Luis A. Juncos

Obesity, hypertension, and chronic kidney disease

Obesity is a major risk factor for essential hypertension, diabetes, and other comorbid conditions that contribute to development of chronic kidney disease. Obesity raises blood pressure by increasing renal tubular sodium reabsorption, impairing pressure natriuresis, and causing volume expansion via activation of the sympathetic nervous system and renin–angiotensin–aldosterone system and by physical compression of the kidneys, especially when there is increased visceral adiposity. Other factors such as inflammation, oxidative stress, and lipotoxicity may also contribute to obesity-mediated hypertension and renal dysfunction. Initially, obesity causes renal vasodilation and glomerular hyperfiltration, which act as compensatory mechanisms to maintain sodium balance despite increased tubular reabsorption. However, these compensations, along with increased arterial pressure and metabolic abnormalities, may ultimately lead to glomerular injury and initiate a slowly developing vicious cycle that exacerbates hypertension and worsens renal injury. Body weight reduction, via caloric restriction and increased physical activity, is an important first step for management of obesity, hypertension, and chronic kidney disease. However, this strategy may not be effective in producing long-term weight loss or in preventing cardiorenal and metabolic consequences in many obese patients. The majority of obese patients require medical therapy for obesity-associated hypertension, metabolic disorders, and renal disease, and morbidly obese patients may require surgical interventions to produce sustained weight loss.

Antioxidants Block Angiotensin II-Induced Increases in Blood Pressure and Endothelin

Chronically infusing a subpressor dose of angiotensin (Ang) II increases blood pressure via poorly defined mechanisms. We found that this hypertensive response is accompanied by increased oxidant stress and is prevented by blocking endothelin (ET) receptors. Thus, we now tested whether blocking oxidant stress decreases both blood pressure and ET levels. We infused Sprague-Dawley rats (via osmotic pumps) with either vehicle (group 1) or Ang II (5 ng · kg−1 · min−1; groups 2 to 4) for 15 days. Groups 3 and 4 also received either tempol in the drinking water (1 mmol/L) or vitamin E (5000 IU/kg diet), respectively, for 15 days. We measured systolic blood pressure (SBP) and urinary nitrite excretion every 3 days, and on day 15 we measured systemic and renal venous plasma levels of ET, isoprostanes, and thiobarbituric acid reactive substances (TBARS). SBP in Group 1 did not change throughout the study, whereas Ang II increased SBP (from 132±5 to 151±7 mm Hg). In addition, Ang II increased the systemic and renal venous levels of isoprostanes, TBARS, and ET and caused a transient decrease in urinary nitrites (that returned to control levels by day 9). Both tempol and vitamin E prevented Ang II-induced hypertension and either prevented or tended to blunt the increase in systemic and renal isoprostanes, TBARS, and ET. Finally, both antioxidants abolished the transient decrease in urinary nitrites. These results together with our previous study suggest that subpressor-dose Ang II increases oxidant stress (and isoprostanes). This in turn increases ET levels, which participate in the hypertensive response to Ang II.

Effects of Captopril on the Renin Angiotensin System, Oxidative Stress, and Endothelin in Normal and Hypertensive Rats

There is substantial evidence suggesting that angiotensin II plays an important role in elevating blood pressure of spontaneously hypertensive rats, despite normal plasma renin activity, and that converting enzyme inhibitors (captopril) can effectively normalize blood pressure in the spontaneously hypertensive rats. One mechanism by which angiotensin II induces hypertension is via oxidative stress and endothelin, as seen in subpressor angiotensin II–induced hypertension. In fact, it has been shown that antioxidants lower mean arterial pressure in spontaneously hypertensive rats. However, the relationship between angiotensin II, oxidative stress, and endothelin in the spontaneously hypertensive rats is still relatively undefined. This study examines the relationship between mean arterial pressure, plasma renin activity, angiotensin II, oxidative stress, and endothelin in spontaneously hypertensive rats compared with normotensive Wistar Kyoto rats, and the effects of captopril on this association. Untreated spontaneously hypertensive rats had increased plasma angiotensin II levels despite normal plasma renin activity, oxidative stress, and endothelin. Captopril treatment in spontaneously hypertensive rats lowered mean arterial pressure, angiotensin II, oxidative stress, and endothelin, and increased plasma renin activity. In contrast, captopril increased plasma renin activity (suggesting effective captopril treatment) but did not significantly alter mean arterial pressure, angiotensin II, oxidative stress, or endothelin of Wistar Kyoto rats. These results suggest that in spontaneously hypertensive rats, angiotensin II is a primary instigator of hypertension, and that captopril selectively lowers angiotensin II, oxidant stress, and endothelin, which in turn may contribute to the blood pressure-lowering efficacy of captopril in spontaneously hypertensive rats.

