Nitric oxide (NO), among several other functions, may play a role in hypoxia and reoxygenation injury due to its free radical nature and high reactivity with the superoxide radical to yield peroxynitrite, an oxidant molecule. The present study was undertaken to evaluate a potential role for NO, either endogenous or exogenous, in a model of hypoxia/reoxygenation (H/R) in freshly isolated rat proximal tubules. NO synthase activity, as assessed by conversion of L-[3H]arginine to L-[3Hlcitrulline, was detected in normoxic tubules. This activity could be inhibited by N-nitro-L-argin ne methyl ester (L-NAME), a NO synthase inhibitor, and was stimulated by 15 min of hypoxia. The injury in proximal tubules caused by 15 min of hypoxia followed by 35 min of reoxygenation was completely prevented by L-NAME as assessed by release of lactate dehydrogenase, whereas D-NAME, which does not inhibit NO synthase, had no effect. In contrast, L-arginune (NO substrate) enhanced the H/R injury. These effects were paralleled by nitrite/nitrate production. In separate experiments, the addition of sodium nitroprusside, a NO donor, to proximal tubules enhanced the H/R injury; this effect could be blocked by hemoglobin, a NO scavenger. Also, addition of nitroprusside reversed L-NAME protection against H/R injury. These results demonstrate that NO is synthesized in rat proximal tubules and participates as one of the mediators in rat tubular H/R injury.Nitric oxide (NO) is a major chemical form of endotheliumderived relaxing factor (1, 2), an important regulator of vascular tone, and is released by endothelial cells (3). However, the role of NO is not restricted to the vascular system; several other functions have been described in recent years. NO has an important role in platelet function, causing inhibition of platelet aggregation; in immunological reactions, as a host defense mechanism against tumor cells and invasive organisms; and in the central and peripheral nervous systems, as a neurotransmitter (4, 5). Moreover, alterations in NO synthesis have been incriminated in several other pathophysiological conditions, including arterial hypertension and progression of renal failure (6), as well as septic shock (7), hypoxia-induced vasodilation (8), the vasospasm that follows subarachnoid hemorrhage due to inhibition of NO by hemoglobin (9), and neuronal destruction in vascular stroke and other neurodegenerative conditions (10).While endothelial cells, neurons, macrophages, neutrophils, and platelets are well-known sources of NO, recent studies have suggested that epithelial cells may constitutively generate NO (11). Furthermore, constitutive NO synthase (NOS) has been identified in the kidney, specifically in macula densa cells (12) and in the inner medullary collecting duct (13). Recently, the inducible form of NOS was identified in the rat proximal tubule and inner medullary collecting duct (14). In the kidney, NO has an important role in renal hemodynamic regulation and sodium and water excretion (15).The use of freshly isolated ...

These results demonstrate a role of TNF in the early renal dysfunction (16 h) in a septic mouse model independent of iNOS, hypotension, apoptosis, leukocyte accumulation, and morphological alterations, thus suggesting renal hypoperfusion secondary to an imbalance between, as yet to be defined, renal vasoconstrictors and vasodilators.

Acute renal failure (ARF) during sepsis is associated with increased nitric oxide (NO) and oxygen radicals, including superoxide (O(2)(-)). Because O(2)(-) reacts with NO in a rapid manner, it plays an important role in modulating NO levels. Therefore, scavenging of O(2)(-) by superoxide dismutase (SOD) may be critical for preserving NO bioavailability. In mice, substantial renal extracellular SOD (EC-SOD) expression implies its important role in scavenging O(2)(-) in the kidney. We hypothesized that during endotoxemic ARF, EC-SOD is decreased in the kidney, resulting in increased O(2)(-) and thus decreased vascular NO bioavailability with resultant renal vasoconstriction and ARF. In the present study, normotensive endotoxemic ARF was induced in mice using lipopolysaccharide (LPS; 5 mg/kg ip). Sixteen hours after LPS, glomerular filtration rate (GFR; 50 +/- 16 vs. 229 +/- 21 microl/min, n = 8, P < 0.01) and renal blood flow (RBF; 0.61 +/- 0.10 vs. 0.86 +/- 0.05 ml/min, n = 8, P < 0.05) were subsequently decreased. EC-SOD mRNA and protein expression in endotoxemic kidneys were decreased at 16 h compared with controls. A catalytic antioxidant, metalloporphyrin, reversed the deleterious effects of endotoxemia on renal function as GFR (182 +/- 40 vs. 50 +/- 16 microl/min, n = 6, P < 0.01) and RBF (1.08 +/- 0.10 vs. 0.61 +/- 0.10 ml/min, n = 6, P < 0.05) were preserved. Similar results were obtained with tempol, a chemically dissimilar antioxidant. Specific inhibition of inducible nitric oxide synthase (iNOS), l-N(6)-(1-iminoethyl)-lysine, reversed the renal protective effect on GFR and RBF observed with antioxidant treatment during endotoxemia. In summary, renal EC-SOD expression is decreased during endotoxemia. Antioxidant therapy preserved GFR and RBF during endotoxemia. The reversal of this protective effect by inhibition of iNOS suggests the importance of the bioavailability of NO for preservation of renal function during early endotoxemia.

