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(The FASEB Journal. 2003;17:636-643.)
© 2003 FASEB

Induction of LOX-1 and iNOS expressions by ischemia-reperfusion of rat kidney and the opposing effect of L-arginine

HIROAKI KOSAKA1, HIROHITO YONEYAMA, LING ZHANG, SHIGEMOTO FUJII, AKIRA YAMAMOTO and JUNSUKE IGARASHI

Second Department of Physiology, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan

1Correspondence: The 2nd Department of Physiology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: hkosaka{at}kms.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a newly identified endothelial cell surface major receptor for oxidatively modified low-density lipoprotein. Progression of arthrosclerosis in the donor organ after organ transplantation is a major problem. We hypothesized that ischemia-reperfusion induces LOX-1. After 1 h ischemia of bilateral kidneys plus 3, 6, or 12 h reperfusion, we first revealed that LOX-1 mRNA expression was increased in renal cortex and medulla at 6 h after reperfusion, which was decreased by L-arginine supplement. Plasma nitric oxide (NO) end-product nitrite plus nitrate and inducible nitric oxide synthase (NOS) expression were increased after reperfusion of 6 h. However, NOS substrate L-arginine did not augment but markedly decreased plasma NO end product, because L-arginine supplement suppressed inducible NOS expression in kidney. We hypothesized that available L-arginine is depleted by ischemia-reperfusion, leading to inducible NOS induction. Ischemia decreased L-arginine levels in kidney and L-arginine supplement increased NO end products in renal cortex in the earliest phase of reperfusion. These results disclosed for the first time that a deficiency in L-arginine by ischemia reperfusion causes uncoupling of constitutive NOS, which induces inducible NOS and LOX-1, implying why L-arginine is effective for stroke or transplantation in preventing atherosclerotic progress.—Kosaka, H., Yoneyama, H., Zhang, L., Fujii, S., Yamamoto, A., Igarashi, J. Induction of LOX-1 and iNOS expressions by ischemia-reperfusion of rat kidney and the opposing effect of L-arginine.


Key Words: NO end product • ox-LDL • renal cortex


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ISCHEMIA REPERFUSION INJURY is a serious concern in a variety of clinical circumstances including organ transplantation, infarction, and stroke (1 2 3) . Generation of reacting oxygen species (ROS) after reperfusion (4 , 5) can result in oxidative damage of lipids, proteins, and nucleic acids.

Accumulating evidence suggests that endothelial activation, dysfunction and injury by oxidized low-density lipoprotein (ox-LDL) are an early key step in the development of atherosclerosis (6 , 7) . Ox-LDL is also a mitogenic activator for macrophage and smooth muscle cells and is taken up by monocytes, macrophages, and smooth muscle cells through a variety of scavenger receptors (8) that are, however, absent or a little in endothelial cells (9) . Endothelial cells internalize ox-LDL through a presumed receptor that does not involve macrophage scavenger receptors (9) .

Lectin-like ox-LDL receptor-1 (LOX-1) (10) is a newly identified cell surface major receptor in endothelial cells specific for ox-LDL. Immunohistochemistry of the atherosclerotic plaque from a patient with unstable angina pectoris revealed accumulation of LOX-1 protein at the site of thrombus as LOX-1 recognizes and binds activated platelets (11) .

