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Simvastatin Attenuates Renal Ischemia/Reperfusion Injury in Rats Administered Cyclosporine A

      ABSTRACT

      Background

      3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors increase renal blood flow independent of their lipid-lowering properties. In organ transplantation, the calcineurin inhibitor cyclosporine A (CyA) is the immunosuppressant of choice. However, its renal vasoconstrictor properties limit its use. This study aimed to determine the effect of an HMG-CoA reductase inhibitor, simvastatin (Zocor), on renal function in rats after ischemia/reperfusion injury (I/R) with concomitant CyA treatment.

      Methods

      Male Wistar rats (250 g) were anesthetized and the suprarenal aorta clamped for 40 minutes. The right kidney was removed. After recovery, the rats were divided into 5 groups: (1) control rats, no ischemia, no treatment; (2) ischemia with no treatment; (3) ischemia plus CyA only; (4) ischemia plus CyA and low-dose simvastatin; and (5) ischemia plus CyA and high-dose simvastatin. Five to 7 days after I/R injury, glomerular filtration rate (GFR) was determined using urinary iohexol clearance.

      Results

      The GFR values (mL/min) for all 5 groups were as follows: (1) 1.23±0.08; (2) 1.05±0.10; (3) 0.44±0.06 (P<0.05 versus groups 1, 2, and 5; one-way analysis of variance); (4) 0.51±0.04 (P<0.05 versus groups 1, 2, and 5; one-way analysis of variance); and (5) 0.85±0.11.

      Conclusions

      After I/R injury and cyclosporine treatment, simvastatin preserved renal function compared with cyclosporine treatment alone because it may not have a direct vasoconstrictor effect on the renal microcirculation. In fact, it may exhibit vasodilator properties on the renal microcirculation mediated by nitric oxide.

      KEY INDEXING TERMS

      Renal transplantation is the most effective and cost-efficient treatment for patients with established renal disease.
      • Eggers P.W.
      Effect of transplantation on the Medicare end-stage renal disease program.
      The low rate of organ donation versus need has led to the increasing use of kidneys from so-called “marginal donors.” These include kidneys from non–heart-beating donors. As pointed out by Cho and coworkers,
      • Cho Y.W.
      • Terasaki P.I.
      • Cecka J.M.
      Transplantation of kidneys from donors whose hearts have stopped beating.
      transplantation of kidneys from non–heart-beating donors is often successful, and the use of such kidneys could increase the overall viability of cadaveric kidney transplants. However, many of these kidneys are exposed to longer warm ischemic times, compared with living-donor transplants, and are consequently prone to ischemia/reperfusion (I/R) injury. I/R injury is a nonimmune factor that is thought to contribute to both short- and long-term dysfunction of the graft.
      Currently, cyclosporine A (CyA) is the immunosuppressant of choice.
      • Abraham J.S.
      • Bentley F.R.
      • Garrison R.N.
      Cyclosporine A directly constricts intrarenal arterioles.
      However, its use is limited because of its nephrotoxic effects. CyA has been shown to cause microvascular vasoconstriction to decrease both glomerular filtration rate (GFR) and renal blood flow.
      • Abraham J.S.
      • Bentley F.R.
      • Garrison R.N.
      Cyclosporine A directly constricts intrarenal arterioles.
      Thus, the intrarenal effects of CyA may further exacerbate the effects of I/R injury.
      • Kanazi G.
      • Stowe N.
      • Steimuller D.
      Effect of cyclosporine upon the function of ischemically damaged kidneys in the rat.
      3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have been shown to have renal hemodynamic effects independent of their lipid-lowering properties.
      • Auer J.
      • Berent R.
      • Weber T.
      Clinical significance of pleiotropic effects of statins: lipid reduction and beyond.
      These effects may be mediated by the ability of the statins to vasodilate blood vessels via nitric oxide.
      • Laufs U.
      • Gertz K.
      • Dirnagl U.
      Rosuvastatin, a new HMG-CoA reductase inhibitor, upregulates endothelial nitric oxide synthase and protects from ischemic stroke in mice.
      • Davignon J.
      • Laaksonen R.
      Low-density lipoprotein independent effects of statins.
      Nitric oxide and endothelin are both released from the endothelial cell. NO may oppose the effect of endothelin-induced vasoconstriction known to occur after I/R injury.
      • Inman S.R.
      • Burns T.E.
      • Plott W.K.
      Addition of an endothelin receptor antagonist to modified Belzer’s perfusion preservation solution mitigates the adverse effect of pre-retrieval warm ischemic injury on post-transplant glomerular filtration rate.
      In a normal kidney, the HMG-CoA reductase inhibitor lovastatin dilates preglomerular arterioles and increases renal blood flow after rats are treated daily with lovastatin for 3 weeks.
      • Stowe N.T.
      • Inman S.R.
      • Tapolyai M.
      Lovastatin has direct renal hemodynamic effects in a rodent model.
      Although plasma lipids were not measured, we have previously shown beneficial renal hemodynamic effects independent of any lipid lowering effect in a puromycin-induced nephrotic syndrome model, a renal ablation model, and a streptozotocin-induced diabetic model.
      • Brouhard B.H.
      • Takamori H.
      • Satoh S.
      The combination of lovastatin and enalapril in a model of progressive renal disease.
      • Hafez K.S.
      • Inman S.R.
      • Stowe N.T.
      The renal hemodynamic effects of lovastatin in a renal ablation model.
      • Inman S.R.
      • Stowe N.T.
      • Brouhard B.H.
      Lovastatin preserves renal function in experimental diabetes.
      Thus, the purpose of this study was to test the hypothesis that using the HMG-CoA reductase inhibitor simvastatin in combination with CyA after I/R injury would attenuate the renal injury after I/R injury (specifically, the renal vasoconstriction caused by CyA).

