Vol. 29, pp.673 ~ 679, 2001 13 12 20 ARF 50% ARF ARF ARF ARF NO ARF NO NOS NO NO2 /NO3 NOx Griess NO NOx 1 72 7 NOx NOS 1 7 NOx µmol/kidney Wet Weight; Sham 0.116 ± 0.005 0.168 ± 0.006 24 0.185 ± 0.004 72 0.181 ± 0.011 7 0.220 ± 0.012 p<0.001 NOS nmol NOx formed/30 min/kidney Wet Weight; 24.98 ± 1.72 28.25 ± 1.81 24 31.23 ± 3.44 72 32.70 ± 2.51 p<0.05 7 37.78 ± 2.36 p<0.001 NOx NOS r=0.809, p=0.002 NO acute renal failure: ARF 50% ARF HO ; hydoxyradicalno; nitric oxide 15
ROS/RNS; reactive oxygen/nitrogen species 1984 Paller 1 ARF ARF ROS NO ARF 2~5 NO ARF NO ARF NOS; nitric oxide synthase Fig. 1 Sample preparation for NOS activity. Dissected kidney was weighed and frozen immediately using liquid nitrogen, then homogenized and centrifuged. ppt; precipitate Table 1 Components of homogenization buffer 1 ARF 250 300 g Sprague-Dawley 22.5 ± 0.5 C 55 ± 5% 6:30 18:30 1 50 mg/kg 3 cm 45 sham Control ARF 1 Day 0: D024 Day 1: D1 72 Day 3: D3168 7 Day 7: D7 4 8 2 NADPH tetrahydrobiopterin BH4leupeptin pepstatin A Sigma St. Louis, MO, U.S.A. Calmodulin L-arginine, dithiothreitol DTT, Dowex 50WX8 H form, Sigma NaOH Na form 3 nitric oxide synthase: NOS 1 Fig. 1 23 A 5 A ULTRA-TURRAX,, 80 B 3 24,000 g Dowex 50W-X8 Na NOS Table 1 16
Table 2 Method of NOS activity determination Table 3 Laboratory data in control and acute renal failure rats (Body weight, plasma creatinine, and urea nitrogen levels, and creatinine clearance) 2 NOS Table 2 NOS NO NO2 /NO3 NOx Yokoi 6 3 15 30 60 3 NOx NOx NOx TCI NOx 1000m,, 7 0.5 µm 30 NOx 3 NO NOx NOS 24,000 g NOx 100 µl 3 0.3N NaOH 5 3 5% ZnSO4 15,000 g, 10 TCI NOx 1000m NOx 4 Cr Cr Cr 5 24 Ccr 10 24 Cr Cr Ccr 100 4 Kruskal-Wallis Bonferroni/Dunn Spearman 5% 1 Table 3 24 232 ± 8 g 72 214 ± 15 g p<0.001 7 223 ± 15 g p<0.05 Cr mg/dl 1 0.67 ± 0.050.40 ± 0.03 p<0.01 24 1.59 ± 0.62 72 2.01 ± 1.62 7 0.57 ± 0.04 Ccr ml/min/100 g BW Cr 1 ARF 24 0.62 ± 0.37p<0.001 7 2.65 ± 0.53 2 NOx Fig. 2 NOx 0.116 ± 0.005 17
Fig. 2 No metabolite ( NOx ) levels in the whole kidney. NOx levels were measured 1 hour, 1, 3, and 7 days after lt. renal artery reperfusion. Values are expressed means ± SD. Fig. 3 NOS activity in the whole kidney. NOS activity was measured 1 hour, 1, 3, and 7 days after lt. renal artery reperfusion. Values are expressed means ± SD. 1 0.168 ± 0.006 24 0.185 ± 0.00472 0.181 ± 0.0117 0.220 ± 0.012 0.116 ± 0.005p<0.001 3 Fig. 3 NOS nmol NOx formed/30 min/kidney Wet Weight 1 28.25 ± 1.81 24.98 ± 1.72 24, 72 31.23 ± 3.44, 32.70 ± 2.51, p<0.05 7 37.78 ± 2.36, p<0.001 4 NOx NOS Fig. 4 NOx NOS r=0.809, p=0.002 ARF ROS/RNS; reactive oxygen/nitrogen species O2 NO HO ROS/RNS DNA DNA Fig. 4 Relationship between kidney NOS activities and NOx levels. These values were measured following 1 hour, 1 day, 3 days, and 7 days after lt. renal artery reperfusion. ARF ROS Paller 1 ROS xanthine oxidase ATP xanthine dehydrogenase xanthine oxidase O2 O2 SOD xanthine oxidase 8 xanthine oxidase 18
xanthine oxidase ARF II NADPH-oxidase O2 9 NO 10 NO NOS enos inos NOS nnos 3 NOS ARF NO Chintala 2 NOS L- NMMA ARF ARF ARF ARF NO Kakoki 5 Wistar 45 24 NO NOS immuno blot NOS cofactor tetrahydrobiopterin BH4 NO enos BH4 NO inos ARF Noiri 4 NO O2 NO peroxinitrite 11 NO NO NOS NO O2 ROS/RNS ARF NO NOS NO ROS/RNS 12 13~15 NOS 1 Fig. 4 NOx NOS 1 NOS 16 17 ROS NOS NO ARF NOS 24 NOS NOS ARF NOS NO inos NO O2 peroxinitrite HO 72 NO NO 19
ARF 1 1 NO NOS NOS 1 Paller MS, Hoidal JR and Ferris TF. Oxygen free radicals in ischemic acute renal failure in the rat. J Cnin Invest 1984; 74: 1156-1164. 2 Chintala MS, Chiu PJ, Vemulapalli S, Watkins RW and Sybertz EJ. Inhibition of endothelial derived relaxing factor (EDRF) aggravates ischemic acute renal failure in anesthetized rats. Naunyn-Schmiedeberg's Arch Pharmacol 1993; 348: 305-310. 3 Yu L, Gengaro PE, Niederberger M, Burke TJ and Schrier RW. Nitric oxide: a mediator in rat tubular hypoxia/reoxygenation injury. Proc Natl Acad Sci USA 1994; 91: 1691-1695. 4 Noiri E, Peresleni T, Miller F and Goligorsky MS. In vivo targeting of inducible NO synthase with oligodeoxynucleotides protect rat kidney against ischemia. J Clin Invest 1996; 97: 2377-2383. 