J Am Soc Nephrol 11: 301–309, 2000 Effects of Tetrahydrobiopterin on Endothelial Dysfunction in Rats with Ischemic Acute Renal Failure

MASAO KAKOKI,* YASUNOBU HIRATA,* HIROSHI HAYAKAWA,* ETSU SUZUKI,* DAISUKE NAGATA,* AKIHIRO TOJO,* HIROAKI NISHIMATSU,* NOBUO NAKANISHI,‡ YOSHIYUKI HATTORI,§ KAZUYA KIKUCHI,† TETSUO NAGANO,† and MASAO OMATA* *The Second Department of Internal Medicine, Faculty of Medicine, and †Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan; ‡Department of Biochemistry, Meikai University School of Dentistry, Saitama, Japan; and §Department of Endocrinology, Dokkyo University School of Medicine, Tochigi, Japan.

Abstract. The role of nitric oxide (NO) in ischemic renal injury 4 fmol/min per g kidney; serum creatinine: control 23 Ϯ 2, is still controversial. NO release was measured in rat kidneys ischemia 150 Ϯ 27, ischemia ϩ BH4 48 Ϯ 6 ␮M; mean Ϯ subjected to ischemia and reperfusion to determine whether SEM). Most of renal NOS activity was calcium-dependent, and (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), a cofactor of NO its activity decreased in the ischemic kidney. However, it was synthase (NOS), reduces ischemic injury. Twenty-four hours restored by BH4 (control 5.0 Ϯ 0.9, ischemia 2.2 Ϯ 0.4, after bilateral renal arterial clamp for 45 min, acetylcholine- ischemia ϩ BH4 4.3 Ϯ 1.2 pmol/min per mg protein). Immu- induced vasorelaxation and NO release were reduced and renal noblot after low-temperature sodium dodecyl sulfate-polyac- excretory function was impaired in Wistar rats. Administration rylamide gel electrophoresis revealed that the dimeric form of of BH4 (20 mg/kg, by mouth) before clamping resulted in a endothelial NOS decreased in the ischemic kidney and that it marked improvement of those parameters (10Ϫ8 M acetylcho- was restored by BH4. These results suggest that the decreased line, ⌬renal perfusion pressure: sham-operated control Ϫ45 Ϯ activity of endothelium-derived NO may worsen the ischemic 5, ischemia Ϫ30 Ϯ 2, ischemia ϩ BH4 Ϫ43 Ϯ 4%; ⌬NO: tissue injury, in which depletion of BH4 may be involved. control ϩ30 Ϯ 6, ischemia ϩ10 Ϯ 2, ischemia ϩ BH4 ϩ23 Ϯ

Nitric oxide (NO) participates in the physiologic regulation of Endothelium-derived NO, which causes vasodilation, re- cardiovascular and renal functions. In this respect, NO has duces intracellular calcium, and inhibits leukocyte adhesion to been suggested to play a pivotal role in ischemic acute renal vessels (4) and platelet aggregation (5), probably exerts a failure (iARF). However, there have been conflicting reports protective action on ischemia-induced tissue damage. On the on the amount of NO itself and its origins. Chintala et al. (1) other hand, NO derived from iNOS has been considered to reported that NO synthase (NOS) inhibition by NG-monometh- enhance the tissue damage observed in ischemia-reperfusion yl-L-arginine worsened renal ischemic injury, whereas L-argi- injury, partly via the formation of peroxynitrite anion, an nine mitigated it, suggesting that a reduction in NO release NO-derived oxidant formed from the interaction of NO with may be involved in ischemia-induced tissue damage. Conger et superoxide (6–8). However, administration of exogenous NO al. (2) observed maximal release of endothelium-derived NO donors has been shown to be unequivocally beneficial against in postischemic rat kidneys because endothelium-dependent ischemia-reperfusion insults (9,10). renal vasodilation was reduced and renal vessels constricted to It has been demonstrated that (6R)-5,6,7,8-tetrahydro-L- ␻ a greater extent by N -nitro-L-arginine methyl ester (L- biopterin (BH4), a cofactor of all isoforms of NOS, is a NAME), but not by aminoguanidine, an antagonist of inducible rate-limiting factor of NO production by iNOS (11). Several NOS (iNOS). On the other hand, Noiri et al. (3) showed that cytokines induce not only iNOS but also guanosine triphos- iNOS-derived NO increased in ischemic kidneys and that in- phate cyclohydrolase-1, which is the rate-limiting enzyme for hibition of iNOS expression decreased the tubular damage. de novo synthesis of BH4 (12), and it may lead to an enhanced production of NO in sepsis (13). It has also been shown that BH4 is essential for NO production by endothelial NOS Received February 18, 1999. Accepted July 11, 1999. (eNOS) (14,15) and that in patients with hypercholesterolemia Correspondence to Dr. Yasunobu Hirata, The Second Department of Internal (16), the administration of BH4 improves endothelial dysfunc- Medicine, The University of Tokyo, 7–3-1 Hongo, Bunkyo-ku, Tokyo 113- tion. ϩ ϩ 8655, Japan. Phone: 81–3-5800–8649; Fax: 81–3-3814–0021; E-mail: Thus, in the present study we investigated whether NO [email protected] release increased or decreased, and examined the origin of NO 1046-6673/1102-0301 Journal of the American Society of Nephrology in rats with iARF. Furthermore, we examined whether BH4 Copyright © 2000 by the American Society of Nephrology exerts beneficial effects on ischemic injury in the rat kidney. 302 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 301–309, 2000

