs also restored M1/M2 ratio 4 weeks later. Conclusions— Intrarenal infusion of EOC after PTRA induced a vascular endothelial growth factor–mediated attenuation in macrophages inflammatory phenotype, preserved microvascular architecture and function, and decreased inflammation and fibrosis in the stenotic kidney, suggesting a novel mechanism and therapeutic potential for adjunctive EOC delivery in experimental RAS to improve PTRA outcomes.

Renal artery stenosis (RAS) is an important cause of chronic renal dysfunction. Recent studies have underscored a critical role for CCL2 (MCP-1)-mediated inflammation in the progression of chronic renal damage in RAS and other chronic renal diseases. In vitro studies have implicated p38 MAPK as a critical intermediate for the production of CCL2. However, a potential role of p38 signaling in the development and progression of chronic renal disease in RAS has not been previously defined. We sought to test the hypothesis that inhibition of p38 MAPK ameliorates chronic renal injury in mice with RAS. We established a murine RAS model by placing a cuff on the right renal artery and treated mice with the p38 inhibitor SB203580 or vehicle for 2 wk. In mice treated with vehicle, the cuffed kidney developed interstitial fibrosis, tubular atrophy, and interstitial inflammation. In mice treated with SB203580, the RAS-induced renal atrophy was reduced (70% vs. 39%, P < 0.05). SB203580 also reduced interstitial inflammation and extracellular matrix deposition but had no effect on the development of hypertension. SB203580 partially blocked the induction of CCL2, CCL7 (MCP-3), CC chemokine receptor 2 (CCR2), and collagen 4 mRNA expression in the cuffed kidneys. In vitro, blockade of p38 hindered both TNF-α and TGF-β-induced CCL2 upregulation. Based on these observations, we conclude that p38 MAPK plays a critical role in the induction of CCL2/CCL7/CCR2 system and the development of interstitial inflammation in RAS.

Abstract Endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) augment tissue repair but possess slightly different properties. How the cellular phenotype affects the efficacy of this approach in renovascular disease is incompletely understood. This study tested the hypothesis that EPC and MSC protect the poststenotic kidney by blunting different disease pathways. Peripheral blood EPC and adipose-derived MSC were expanded and characterized by cell surface markers (e.g., CD34/kinase insert domain receptor, or CD44/CD90). Single-kidney hemodynamics and function were assessed in pigs after 10 weeks of renal artery stenosis (RAS) treated 4 weeks earlier with an intrarenal infusion of vehicle (n = 7), EPC (RAS+EPC) or MSC (RAS+MSC) (both 10 × 106, n = 6), and normal controls (n = 7). Kidney disease mechanisms were evaluated ex vivo. The ability of EPC and MSC to attenuate endoplasmic reticulum (ER) stress was also studied in isolated ER and in tubular cells cocultured with EPC and MSC. Glomerular filtration rate in RAS was lower than controls, increased in RAS+EPC, and further improved in RAS+MSC, although both improved renal blood flow similarly. EPC prominently enhanced renal growth factor expression and decreased oxidative stress, while MSC more significantly attenuated renal inflammation, ER stress, and apoptosis. Furthermore, MSC induced a greater decrease in caspase-3 and CHOP expression in cultured tubular cells through mechanisms involving cell contact. EPC and MSC achieve a comparable decrease of kidney injury in RAS by different mechanisms, although MSC elicited slightly superior improvement of renal function. These results support development of cell-based approaches for management of renovascular disease and suggest cell selection based on the underlying pathophysiology of kidney injury.

Background— The selection of patients with renal artery stenosis (RAS) likely to improve glomerular filtration rate (GFR) after percutaneous transluminal renal angioplasty is difficult. We examined basal hemodynamic and inflammatory factors linked to improved stenotic kidney (STK) function after percutaneous transluminal renal angioplasty in swine RAS. Methods and Results— Fifteen pigs after 6 weeks of hemodynamically significant RAS were studied before and 4 weeks after technically successful percutaneous transluminal renal angioplasty+stenting. STK and contralateral kidney hemodynamics and function were evaluated by multidetector computed-tomography before and after acetylcholine challenge. Single-kidney deoxyhemoglobin (R2*, reciprocal to blood relaxation) and energy-dependent tubular function were assessed using blood-oxygen-level–dependent magnetic resonance imaging before and after furosemide. Baseline renal vein and inferior vena cava levels of inflammatory markers were measured and their gradient and net release calculated. Baseline parameters were compared with normal (n=7) and sham-RAS (n=7) pigs and correlated with the change in STK-GFR after revascularization (ΔGFR). Four weeks after percutaneous transluminal, renal angioplasty blood pressure was normalized in all animals, but STK-GFR improved in 10 of 15 (ΔGFR =+22.0±8.5 mL/min). ΔGFR correlated inversely with basal STK-GFR, renal release of inflammatory markers, and medullary R2* response to furosemide, but directly with GFR response to acetylcholine. Basal contralateral kidney GFR correlated directly with ΔGFR. Conclusions— Low basal STK-GFR with preserved response to acetylcholine may predict benefit from revascularization in RAS, whereas renal inflammation and robust STK-R2* responses to furosemide (possibly reflecting avid tubular oxygen consumption) are associated with less favorable outcomes. These tools may be useful for identification of patients likely to improve renal function after revascularization.

Revascularization improves blood pressure but not renal function in most patients with atherosclerotic renal artery stenosis (ARAS), possibly related to injury incurred during renal reperfusion. Bendavia, a novel tetrapeptide that inhibits mitochondrial permeability transition pore opening, reduces apoptosis, oxidative stress, and ischemia-reperfusion injury in experimental models. However, its potential for improving renal response to revascularization of chronic ARAS is unknown. We hypothesized that adjunct Bendavia would improve renal structure and function after percutaneous transluminal renal angioplasty (PTRA). Pigs were treated after 6 weeks of ARAS or control with PTRA+stenting (or sham), adjunct continuous 4-hour infusion of Bendavia (0.05 mg/kg IV) or vehicle (n=7 each) during PTRA. Single-kidney renal blood flow and glomerular filtration rate were studied 4 weeks later and renal mitochondrial biogenesis, microvascular architecture, and injurious pathways evaluated ex vivo. Monocyte chemoattractant protein-1 levels rose after PTRA, suggesting inflammatory injury. Bendavia did not immediately affect inflammatory cytokine levels, yet 4 weeks later, stenotic kidney renal blood flow and glomerular filtration rate both improved (44.00 ± 0.21% and 36.40 ± 10.21%, respectively) in ARAS+PTRA+Bendavia compared with ARAS+PTRA+vehicle. Renal mitochondrial biogenesis was restored after PTRA+Bendavia, and microvascular rarefaction, apoptosis, oxidative stress, tubular injury, and fibrosis decreased. Infusion of Bendavia during PTRA preserved mitochondrial biogenesis, renal hemodynamics, and function, and attenuated tissue injury in swine ARAS. Thus, functional mitochondrial injury during renal reperfusion may sustain renal inflammatory injury and limit kidney recovery after PTRA. Potent antiapoptotic and antioxidant effects provide Bendavia a novel therapeutic potential for improving kidney outcomes after PTRA in experimental ARAS.