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& 2008 International Society of Nephrology

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Pravastatin improves renal ischemia–reperfusion injury by inhibiting the mevalonate pathway Satoru Sharyo1,3,6, Naoko Yokota-Ikeda2,6, Miyuki Mori1,6, Kazuyoshi Kumagai3, Kazuyuki Uchida4, Katsuaki Ito1, Melissa J. Burne-Taney5, Hamid Rabb5 and Masahiro Ikeda1 1

Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan; 2Nephrology Division, Miyazaki Prefectural Miyazaki Hospital, Kita-Takamatsu 5-30, Miyazaki, Japan; 3Medicinal Safety Research Laboratories, R&D Division, Daiichi-Sankyo Co., Ltd., Horikoshi 717, Shizuoka, Japan; 4Department of Veterinary Pathology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan and 5Nephrology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Statins are known to lessen the severity of renal ischemia-reperfusion injury. The present study was undertaken to define the mechanism of renoprotective actions of statins using a mouse kidney injury model. Treatment of mice with pravastatin, a widely used statin, improved renal function after renal ischemia-reperfusion without lowering the plasma cholesterol level. Administration of pravastatin with mevalonate, a product of HMG-CoA reductase, eliminated renal protection suggesting an effect of pravastatin on mevalonate or its metabolism. In hypercholestrolemic apolipoprotein E knockout mice with reduced HMG-CoA reductase activity; the degree of injury was less severe than in control mice, however, there was no protective action of pravastatin on renal injury in the knockout mice. Treatment with a farnesyltransferase inhibitor (L-744832) mimicked pravastatin’s protective effect but co-administration with the statin provided no additional protection. Both pravastatin and L-744832 inhibited the injury-induced increase in plasma IL-6 concentration to a similar extent. Our results suggest the protective effect of pravastatin on renal ischemia-reperfusion injury is mediated by inhibition of the mevalonate-isoprenoid pathway independent of its lipid lowering action. Kidney International (2008) 74, 577–584; doi:10.1038/ki.2008.210; published online 28 May 2008 KEYWORDS: ischemia–reperfusion injury; the mevalonate pathway; pravastatin; apolipoprotein E knockout mouse; farnesyltransferase inhibitor

Correspondence: Masahiro Ikeda, Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Gakuenkibanadai-nishi 1-1, Miyazaki 889-2192, Japan. E-mail [email protected] 6

These authors contributed equally to this work.

Received 6 August 2007; revised 3 March 2008; accepted 26 March 2008; published online 28 May 2008 Kidney International (2008) 74, 577–584

Acute kidney injury (AKI), previously referred to as acute renal failure, is a common clinical syndrome resulting from a rapid reduction of glomerular filtration rate. AKI has been reported to occur in approximately 5% of hospitalized patients and 30–50% of patients in intensive care units. Despite the significant improvements in supportive care, the mortality and morbidity associated with AKI continue to be alarmingly high over the past three decades.1–3 A major reason for this is a lack of specific pharmacological interventions against AKI.1 The mevalonate pathway mediates the sequential biochemical reactions leading to the synthesis of cholesterol.4,5 Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, statins, block the conversion of HMG-CoA to mevalonate, which is a rate-determining step in the mevalonate pathway, resulting in decreased cholesterol production.6 As hyperlipidemia causes tissue injury through direct toxic effects of lipids on cells and a formation of intratissue atherosclerosis, statins are widely used to lower plasma cholesterol levels in hyperlipidemic patients.5,7–10 Blocking cholesterol synthesis was thought to be the primary statins’ mechanism of action. However, reports about plasma cholesterol-independent tissue-protective effects of statins have been increasing, and it is now recognized that statins have clinical benefits that appear to be greater than those one would expect from improving the lipid profile alone.5,8–12 The plasma cholesterol-independent tissue-protective effects of statins are thought to be mediated by their immunomodulatory and anti-inflammatory effects that relate to statin’s ability to block the synthesis of important intermediate products, including the isoprenoids in the mevalonate pathway.5,13–15 Renal ischemia–reperfusion (I/R) injury is an important cause for AKI. There is growing evidence that intrarenal inflammation is involved in renal I/R injury using in vitro and in vivo models, and therefore intrarenal inflammation is an attractive target for the development of drug therapies for AKI.16–18 Given that statins have immunomodulatory effects, they would be expected to protect against renal I/R injury. 577