Materials and Methods Measurement of NOS Activity Animals The activity of NOS in vitro was determined by the conversion of 14 14 The in vivo model of renal ischemia was prepared in 12-wk-old L-[ C] arginine to L-[ C] citrulline, according to the method de- Wistar rats weighing 313 Ϯ 10 g by clamping both renal arteries for scribed by Bredt and Snyder (27). The renal medulla was dissected 45 min; then clamps were removed and the rats developed ischemic after sagittal section of the left kidney in ice-cold phosphate-buffered Ϫ renal failure 24 h later. The NOS substrate L-arginine (1 g/kg), the saline and then frozen in liquid nitrogen. Samples were kept at 70°C NOS cofactor BH4 (5 or 20 mg/kg; Allexis, San Diego, CA), 2,4- until assayed. Frozen samples were homogenized using a Polytron diamino-6-hydroxypyrimidine (DAHP; 1 g/kg), which is an inhibitor homogenizer in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.1 mM of guanosine 1,4,5-triphosphate cyclohydrolase-1, or the NOS inhib- mercaptoethanol, 0.1 mM ethylenediaminetetra-acetic acid, 0.1 mM ␤ Ј itor L-NAME (30 mg/kg) was administered by gavage to some of the ethyleneglycol-bis( -aminoethyl ether)-N,N -tetra-acetic acid [EGTA], 2 rats just before anesthesia with pentobarbital, i.e., 5 min before ␮M leupeptin, 1 ␮M pepstatin A, 1 mM phenylmethylsulfonyl fluo- bilateral renal arterial clamping. The effects of BH4 (20 mg/kg) when ride). Forty microliters of the sample was incubated in duplicate in 14 administered 1 h after reperfusion were also examined. The doses of 100 ␮l of assay buffer containing 0.5 ␮Ci/ml L-[ C] arginine and BH4 and DAHP were determined as described in previous reports incubated for 20 min at 37°C. The reaction was stopped with the (13,17). All animal studies were performed in conformance with the addition of 500 ␮l of ice-cold buffer (20 mM Hepes, 2 mM ethyl- Guide for Animal Experimentation (Faculty of Medicine, The Uni- enediaminetetra-acetic acid, 2 mM EGTA, pH 5.5). To separate 14 14 versity of Tokyo). L-[ C] arginine from L-[ C] citrulline, samples were loaded onto 1-ml columns of Dowex resin (AG50WX-8 Naϩ form) and eluted with 500 ␮l of distilled water. Aliquots were used for liquid scintil- Measurement of Biopterin Levels in Plasma and Renal lation counting. Calcium-dependent activity was determined as the 14 Tissues in Rats with iARF difference between the L-[ C] citrulline produced in control samples To confirm that orally administered BH4 reached the kidneys of and samples containing 3 mM EGTA to bind calcium. Calcium- rats with iARF, we measured the levels of biopterin in the plasma and independent activity was determined as the difference between the 14 kidneys after administration of BH4 or vehicle by gavage to rats L-[ C] citrulline produced from samples containing 3 mM EGTA and subjected to 45 min ischemia and 3 h reperfusion, by differential samples containing both 3 mM EGTA and 1 mM L-NNA. Activity of iodine oxidation in acid and base, using reversed-phase high perfor- the enzyme was normalized by protein content determined by the mance liquid chromatography (18–20). Bradford method (Bio-Rad, Richmond, CA), using bovine serum albumin as the standard. Isolated Perfused Kidney and Measurement of NO The kidneys of rats with iARF were isolated and perfused as Immunoblot Analysis for NOS Protein described elsewhere to evaluate renovascular endothelial function Each kidney was excised and homogenized on ice in 3 ml of lysis (21,22). The kidneys were perfused with Krebs-Henseleit buffer at Ϫ6 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 37°C and saturated with 95% O2/5% CO2, which contained 10 M Ϫ and 10% glycerol, 2 ␮g/ml leupeptin, 2 ␮g/ml aprotinin, 1 mM phenylephrine and 10 5 M indomethacin, at a constant flow of 5 Ϫ phenylmethylsulfonyl fluoride), using a Polytron homogenizer. The ml/min. Phenylephrine at 10 6 M was used for the precontraction of homogenates were centrifuged at 12,000 ϫ g at 4°C for 20 min. After renal vasculature to efficiently examine the effects of acetylcholine determining the protein content, appropriate volumes of the superna- (ACh) on the change in renal perfusion pressure (RPP). This dose was tant (200 ␮g/lane) were mixed with an equal volume of 2ϫ sample selected to maintain the RPP at a level similar to the in vivo RPP. buffer (100 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate [SDS], Indomethacin was added to exclude the effects of prostanoids on RPP 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromphenol blue) and because previous studies have suggested that the effect of ACh on the subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using tone of vasculature is partly mediated by prostanoids (23,24). Renal 6% acrylamide gels. For immunoblot analysis of monomer of eNOS arterial pressure was continuously monitored with a polygraph re- and iNOS, samples were heated at 98°C for 5 min before electro- corder and maintained at about 100 mmHg. After a 60-min equilibra- phoresis (28). For immunoblot analysis of the dimeric form of eNOS, tion period, we investigated the effects of the vehicle, cumulative ␻ samples were not heated and the temperature of the gel was main- doses of ACh, and N -nitro-L-arginine (L-NNA) on RPP and NO tained below 15°C during electrophoresis (low temperature SDS- release. The effluent perfusate from the renal vein was collected PAGE [LT-PAGE]) (29). The proteins were transferred by semidry continuously (2 ml/min), mixed with a chemiluminescence probe (0.5 electroblotting to polyvinylidene difluoride membranes for 120 min. ml/min; 2 mM H O ,18␮M luminol, 2 mM potassium carbonate, and 2 2 The blots were then blocked and incubated with either anti-eNOS 150 mM desferrioxamine), and then forwarded into a chemilumines- antibody (IgG monoclonal antibody; 1:100 dilution; Transduction cence analyzer (825-CL; Jasco Corp., Tokyo, Japan) to measure the 1 Laboratories, Lexington, KY) or anti-iNOS antibody (IgG mono- NO output (25). 2a clonal antibody, 1:500 dilution; Transduction Laboratories) for 60 min at room temperature. To detect bound antibodies, the blots were Histologic Studies incubated with horseradish peroxidase-linked rabbit anti-mouse sec-

