... PATRICIA E. GENGARO, MICHEL NIEDERBERGER, THOMAS J. BURKE, AND ROBERT W. SCHRIERt ... to its free radical nature and high reactivity with the superoxide ...... Palmer, R. M., Ferrige, A. G. & Moncada, S. (1987) Nature (Lon-.
Proc. Nati. Acad. Sci. USA Vol. 91, pp. 1691-1695, March 1994 Medical Sciences
Nitric oxide: A mediator in rat tubular hypoxia/ reoxygenation injury (nitric oxide synthasc/L-arginine/L-citrulfline)
Luis Yu*, PATRICIA E. GENGARO, MICHEL NIEDERBERGER, THOMAS J. BURKE, AND ROBERT W. SCHRIERt Department of Medicine, Box C281, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, CO 80262
Communicated by Carl Gottschalk, November 5, 1993
ABSTRACT 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 (LNAME), 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.
(14). In the kidney, NO has an important role in renal hemodynamic regulation and sodium and water excretion (15). The use of freshly isolated proximal tubules in suspension permits the study of tubular epithelial cells in the absence of other sources of NO, such as endothelial cells and neutrophils. Using a model of hypoxia/reoxygenation (H/R) in rat proximal tubules, we have shown that oxygen radicals may participate in the H/R injury (16). Others have shown that superoxide anions inactivate NO and that superoxide dismutase doubles the half-life of NO (17). Superoxide and NO rapidly react to form the stable peroxynitrite, which decomposes and generates strong oxidant molecules (18). On this background, we hypothesized that NO, possibly by combining with superoxide radical to generate peroxynitrite, may play a role as one of the mediators of H/R injury in rat proximal tubules. The present study shows that NO production is stimulated by hypoxia in epithelial cells and its inhibition is protective against H/R injury in rat proximal tubule cells.t
MATERIALS AND METHODS Isolation of Proximal Tubules. Proximal renal tubules were isolated as previously described (19). In brief, male SpragueDawley rats (150-300 g) were anesthesized with sodium pentobarbital (60 mg/kg of body weight, i.p.) and underwent laparotomy. The aorta was cannulated and kidneys were perfused with 60 ml of cold heparinized (4000 units) oxygenated solution (112 mM NaCl/25 mM NaHCO3/5 mM KCl/1.6 mM CaCl2/2.0 mM NaH2PO4/1.2 mM MgSO4/5 mM glucose/2.5 mM Hepes/10 mM mannitol/1 mM glutamine/1 mM sodium butyrate/1 mM sodium lactate, pH 7.4). Subsequently, the kidneys were perfused with 30 ml of solution A containing 15 mg of collagenase (type B, Boehringer Mannheim) and 15 mg of hyaluronidase (Sigma). After perfusion, kidneys were placed in ice-cold solution A. Renal cortices were minced, washed once with cold solution A, and incubated in 55 ml of oxygenated solution A containing 40 mg of collagenase and 10 mg of hyaluronidase. Tissue digestion was performed at 370C under 95% 02/5% CO2 in a shaking water bath. The digestion was halted at 15, 20, 25, and 30 min to remove the suspended digested tubules, which were placed in 30 ml of solution A containing 1 g of fatty acid-free bovine serum albumin (ICN). Meanwhile, the larger particles were returned to the water bath for further digestion. At 30 min of digestion, all tissue was washed free of collagenase and
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
Abbreviations: NOS, nitric oxide synthase; L-NAME, N-nitro-Larginine methyl ester; H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; SNP, sodium nitroprusside. *Present address: Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo-Nefrologia, Av. Dr. Eneas C. Aquiar 255-S. 711 F, Sao Paulo, Brazil 05403. tTo whom reprint requests should be addressed. tPortions of this work were presented at the 12th International Congress of Nephrology, June 13-18, 1993, Jerusalem.