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Thus far, several groups have tested the protective effects of statins, including pravastatin,19 cerivastatin,20,21 and atorvastatin,22 on experimental renal I/R injury, and these data have consistently shown that statins improve the course of the injury. However, it is not yet clear whether the protective action of statins on renal I/R injury is mediated by their lipid-lowering action or the inhibition of the mevalonate pathway in any in vivo studies. To clarify the mechanism of statin’s action in vivo, this study examined the effect of mevalonate on the protective effect of pravastatin from renal I/R injury using a mouse model. The renoprotective effect of pravastatin was also evaluated using apolipoprotein E-deficient mice (ApoE/) that is known to exhibit hypercholesterolemia.23,24 Furthermore, to dissect further the mechanism of statin’s renoprotection, we examined whether prenylation inhibitors, including L-744832 and GGTI-2133, protected mice against renal I/R injury. Our results indicate that the protective effect of pravastatin on renal I/R injury is dependent on the mevalonate–isoprenoid pathway, but independent of its lipid-lowering action.

S Sharyo et al.: Mechanism of pravastatin’s renoprotection

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First, we confirmed the protective effect of pravastatin on renal I/R injury. The mice were divided into three groups. Control group was treated with vehicle, low-dose group was given 50 mg per kg body weight of pravastatin and high-dose group was given 150 mg per kg body weight of pravastatin. As shown in Figure 1a, although no significant effect was observed in the low-dose group at any time points and in the high-dose group at 48 and 72 h after I/R, plasma creatinine level of the high-dose group was significantly lower than the control group at 24 h after I/R. Histopathological analysis with periodical acid-Schiff (PAS) staining revealed that I/R caused cast formation in tubules, tubular dilatation, and leukocyte infiltration in the outer medulla (Figure 1b). Pravastatin ameliorated these pathological changes (Figure 1c). As the histopathological changes were semiquantified by the measurement of PAS-positive area (Figure 1d), the improvement by pravastatin was dose dependent. These data clearly collaborated with the previous observation that pravastatin had protective effect in a rat renal I/R model.19 Effect of mevalonate on renal I/R

Next, we studied the effect of mevalonate, a product of HMG-CoA reductase, on renal I/R injury. The mice were divided into four groups: control group (vehicle treatment and sham operation); mevalonate treatment and sham operation group; vehicle treatment and I/R group; mevalonate treatment and I/R group. Figure 2 summarizes the data for plasma total cholesterol and creatinine concentrations. Mevalonate tended to increase the plasma total cholesterol in sham-operated mice and significantly increased it in I/R-operated mice. Approximately 40% of administered mevalonate has been reported to be eliminated into urine.25 578

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Figure 1 | Effect of pravastatin on plasma creatinine concentration and histology after renal I/R. (a) Pravastatin (50 or 150 mg/kg) or vehicle was administered for 3 consecutive days before mice were subjected to renal I/R. Plasma for creatinine concentration measurement was collected before I/R (0 h) and 24, 48, and 72 h after I/R. Open circle, open square, and closed circle indicate vehicle group, 50 mg pravastatin per kg body weight group, and 150 mg pravastatin per kg body weight group, respectively. Values are mean±s.e. (N ¼ 7–14 per group). *Po0.05 vs vehicle. PAS staining was performed with kidney sections 24 h after I/R from mouse treated with (b) vehicle or (c) 150 mg pravastatin per kg body weight. The outer strip of outer medulla is shown at the left and the inner strip of outer medulla is at the right. Bar ¼ 50 mm. (d) The PAS-positive area was measured (see Materials and Methods) in kidney sections 24 h after I/R from mouse treated with vehicle, 50 mg pravastatin per kg body weight , or 150 mg pravastatin per kg body weight. Values are mean±s.e. (N ¼ 5–6 per group). *Po0.05 vs vehicle.