Samples of renal tissue from the control, ischemic, and BH4- ond antibody (IgG1 polyclonal antibody; 1:7000 dilution; Amersham, treated (20 mg/kg, orally, before ischemia) animals were fixed in 10% Little Chalfont, United Kingdom) for 60 min at room temperature, and formaldehyde, stained with periodic acid-Schiff reagent, and exam- the antibody was visualized using an enhanced chemiluminescence ined under an optical microscope in a blinded manner by a pathologist method (ECL; Amersham). The integrated density (density ϫ area) of (A.T.). The kidneys were examined for the presence of tubular dila- the bands was quantified using NIH Image 1.61 software. For the tion, necrosis, casts, interstitial edema, and brush border loss. The immunoblot analysis of iNOS, we prepared two kinds of positive degree of renal injury was evaluated using the criteria established by controls; i.e., mouse macrophages and the kidney of rats treated with Solez et al. (26). lipopolysaccharide (20 mg/kg, intraperitoneally) for 6 h. J Am Soc Nephrol 11: 301–309, 2000 BH4 in Ischemic Renal Failure 303

Figure 1. Levels of biopterin in the plasma and kidneys of rats with ischemic acute renal failure (iARF) 3 h after oral administration of ‡ (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4). *P Ͻ 0.05, P Ͻ 0.01 versus vehicle.

Statistical Analyses Data are expressed as the mean Ϯ SEM. Statistical comparisons were made with ANOVA, followed by the Student-Neumann-Keuls test. To compare tissue damage scores, the Mann–Whitney U test was used. Differences with a P value Ͻ0.05 were considered statistically significant.

Results Biopterin Levels in Plasma and Renal Tissues in Rats with iARF As shown in Figure 1, the levels of biopterin in the plasma and kidneys of rats with iARF increased in a dose-dependent manner after oral administration of BH4, which confirmed that orally administered BH4 reached the kidneys of rats with iARF during the reperfusion period.

Effects of Ischemia on RPP and NO in Wistar Rats Figures 2 and 3 illustrate the changes in RPP and NO in response to ACh and L-NNA in the isolated perfused kidney after 45 min ischemia and 24 h reperfusion. As shown in Table 1, the RPP in the ischemic group was slightly lower. However, the differences in the achieved RPP values among groups were not significant when assessed by one-way ANOVA. In the control, sham-operated rat kidney, ACh increased NO release and reduced RPP in a dose-dependent manner; the effects of ACh were antagonized by L-NNA. On the other hand, the changes in RPP and NO induced by ACh or L-NNA were significantly attenuated in ischemic kidneys. Pretreatment with Figure 2. Tracings of nitric oxide (NO) release and renal perfusion BH4 potentiated the effects of ACh on RPP and NO release in pressure (RPP) during infusion of acetylcholine (ACh) and N␻-nitro- a dose-dependent manner. In particular, 20 mg/kg BH4 re- L-arginine (L-NNA) in the isolated kidney of Wistar rats with iARF. stored the parameters of endothelial function to levels similar control, sham-operated rats; ischemia ϩ BH4, rats administered 20 to those of the control kidney. Administration of BH4 1 h after mg/kg BH4 orally before ischemia. reoxygenation also significantly restored renal responses to ACh, although the extent of the improvement was smaller. However, pretreatment with BH4 did not influence the re- These effects of BH4 were confirmed by changes in serum sponses to ACh in sham-operated controls. Pretreatment with urea nitrogen and creatinine levels. As shown in Figure 4, the L-arginine did not affect the responses to ACh in ischemic level of urea nitrogen in rats with iARF was much higher than kidneys. in the control rats (35 Ϯ 2 versus 8 Ϯ 1mM,P Ͻ 0.01). 304 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 301–309, 2000

Figure 3. Effects of ACh and L-NNA on RPP and NO in Wistar rats with iARF. pre 5 and pre 20, rats administered 5 and 20 mg/kg BH4 orally before ischemia, respectively; post 20, rats administered 20 mg/kg BH4 by gavage 1 h after reoxygenation; *P Ͻ 0.05, ‡P Ͻ 0.01 versus sham-operated control; §P Ͻ 0.05, ʈP Ͻ 0.01 versus ischemia. Each group consists of five or six rats.