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placed in the albumin-supplemented solution A for 10 min. After filtering through a coarse wire mesh, the tissue was washed free ofalbumin three times with solution A, suspended in 60 ml of oxygenated cold (40C) 45% Percoll layered onto 5 ml of 100%o Percoll. The tissue was centrifuged at 15,000 x g for 10 min and the lowest band, which was composed primarily (95%o) of proximal tubules, was removed and washed three times with solution A. The tubules were subsequently incubated in a cold oxygenated solution B. (106 mM NaCl/18 mM NaHCO3/5 mM KCl/1 mM CaCl2/2 mM NaH2PO4/1 mM MgSO4/5 mM glucose/2.5 mM Hepes/2 mM glutamine/10 mM sodium butyrate/4 mM sodium lactate, pH 7.2). Aliquots (containing 1.0-1.5 mg of protein per ml) were placed in ice cold siliconized Erlenmeyer flasks and superfused with 95% 02/5% CO2 for 5 min. Thereafter, the flasks were capped and placed in a shaking water bath at 370C for 15 min. After this equilibration period, experiments were initiated. Determination of NOS Activity. NOS activity was assessed by measuring the conversion Of L-arginine to L-citrulline by a method similar to that of Rengasamy and Johns (20). The tubule pellet from either normoxic or 15-min hypoxic tubules was reconstituted in S ml ofcold homogenizing buffer (50 mM Hepes, pH 7.5/0.5 mM EDTA/1 mM dithiothreitol/0.01% phenylmethylsulfonyl fluoride) and sonicated for 2 min (30 sec on/30 sec off) on ice. The homogenate was then centrifuged at 20,000 g for 1 hr at 40C and the supernatant was passed over a Dowex AG5OWX-8 (Na+ form) column to remove endogenous L-arginine. Protein content was determined with the Bio-Rad protein reagent with bovine serum albumin as the standard. The NOS reaction mixture (400 A) consisted of 320 /1 of enzyme extract, 100 pM L-[3H]arginine (1 pCi/ml; 1 uCi = 37 kBq), 1 mM NADPH, and 2 mM CaCl2. The reaction was carried out at 370C and stopped at 40 and 80 min by removing 200 of the reaction mixture and adding it to 2 ml of 20 mM Hepes, pH 5.5/2 mM EDTA/2 mM EGTA. The mixture was then passed over a 1-ml Dowex AG50WX-8 (Na+ form) column and citrulline was eluted with an additional 2 ml of water. Radioactivity in the eluate was measured by liquid scintillation counting. When the eluate was analyzed by thin-layer chromatography [silica gel 60; chloroform/methanol/ammonium hydroxide/water, 1:4:2:1 (vol/vol)], >70%o of the radioactivity comigrated with authentic citrulline. NOS activity was measured in both normoxic and 15-min hypoxic tubules. The assay was also carried out in the presence or absence of EGTA (2 mM) and N-nitro-L-arginine methyl ester (L-NAME, 5 mM). Results are expressed as nmol of [3H]citrulline produced per mg of protein in 40 and 80 min. H/R Protocol. After the equilibration period, isolated tubules were divided into an experimental group and a time control group. The Po2 in the time control group was kept throughout the experiment in the range 300-400 mmHg (1 mmHg = 133 Pa). The experimental group was made hypoxic (Po2 averaged 20-40 mmHg) by gassing with 95% N2/5% CO2 for 5 min. The duration of hypoxia was 15 min. After hypoxia, the tubules were reoxygenated by gassing with 95% 02/5% CO2 for 5 min. The Po2 after reoxygenation returned to 300-400 mmHg. The flasks were then resealed and kept reoxygenated for 35 min. The effects of the following substances were evaluated in this H/R model: L-NAME (Calbiochem), a NOS inhibitor; N-nitro-D-arginine methyl ester (D-NAME, Calbiochem), as a control because the NOS inhibition is stereospecific; and L-arginine (Sigma), the substrate for NOS. Samples were obtained at baseline (normoxic at 0 min, N-0), after hypoxia (H-15), and after 35 min ofreoxygenation X
(R-35). Samples from time control groups (C) were obtained at the same time points. Lactate dehydrogenase (LDH) release, which increases during cell membrane injury as occurs with H/R, and the concentrations of nitrite (NO-) and
