Human glomerular endothelium: interplay among glucose, free fatty acids, angiotensin II, and oxidative stress.

Glomerular endothelial cells (GEC) are strategically situated within the capillary loop and adjacent to the glomerular mesangium. GEC serve as targets of metabolic, biochemical, and hemodynamic signals that regulate the glomerular microcirculation. Unequivocally, hyperglycemia, hypertension, and the local renin-angiotensin system partake in the initiation and progression of diabetic nephropathy (DN). Whether free fatty acids (FFA) and reactive oxygen species (ROS) that have been associated with the endothelial dysfunction of diabetic macrovascular disease also contribute to DN is not known. Since endothelial cells from different organs and from different species may display different phenotypes, we employed human GEC to investigate the effect of high glucose (22.5 mmol/l), FFA (800 micromol/l), and angiotensin II (ANG II; 10(-7) mol/l) on the genesis of ROS and their effects on endothelial nitric oxide synthase (eNOS), cyclooxygenase-2 (COX-2), and the synthesis of prostaglandins (PGs). We demonstrated that high glucose but not high FFA increased the expression of a dysfunctional eNOS as well as increased ROS from NADPH oxidase (100%) and likely from uncoupled eNOS. ANG II also induced ROS from NADPH oxidase. High glucose and ANG II upregulated (100%) COX-2 via ROS and significantly increased the synthesis of prostacyclin (PGI(2)) by 300%. In contrast, FFA did not upregulate COX-2 but increased PGI(2) (500%). These novel studies are the first in human GEC that characterize the differential role of FFA, hyperglycemia, and ANG II on the genesis of ROS, COX-2, and PGs and their interplay in the early stages of hyperglcyemia.

Nicotine augments glomerular injury in a rat model of acute nephritis.

BACKGROUND/AIMS: Epidemiologic studies suggest that cigarette smoke worsens the progression of renal injury in patients with glomerular diseases. The mechanisms involved have not been elucidated. These studies were designed to determine whether nicotine worsens markers of inflammation including glomerular cell proliferation and fibronectin deposition in an in vivo model of glomerular injury. METHODS: Sprague-Dawley rats were injected with anti-Thy1 antibody and given either tap water or nicotine in the drinking water until sacrifice at day 14. Fibronectin expression was measured by Western blot and immunohistochemistry. COX-2 expression was also determined by Western blot in the kidney cortex of rats treated with nicotine and in cultured human mesangial cells treated with nicotine. RESULTS: Anti-Thy1 antibody administration resulted in a significant increase in the number of cells per glomerulus that was further increased by the administration of nicotine. In nephritic rats, the administration of nicotine significantly increased fibronectin and COX-2 expression. In cultured human mesangial cells we also demonstrated that nicotine increases COX-2 expression and activity and that COX-2 mediates mesangial cell proliferation in response to nicotine. CONCLUSION: Either in vivo or in vitro treatment with nicotine leads to activation of inflammatory mediators and hallmarks of glomerular injury, which may explain the mechanisms involved in the deleterious effects of cigarette smoking on renal disease.

Angiotensin II increases the expression of the transcription factor ETS-1 in mesangial cells.

Maladaptive activation of the renin-angiotensin system (RAS) has been shown to play a critical role in the pathogenesis of chronic kidney disease. Reactive oxygen species (ROS) are critical signals for many of the nonhemodynamic effects of angiotensin II (ANG II). We have demonstrated that ANG II increases mesangial and cortical cyclooxygenase-2 (COX-2) expression and activity via NADPH oxidase-derived ROS. The transcription factor ETS-1 (E26 transformation-specific sequence) has been identified as a critical regulator of growth-related responses and inflammation. The present studies were designed to determine: 1) whether ANG II induces ETS-1 expression in vitro in cultured rat mesangial cells and in vivo in rats infused with ANG II; and 2) whether ROS and COX-2 are mediators of ETS-1 induction in response to ANG II. Mesangial cells stimulated with ANG II (10(-7) M) exhibited a significant increase in ETS-1 expression that was prevented by the angiotensin type 1 receptor blocker candesartan. NADPH oxidase inhibition with dyphenilene iodinium or apocynin also prevented ETS-1 induction, establishing the role of ROS as mediators of ETS-1 expression in response to ANG II. COX-2 inhibition prevented ETS-1 expression in response to ANG II, suggesting that COX-2 is required for ETS-1 induction. By utilizing short interfering RNAs against ETS-1, we have also determined that ETS-1 is required to induce the production of fibronectin in response to ANG II. Furthermore, rats infused with ANG II manifested increased glomerular expression of ETS-1. These studies unveil novel pathways that may play an important role in the pathogenesis of renal injury when RAS is activated.