Therefore, this increase in cholesterol may be explained by the impairment of mevalonate excretion into the urine by I/R injury, thereby increasing blood mevalonate concentration with consecutive increase in the cholesterol synthesis in liver. I/R significantly increased plasma creatinine level at 24 h after the operation, and this increase was significantly enhanced by the pretreatment of mice with mevalonate. These raise a possibility that mevalonate worsens the renal I/R injury. Kidney International (2008) 74, 577–584

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Figure 2 | Effect of mevlonate on plasma total cholesterol and creatinine concentrations after renal I/R. Mevalonate or vehicle was administered 25, 13, and 1 h before sham or renal I/R operation. (a) Plasma total cholesterol concentration was measured 24 h after operation and (b) plasma creatinine concentration was measured before (pre) and 24 h after operation. Values are mean±s.e. (N ¼ 4–5 per group). **Po0.01 vs vehicle and sham. #Po0.05 vs vehicle and I/R.

The combined administration of pravastatin and mevalonate eliminated the protective effect of pravastatin

The effect of mevalonate on the renoprotective action of pravastatin was examined. The mice were divided into three groups: control group (both vehicles for pravastatin and mevalonate treatment); pravastatin and vehicle for mevalonate treatment group; pravastatin and mevalonate treatment group. Plasma cholesterol levels 24 h after I/R did not differ between the three groups (Figure 3a). At 24 h after I/R, plasma creatinine concentration in the pravastatin and vehicle for mevalonate treatment group was significantly lower than that in the control group. In contrast, the protective effect of pravastatin from renal I/R injury was not observed in the pravastatin and mevalonate treatment group. These data suggest that the renoprotective effect of pravastatin is mediated by an inhibition of the mevalonate pathway. Effect of hyperlipidemia on renal I/R injury in apolipoprotein E knockout mice (ApoE/)

Apolipoprotein E knockout mice (ApoE/) have been reported to exhibit hypercholesterolemia insensitive to statins and a lower activity of HMG-CoA reductase.23,24,26,27 We used these mice and evaluated the effect of hyperlipidemia on renal I/R injury. Mean±s.e. values of the plasma total cholesterol concentration 24 h after sham operation or I/R were 78.0±8.5 (sham) and 137.8±33.9 mg/100 ml (I/R) for the genetic control (C57BL/6J) mice (n ¼ 4), and 842.3±129.4 (sham) and 776.5±51.6 mg/100 ml (I/R) for Kidney International (2008) 74, 577–584

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Figure 3 | Effect of mevlonate on renoprotection by pravastatin. Vehicles for mevalonate and pravastatin, for mevlonate and pravastatin, or for mevalonate and pravastatin were administered to mice before I/R as in Figures 1 and 2, except for the treatment protocol for pravastatin (100 mg/kg/day for 5 consecutive days before I/R operation). (a) Plasma total cholesterol concentration was measured 24 h after I/R and (b) plasma creatinine concentration was measured before (pre) and 24 h after I/R. Values are mean±s.e. (N ¼ 7–8 per group). *Po0.05 vs vehicles for mevalonate and pravastatin.

ApoE/ (n ¼ 4), respectively. Mean±s.e. values of the creatinine concentrations 24 h after sham operation and I/R were 0.1±0.01 (sham) and 2.1±0.2 mg/100 ml (I/R) for the control mice, and 0.1±0.01 (sham) and 2.1±0.5 mg/100 ml (I/R) for ApoE/, respectively. These values were not significantly different between the control mice and ApoE/. Figure 4 shows the results from histopathological analysis with PAS staining. I/R dramatically caused cast formation in the control mice, whereas the change in ApoE/ was somewhat reduced. The PAS-positive area in ApoE/ after I/R was significantly smaller than that in the control mice. These data show that hyperlipidemia of ApoE/ does not worsen the renal I/R injury but seems to rather weaken it. Effect of pravastatin in ApoE/

We checked the protective effect of pravastatin on renal I/R injury in ApoE/. Figure 5 summarizes the data for plasma total cholesterol and creatinine concentrations. In the control mice, increase in plasma total cholesterol level by renal I/R was observed. On the contrary, I/R decreased plasma total cholesterol concentration in ApoE/. These changes in response to I/R were not affected by the treatment with pravastatin. The mechanism underlying the I/R-induced plasma cholesterol changes in both the control mice and ApoE/ is not clear at present. In the control mice, renal function after I/R was significantly improved by the treatment with pravastatin compared with mice treated with vehicle (Figure 5c). This renal protective action of pravastatin was not seen in ApoE/ (Figure 5d). 579

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Figure 4 | Renal histology after sham or renal I/R operation in the genetic control mice and ApoE/. PAS staining was performed with kidney sections from the genetic control mice (a) before and (b) 24 h after renal I/R. Similarly, kidney sections from ApoE/ (c) before and (d) 24 h after renal I/R were stained with PAS. Bar ¼ 50 mm. (e) The PAS-positive area was measured in kidneys from the genetic control mice and ApoE/. Values are mean±s.e. (N ¼ 4 per group). *Po0.05 vs control with I/R.