Proc. Natl. Acad. Sci. USA 91 (1994)
nitrate (NO-), the stable metabolites of NO, were measured as follows. LDH release. One milliliter of tubule suspension was centrifuged for 10 sec in a refrigerated centrifuge at 1500 x g. The pellet was lysed with 1.5% (vol/vol) Triton X-100. LDH activity was measured in the supernatant and pellet according to Bergmeyer (21). LDH activities were converted to percent release by dividing supernatant activity by total activity. NO1/NO3 determinations. Samples of 1 ml of tubule suspension were obtained at baseline (N-0), 15 min (H-15), and 50 min (R-35) of the experiment. The suspension was immediately centrifuged for 10 sec at 1500 x g and the supernatant was frozen until assay. The pellet was assayed for protein determination by the Lowry method (22). NO3 was determined by a modification of the method ofConrad et al. (23). In brief, 200 /4 of supernatant was incubated for 1 hr at 250C with 100 /4 of nitrate reductase (90 milliunits/ml, Boehringer Mannheim), 100 ,ul ofNADPH (0.28 mM, Sigma), 100 p4 of FAD (35 AM, Sigma), and 200 /4 of potassium phosphate buffer (0.1 M, pH 7.5). The reaction was stopped by boing, for 3 min. An equal volume (700 /4) of Griess reagent (1:1 mixture of 2% sulfanilamide in 5% H3P04 and 0.2% N41-naphthyl)ethylenediamine dihydrochloride in water) was then added to the reduced samples and incubated at 600C for 10 min. The NO2 reacts with the Griess reagent to form a chromophore (24) and its absorbance at 546 nm was measured in a Beckman model 25 spectrophotometer. A standard curve (0-10 nmol per tube) of sodium nitrate in 200 /ul of buffer C was included with each assay. Values are expressed as nmol/mg of protein at 15 and 50 min, after subtraction of basal production of NO /NOj-. Generati ofExogenous NO. Rat proximal tubules isolated and equilibrated as described above were subjected to the same H/R protocol, except for a longer reoxygenation period (45 min). Exogenous NO was generated by the addition of sodium nitroprusside (SNP, Sigma). SNP dissolved in buffer C (0.2 mM or 1 mM) was added at baseline. SNP (0.2 mM) was added to normoxic controls and H/R tubules in the presence or absence of bovine hemoglobin (Hb, Sigma), a NO scavenger. Hb dissolved in distilled water (20 pM) was also added at baseline. In separate groups, SNP (1 mM) was added to normoxic and H/R tubules in the presence or absence of L-NAME (10 mM). Samples were obtained at baseline (N-0) and after H/R at 60 min (R-45) and were analyzed for LDH release and NO /NO- production as described above. Statistical Analyses. All values are reported as means ± SE. Data were analyzed by analysis of variance and the StudentNewman-Keuls multiple comparison test. Statistical significance was considered as P < 0.05.
RESULTS Determinatio of NOS Activity. NOS activity was deter-
mined at 40 and 80 min of assay incubation time. NOS activity was initially determined in normoxic tubules (n = 5) in the presence or absence of L-NAME (5 mM). The production of [3H]citrulline (nmol per mg of protein) in normoxic tubules compared with L-NAME-treated tubules (n = 4) was 2.6 ± 0.2 vs. 1.0 ± 0.1 at 40 min (P < 0.001) and 6.2 ± 0.4 vs. 2.4 + 0.1 at 80 min (P < 0.001). NOS activity was also measured in hypoxic tubules (n = 5) and compared with that in normoxic tubules in the presence or absence of EGTA. NOS activity (nmol/mg) was 6.3 ± 0.9 vs. 3.4 ± 0.1 at 40 min (P < 0.05) and 9.7 + 0.9 vs. 6.3 ± 0.3 at 80 min (P < 0.05). EGTA (2 mM) had no effect on NOS activity in either normoxic or hypoxic tubules (Fig. 1). Therefore, NOS activity, as assessed by the conversion of L-[3Hlarginine to L-[3H]citrulhine, was shown to be present in our rat proximal tubule preparation. Moreover, this NOS activity was stimulated by 15 min
Medical Sciences: Yu et al.