However, administration of BH4 prevented the increase in urea Effects of Ischemia on NOS Activity and Expression of nitrogen substantially (16 Ϯ 2 mM). Similar results were eNOS and iNOS obtained in the case of serum creatinine levels. In contrast, In Wistar rats, calcium-dependent NOS activity, which was L-arginine did not affect urea nitrogen or creatinine in iARF. measured based on the conversion of L-arginine to L-citrulline, As shown in Figure 5, DAHP, an inhibitor for de novo significantly decreased in the medulla of the kidney in iARF, synthesis of BH4, attenuated the effects of ACh on RPP and and it was partially reversed by treatment with BH4 (Figure 7). NO release in the sham-operated kidney. However, DAHP did On the other hand, calcium-independent NOS activity was not not affect the responses to ACh in iARF. The NOS inhibitor detected in the kidney of any group of animals. L-NAME diminished the responses to ACh in both the sham- The expression of eNOS and iNOS protein in iARF was operated kidney and in iARF. This effect of L-NAME was evaluated in the whole kidney of Wistar rats. In conventional associated with further increases in urea nitrogen and creati- immunoblot analysis (Figure 8A), the immunoreactivity of nine in iARF (Figures 4 and 5). total eNOS protein significantly increased in iARF, and it was not significantly affected by BH4 (n ϭ 8 each; iARF 196 Ϯ 26; Histopathologic Analysis of Kidneys with Ischemia- iARF ϩ BH4 250 Ϯ 48% of optical densities in the control; Reperfusion Injury both P Ͻ 0.05 versus control). However, in immunoblot anal- Ischemia-induced morphologic changes, including tubular ysis after LT-PAGE (Figure 8B), the immunoreactivity of the necrosis, tubular casts, interstitial edema, tubular dilatation, eNOS dimer significantly decreased in iARF, which was re- and brush border loss, were clearly observed in the ischemic stored by BH4 (n ϭ 6 each; iARF 64 Ϯ 9; P Ͻ 0.01 versus kidney of Wistar rats. BH4 significantly reduced the scores for sham-operated control; iARF ϩ BH4 124 Ϯ 20% of optical the renal tissue damage (Figure 6). densities in the control; P Ͻ 0.05 versus iARF). iNOS was not J Am Soc Nephrol 11: 301–309, 2000 BH4 in Ischemic Renal Failure 305

Table 1. Values of body weight (BW), kidney weight (KW), baseline renal perfusion pressure (RPP), and renal perfusion rate (RPR) in all groups of ratsa

Group n BW (g) KW (g) RPP (mmHg) RPR (ml/min)

Sham 5 313 Ϯ 10 1.36 Ϯ 0.07 107 Ϯ 5 5.0 ϩ Ϯ Ϯ Ϯ Sham BH4 5 339 14 1.45 0.01 97 2 5.0 Sham ϩ DAHP 4 333 Ϯ 10 1.45 Ϯ 0.09 97 Ϯ 4 5.0 Sham ϩ L-NAME 6 302 Ϯ 11 1.37 Ϯ 0.06 95 Ϯ 5 5.0 Ischemia 5 309 Ϯ 9 1.36 Ϯ 0.08 87 Ϯ 2 5.0 ϩ Ϯ Ϯ Ϯ Ischemia BH4 (pre; 5) 5 332 10 1.36 0.04 88 2 5.0 ϩ Ϯ Ϯ Ϯ Ischemia BH4 (pre; 20) 5 302 7 1.20 0.03 99 3 5.0 ϩ Ϯ Ϯ Ϯ Ischemia BH4 (post; 20) 6 322 7 1.43 0.03 95 5 5.0 Ischemia ϩ DAHP 5 313 Ϯ 5 1.36 Ϯ 0.03 85 Ϯ 6 5.0 Ischemia ϩ L-NAME 5 288 Ϯ 12 1.57 Ϯ 0.08 90 Ϯ 6 5.0 Ischemia ϩ L-arginine 5 319 Ϯ 16 1.53 Ϯ 0.07 97 Ϯ 2 5.0

a Ϯ Values are expressed as mean SEM. Sham, sham-operated rats; BH4, tetrahydrobiopterin; DAHP, 2,4-diamino-6-hydroxypyrimidine; ␻ L-NAME, N -nitro-L-arginine methyl ester; (pre; 5 or 20), oral administration of 5 or 20 mg/kg before ischemia; (post; 20), oral administration of 20 mg/kg after ischemia.