Effects of prenylation inhibitors on renal I/R injury

It is known that the mevalonate pathway regulates the biosynthesis of cholesterol as well as isoprenoids including farnesyl pyrophosphate and geranylgeranyl pyrophosphate. We thus evaluated the renoprotective effects of L-744832 (farnesyltransferase inhibitior) and GGTI-2133 (geranylgeranyl transferase I inhibitior) on renal I/R injury.28,29 L-744832 and GGTI-2133 were administered to mice just after I/R and plasma creatinine levels were measured 24 h after I/R (Figure 6a). Serum creatinine level in L-744832 treatment group was significantly lower than that of vehicle control group (Figure 6a). In contrast, GGTI-2133 had no significant effect on the increase in plasma creatinine concentration after I/R. To examine whether the effect of L-744832 resulted from the inhibition of the mevalonate pathway, we compared the effect of treatment with L-744832 alone with that of combination of L-744832 and pravastatin on I/R-induced dysfunction in normal mice. As shown in Figure 6b, no 580



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Figure 5 | Effect of pravastatin on plasma total cholesterol and creatinine concentrations after renal I/R in the genetic control mice and ApoE/. Pravastatin (100 mg/kg) or vehicle was administered to the (a and c) genetic control mice and (b and d) ApoE/ for 5 consecutive days before I/R. Plasma for measurements of (a and b) total cholesterol and (c and d) creatinine concentrations was collected before I/R (Pre) and 24 h after I/R. Values are mean±s.e. (N ¼ 5 per group). *Po0.01 vs vehicle with I/R.

significant additive effect of the coadministration was observed, indicating that two drugs share the same pathway. Effects of pravastatin and L-744832 on serum IL-6 concentration after renal I/R

Interleukin-6 is primarily involved in the regulation of inflammation.30 Furthermore, serum IL-6 level is reported to increase after renal I/R.31 To confirm whether the pravastatin’s renoprotection is mediated by its anti-inflammatory effect and L-744832 has a similar effect to pravastatin on serum IL-6 level or not, we measured serum IL-6 levels 24 h after I/R in mice treated with pravastatin or L-744832. Serum IL-6 concentration in mice subjected to I/R was significantly higher than that in intact mice. When mice were treated with Kidney International (2008) 74, 577–584

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Figure 6 | Effects of prenylation inhibitors on I/R-induced renal dysfunction. (a) L-744832 (40 mg/kg), its vehicle (saline), GGTI2133 (40 mg/kg), or its vehicle (dimethyl sulfoxide) was intraperitoneally administered to mice just after renal I/R. (b) L-744832 and vehicle for pravastatin or L-744832 and pravastatin were administered to mice as in Figures 5 and 6a. Plasma for creatinine concentration measurement was collected 24 h after I/R. Values are mean±s.e. (N ¼ 8). *Po0.05 vs vehicle. N.S., not significant.

pravastatin or L-744832, significantly lower serum IL-6 levels were observed compared with that in mice treated with vehicle (Figure 7). DISCUSSION

This study clearly showed that pravastatin, a widely used statin, protected normal mice from renal I/R injury without any reduction in plasma cholesterol level. This renoprotective effect of pravastatin was diminished by the coadministration of mevalonate with pravastatin. When we used ApoE/ that had a 10-times higher plasma total cholesterol concentration compared with that of their genetic control and had been shown to exhibit a reduced activity of HMGCoA reductase,23,24 pravastatin had no effect on renal I/R injury. Treatment with mevalonate alone worsened the degree of renal I/R injury. Treatment with L-744832, an isoprenylation inhibitor, significantly ameliorated renal dysfunction after I/R. The combination of pravastatin and L-744832 did not further improve renal dysfunction after I/R, compared with treatment with L-744832 alone. Pravastatin as well as L-744832 inhibited I/R-induced increase in plasma IL-6 concentration to a similar extent. Together, these data strongly suggest that the inhibition of the mevalonate– isoprenoid pathway, which leads to anti-inflammation, is important for the renoprotective action of pravastatin. Moreover, the lipid-lowering action of pravastatin is unlikely to be related to its protective effect on renal I/R injury. Kidney International (2008) 74, 577–584