Low-Dose Angiotensin II Increases Free Isoprostane Levels in Plasma

Chronic intravenous infusion of subpressor doses of angiotensin II causes blood pressure to increase progressively over the course of several days. The mechanisms underlying this response, however, are poorly understood. Because high-dose angiotensin II increases oxidative stress, and some compounds that result from the increased oxidative stress (eg, isoprostanes) produce vasoconstriction and antinatriuresis, we tested the hypothesis that a subpressor dose of angiotensin II also increases oxidative stress, as measured by 8-epi-prostaglandin F(2alpha) (isoprostanes), which may contribute to the slow pressor response to angiotensin II. To test this hypothesis, we infused angiotensin II (10 ng/kg per minute for 28 days via an osmotic pump) into 6 conscious normotensive female pigs (30 to 35 kg). We recorded mean arterial pressure continuously with a telemetry system and measured plasma isoprostanes before starting the angiotensin II infusion (baseline) and again after 28 days with an enzyme immunoassay. Angiotensin II infusion significantly increased mean arterial pressure from 121+/-4 to 153+/-7 mm Hg (P<0. 05) without altering total plasma isoprostane levels (180.0+/-24.3 versus 147.0+/-29.2 pg/mL; P=NS). However, the plasma concentrations of free isoprostanes increased significantly, from 38.3+/-5.8 to 54.7+/-10.4 pg/mL (P<0.05). These results suggest that subpressor doses of angiotensin II increase oxidative stress, as implied by the increased concentration of free isoprostanes, which accompany the elevation in mean arterial pressure elevation. Thus, isoprostane-induced vasoconstriction and antinatriuresis may contribute to the hypertension induced by the slow pressor responses of angiotensin II.

Role of Endothelin and Isoprostanes in Slow Pressor Responses to Angiotensin II

We tested the hypothesis that angiotensin II (Ang II)–induced stimulations of endothelin (ET) and isoprostanes are implicated in the slow pressor responses to Ang II. We infused either vehicle (group 1) or Ang II (groups 2 to 4) intravenously at 5 ng/kg per minute via osmotic pumps for 15 days into Sprague-Dawley rats. Groups 3 and 4 received 30 mg/kg per day of either losartan (Ang II type 1 receptor blocker) or bosentan (ETA and ETB receptor blocker) in their drinking water. We measured systolic blood pressure (SBP) every 3 days during the infusion. Plasma levels of Ang II, ET, isoprostanes, and urinary nitrites were determined at 15 days. Vehicle infusion did not change SBP (from 138±13 to 136±2 mm Hg at day 15). Circulating Ang II, ET, and isoprostane levels were 35±9, 39±3, and 111±10 pg/mL, respectively, whereas urinary nitrites were 2.3±0.4 &mgr;g/d. Ang II increased SBP (from 133±10 to 158±8 mm Hg), plasma Ang II (179±77 pg/mL), and isoprostanes (156±19 pg/mL) without altering ET levels (38±5 pg/mL) or urinary nitrites (1.8±0.5 &mgr;g/d). Losartan prevented Ang II–induced increases in SBP and isoprostanes (SBP went from 137±5 to 120±4 mm Hg; isoprostanes were 115±15 pg/mL) while increasing urinary nitrite levels (5.2±1.1 &mgr;g/d). Losartan did not alter Ang II (141±57 pg/mL) or ET (40±4 pg/mL) levels. Bosentan also blocked Ang II–induced hypertension (from 135±4 to 139±3 mm Hg) but did not decrease isoprostanes (146±14 pg/mL). Ang II (63±11 pg/mL), ET levels (46±2 pg/mL), and urinary nitrites (2.8±0.4 &mgr;g/d) were not altered. In conclusion, our results suggest that low-dose Ang II increases isoprostanes via its Ang II type 1 receptor and causes an ET-dependent hypertension, without altering circulating ET levels.

Endothelium-derived relaxing factor modulates endothelin action in afferent arterioles.