      Materials and Methods

      All experiments were performed with the approval of the Institutional Animal Care and Use Committee for Ohio University. Male Wistar rats (250 g) were obtained from Harlan (Indianapolis, IN). The rats were randomized into 5 groups: (1) control group, (2) ischemic group receiving no treatment, (3) ischemic group receiving CyA (30 mg/kg/day, i.p.), (4) ischemic group receiving CyA plus simvastatin (30 mg/kg/day, i.p; 5 mg/kg/day, gavage), and (5) ischemic group receiving CyA plus simvastatin (30 mg/kg/day, i.p; 10 mg/kg/day, gavage). All rats were treated for 5 to 7 days.

      Induction of Warm Ischemia

      To induce warm ischemia, the rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). After induction of anesthesia, a midline incision was made. The suprarenal aorta was then identified and a clamp was placed at this location. Confirmation of ischemia distally was made by visual inspection of both kidneys. The kidneys would turn a pale color.
      The clamp remained in place for 40 minutes. In the control group, the clamp was placed but not tightened. Five minutes before clamp removal, a right nephrectomy was performed. The midline incision was sutured and the rats were placed back into their cages so recovery could be monitored.
      • Inman S.R.
      • Burns T.E.
      • Plott W.K.
      Addition of an endothelin receptor antagonist to modified Belzer’s perfusion preservation solution mitigates the adverse effect of pre-retrieval warm ischemic injury on post-transplant glomerular filtration rate.
      • Inman S.R.
      • Burns T.E.
      • Plott W.K.
      Endothelin receptor blockade during hypothermic perfusion preservation mitigates the adverse effect of pre-retrieval warm ischemic injury on post-transplant glomerular filtration rate.
      If a rat was in the control group, no further intervention was done. If the rat was randomized to the CyA group or CyA plus simvastatin groups, treatment was started the following day, continuing for 5 to 7 days.