After organ transplantation, progression of arthrosclerosis in the donor organ a major problem (12) . We hypothesized that LOX-1 is up-regulated after the reperfusion. In the current study, we investigated first the possibility by exerting the ischemia reperfusion of rat kidney. Next, we hypothesized that uncoupling of constitutive nitric oxide synthase (NOS) due to a deficiency in the substrate L-arginine increases LOX-1 and inducible NOS after reperfusion. We also examined the effects of superoxide dismutase (SOD) and tetrahydrobiopterin (BH4).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ischemia reperfusion of both kidneys
Sixty-two male Wistar rats (8 wk old, Charles River, Wilmington, MA, USA) were used. All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH Publication No.85–23, revised 1996) and were approved by the Animal Research Committee of Kagawa Medical University. After pentobarbital anesthesia [50 mg/kg, intraperitoneally (i.p.)], bilateral renal arteries were clamped by microvascular clips for 1 h, then recirculated by removing both clips. The rat received 50 units heparin intravenously (i.v.) before the renal ischemia and 10 mL of sterilized Ringer’s solution (NaCl 8.6 g/L, KCl 0.3 g/L, CaCl2 0.33 g/L) i.p. concurrent with clamping to prevent dehydration. Some of the rats received L-arginine hydrochloride (645 mg/kg) dissolved in the Ringer’s solution i.p.. Recombinant human Cu, Zn-SOD (5 mg/kg) (endotoxin-free, Nihon Kayaku Co. Ltd., Tokyo, Japan) or BH4 (20 mg/kg, Cayman Chem. Co. Ann Arbor, MI, USA) was dissolved in sterilized saline (0.2 mL) and immediately injected i.v. concurrent with clamping of bilateral renal arteries. The dose and timing of BH4 administration and the dose of SOD were designed based on the study by Kakoki et al. (13) and our previous study (5) , respectively. At 3, 6, and 12 h after reperfusion, renal arteries were clamped to prevent bleeding under pentobarbital anesthesia and kidneys were isolated. Parts of the renal cortex and medulla were immediately soaked in TRIzol reagent (Life Technologies, Rockville, MD, USA) and homogenized for RNA extraction. The other renal tissues were immediately frozen in liquid nitrogen and kept at –80°C. Then blood was sampled with heparinized syringe from abdominal artery. Rats in sham group also received 10 mL of sterilized Ringer’s solution after sham operation and were sampled at 4, 7, and 13 h in the same way as ischemia-reperfused groups.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the rat renal cortex and medulla using the guanidine isothiocyanate method with TRIzol reagent. RNA was transcribed with oligo dT primer and Moloney murine leukemia virus reverse transcriptase (Life Technologies). Product of reverse transcription was subjected to PCR with Taq DNA polymerase (Life Technologies). The reaction mixture contained 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide mixture, 0.2 µM each PCR primer, and 25 U/mL Taq DNA polymerase.

The primers used for LOX-1 mRNA were as follows (14) : sense primer 5'-GACTGGATCTGGCATAAAGA-3' and antisense primer 5'-CCTTCTTCTGACATATGCTG-3'. The PCR condition was as follows: after initial melting at 94°C for 2 min, there were 1 min of denaturation at 94°C, 1 min of annealing at 54°C, and 1 min of extension at 72°C for repeat of amplification. There were 28 amplification cycles for LOX-1, confirmed in pilot experiments as exponential phase of amplification. The PCR product was separated on 1.5% agarose gel and visualized with ethidium bromide under UV light. Gel image was captured with CCD camera system and subjected to densitometry analysis using NIH image software. The amount of cDNA was normalized by the amount of G3PDH cDNA.

The primers used for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA, a housekeeping gene product, were sense primer 5'-AAACCCATCACCATCTTCCA-3' and antisense primer 5'-CAGGGGTTTCTTACTCCTTG-3'. The PCR condition was as follows: after initial melting at 94°C for 2 min, there were 45 s of denaturation at 94°C, 30 s of annealing at 60°C, 90 s of extension at 72°C for repeat of amplification. The numbers of the amplification cycle were 16 cycles for G3PDH, which were confirmed in pilot experiments as exponential phase of amplification.

The following primers were used for inducible nitric oxide synthase (iNOS) mRNA,: sense primer 5'-GTCGACCTTCCGAAGTTTCTGGCAGCAGCG-3' and antisense primer 5'-GTCGACGAGCCTCGTGGCTTTGGGCTCCTC-3'. The PCR condition for iNOS was as follows: after initial melting at 94°C for 3 min, there were 45 s of denaturation at 94°C, 30 s of annealing at 60°C, and 90 s of extension at 72°C for 34 cycles repeat of amplification, followed by a final extension at 72°C for 5 min. The amount of iNOS cDNA was quantified by densitometric analysis described as above and normalized by the amount of G3PDH cDNA.