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Figure 7 | Effect of pravastatin or L-744832 on renal I/R-induced serum IL-6 elevation. Vehicle for pravastatin and vehicle for L-744832 (vehicle), pravastatin and vehicle for L-744832 (pravastatin), or vehicle for pravastatin and L-744832 (L-744832) were administered to mice (pravastatin, 100 mg/kg/ day, 5 consecutive days before I/R operation; L-744832, 40 mg/kg, just after I/R operation). Plasma IL-6 concentration was measured 24 h after I/R. Plasma IL-6 concentration from intact control mice (intact) was also measured. Values are mean±s.e. (N ¼ 6–8 per group). *Po0.05, **Po0.01 vs vehicle.

Biologically important products other than cholesterol in the mevalonate pathway are dolichols, ubiquinone, and the isoprenoids, including farnesyl pyrophosphate and geranylgeranyl pyrophosphate.4,5 Isoprenoids are known to play an important role in the post-translational modification of G proteins such as Ras, Rho, and Rab, for their proper intracellular localization.5,13 The protein isoprenylation reaction is catalyzed by farnesyl or geranylgeranyl transferase. In this study, the treatment with L-744832, a farnesyl transferase inhibitor,28 significantly improved the renal deficit after I/R. On the other hand, GGTI-2133, a geranylgeranyl transferase I inhibitor,29 had not significant effect on renal I/R injury. Recently, chaetomellic acid A, a farnesyltransferase inhibitor, was also reported to produce protection against I/R-induced renal dysfunction.32 These data suggest that protein farnesylation is involved in pathobiology of renal I/R injury, and this leads to the development of specific pharmacological interventions. We demonstrate here that serum IL-6 level was significantly increased after renal I/R, and this increase was prevented by pravastatin or L-744832 treatment. IL-6 is a pleiotropic cytokine and a major regulator of inflammation.30 Growing evidence suggests that IL-6 is a critical contributor to renal I/R injury. In IL-6 knockout mice, the degree of renal I/R injury was less severe compared with that of the genetic control mice.30 Normal mice administered a monoclonal antibody against IL-6 were resistant to renal I/R injury.30,31 Increased production of IL-6 in the ischemic kidney originated largely from macrophages that infiltrated into the outer medulla.31 Resistance of IL-6 knockout mice to renal I/R injury was reduced by the reconstitution of IL-6 knockout mice with bone marrow from wild-type mice.31 Moreover, a genetic heme oxygenase-1 deficiency in mice 581

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increased the sensitivity to renal I/R injury, and this increase was lowered by the treatment of mice with a monoclonal antibody against IL-6.33 Hence, the renoprotective effects of paravastatin and L-744832 against I/R injury may be mediated through the suppression of IL-6 production. We note that Xue et al.34 recently reported that pretreatment with tipifarnib, a farnesyl transferase inhibitor, resulted in a significant inhibition of IL-6 production in a murine model of LPS-induced inflammation. In this study, pravastatin and L-744832 could not completely protect mice against renal I/R injury. Similar partial effects of other statins including cerivastatin and atorvastatin on renal I/R injury have been reported.20–22 Although the exact reason for the insufficient effect of statins is not clear at present, this might be explained by an inhibitory activity of statins on a mammalian target of rapamycin (mTOR) pathway. Recently, Finlay et al.35 observed that atorvastatin could inhibit the mTOR pathway. An inhibition of the mTOR pathway by sirolimus has been shown to contribute to the development of renal I/R injury.36,37 Therefore, decrease in the mTOR response as a consequence of statin treatment may partially mask statin’s beneficial effect on renal I/R injury. Alternatively, a complicated role of nitric oxide (NO) in the pathobiology of renal I/R injury may explain the partial effect of statin on renal I/R injury. Statin is known to increase NO production that is mediated by inhibiting isoprenylation of Rho.5,13 It has been observed that NO can either ameliorate or exacerbate renal injury depending on the origin, site, and rate of production and its chemical fate.38 Therefore, it is possible that improper production of NO by statins leads to the exacerbation of renal I/R injury. The detailed molecular mechanisms of partial effect of statins on renal I/R injury await clarification in future studies. When we used ApoE/, renal function after I/R was comparable to those of the control mice. Similarly, Buzello et al. have reported that the degree of nephrectomy-induced renal injury is not so severe in ApoE/, compared with that of the genetic control mice.39 Furthermore, 3 weeks after renal I/R, kidney functions of rats fed with a cholesterolsupplemented diet for 3 weeks before I/R was similar to those of rats fed with a regular diet.40 Although dyslipidemia has been suggested to accelerate the progression of chronic kidney disease,41 these data indicate that dyslipidemia does not appear to worsen the AKI. A series of experiments done by Zager et al.42,43 have revealed that renal I/R induces cholesterol accumulation in the kidney cortex including proximal tubules that is susceptible to I/R injury, and this accumulation contributes to the socalled ‘cytoresistant state’ (renal resistance to additional ischemic damage). The mechanism underlying ‘cytoresistant state’ is thought to be an increase in plasma membrane rigidity, which lessens membrane rupture during the evolution of necrotic cell death.44 In this study, although we did not see significant difference in renal function in between the control mice and ApoE/, less severe histopathological 582