      Renal Function Studies

      To assess renal function at 5 to 7 days after ischemia/reperfusion injury, GFR was determined using urinary iohexol clearance. The rat was anesthetized with ethyl-(1-methyl-propyl)-malonyl-thio-urea (Inactin; 100 mg/kg, i.p.), intubated, and placed on a heating pad. The jugular vein was cannulated for continuous infusion of saline and iohexol (2.5-mL bolus containing an iohexol-heparinized saline solution, 372.5 mg of iohexol, 149 mg/mL, followed by a 3 mL/hr infusion). A cannula was inserted into the carotid artery for measurement of blood pressure via a pressure transducer (Spectromed, Oxford, CA). The left ureter was exposed through an abdominal incision and cannulated for urine collection. After a 30-minute equilibration period, at least 3 successively timed 20-minute urine samples and a midpoint plasma sample were taken during each collection for determination of urinary iohexol clearance as an estimate of GFR using the Renalyzer machine.
      • Lewis R.M.
      • Kerr N.
      • Van Buren C.
      Comparative evaluation of urographic contrast media, inulin, and 99m Tc-DPTA clearance methods for determination of glomerular filtration rate in clinical transplantation.
      The Renalyzer machine (Porvalid AB, Lund, Sweden) is an x-ray fluorescence apparatus used to determine the clearance rates of the urographic contrast media, iohexol. The machine contains 2 Americium-241 sources emitting 60-kV photons that irradiate the contrast medium containing plasma and urine sample. Because of the irradiation, the iodine atoms emit characteristic x-rays. The intensity of these is proportional to the quantity of iodine.
      • Lewis R.M.
      • Kerr N.
      • Van Buren C.
      Comparative evaluation of urographic contrast media, inulin, and 99m Tc-DPTA clearance methods for determination of glomerular filtration rate in clinical transplantation.
      The values for iohexol clearance, urine flow, and blood pressure represent the average value obtained for the 3 consecutive 20-minute urine collection periods. At the conclusion of the experiment, plasma was saved for plasma creatinine measurements (Sigma Diagnostics, St. Louis, MO).

      Spot Urine Protein Determination

      Total protein collected in the urine during the renal clearance periods was measured by a spectrophotometric assay using bicinchoninic acid reagent (Pierce, Rockford, IL) and a modification of the method of Lowry et al.
      • Lowry O.H.
      • Rosebrough N.J.
      • Fall A.L.
      Protein measurement with the Folin phenol reagent.
      • Tofovic S.P.
      • Kost C.K.
      • Jackson E.K.
      Long-term caffeine consumption exacerbates renal failure in obese, diabetic, ZSF1 (fa-fa (cp)) rats.

      Statistical Analysis

      GFR, mean arterial pressure, plasma creatinine, and urine protein were compared using the analysis of variance (one-way) to determine differences between the groups.
      • Zar J.H.
      Multicentral hypothesis: the analysis of variance.
      The 0.05 level of probability was used as the criterion of significance. The data are reported as the mean and SEM.