Immunoprecipitation/Western blot analysis
The degree of iNOS protein expression in rat kidney was determined by Western blot analyses following an immunoprecipitation procedure. Frozen renal tissues were homogenized with the Polytron in RIPA buffer containing 20 mM tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% Triton X-100, 1 mM EDTA and 0.1% protease inhibitor cocktail set III (Calbiochem, La Jolla, CA) in volumes of ninefold the tissue weight. The homogenates were centrifuged at 22,000 g for 10 min at 4°C. Equal amounts of tissue protein in the supernatant fractions (typically ~700 µg) were incubated with 4 µg of an anti-iNOS polyclonal antibody (Transduction Laboratories, Lexington, KY, USA), followed by an incubation with protein A/G Sepharose with rocking, each at 4°C for 1 h. After the Sepharose beads being extensively washed, they were boiled with 50 µL of 2 x sample buffer (Laemmli’s condition) for 5 min. After the Sepharose pellets had been discarded, samples were separated by SDS-PAGE on a 7% gel and electroblotted to a nitrocellulose membrane. The membrane was incubated with an anti-iNOS monoclonal antibody (Transduction Laboratories), followed by incubation with secondary antibody conjugated to horseradish peroxidase, each in Tris-buffered saline containing 1% milk, for 1 h at room temperature. Immunoreactive bands were visualized by using SuperSignal substrate (Pierce, Rockford, IL, USA) with exposure to X-ray films (Fuji, Tokyo, Japan) following the supplier’s protocol.

Measurement of nitrate plus nitrite and L-arginine
Blood samples were centrifuged for 5 min at 1200 x g. The plasma fraction and supernatant of kidney tissue homogenates were used for nitrate plus nitrite and arginine assay. For nitrate plus nitrite assay, sample was diluted 1:3 with nitrite/nitrate-free distilled water, then 0.4 mL of diluted sample was centrifuged at 5000 x g with a micropore filter (Ultrafree MC microcentrifuge device, UFC3, Millipore, MA, USA) to remove substances larger than 10 kDa. The micropore filter had been washed with distilled water before applying the sample. The filtrates were analyzed with an automated procedure based on the Griess reaction (Flow Injector Analyzer model TCI NOx, Tokyo Kasei Co., Tokyo, Japan) (15) . After reducing nitrate to nitrite through a copper-plated cadmium column, the absorbance at 540 nm was measured after the reaction with the Griess reagent. The value was expressed as the total of NO end products, nitrite plus nitrate. L-arginine concentration was analyzed by reversed-phase HPLC (Pico-TagR, Waters, MA, USA), after precolumn derivatization with phenylisothiocyanate using UV detection. Nitrite plus nitrate and L-arginine concentrations were expressed as µM based on tissue weight.

Statistical analyses
Data were given as mean ± SE. Data were analyzed with ANOVA, followed by Fisher’s post hoc test. Significance was taken at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We obtained a preliminary result by performing RT-PCR that supports our hypothesis that LOX-1 is up-regulated after ischemia reperfusion of bilateral kidneys. Then we followed the time course. After 1 h ischemia of bilateral kidneys and 3 h reperfusion (denoted as 4 h), the mRNA of LOX-1 was significantly expressed in renal cortex compared with that of time 0 and sham operation (Fig. 1A ). The mRNA level after reperfusion of 12 h (expressed as 13 h) was further increased compared with that at 4 h. There was no increase among sham groups. The increase in LOX-1 mRNA expression in the renal medulla was slow (Fig. 1B ) compared with the cortex (P<0.005 at 4 h). The level was substantially increased at 7 h after the ischemia reperfusion. However, it decreased at 13 h as contrasted with the cortex.



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  		Figure 1. Time course of renal cortex (A) and medulla (B) LOX-1 mRNA. Time 4, 7, and 13 h denote bilateral renal ischemia of 1 h plus reperfusion ({blacksquare}) of 3 (n=3), 6 (n=4), and 12 h (n=3), respectively. Sham groups ({square}) were analyzed at 0, 4, 7, and 13 h (n=3 each) after sham operation. *P < 0.05, ** P < 0.005, and ***P < 0.0001 vs. both time 0 and sham; #P < 0.05 vs. time 4; ##P < 0.005 vs. time 4 h and 13 h; =P < 0.05 vs. sham.

We previously revealed that superoxide and nitric oxide (NO) are generated early after recirculation after middle cerebral arterial occlusion (5) . Superoxide and NO would be generated early after recirculation of renal arteries, which may increase and decrease oxidized LDL, respectively. We therefore examined effects of SOD, NOS cofactor BH4, and the substrate L-arginine on LOX-1 mRNA expression at 7 h after the ischemia reperfusion. SOD did not statistically lower the LOX-1 mRNA expression in cortex and medulla (n=3, data not shown). BH4 decreased the LOX-1 mRNA expression in cortex, but not in medulla (Fig. 2 A). L-Arginine decreased the LOX-1 mRNA expression in cortex and medulla (Fig. 2B ).