␻ ‡ Figure 4. Values of serum urea nitrogen and creatinine in rats with iARF. L-NAME, N -nitro-L-arginine methyl ester. *P Ͻ 0.05, P Ͻ 0.01 versus sham-operated control; §P Ͻ 0.05, ʈP Ͻ 0.01 versus ischemia. Each group consists of five or six rats.

detected in any group of rats, whereas it was clearly detected in constriction in response to L-NNA was attenuated. Calcium- mouse macrophages and in the kidneys from lipopolysaccha- dependent NOS activity was also attenuated. Furthermore, the ride-treated rats (Figure 8C). activity and expression of iNOS were very little in the kidney with iARF. These results indicate that basal and stimulated NO Discussion release from eNOS is reduced in the ischemic kidneys, al- In the present study, we found that in the isolated rat kidney though there have been conflicting reports about the amount of exposed to ischemia-reperfusion, vasorelaxation and NO re- NO and the isoform of NOS responsible for NO in ischemia. lease in response to ACh were markedly decreased, and vaso- It is striking that preischemic administration of BH4, a 306 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 301–309, 2000

Figure 7. Effects of ischemia and treatment with BH4 (20 mg/kg, orally) on NO synthase (NOS) activity in the kidneys of Wistar rats. ‡P Ͻ 0.01 versus sham-operated control; §P Ͻ 0.05 versus ischemia. Each group consists of five rats.

also significant when given 1 h after reperfusion, in contrast to a reported finding that preischemic administration of L-argi- nine, a NOS substrate, had a beneficial effect, whereas its Figure 5. Effects of ACh on RPP and NO in the isolated perfused postischemic administration was harmful (30). On the other kidney of rats administered 2,4-diamino-6-hydroxypyrimidine hand, BH4 had little effect on the endothelial function in (DAHP), an inhibitor of de novo synthesis of BH4, and L-NAME nonischemic controls. These results are in accord with the idea Ͻ ‡ Ͻ before induction of ischemia. *P 0.05, P 0.01 versus sham- that vessels exposed to ischemia-reperfusion are depleted from operated control; §P Ͻ 0.05, ʈP Ͻ 0.01 versus ischemia. Each group BH4 (31). It has been suggested that BH4 has a direct antiox- consists of five rats. idant effect and reduces reactive oxygen species (32), which would also contribute to its cytoprotective action in ischemic injury. However, it has also been shown that BH4 generates superoxide during its autooxidation (33,34) and exerts a sup- pressive effect on endothelium-dependent vasodilation under normal conditions (35). Therefore, the content of BH4 as a cofactor of NOS is intact in normal vessels, and the supple- mentation of BH4 does not have additional effects. However, the fact that administration of DAHP attenuated endothelial function under nonischemic conditions suggested that production of BH4 was important for the maintenance of endothelium-dependent relaxation, as previous studies have already demonstrated (31,36). Furthermore, DAHP did not affect the changes in RPP and NO release in response to ACh under ischemic conditions, indicating that de novo production Figure 6. Effects of ischemia and preischemic treatment with BH4 (20 of BH4 might be impaired in iARF. In contrast, administration mg/kg, orally) on the renal injury scores in the kidneys of Wistar rats. of L-NAME resulted in a deterioration of the endothelial func- *P Ͻ 0.05, ‡P Ͻ 0.01 versus ischemia. Each group consists of five tion under both nonischemic and ischemic conditions to a rats. similar extent. Thus, the induction of endogenous NOS inhib- itors (37,38) is not the main cause for endothelial dysfunction after ischemia. cofactor of NOS, dose-dependently restored not only endothe- The regulation of eNOS expression in hypoxia remains lium-dependent vasorelaxation, but also renal excretory func- controversial. Several studies have shown that hypoxia de- tion and morphologic changes in iARF. In contrast, adminis- creases eNOS expression in human umbilical vein endothelial tration of L-NAME resulted in a deterioration of the endothelial cells (39), in human pulmonary artery endothelial cells (40), function and renal excretory function under ischemic condi- and in bovine pulmonary artery endothelial cells (41). On the tions. These findings suggest that the reduction in endotheli- other hand, it has been reported that hypoxia increases eNOS um-derived NO is an important factor that accelerates renal protein in bovine aortic endothelial cells (42), hypoxic rat damage in iARF. lungs (43,44), and hypoxic rat systemic organs (45). The The beneficial effect of BH4 on endothelial function was discrepancies in the regulation of eNOS expression among J Am Soc Nephrol 11: 301–309, 2000 BH4 in Ischemic Renal Failure 307