      Results

      GFR values at 5 to 7 days after I/R injury for each of the 5 groups were as follows (Figure 1): nonischemic control rats, 1.23±0.08 mL/min; ischemic only, 1.05±0.10 mL/min; ischemic plus CyA alone, 0.44±0.06 mL/min; and ischemic plus CyA and low-dose simvastatin, 0.51±0.04 mL/min; and ischemic plus CyA and high-dose simvastatin, 0.85±0.11 mL/min (P<0.05, CyA alone group versus other 4 groups). The ischemic only group had 2 rats with GFRs at 0.44 mL/min the rest were all approximately 0.8 to 1.0 mL/min. The kidneys were all immediately removed after anesthesia and were all weighed in the same fashion. The values for GFR/g of kidney weight and GFR/g of body weight are shown in Table 1. The CyA group had significantly lower body weights than all the other groups except the CyA group receiving low-dose simvastatin (P<0.05). No significant differences could be detected in plasma creatinine or in the spot protein urine measurements (Table 1).
      Figure thumbnail gr1
      Figure 1GFR as measured by urinary iohexol clearance 5 to 7 days after I/R injury for nonischemic control rats (control), rats with ischemic kidneys not receiving any treatment (ischemic), rats with ischemic kidneys receiving only CyA (CyA; 30 mg/kg/day, i.p.), ischemic group receiving CyA plus low-dose simvastatin (CyA/LDS; 5 mg/kg/day, gavage), and the ischemic group receiving CyA plus high-dose simvastatin (CyA/HDS; 10 mg/kg/day, gavage) *, P<0.05 versus control, ischemic, HDS; **, P<0.05 versus control, ischemic, HDS. No significance was found between ischemic alone and CyA/HDS.
      Table 1Study Groups
      Control (n=10)Ischemic (n=8)Ischemic+CyA only (n=9)Ischemic+CyA+Low-Dose Simvastatin (n=11)Ischemic+CyA+HighDose Simvastatin (n=10)
      Body weight (g)273±8.7272±9.8230±5.6240±11.2242±6.1
      Kidney weight (g)1.4±0.051.7±0.161.8±0.121.6±0.141.6±0.09
      GFR (mL/min)1.23±0.081.05±0.100.44±0.06
      p<0.05 vs control, ischemic alone, and high-dose simvastatin
      0.51±0.04
      p<0.05 vs control, ischemic alone, and high-dose simvastatin
      0.85±0.11
      GFR/g of kidney weight0.91±0.070.66±0.080.26±0.03
      p<0.05 vs control, ischemic alone, and high-dose simvastatin
      0.4±0.060.57±0.09
      GFR/g of body weight×1000.50±0.010.65±0.080.77±0.060.70±0.070.66±0.05
      Urine flow (mL/min)0.01±0.0020.03±0.0030.01±0.0010.02±0.0010.02±0.001
      Mean arterial pressure (mm Hg)111±5119±391±282±8105±4
      Hematocrit0.45±0.020.46±0.010.38±0.020.45±0.010.48±0.02
      Plasma creatinine (mg/dL)1.3±0.11.3±0.11.3±0.11.4±0.031.2±0.02
      Urine protein (mg/mL)0.54±0.050.81±0.140.57±0.040.56±0.070.46±0.05
      * p<0.05 vs control, ischemic alone, and high-dose simvastatin
      The CyA-only group gained the least weight and, in many cases, lost weight. When Simvastatin was given in conjunction with CyA, the rats gained more weight. The weight loss was probably not caused by volume contraction because their hematocrit values were low; instead, they were just not as healthy without the simvastatin.