Nicotine: the link between cigarette smoking and the progression of renal injury?

Cigarette smoke (CS) is the most important source of preventable morbidity and mortality in the United States. Recent clinical studies have suggested that, in addition to being a major cardiovascular risk factor, CS promotes the progression of kidney disease. The mechanisms by which CS promotes the progression of chronic kidney disease have not been elucidated. Here we demonstrate for the first time that human mesangial cells (MCs) are endowed with the nicotinic ACh receptors (nAChRs) alpha4, alpha5, alpha7, beta2, beta3, and beta4. Studies performed in other cell types have shown that these nAChRs are ionotropic receptors that function as agonist-regulated Ca(2+) channels. Nicotine induced MC proliferation in a dose-dependent manner. At 10 (-7) M, a concentration found in the plasma of active smokers, nicotine induced MC proliferation [control, 1,328 +/- 50 vs. nicotine, 2,761 +/- 90 counts/minute (cpm); P < 0.05] and increased the synthesis of fibronectin (50%), a critical matrix component involved in the progression of chronic kidney disease. We and others have shown that, in response to PKC activation, MC synthesize reactive oxygen species (ROS) via NADPH oxidase. In the current studies we demonstrate that PKC inhibition as well as diphenyleneiodonium and apocynin, two inhibitors of NADPH oxidase, prevented the effects of nicotine on MC proliferation and fibronectin production, hence establishing ROS as second messengers of the actions of nicotine. Furthermore, nicotine increased the production of ROS as assessed by 2',7'-dichlorofluorescein diacetate fluorescence [control, 184.4 +/- 26 vs. nicotine, 281.5 +/- 26 arbitrary fluorescence units (AFU); n = 5 experiments, P < 0.05]. These studies unveil previously unrecognized mechanisms that indict nicotine, a component of CS, as an agent that may accelerate and promote the progression of kidney disease.

Up-regulation of glomerular COX-2 by angiotensin II: role of reactive oxygen species.

BACKGROUND: Prostaglandins such as prostaglandin E(2) (PGE(2)) and prostaglandin I(2) (PGI(2)) counteract the angiotensin II (Ang II)-induced vasoconstriction in the glomerular microcirculation. We have shown that Ang II promotes mesangial cell hypertrophy via reactive oxygen species (ROS), which originate from nicotinamide adenine dinucleotide phosphate and its reduced form (NADH/NADPH) oxidase. It has been reported that conditions associated with activation of the renin-angiotensin system result in increased glomerular cyclooxygenase-2 (COX-2) expression and activity. METHODS: We designed studies to determine (1) whether Ang II induces COX-2 in the glomerulus in vivo in the glomerulus as well as in vitro in mesangial cells, (2) whether ROS originated from Ang II are involved, and (3) whether COX-2-derived prostaglandins modulate the growth promoting effects of Ang II in mesangial cells. Rats were infused with Ang II (0.7 mg/kg/day) for 5 days and glomerular COX-2 expression and activity assessed in isolated glomeruli. RESULTS: Ang II increased glomerular PGE(2) production (100%) accompanied by a concomitant increase in glomerular COX-2 expression at the mRNA (1.7-fold) and protein level (sixfold). In mesangial cells, Ang II significantly increased mesangial cell PGE(2) (200%) and PGI(2) (100%) production as well as COX-2 mRNA that was prevented by the angiotensin type 1 (AT1) receptor blocker irbesartan and the COX-2 inhibitor NS-398. The NADPH oxidase inhibitor diphenyleneiodonium (DPI), the ROS scavenger tiron as well as catalase, inhibited Ang II-induced PGE(2) production suggesting that Ang II-induced ROS mediate COX-2 up-regulation. Strikingly, COX-2 inhibition as well as blockade of the type 1 PGE(2) receptor (EP1) prevented Ang II-induced mesangial cell hypertrophy suggesting that COX-2-derived prostaglandins, and specifically PGE(2), importantly contribute to the growth promoting effects of Ang II. CONCLUSION: These studies suggest that blockade of specific PGE(2) receptors may be a novel strategy to modulate the pathologic effects of COX-2-derived prostaglandins without simultaneously affecting protective vasodilatory mechanisms.

Stable compounds of cigarette smoke induce endothelial superoxide anion production via NADPH oxidase activation.