Endothelin is a potent vasoconstrictor, whereas endothelium-derived relaxing factor (EDRF) is a potent vasodilator. Both are produced by the endothelium. Although they have been studied extensively in large vessels, little is known about their actions in renal microvessels. Using microdissected rabbit afferent arterioles, we studied the vascular response to synthetic endothelin and its interaction with EDRF and the effect of endothelin on renin release. Afferent arterioles were either microperfused in vitro at 60 mm Hg to measure luminal diameter or incubated without microperfusion to assess renin release. When added to the bath, 10−10 or 10−9 M endothelin decreased the diameter by 32±8% (n=7, p<0.01) or 76±7% (p<0.0001), respectively. Pretreatment with NW-nitro L-arginine, which inhibits synthesis of EDRF, decreased basal diameter by 15±1% (p<0.001) and augmented endothelin-induced constriction; decrease in diameter with 10−10 M endothelin was 78±10% (n=4, p<0.01 versus nontreated). In afferent arterioles preconstricted by endothelin, acetylcholine at concentrations of 10−8 to 10−5 M increased the diameter in a dose-dependent manner. Basal renin release was 0.62±0.15 ng angiotensin I/hr/afferent arterioles/hr (n=13) and was not affected by endothelin (10−10 to 10−6 M). Increase in renin release by isoproterenol was the same in afferent arterioles pretreated with vehicle or endothelin (10−7 M; Δ, 0.49±0.21 versus 0.42±0.19; n=13). In summary, endothelin constricts afferent arterioles but, at the same doses, does not inhibit renin release, and afferent arterioles, small resistant vessels, produces EDRF, which in turn participates in the control of basal tone and opposes vasoconstrictor action of endothelin.

Volume-related weight gain and subsequent mortality in acute renal failure patients treated with continuous renal replacement therapy.

Fluid overload is a frequent finding in critically ill patients suffering from acute kidney injury (AKI). To assess the impact of fluid overload on the mortality of AKI patients treated with continuous renal replacement therapy (CRRT), we used a registry of 81 critically ill patients with AKI initiated on CRRT assembled over an 18-month period to conduct a cross- sectional analysis using volume-related weight gain (VRWG) of ≥10% and ≥20% of body weight and oliguria (≤20 ml/h) as the principal variables, with the primary outcome measure being mortality at 30 days. Mean Apache II scores were 27.5 ± 6.9 with overall cohort mortality of 50.6%. Mean (±SD) VRWG was 8.3 ± 9.6 kg, representing a 10.2% ± 13.5% increase since admission. Oliguria was present in 65.4% of patients. Odds ratio (OR) for mortality on univariate analysis was increased to 2.62 [95% confidence interval (CI): 1.07–6.44] by a VRWG ≥10% and to 3.22 (95% CI: 1.23–8.45) by oliguria. VRWG ≥20% had OR of 3.98 (95% CI: 1.01–15.75; p = 0.049) for mortality. Both VRWG ≥10% (OR 2.71, p = 0.040) and oliguria (OR 3.04, p = 0.032) maintained their statistically significant association with mortality in multivariate models that included sepsis and Apache II score. In conclusion, fluid overload is an important prognostic factor for survival in critically ill AKI patients treated with CRRT. Further studies are needed to elicit mechanisms and develop appropriate interventions.

Increased shear stress with upregulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts

Venous injury and subsequent venous stenosis formation are responsible for hemodialysis graft failure. Our hypothesis is that these pathological changes are in part related to changes in wall shear stress (WSS) that results in the activation of matrix regulatory proteins causing subsequent venous stenosis formation. In the present study, we examined the serial changes in WSS, blood flow, and luminal vessel area that occur subsequent to the placement of a hemodialysis graft in a porcine model of chronic renal insufficiency. We then determined the corresponding histological, morphometric, and kinetic changes of several matrix regulatory proteins including VEGF-A, its receptors, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and TIMP-2. WSS was estimated by obtaining blood flow and luminal vessel area by performing phase-contrast MRI with magnetic resonance angiography in 21 animals at 1 day after graft placement and prior to death on day 3 (n = 7), day 7 (n = 7), and day 14 (n = 7). At all time points, the mean WSS at the vein-to-graft anastomosis was significantly higher than that at the control vein (P < 0.05). WSS had a bimodal distribution with peaks on days 1 and 7 followed by a significant reduction in WSS by day 14 (P < 0.05 compared with day 7) and a decrease in luminal vessel area compared with control vessels. By day 3, there was a significant increase in VEGF-A and pro-MMP-9 followed by, on day 7, increased pro-MMP-2, active MMP-2, and VEGF receptor (VEGFR)-2 (P < 0.05) and, by day 14, increased VEGFR-1 and TIMP-1 (P < 0.05) at the vein-to-graft anastomosis compared with control vessels. Over time, the neointima thickened and was composed primarily of alpha-smooth muscle actin-positive cells with increased cellular proliferation. Our data suggest that hemodialysis graft placement leads to early increases in WSS, VEGF-A, and pro-MMP-9 followed by subsequent increases in pro-MMP-2, active MMP-2, VEGFR-1, VEGFR-2, and TIMP-1, which may contribute to the development of venous stenosis.