      Discussion

      The present study demonstrates that the administration of the HMG-CoA reductase inhibitor simvastatin preserved renal function when administered in conjunction with CyA after I/R injury. The rats with ischemic kidneys receiving no treatment had GFR values that were 15% lower than the control rats. Some of the rats with ischemic kidneys not receiving any CyA recovered to almost normal renal function values, whereas others did not. The rats with ischemia that were administered CyA had a decrease in GFR of ~58% compared with the ischemic rats receiving no treatment. When simvastatin (10 mg/kg/day) was given to the rats in conjunction with CyA, the GFR values were not different from those of ischemic rats not receiving CyA; however, the GFR values were significantly higher (approximately 92%) than those rats with ischemia administered CyA only. There were no differences detected in plasma creatinine levels or in the spot protein measurements. This is of interest because at 5 to 7 days after I/R injury, GFR was the most sensitive measurement of renal function in these studies.
      Preretrieval warm ischemia or I/R injury is considered to be an immune-independent factor contributing to both short- and long-term dysfunction of renal allografts.
      • Tilney N.L.
      • Paz D.
      • Ames J.
      Ischemia-reperfusion injury.
      With the demand far exceeding the need for kidneys, more “marginal” kidneys are being considered for transplantation. These include kidneys from donors with hemodynamic instability and non–heart-beating donors. These kidneys are more likely to be exposed to longer ischemic times. The immunosuppressive agent of choice today is CyA. CyA is known to have profound vasoconstrictor actions on the renal microcirculation.
      • Abraham J.S.
      • Bentley F.R.
      • Garrison R.N.
      Cyclosporine A directly constricts intrarenal arterioles.
      Thus, the combination of I/R injury with CyA treatment may exacerbate the injury, which can progress to loss of the graft. Endothelin-1, an amino acid peptide released by the endothelial cell, also has vasoconstrictor effects on both pre- and postglomerular arterioles.
      • Bloom I.T.
      • Bentley F.R.
      • Wilson M.A.
      In vivo effects of endothelin in the renal microcirculation.
      Endothelin has been clearly implicated in I/R injury.
      • Buyukgebiz O.
      • Aktan A.O.
      • Haklar G.
      BQ-123, a specific endothelin (ETA) receptor antagonist, prevents ischemia-reperfusion injury in kidney transplantation.
      • Herrero I.
      • Torras J.
      • Riera M.
      Prevention of cold ischaemia-reperfusion injury by an endothelin receptor antagonist in experimental renal transplantation.
      • Mino N.
      • Kobayashi M.
      • Nakajima A.
      Protective effect of a selective endothelin receptor antagonist, BQ 123, in ischemic acute renal failure in rats.
      • Inman S.R.
      • Burns T.E.
      • Plott W.K.
      Addition of an endothelin receptor antagonist to modified Belzer’s perfusion preservation solution mitigates the adverse effect of pre-retrieval warm ischemic injury on post-transplant glomerular filtration rate.
      • Inman S.R.
      • Burns T.E.
      • Plott W.K.
      Endothelin receptor blockade during hypothermic perfusion preservation mitigates the adverse effect of pre-retrieval warm ischemic injury on post-transplant glomerular filtration rate.
      The actions of endothelin and CyA vasoconstriction can be dampened by the action of a vasodilator also released from the endothelial cell, nitric oxide. One pharmacologic approach to the injury observed with preretrieval warm ischemia would be to increase nitric oxide production to counter the vasoconstrictor effects of endothelin and CyA.
      HMG-CoA reductase inhibitors have, in fact, been shown to mediate some of their effects through nitric oxide. Specifically, Laufs et al
      • Davignon J.
      • Laaksonen R.
      Low-density lipoprotein independent effects of statins.
      • Laufs U.
      • La Fata V.
      • Plutzky J.
      Upregulation of endothelial nitric oxide synthase by HMG-CoA reductase inhibitors.
      demonstrated a direct effect of simvastatin on constitutive endothelin nitric-oxide synthase protein mass and on mRNA levels on human veins exposed to oxidized low-density lipoprotein. Simvastatin was also shown to increase vasorelaxation in coronary arteries in rat aortic rings.
      • Davignon J.
      • Laaksonen R.
      Low-density lipoprotein independent effects of statins.
      • Kaesemeyer W.H.
      • Caldwell R.B.
      • Huang J.
      Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol lowering actions.
      Thus, nitric oxide seems a likely candidate in mediating the protective effects observed in our study with simvastatin.
      Though the statins preserve renal function in various disease models by lowering plasma lipids,
      • Wheeler D.C.
      Statins and the kidney.
      other nonlipid-lowering actions of statins may also prove to be important in preserving deterioration of renal function in these experimental models. HMG-CoA reductase inhibitors also impair the synthesis of nonsterol metabolites derived from the same metabolic pathway. Statins may prevent necrosis, tubular epithelial cell attachment and tubular obstruction in the S3 segment of the outer medullary stripe. The satins have also been shown to inhibit the production of chemotactic factors, which include growth-promoting cytokines, and to modulate the expression of mRNA coding for matrix components including collagen and fibronectin.
      • Wheeler D.C.
      Statins and the kidney.
      • Kim S.-Y.
      • Guijarro C.
      • O’Donnell M.P.
      Human mesangial cell production of monocyte chemoattractant protein-1modulation by lovastatin.