OBJECTIVE: Endothelial dysfunction is an early manifestation of cigarette smoke (CS) toxicity. We have previously demonstrated that CS impairs nitric oxide (NO)-mediated endothelial function via increased generation of superoxide anion (O2*). In these studies, we investigated whether stable compounds present in CS activate specific pathways responsible for the increased endothelial O2* production. METHODS AND RESULTS: Short exposure of bovine pulmonary artery endothelial cells (BPAECs), human pulmonary artery endothelial cells, and rat pulmonary arteries to CS extracts (CSEs) resulted in a large increase in O2* production (20-fold, 3-fold, and 2-fold increase, respectively; P<0.05 versus control), which was inhibited by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenyleneiodinium, apocynin, and gp91 docking sequence-tat peptide but not by oxypurinol, the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester, or the mitochondrial respiration inhibitor rotenone. Exposure of BPAECs to acrolein, a stable thiol-reactive agent found in CS, increased O2* production 5-fold, which was prevented by prior inhibition of NADPH oxidase. CONCLUSIONS: These studies demonstrate that thiol-reactive stable compounds in CS can activate NADPH oxidase and increase endothelial O2* production, thereby reducing NO bioactivity and resulting in endothelial dysfunction. Clinically, these studies may contribute to the development of agents able to mitigate CS-mediated vascular toxicity.

Development of (17)O NMR approach for fast imaging of cerebral metabolic rate of oxygen in rat brain at high field.

A comprehensive technique was developed for using three-dimensional (17)O magnetic resonance spectroscopic imaging at 9.4T for rapidly imaging the cerebral metabolic rate of oxygen consumption (CMRO(2)) in the rat brain during a two-min inhalation of (17)O(2). The CMRO(2) value (2.19 +/- 0.14 micromol/g/min, n = 7) was determined in the rat anesthetized with alpha-chloralose by independent and concurrent (17)O NMR measurements of cerebral H(2)17O content, arterial input function, and cerebral perfusion. CMRO(2) values obtained were consistent with the literature results for similar conditions. Our results reveal that, because of its superior sensitivity at ultra-high fields, the (17)O magnetic resonance spectroscopic imaging approach is capable of detecting small dynamic changes of metabolic H(2)17O during a short inhalation of (17)O(2) gas, and ultimately, for imaging CMRO(2) in the small rat brain. This study provides a crucial step toward the goal of developing a robust and noninvasive (17)O NMR approach for imaging CMRO(2) in animal and human brains that can be used for studying the central role of oxidative metabolism in brain function under normal and diseased conditions, as well as for understanding the mechanisms underlying functional MRI.

Systemic and Regional Hemodynamic Responses to Tempol in Angiotensin II-Infused Hypertensive Rats.

-Recent studies have indicated that angiotensin II (Ang II) can stimulate oxidative stress. The present study was conducted to assess the contribution of oxygen radicals to hypertension and regional circulation during Ang II-induced hypertension. With radioactive microspheres, the responses of systemic and regional hemodynamics to the membrane-permeable, metal-independent superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol) were assessed in conscious Ang II-infused hypertensive rats. Ang II-infused rats (80 ng/min SC for 12 days: n=25) showed higher mean arterial pressure (MAP: 161+/-4 mm Hg) and total peripheral resistance (TPR: 1.59+/-0.08 mm Hg. min(-1). mL(-1)) than vehicle-infused normotensive rats (116+/-3 mm Hg and 0.95+/-0.04 mm Hg. min(-1). mL(-1), respectively; n=23). The blood flow rates in the brain, spleen, large intestine, and skin were significantly reduced in Ang II-infused rats compared with vehicle-infused rats, whereas rates in the lung, heart, liver, kidney, stomach, small intestine, mesenterium, skeletal muscle, and testis were similar. Vascular resistance was significantly increased in every organ studied except the lung, in which the resistance was similar. Tempol (216 µmol/kg IV) significantly reduced MAP by 30+/-4% from 158+/-7 to 114+/-5 mm Hg and TPR by 35+/-6% from 1.57+/-0.17 to 0.95+/-0.04 mm Hg. min(-1). g(-1) in Ang II-infused rats (n=9) but had no effect on these parameters in vehicle-infused rats (n=8). In Ang II-infused rats, tempol did not affect regional blood flow but significantly decreased vascular resistance in the brain (29+/-6%), heart (31+/-6%), liver (37+/-7%), kidney (30+/-7%), small intestine (38+/-6%), and large intestine (47+/-7%). Ang II-infused hypertensive rats showed doubled vascular superoxide production (assessed with lucigenin chemiluminescence), which was normalized by treatment with tempol (3 mmol/L, n=7). Further studies showed that the NO synthase inhibitor, N:(omega)-nitro-L-arginine methyl ester (11 µmol. kg(-1). min(-1) IV, n=11) markedly attenuated the systemic and regional hemodynamic responses of tempol in Ang II-infused rats. These results suggest that in this model of hypertension, oxidative stress may have contributed to the alterations in systemic blood pressure and regional vascular resistance through inactivation of NO.