Figure 8. Effects of ischemia and pretreatment with BH4 (20 mg/kg, orally) on the expression of endothelial NOS (eNOS) and inducible NOS (iNOS) protein in the whole kidney of Wistar rats. Conventional immunoblot for eNOS (A) and iNOS (C) and immunoblot after low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis for eNOS (B) are shown. M␾, mouse macrophages; LPS, the kidney of lipopolysaccharide-treated rats.

these studies may be due to differences in the experimental iNOS in rat cultured cardiac myocytes (49); rat cultured mes- preparation or the duration and severity of hypoxia. angial cells (50); rat liver, ventricles, lungs, and kidneys (45); In the present study, conventional immunoblot analysis re- and rat splanchnic artery (51). Additional studies are required vealed that total eNOS expression was increased in iARF, to elucidate the role of iNOS in iARF. which was unaltered by the supplementation of BH4. In con- In conclusion, endothelium-dependent vasorelaxation and trast, LT-PAGE revealed that the immunoreactivity for dimeric NO release were impaired in rat kidneys with iARF. The eNOS was decreased in iARF, which was restored by the endothelial dysfunction in iARF was associated with decreased supplementation of BH4. BH4 has been shown to stabilize NOS activity and decreased immunoreactivity of eNOS eNOS in its dimeric form, which is an active form (46,47). dimers. The expression of iNOS and calcium-independent Therefore, BH4 may determine the effective amount of eNOS NOS activity was very little in iARF. Exogenous supplemen- regardless of the total quantity of eNOS protein. These results tation of BH4 markedly restored endothelial function, renal suggest that the impairment of dimerization of eNOS by de- morphology, renal excretory function, NOS activity, and im- pletion in BH4 plays an important role in endothelial dysfunc- munoreactivity of eNOS dimers. These results suggest that the tion in iARF. It has also been demonstrated that L-arginine impairment of dimerization of eNOS by depletion in BH4 facilitates eNOS dimerization (29). However, administration of plays an important role in endothelial dysfunction and tissue L-arginine did not improve NO release in ischemic kidneys, damage in iARF. suggesting that L-arginine is not depleted in iARF. In view of the evidence provided by previous studies con- cerning the role iNOS plays in the pathogenesis of iARF, the Acknowledgments present results seem to be inconsistent with them. Because This study was supported in part by Grants-in-Aid 09281206 and BH4 has been shown to be a rate-limiting factor of the activity 10218202 from the Japanese Ministry of Education, Culture, and of iNOS (11), supplementation of BH4 would aggravate tissue Science. damage. However, at least in the present preparation, BH4 restored endothelium-dependent relaxation, renal excretory References function, and renal morphology. Moreover, iNOS protein and 1. Chintala MS, Chiu PJ, Vemulapalli S, Watkins RW, Sybertz EJ: calcium-independent NOS activity were very scant in iARF Inhibition of endothelial derived relaxing factor (EDRF) aggra- and in BH4-treated iARF rats. It has been suggested that iNOS vates ischemic acute renal failure in anesthetized rats. Naunyn- is induced in hypoxia and that hypoxia-responsive elements Schmiedebergs Arch Pharmacol 348: 305–310, 1993 exist in the promoter region of mouse iNOS gene (48). How- 2. Conger J, Robinette J, Villar A, Raij L, Shultz P: Increased nitric ever, some authors have shown that hypoxia fails to induce oxide synthase activity despite lack of response to endothelium- 308 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 301–309, 2000

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