Proc. Natl. Acad. Sci. USA 91 (1994)
of hypoxia, inhibited by L-NAME treatment, and not affected by calcium chelation. H/R Protocol. Effect of L-NAME on LDH release in HIR tubules (Fig. 2A). LDH release in control tubules compared with H/R tubules (n = 5) was 9.0 ± 0.7% vs. 8.2 ± 0.7% at N-0; 12.2 ± 1.0% vs. 32.6 ± 4.3% at H-15 (P < 0.001), and 18.6 ± 0.6% vs. 44.7 ± 3.5% at R-35 (P < 0.001). L-NAME (10 mM) completely protected against H/R injury. LDH release in L-NAME-treated vs. untreated H/R tubules (n = 5) averaged 8.0 ± 0.6% vs. 8.2 ± 0.7% at N-0; 13.8 ± 0.6% vs. 32.6 ± 4.3% at H-15 (P < 0.001), and 21.5 ± 1.3% vs. 44.7 ± 3.5% at R-35 (P < 0.001). D-NAME (10 mM) had no effect on H/R tubules. LDH release in D-NAME-treated vs. untreated H/R tubules was 8.0 ± 0.6% vs. 8.2 ± 0.7% at N-0; 25.7 ± 3.2% vs. 32.6 ± 4.3% at H-15 (not significant), and 37.8 + 3.3% vs. 44.7 ± 3.5% at R-35 (not significant). Neither L-NAME nor D-NAME had any effect on the LDH release of time control tubules. Effect of L-arginine on LDH release in H/R tubules (Fig. 2B). LDH release in control tubules compared with H/R tubules (n = 6) was 9.0 ± 1.0% vs. 8.4 ± 1.3% at N-0; 12.0 ± 0.6% vs. 36.7 ± 2.5% at H-15 (P < 0.001), and 19.9 ± L.0o vs. 47.5 ± 2.4% at R-35 (P < 0.001). L-Arginine (5 mM) enhanced H/R injury. LDH release in L-argmine-treated vs. untreated H/R tubules (n = 4) averaged 7.1 ± 0.9%6 vs. 8.4 ± 1.3% at N-0; 47.4 + 2.6% vs. 36.7 ± 2.5% at H-15 (P < 0.01), and 57.0 ± 3.0% vs. 47.5 ± 2.4% at R-35 (P < 0.05). L-Arginine addition had no effect on time control tubules. NO2j/NOj results (Fig. 3A). NO /NO- levels (nmol/mg of protein) in control vs. H/R tubules (n = 5) averaged 0.8 + 0.3 vs. 4.4 + 1.2 at H-15 (P < 0.001) and 2.8 ± 0.7 vs. 6.8 + 1.0 at R-35 (P < 0.05). L-NAME (10 mM) significantly decreased NOj/NO- levels in H/R tubules. NO /NOlevels (nmol/mg of protein) in L-NAME-treated vs. untreated H/R tubules (n = 5) were 2.0 ± 0.1 vs. 4.4 ± 1.2 at H-15 (P < 0.05) and 4.1 ± 0.5 vs. 6.8 ± 1.0 at R-35 (P < 0.05). D-NAME (10 mM) did not decrease NOj1NOj levels in H/R tubules. In contrast, L-arginine (5 mM) (n = 4) increased NO /NOj in H/R tubules (Fig. 3B). NO /NO- levels in L-arginine-treated vs. untreated H/R tubules were 6.1 ± 1.0 vs. 4.0 ± 0.4 at H-15 (P < 0.05) and 11.0 ± 1.0 vs. 8.4 ± 1.3 at R-35 (not significant). In control tubules, NO /NO- levels were 0.8 ± 0.1 at N-0 (P < 0.001 vs. H/R alone) and 2.9 + 0.4 at R-35 (P < 0.001 vs. H/R alone). L-Arginine addition had no effect on NOj/NO- levels in time control tubules. Exogenous NO. Effect of SNP and Hb on LDH release in H/R tubules (Fig. 4A). LDH release in control tubules compared with H/R tubules (n = 4) was 7.8 ± 0.9%6 vs. 7.3 ± 0.5% at N-0 and 19.4 ± 0.9% vs. 40.8 ± 3.2% at R-45 (P Control
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