changes in ApoE/ post I/R was observed. This observation might result from ‘cytoresistant state’ induced by hyperlipidemia in ApoE/. Alternatively, as ApoE/ has a reduced activity of HMG-CoA reductase,23,24 a less production of intermediate products in the mevalonate pathway after renal I/R may contribute to the resistance of ApoE/ to renal I/R injury. Simvastatin, one of clinically used statin, has been reported to attenuate cardiac I/R injury in ApoE/.26 In contrast, we failed to observe the protective effect of pravastatin on renal I/R injury in ApoE/ and interpreted the lack of the effect on their reduced HMG-CoA reductase activity. The reason for the apparent loss in potency of pravastatin compared with that of simvastatin is not clear at present. Tissue-dependent effect of statins is one possibility. Other possible explanation for this is that simvastatin could have additional effects other than the inhibition of HMG-CoA reductase. In fact, it has been reported that simvastatin, but not pravastatin, binds to the inserted domain of leukocyte function antigen (LFA)-1 and prevents the function of LFA-1.45 This effect was independent of inhibition of HMG-CoA reductase.45 It is important to note that inhibition of LFA-1 by monoclonal antibodies specific for the antigen has been shown to be effective in animal models for renal I/R injury.46 In this study, we administered pravastatin to mice with a dose of 100 or 150 mg/kg. These doses were higher than daily therapeutic dose in humans (20–80 mg/kg/day). But the dose for mice is not extremely high because, when these doses were given to rodents, serum concentration of pravastatin was equivalent to those in humans.6,47 This species difference might be explained by more rapid metabolism from circulation in rodents.48 In addition, we did not observe serious adverse effects after the administration of such high doses of pravastatin to mice (unpublished data). Therefore, it is unlikely that clinically irrelevant blood concentration of pravastatin causes renoprotection from I/R injury observed in this study. Pravastatin is unique among statins in that it is hydrophilic and is the only statin that is selectively taken up into hepatocytes, thereby efficiently inhibiting liver HMG-CoA reductase with minimal inhibition of the enzyme in other tissues such as skeletal muscle.6,49 Therefore, myositis and rhabdomyolysis are scarcely seen in patients treated with pravastatin. Furthermore, a single-dose study demonstrated that pravastatin pharmacokinetics was not affected in patients with renal impairment50 and there was no accumulation of pravastatin or its metabolites over an interdialytic interval in hemodialysis patients when administered with a usual clinical dose.51 Based on these, pravastatin can be safely administered with a usual dose to patients with renal failure. Our study clearly showed that pravastatin had the potential to be effective in renal I/R injury. These features indicate that pravastatin would be an attractive drug against AKI. To this end, we need further clinical evaluations in humans. Kidney International (2008) 74, 577–584