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  		Figure 2. Effect of tetrahydrobiopterin (BH4) (A) and L-arginine (B) on renal cortex and medulla LOX-1 mRNA. Sham, IR, + BH4, and + Arg denote 7 h after sham operation (n=3), bilateral renal ischemia of 1 h plus reperfusion of 6 h (n=4), IR with BH4 (n=3), and IR with the L-arginine supplement (n=4), respectively. *P < 0.05 vs. IR (cortex); **P < 0.01 vs. IR (medulla).

Because NOS substrate L-arginine decreased LOX-1 mRNA expression, we assumed that the L-arginine supply increased NO, which works as a chain breaker of lipid peroxidation. We examined this effect by measuring the plasma NO end product, nitrite plus nitrate. Before that, we examined whether the renal ischemia reperfusion itself increases plasma NO. Plasma nitrite plus nitrate was increased by the ischemia reperfusion at 7 and 13 h vs. 0 h. Significant increase in plasma nitrite plus nitrate compared with sham was noticed 7 h after the ischemia reperfusion (Fig. 3A ). There was no statistical difference in the plasma nitrite plus nitrate among sham groups. We examined at 7 h (ischemia of 1 h plus reperfusion of 6 h) whether addition of BH4, SOD, or L-arginine modulates the amount of plasma NO end product. SOD did not significantly decrease plasma nitrite plus nitrate level. NOS cofactor BH4 did not significantly modify the level of plasma NO end product. Surprisingly, NOS substrate L-arginine did not increase, but notably decreased, plasma nitrite plus nitrate below to the sham level (Fig. 3B ).



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  		Figure 3. Time course of plasma nitrate (A) and effect of arginine, tetrahydrobiopterin (BH4), and superoxide dismutase (SOD) on plasma nitrate (B) after ischemia (1 h) reperfusion (6 h). A) Times 4, 7, and 13 h denote bilateral renal ischemia of 1 h plus reperfusion ({blacksquare}) of 3 (n=3), 6 (n=4), and 12 h (n=3), respectively. Sham groups ({square}) were analyzed at 0, 4, 7, and 13 h (n=3 each) after sham operation. **P < 0.005 vs. time 0 h; #P < 0.05 vs. time 4 h; =P < 0.05 vs. sham. B) Sham, IR, + Arg, + BH4, and + SOD denote 7 h after sham operation (n=3), ischemia of 1 h plus reperfusion of 6 h (n=4), IR with L-arginine (n=4), IR with BH4 (n=3), and IR with SOD supplement (n=3), respectively. *P < 0.05 and **P < 0.01 vs. IR.

We thus pursued iNOS mRNA expression to reveal why this arginine effect occurred. We first examined the time course of iNOS mRNA expression. The mRNA of iNOS was highly expressed at 7 h after the ischemia reperfusion in cortex (Fig. 4A ) and medulla (Fig. 4B ). The mRNA expression of iNOS was entirely decreased in medulla at 13 h after the ischemia reperfusion, but only partially in cortex. We examined the effect of L-arginine supply at 7 h after the ischemia reperfusion. L-Arginine extensively decreased the iNOS mRNA expression in cortex and in medulla at 7 h after the ischemia reperfusion (Fig. 5 ). Furthermore, L-arginine decreased iNOS protein expressed in kidney after ischemia of 1 h plus reperfusion of 6 h (Fig. 6 ).



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  		Figure 4. Time course of renal cortex (A) and medulla (B) iNOS mRNA (n=3). Times 4, 7, and 13 denotes bilateral renal ischemia of 1 h plus reperfusion ({blacksquare}) of 3, 6 and 12 h, respectively. Sham groups ({square}) were analyzed at 0, 4, 7, and 13 h after sham operation. **P < 0.005 vs. sham, time 0 and 4 h; ***P < 0.0001 vs. sham, time 0, 4, and 13 h; #P < 0.05 vs. time 0, 4, and 7 h.



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  		Figure 5. Effect of L-arginine on renal cortex ({square}) and medulla iNOS mRNA ({blacksquare}) (n=3). Sham, IR, and + Arg denote 7 h after sham operation, bilateral renal ischemia of 1 h plus reperfusion of 6 h, and IR with the L-arginine supplement, respectively. **P < 0.01 vs. IR (cortex); *P < 0.05 vs. IR (medulla).