5 Kakoki M, Hirata Y, Hayakawa H, Suzuki E, Nagata D, Tojo A, Nishimatsu H, Nakanishi N, Hattori Y, Kikuchi K, Nagano T and Omata M. Effects of tetrahydrobiopterin on endothelial dysfunction in rats with ischemic acute renal failure. J Am Soc Nephrol 2000; 11: 301-309. 6 Yokoi I, Habu H, Kabuto H and Mori A. Analysis of nitrite, nitrate, and nitric oxide synthase activity in brain tissue by automated flow injection technique. Methods in Enzymology 1996; 268: 152-159. 7 Higuchi K and Motomizu S. Flow-injection spectrophotometric determination of nitrite and nitrate in biological samples. Anal Sci 1999; 15: 129-134. 8,.. 1988; 24: 743-748. 9 Zafari AM, Ushio FM, Arkes M, Lassegue B and Griendling KK. Arachidonic acid metabolites mediate angiotensin-induced NADH/NADPH oxidase activity and hypertrophy in vascular smooth muscle cells. Antioxidants and Redox Signaling 1999; 1: 167-179. 10. 3 NO, 4 NO., NO,,, 1993: 34-56. 11. 1 NO., (NO),,, 2000: 3-11. 12 Lauriat S and Linas SL. The role of neutrophils in acute renal failure. Seminars in Nephrology 1998; 18: 498-504. 13. NO. 2000; 49: 354-357. 14. NO. 1998; 45: 781-784. 15,,,. 10.,,,, 1998: 280-288. 16 Milhoan KA, Lane TA and Bloor CM. Hypoxia induces endothelial cells to increase their adherence for neutrophils: role of PAF. Am J Physiol 1992; 263: H965-H962. 17 Palluy O, Morliere L, Gris JC, Bonne C and Modat G. Hyopoxia/reoxygenation stimulates endothelium to promote neutrophil adhesion. Free Rad Biol Med 1992; 13: 21-30. 20
Abstract Alteration of Nitric Oxide Synthase Activity in Experimental Acute Renal Failure Model Rats Fumiaki Matsuoka, Katsuhide Toyama, Masayuki Ominato, Takeo Satoh, and Teruhiko Maeba Despite the improvement of dialysis therapy, the mortality rate in acute renal failure (ARF) patients is still more than 50%. One of reasons of this phenomenon is that ARF is a heterogeneous disease and that ARF itself could deteriorate various systemic conditions. Elucidation of the precise mechanism in the pathogenesis of ARF and its complications has been required for improving the prognosis of ARF. There has been a growing body of evidence for the roles of reactive oxygen species (ROS) and reactive nitrogen species (nitric oxide; NO) in the pathogenesis of a variety of renal diseases including acute renal failure (ARF). However, there are controversial reports of the pathogenetic roles of ROS and NO in experimental ischemic ARF. Therefore, time course changes of NOS activity will provide us with some important information needed to clarify the pathophysiology in ARF. This study was designed to clarify this issue by examining the change of nitric oxide production (measured final NO metabolites, NOx; NO2 /NO3 ) and nitric oxide synthase (NOS) activity using the experimental ARF model rats with ischemia and reperfusion. NOx concentration and NOS activity were measured by the flow injection method. Renal function was already deteriorated 1 hour after the reperfusion followed by further reduction and was most decreased between 1 and 3 days after the induction of ARF. Thereafter it gradually recovered but did not reach the control level. Both NOx level and NOS activity were significantly increased in ARF model in a timedependent fashion (NOx: Control 0.116 ± 0.005; Day 0 0.168 ± 0.006; Day 1 0.185 ± 0.004; Day 3 0.181 ± 0.011; Day 7 0.220 ± 0.012 µmol/kidney Wet Weight, NOS: Control 24.98 ± 1.72; Day 0 28.25 ± 1.81; Day 1 31.23 ± 3.44; Day 3 32.70 ± 2.51; Day 7 37.78 ± 2.36 nmol NOx formed/30 min/ Kidney Wet Weight). Moreover, a significantly positive correlation (r=0.809, p=0.002) was observed between NOx level and NOS activity in the kidney. These findings suggest that NO plays some role in the pathogenesis of ARF. Production sites of NO and responsible NOS isoforms in the kidney should be further determined by a longer period examination using this ARF model. (St. Marianna Med. J., 29: 673-679, 2001) Division of Nephrology and Hypertension, Department of Internal Medicine (Director: Prof. Kenjiro Kimura) St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8511, Japan 21