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MATERIALS AND METHODS Animals All animal studies were in accordance with Guide for the Care and Use of Laboratory Animals in University of Miyazaki and SOP for the experimental animals of Daiichi-Sankyo Co., Ltd. and were conducted in compliance with Law Concerning the Protection and Control of Animals (Japanese Law No. 105, October 1, 1973, revised on June 22, 2005), Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Notification No.88 of the Ministry of the Environment, Japan, April 28, 2006) and Guidelines for Animal Experimentation (the Japanese Association for Laboratory Animal Science, May 22, 1987). Male ddy mice (ddy) were purchased from Japan SLC Inc. (Shizuoka, Japan). Male apolipoprotein E knockout mice (ApoE/) originated from the Jackson laboratory (Bar Harbor, ME, USA) were breaded in our laboratory. Male C57BL/6J mice were purchased from Clea Japan Inc. (Tokyo, Japan) as control mice for the ApoE/. Renal ischemia–reperfusion (I/R) injury In this study, an established mouse model of renal ischemia– reperfusion was used as described previously. Mice (25–35 g) were anesthetized with pentobarbital (65–75 mg/kg) and underwent abdominal incision and dissection of the bilateral renal pedicles. A microvascular clamp (Roboz, Gaithersburg, MD, USA) was placed on each renal pedicle for 30 min for ddy mice, and for 20 min for both ApoE/ and its wild type of C57BL/6J mice, respectively. After ischemia period, the clamps were removed, the wound was sutured, and animals were allowed to recover. The animals were kept at constant body temperature with continuous monitoring during ischemia–reperfusion period, and warmed physiological saline was given to maintain the body fluid volume just after being clamped and before being sutured.

Serum IL-6 concentration was measured using the Bio-Plex Mouse Cytokine Panel (Bio-Rad Laboratories Inc., Tokyo, Japan). IL-6 concentration was calculated by Bio-Plex Manager 4.0 software using a standard curve derived from a known concentration of a recombinant IL-6 solution. For the histopathological evaluation, the left kidney was removed at indicated time points and fixed in 10 % (v/v) neutral-buffered formalin. Serial sections of 2 mm thickness were stained with PAS. The semiquantitative analysis was performed by measuring the area of PAS-positive hyaline cast in the renal tubules of the inner stripe of the outer medulla. Twelve microscope fields (75,436 mm2 per field) were scanned in each mouse using an image-processing apparatus (IPAP-WIN, Sumika Technoservice Corporation, Osaka, Japan). Statistical analysis Data are expressed as mean±s.e.m. values. Statistical comparisons of the mean values between two groups were performed using Student’s t-test; Dunnett’s method was used to test comparisons of the means between three or more groups. A paired t-test was used for comparisons within groups. Significant differences were accepted at Po0.05 and Po0.01. DISCLOSURE

All the authors declared no competing interests. ACKNOWLEDGMENTS

We thank Ms Kyoko Miwa, Ms Shiho Ito, Ms Kumi Honda, and Ms Fusako Atsumi for their excellent technical assistance. This work was supported in part by JSPS KAKENHI, 19580342 (M.I.). REFERENCES 1. 2.

Reagents and administration regimen Pravastatin sodium was generously donated by Daiichi-Sankyo Co., Ltd. (Tokyo, Japan). Mevalonolactone was from Sigma Chemical Co. (St Louis, MO, USA). L-744832 and GGTI-2133 were from Calbiochem (San Diego, CA, USA). Pravastatin, mevalonolacton, and L-744832 were dissolved in physiological saline. GGTI-2133 was dissolved in dimethyl sulfoxide. Pravastatin was administered orally by gavage. Mevalonate, L-744832, and GGTI-2133 were administered intraperitoneally. Pravastatin was administered to ddy mice for 3 or 5 consecutive days before I/R at a dosage level of 50, 100, or 150 mg/kg. Mevalonate (20 mg/kg) was consecutively administered to ddy mice at 25, 13, and 1 h before I/R. Pravastatin was administered to ApoE/ and its control (C57BL/6J) mice at a dosage level of 100 mg/kg for 5 consecutive days before I/R. The control group was administered physiological saline in the same manner. L-744832 and GGTI-2133 were administered to C57BL/6J mice at a dose of 40 mg/kg before closing the peritoneum. The vehicle control groups for L-744832 and GGTI-2133 were administered physiological saline or dimethyl sulfoxide, respectively, in the same manner.

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Kidney International (2008) 74, 577–584