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  		Figure 6. Immunoblots of iNOS protein. Rats were subjected to sham operation (sham, n=3), or bilateral renal ischemia of 1 h plus reperfusion of 6 h of kidneys without (IR, n=3) or IR with L-arginine supplementation (+ Arg, n=3). Protein samples derived from those kidneys were processed as described in detail in Materials and Methods using a standard immunoprecipitation technique. Immunoprecipitates were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with an anti-iNOS monoclonal antibody. Arrow indicates immunoreactive bands corresponding to iNOS proteins (~130 kDa), where we also detected robust signals using positive human macrophage-derived control samples provided by the supplier of the antibody (data not shown). Shown are the results from a representative data from an experiment repeated independently 3 times with equivalent results.

To explain these results, we proposed a hypothesis that a deficiency in L-arginine due to ischemia induces uncoupling of constitutive NOS during early reperfusion that triggers iNOS induction. To verify this possibility, we examined both arginine and NO levels directly in renal tissues in the earliest phase of ischemia-reperfusion. Ischemia of 1 h plus 5 min reperfusion decreased L-arginine concentrations in renal cortex and medulla compared with sham group (Fig. 7 ). L-Arginine supplementation increased the plasma concentration of L-arginine about ninefold at 5 min and fivefold at 45 min after the reperfusion (Fig. 8 ). The inflow of at least ninefold levels of L-arginine after opening the occlusions restored the decreased L-arginine levels in renal cortex and medulla within 5 min after reperfusion (Fig. 7) . However, the effect of the L-arginine supplement was more distinct in renal cortex than in medulla: the inflow of high L-arginine concentration strongly reversed L-arginine levels at 5 and 45 min after reperfusion in cortex. On the other hand, the recovery of medulla in L-arginine levels was weak. The high L-arginine tissue levels increased NO end products in renal cortex after reperfusion of 45 min (Fig. 9 ) whereas there was no difference in the NO end products in renal medulla (data not shown).



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  		Figure 7. L-Arginine concentration (µM) in renal cortex and medulla. Rats were subjected to sham operation (sham, n=3) or bilateral renal ischemia of 1 h plus reperfusion of 5 (IR 5 min, n=3) or 45 min (IR 45 min, n=4) without or with L-arginine supplementation (+ Arg). **P < 0.01, *P < 0.05 vs. sham; ###P < 0.0001 vs. IR 5 min and sham, ##P <0.005, #P < 0.05 vs. IR 5 min.



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  		Figure 8. Effect of an L-arginine supplement during ischemia reperfusion on plasma L-arginine concentration (µM). Rats were not (before IR, n=4) or were subjected to bilateral renal ischemia of 1 h plus reperfusion of 5 (IR 5 min, n=3), 45 min (IR 45 min, n=4), or 6 h (IR 6 h, n=4) without or with L-arginine supplementation (+ Arg). ***P < 0.0001 vs. all except IR 45 min + Arg (**P < 0.005).



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  		Figure 9. Effect of L-arginine supplement (+ Arg) on nitrate plus nitrite concentration (µM) in renal cortex after bilateral renal ischemia of 1 h plus reperfusion of 5 (IR 5 min, n=3) or 45 min (IR 45 min, n=4). *P < 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we found that ischemia-reperfusion in kidney increased mRNA expression of LOX-1 at 7 h, which was down-regulated by the L-arginine supplementation. We then examined plasma NO end products to explain this L-arginine effect. Plasma nitrite plus nitrate was increased at 7 h, which was unexpectedly suppressed by the L-arginine supplement. Furthermore, L-arginine addition decreased the iNOS mRNA and protein expression at 7 h. We imagined ischemia causes intracellular deficiency of available L-arginine, which results in uncoupling of constitutive NOS after reperfusion. In fact, ischemia decreased L-arginine concentrations in renal cortex and medulla. By L-arginine supplementation, the inflow of high levels of L-arginine in the earliest phase of reperfusion powerfully raised L-arginine levels and NO end products in renal cortex. To our knowledge, this is the first report revealing that ischemia-reperfusion causes a deficiency of L-arginine available for NOS and subsequent uncoupling of constitutive NOS that triggers iNOS and LOX-1 induction.

Ox-LDL leads to endothelial activation, dysfunction, and injury. Activation of endothelial cells results in expression of a variety of genes and of adhesion molecules to which inflammatory cells attach, followed by cell rolling and subendothelial migration of inflammatory cells (16) . Ox-LDL induces inhibition of endothelial NOS (eNOS) localization/activation in caveolae, and high-density lipoprotein binding to scavenger receptor activates eNOS, which might be critical to the atheroprotective properties (7 , 17) . LOX-1 is expressed in proatherogenic circumstances such as hypertension, hyperlipidemia and diabetes and is indeed accumulated in atherosclerotic lesions (14 , 18 , 19) . This receptor can support specific binding, internalization, and proteolytic degradation of ox-LDL. It does not bind for acetylated LDL (20) . LOX-1 is present in the endothelium of human coronary arteries, rabbit and rat aortae and bovine endothelial cells (10 , 19 , 21 , 22) .