Isoforms and Functions of NAD(P)H Oxidase at the Macula Densa

Macula densa cells produce superoxide (O2−) during tubuloglomerular feedback primarily via NAD(P)H oxidase (NOX). The purpose of the present study was to determine NOXs expressed by the macula densa and the role of each one in NaCl-induced O2− production. To identify which isoforms are expressed, we applied single-cell RT-PCR to macula densa cells isolated by laser capture microdissection and to MMDD1 cells (a macula densa-like cell line). The captured cells expressed neuronal NOS (marker of macula densa), NOX2, and NOX4 but not NOX1. Expression of the NOXs and neuronal NOS was essentially identical in the MMDD1 cells. Thus, we used MMDD1 cells to investigate which isoform is responsible for NaCl-induced O2− production. We used small-interfering RNA to knock down NOX2 or NOX4 in MMDD1 cells and measured O2− exposed to low-salt solution (LS; 70 mmol/L of NaCl) or high-salt solution (HS; 140 mmol/L of NaCl). Exposing control cells (scrambled small-interfering RNA) to HS increased O2− concentrations from 0.75±0.28 to 1.48±0.46 U/min per 105 cells in LS and HS, respectively (P<0.001). Inhibiting NOX2 blocked the HS-induced increase in O2− (0.62±0.39 versus 0.76±0.31 U/min per 105 cells in LS and HS groups, respectively). Blocking NOX4 did not affect HS-induced O2− levels. O2− levels in the control cells during LS and HS were 0.80±0.30 and 1.56±0.49 U/min per 105 cells, respectively (P<0.001); whereas O2− levels in NOX4-small-interfering RNA–treated cells during LS and HS were 0.40±0.25 and 1.26±0.51 U/min per 105 cells, respectively (P<0.001). We conclude that, whereas macula densa cells express the NOX2 and NOX4 isoforms, NOX2 is primarily responsible for NaCl-induced O2− generation.

Expression of Hypoxia Inducible Factor–1α, Macrophage Migration Inhibition Factor, Matrix Metalloproteinase–2 and −9, and Their Inhibitors in Hemodialysis Grafts and Arteriovenous Fistulas

PURPOSE It is well recognized that arteriovenous fistulas (AVFs) used for hemodialysis access have better primary patency rates with less restenosis than polytetrafluoroethylene (PTFE) grafts; however, the mechanism responsible for this is not known. Recent data suggest that hypoxia inducible factor-1 alpha (HIF-1 alpha) is associated with vascular restenosis, possibly through mechanisms that increase the production of macrophage migration inhibition factor (MIF), matrix metalloproteinase-2 (MMP-2) and MMP-9, and their inhibitors (tissue inhibitor of MMPs; TIMP). The present study tested the hypothesis that there are differences in the expression patterns of HIF-1 alpha, MIF, MMP-2, MMP-9, and TIMPs in specimens removed from patients with AVFs and PTFE grafts. MATERIALS AND METHODS Whole-vessel tissue samples were obtained from the vein distal to the vein-to-PTFE graft anastomosis and the proximal outflow vein (within 6 cm of the arteriovenous anastomosis) of AVFs from 17 patients who required a surgical revision for thrombosis and stenosis. Nonstenotic veins of four patients undergoing hemodialysis vascular access placement were used as controls. PTFE grafts (n = 6), AVFs (n = 6), and control samples (n = 3) underwent Western blot analysis and zymography. A separate group of five patients with PTFE hemodialysis grafts and one control subject were used for immunohistochemical analysis. RESULTS Specimens from patients with PTFE grafts had significantly higher expression of HIF-1 alpha (P = .03), MIF (P = .02), TIMP-1 (P = .0006), pro-MMP-2 (P = .02), and pro-MMP-9 (P = .046) compared with control veins. The expression of only pro-MMP-9 was significantly higher in AVFs compared with control samples (P = .004). There was a significant increase in the expression of MIF (P = .007) and TIMP-1 (P < .0001) in PTFE graft specimens compared with AVFs. MIF and TIMP-1 were localized to the adventitia of the vein distal to the vein-to-PTFE graft anastomosis. CONCLUSIONS There were major differences in the expression patterns of hypoxia (ie, HIF-1 alpha) and proteins regulated by HIF-1?, including MIF, pro-MMP-2, pro-MMP-9, and TIMP-1, in specimens removed from patients with PTFE grafts and AVFs. Understanding the role of HIF-1 alpha and these proteins in hemodialysis access failure can help improve outcomes.