Renal ischemia-reperfusion (I/R) injury was investigated in inducible nitric oxide synthase (iNOS) knockout mice. After a 26-min bilateral renal pedicle clamp, serum creatinine concentrations (in mg/dl) in wild-type mice after a 24-h reperfusion were 0.25 ± 0.03 in sham-operated controls and 2.3 ± 0.38 in ischemic mice ( P < 0.01); after 48 h, concentrations (in mg/dl) were 0.25 ± 0.03 in controls and 2.0 ± 0.18 in ischemic mice ( P < 0.01). iNOS knockout mice demonstrated an attenuation of serum creatinine concentration after renal I/R injury. Serum creatinine concentrations (mg/dl) after a 24-h reperfusion were 2.3 ± 0.22 in wild-type ischemic and 1.21 ± 0.25 in iNOS knockout ischemic mice ( P < 0.05); after 48 h, concentrations were 2.0 ± 0.18 in wild-type ischemic and 0.96 ± 0.25 in iNOS knockout ischemic mice ( P< 0.01). Histological scoring of acute tubular necrosis in iNOS knockout mice was decreased compared with that in wild-type controls (0.88 ± 0.2 vs. 3.3 ± 0.3, P< 0.05). iNOS protein in the renal cortex of wild-type mice subjected to renal I/R injury was undetectable up to 48 h. However, a strong upregulation of heat shock protein 72 expression was observed in renal cortex of iNOS knockout mice under basal conditions. In conclusion, kidneys of iNOS knockout mice were protected against ischemic acute renal failure. This protective effect may be related to a compensatory upregulation of heat shock protein 72.

Protective effect of renal denervation on normotensive endotoxemia-induced acute renal failure in mice. Am J Physiol Renal Physiol 283: F583-F587, 2002. First published March 12, 2002 10.1152/ajprenal.00270.2001.-Acute renal failure (ARF) contributes substantially to the high morbidity and mortality observed during endotoxemia. We hypothesized that selective blockade of the renal nerves would be protective against ARF during the early (16 h) stage of endotoxemia [5 mg lipopolysaccharide (LPS)/kg ip in mice]. At 16 h after LPS, there was no change in mean arterial pressure, but plasma epinephrine (4,604 Ϯ 719 vs. 490 Ϯ 152 pg/ml, P Ͻ 0.001), norepinephrine (2,176 Ϯ 306 vs. 1,224 Ϯ 218 pg/ml, P Ͻ 0.05), and plasma renin activity (40 Ϯ 5 vs. 27 Ϯ 2 ng⅐ ml Ϫ1 ⅐ h Ϫ1 , P Ͻ 0.05) were higher in the LPStreated vs. control mice. The high plasma renin activity level decreased to the control level with renal denervation in endotoxemic mice. After intravenous injection of phentolamine (200 g/kg), the decrement in mean arterial pressure was significantly greater in LPS-treated vs. control mice (19.4 Ϯ 3.5 vs. 8.1 Ϯ 1.5 mmHg, P Ͻ 0.01). Sixteen hours after LPS administration, there were significant decreases in glomerular filtration rate (52 Ϯ 18 vs. 212 Ϯ 23 l/min, P Ͻ 0.01) and renal blood flow (0.58 Ϯ 0.08 vs. 0.85 Ϯ 0.06 ml/min, P Ͻ 0.01) in sham-operated mice. The decrement in glomerular filtration rate during endotoxemia was significantly attenuated in mice with denervated kidneys (32 vs. 79%). Moreover, there was no change in renal blood flow during endotoxemia in mice with renal denervation. The present results therefore demonstrate a protective role of renal denervation during normotensive endotoxemia-related ARF in mice, an effect that may be, at least in part, due to a diminished activation of the renin-angiotensin system. glomerular filtration rate; renal blood flow; epinephrine; norepinephrine; sepsis SEPSIS IS THE MOST FREQUENT CAUSE of acute renal failure (ARF) in intensive care units (1, 2, 9). When sepsis is associated with ARF, the mortality may be as high as 80%. The pathogenetic factors responsible for sepsisrelated ARF, however, are incompletely defined. Although ARF may occur with septic shock, it is also clear that sepsis-related ARF can occur in the absence of hypotension (1, 11).In recent studies from our laboratory, a mouse model of endotoxemia-related ARF has been studied. A significant decrease in glomerular filtration rate (GFR) and renal blood flow (RBF) occurs in the absence of a fall in blood pressure (7). We hypothesized that endotoxemia may be associated with normal blood pressure because of activation of the sympathetic nervous system and renin-angiotensin system (RAS), which secondarily causes renal vasoconstriction. The roles of renal nerves and the RAS in this early renal vasoconstriction during endotoxemia, however, have not been investigated. The present investigation was therefore undertaken in a normotensive mouse model of endotoxemia-induced ARF to examine the effect of selective r...