      Gueler and coworkers,
      • Gueler F.
      • Rong S.
      • Park J.-K.
      Postischemic acute renal failure is reduced by short-term statin treatment in a rat model.
      in a rat model in which they measured renal function 24 hours after I/R injury, clearly demonstrated that treatment with cerivastatin reduced serum creatinine levels by 40%. In addition, cerivastatin treatment prevented the occurrence of tubular obstruction. In addition, monocyte and macrophage infiltration was almost completely prevented. Their conclusion was that the statins may protect by a chain of events that might involve anti-inflammatory events, with inhibition of mitogen-activated protein kinase activation and the redox-sensitive transcription factors nuclear factor κB and activator protein-1.
      In experimental models of progressive renal deterioration, it has been demonstrated that the HMG-CoA reductase inhibitors reduce glomerulosclerosis and thus slow the progression of renal failure.
      • Buemi M.
      • Senatore M.
      • Corica F.
      Statins and progressive renal disease.
      HMG-CoA reductase is the main enzyme in the prevention of cholesterol synthesis, which is the step that converts HMG-CoA into mevalonate. This enzyme is also a precursor of nonsterol complexes, which are essential for cell activity, particularly cell-cycle progression.
      • Buemi M.
      • Senatore M.
      • Corica F.
      Statins and progressive renal disease.
      Statins modify monocyte infiltration, mesangial cell proliferation, and tubulointerstitial inflammation and fibrosis.
      • Kaesemeyer W.H.
      • Caldwell R.B.
      • Huang J.
      Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol lowering actions.
      Thus, there are many different mechanisms by which the statins are protective in the kidney.
      Other studies have examined the effect of an HMG-CoA reductase inhibitor and I/R injury. These studies looked at renal function between 24 and 72 hours after I/R injury. Yokota et al
      • Yokota N.
      • O’Donnell M.
      • Daniels F.
      Protective effect of HMG-CoA reductase inhibitor on experimental renal ischemia-reperfusion injury.
      demonstrated that cerivastatin pretreatment for 3 days led to significant improvement in renal function as measured by serum creatinine. They also saw improvement in tubular injury as well as survival at 72 hours. In another study, Myles et al
      • Joyce M.
      • Kelly C.
      • Winter D.
      Pravastatin, a 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, attenuates renal injury in an experimental model of ischemia/reperfusion.
      pretreated rats with pravastatin for 5 days before induction of I/R injury. They demonstrated that pravastatin significantly attenuated I/R-induced renal injury, improving both urine production and GFR.
      To our knowledge, our study is the first to report that treatment after I/R injury with an HMG-CoA reductase inhibitor is also beneficial in preserving renal function. To gain the benefits reported by Yokota and coworkers
      • Yokota N.
      • O’Donnell M.
      • Daniels F.
      Protective effect of HMG-CoA reductase inhibitor on experimental renal ischemia-reperfusion injury.
      and Myles and coworkers
      • Joyce M.
      • Kelly C.
      • Winter D.
      Pravastatin, a 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, attenuates renal injury in an experimental model of ischemia/reperfusion.
      would require the donor to be pretreated with the statins before retrieval. However, this is most unlikely when the transplant is performed with a cadaveric kidney, which in most cases is more prone to I/R injury. Our study demonstrates that treatment after I/R injury is beneficial. Clinically, this may translate to postoperative treatment of the transplant recipient with an HMG-CoA reductase inhibitor, especially in transplants in which the transplanted kidney was exposed to longer ischemic periods.
      In patients, the statins have been demonstrated to be safe.
      • Martinez-Castelao A.
      • Grinyo J.M.
      • Gil-Vernet S.
      Lipid lowering long-term effects of six different statins in hypercholesterolemic renal transplant patients under cyclosporine immunosuppression.
      • Martinez-Fernandez B.E.
      • Persaud J.W.
      • Varghese Z.
      Low-dose simvastatin is safe in hyperlipidemic renal transplant patients.
      • Cosio F.G.
      • Pesavento T.E.
      • Pelletier R.P.
      Patient survival after renal transplantation III: The effects of statins.
      However, rhabdomyolysis is a potential risk factor with the use of statins, so this needs to be monitored.
      • Sica D.A.
      • Gehr T.W.
      3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and rhabdomyolysis.
      Despite this potential side effect, in a study by Cosio et al,
      • Cosio F.G.
      • Pesavento T.E.
      • Pelletier R.P.
      Patient survival after renal transplantation III: The effects of statins.
      there was an increase of ~4-fold in the use of statins in transplant recipients from 1982 to 1996. During the same period, they showed that there was a significant increase in patient survival. This study suggested that the increased use of statins contributed to this favorable trend.
      In summary, simvastatin preserved renal function when given in conjunction with CyA after I/R injury. There seems to be a dose-dependent property to this action. Pharmacologic treatment of the transplant recipient with these statins to attenuate the ischemic damage could expand the donor pool. Future studies need to asses the direct effect of simvastatin on renal hemodynamics and on the renal microcirculation to assess the mechanism by which simvastatin exerts its beneficial effect.

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