Reperfusion after ischemia of organ accompanies superoxide generation that will accelerate lipid peroxidation and ox-LDL generation. In addition, ox-LDL induces proatherosclerotic NAD(P)H oxidase expression and thus superoxide anion formation in human endothelial cells (23) , although NADPH oxidase does not contribute to the development of myocardial injury and dysfunction after ischemia reperfusion compared with NADPH oxidase-deficient mice (24) . NO is working as a terminator of radical chain propagation reactions in lipid peroxidation (25) . NO generated from added L-arginine can suppress oxidation of LDL by working as a terminator of lipid peroxidation and can decrease LOX-1 mRNA expression. Our data show that scavenging extracellular superoxide by SOD was without effect; BH4 decreased LOX-1 mRNA expression only in cortex, and L-arginine addition was more effective, i.e., L-arginine decreased LOX-1 mRNA expression in medulla, though not entirely.

Previously we showed that supplementation with L-arginine, the substrate for NOS, increased NO end product nitrate and improved hypertension (26) . On the contrary, L-arginine fully decreased the plasma NO end product as well as iNOS mRNA and protein expression in the present study. In the ischemia reperfusion, since iNOS induction is the late event (Fig. 4) , constitutive NOS must play major roles in the earliest reperfusion phase. Immunohistochemistry and in situ hybridization evidence indicates that nNOS is expressed at high levels in the macula densa (27) . The renovascular endothelium is the major site of renal expression of eNOS. We speculate that a deficiency in L-arginine early after ischemia reperfusion induces uncoupling of constitutive NOS, which results in ROS generation (28 29 30) and leads to oxidation of LDL and to triggering iNOS induction, and that L-arginine supplementation inhibits the compensatory iNOS induction by stopping the uncoupling of constitutive NOS. Ischemia decreased L-arginine concentrations in renal cortex and medulla (Fig. 7) . L-Arginine supplementation counteracted the decrease in L-arginine levels and increased NO end products in renal cortex in the earliest phase of ischemia-reperfusion (Fig. 9) . The results also demonstrate the so-called "L-arginine paradox," i.e., L-arginine supplementation stimulates NO synthesis despite saturating intracellular concentrations (31) . The results thus suggest directed delivery of extracellular L-arginine can stop the uncoupling of constitutive NOS. Since iNOS generates large amounts of NO independent of calcium compared with constitutive NOS, L-arginine inhibition of iNOS induction would lead to an obvious decrease in plasma NO end product. Our data that not only SOD but also BH4 failed to decrease plasma NO end products at 7 h after ischemia reperfusion (Fig. 3B ) suggest that BH4 did not inhibit uncoupling of NOS in the earliest phase of ischemia-reperfusion, similar to a recent in vitro study that, in the absence of or under nonsaturating levels of L-arginine, activated NOS generate superoxide whether or not the enzyme contained bound BH4 (32) . Atherosclerotic changes involve iNOS expression (33 , 34) . There have been a variety of reports (35 36 37 38) regarding whether iNOS is harmful or beneficial. Our results suggest that a deficiency in L-arginine and thus uncoupling of constitutive NOS induces iNOS as a compensatory mechanism to supply NO. In this sense, we assume iNOS is beneficial.

In the present study, we revealed that ischemia reperfusion of kidney increased expression of LOX-1 mRNA, plasma NO end product, and iNOS at 7 h, which were suppressed by the L-arginine supplement. In the earliest phase of reperfusion, L-arginine was decreased in renal tissues, and L-arginine supply increased NO end products in renal cortex. These results suggest that ischemia-reperfusion causes a deficiency of L-arginine available for NOS and subsequent uncoupling of constitutive NOS that induces iNOS and LOX-1. Our results imply that L-arginine is effective in preventing atherosclerotic progress in transplantation and stroke, even if NO production is not amended by the L-arginine supplement, and that effects of NOS substrate L-arginine and exogenous therapeutic NO donors are different.

Received for publication June 19, 2002. Accepted for publication December 20, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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