Postischemic Acute Renal Failure Is Reduced by Short-Term Statin Treatment in a Rat Model
Faikah Gueler*,
Song Rong*,
Joon-Keun Park*,
Anette Fiebeler*,
Jan Menne,
Marlies Elger*,
Dominik N. Mueller,
Franziska Hampich,
Ralf Dechend,
Uta Kunter,
Friedrich C. Luft and
Hermann Haller*
*Department of Internal Medicine-Nephrology, Hannover Medical School, Hannover, Germany; Phenomiques GmbH, Hannover, Germany; Franz Volhard Clinic HELIOS Klinikum-Berlin, Max Delbrueck Center of Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany; and Division of Nephrology, Department of Medicine, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen, Aachen, Germany.
Correspondence to Dr. Hermann Haller, Department of Nephrology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Phone: 49-511-6319; Fax: 49-511-552366; E-mail: haller.hermann{at}mh-hannover.de
ABSTRACT. Postischemic acute renal failure (ARF) is common andoften fatal. Cellular mechanisms include cell adhesion, cellinfiltration and generation of oxygen free radicals, and inflammatorycytokine production. Hydroxy-3-methylglutaryl coenzyme A reductaseinhibitors ("statins") directly influence inflammatory mechanisms.The hypothesis that ischemia-induced ARF could be amelioratedwith statin treatment was investigated and possible molecularmechanisms were analyzed in a uninephrectomized rat model. MaleSprague-Dawley rats were pretreated with cerivastatin (0.5 mg/kg)or vehicle for 3 d. Ischemic ARF was induced by left renal arteryclipping for 45 min, while the right kidney was being removed.After 24 h of ARF, serum creatinine levels were increased 7.5-foldin vehicle-treated control animals with ARF, compared with sham-operatedanimals (P < 0.005). Statin treatment reduced the creatininelevel elevation by 40% (P < 0.005). Simultaneously, ischemia-inducedsevere decreases in GFR were significantly ameliorated by statintreatment (sham operation, 0.95 ± 0.09 ml/min, n = 13;ischemia without treatment, 0.06 ± 0.02 ml/min, n = 9;ischemia with statin pretreatment, 0.21 ± 0.03 ml/min,n = 11; P < 0.001). Furthermore, statin pretreatment preventedthe occurrence of tubular necrosis, with marked loss of thebrush border, tubular epithelial cell detachment, and tubularobstruction in the S3 segment of the outer medullary stripe.In addition, monocyte and macrophage infiltration was almostcompletely prevented, intercellular adhesion molecule-1 upregulationwas greatly decreased, and inducible nitric oxide synthase expressionwas reduced. Fibronectin and collagen IV expression was reduced,approaching levels observed in sham-operated animals. In vehicle-treatedrats with ARF, mitogen-activated protein kinase extracellularactivated kinase-1/2 activity was increased and the transcriptionfactors nuclear factor-B and activator protein-1 were activated.Statin treatment reduced this activation toward levels observedin sham-operated rats. The data suggest that hydroxy-3-methylglutarylcoenzyme A reductase inhibition protects renal tissue from theeffects of ischemia-reperfusion injury and thus reduces theseverity of ARF. The chain of events may involve anti-inflammatoryeffects, with inhibition of mitogen-activated protein kinaseactivation and the redox-sensitive transcription factors nuclearfactor-B and activator protein-1.
Renal ischemia-reperfusion injury is a major cause of acuterenal failure (ARF) after major surgery or renal transplantation(1,2). Several experimental ameliorative strategies have beentested (3). However, there is still a remarkable lack of definitiveevidence supporting specific prophylactic therapies in any setting(4). Postischemic ARF seems to be a consequence of hypoxia attributableto impaired perfusion, with subsequent reperfusion leading toacute inflammatory changes. Pathophysiologic mechanisms includeintracellular damage, with ATP depletion (5), and intracellularCa2+ accumulation (6). Cellular activation leads to reactiveoxygen species generation, phospholipase activation, and membranelipid alterations (7). Inflammatory reactions are characterizedby surface adhesion molecule expression, followed by leukocyteinfiltration, protease activation, and cytokine production (8).Reorganization of integrins from basal to apical surfaces ofinjured tubular epithelial cells has been demonstrated to facilitatedetachment, contributing to tubular obstruction (9). These changesare most pronounced in the outer medullary stripe, i.e., a partof the kidney that is extremely susceptible to hypoperfusionand hypoxic damage (10). Other compartments, such as the corticomedullaryjunction, are also injured, however. We and others have demonstratedthat inhibition of inflammatory pathways, such as inhibitionof redox-sensitive nuclear factor-B (NF-B) or blockade of intercellularadhesion molecule-1 (ICAM-1) expression, is a beneficial strategyin ARF models (11,12). Recent evidence suggests that statinscan prevent vascular inflammatory reactions in human subjectsand in animal models, independently of their LDL cholesterol-loweringeffects (13). In earlier studies involving a hypertensive ratmodel of angiotensin II-induced renal and cardiac damage, weobserved that statin treatment was protective and NF-B and activatorprotein-1 (AP-1) activation was reduced (14,15). Because statinscould possibly have prophylactic utility among patients at riskfor ischemic ARF, we tested whether similar amelioration mightoccur in an animal model.
Experiments were performed with male Sprague-Dawley rats (250to 300 g) purchased from Charles River (Sulzfeld, Germany).The rats received a standard diet, with free access to tap water.All procedures were performed according to the guidelines ofthe American Physiological Society and were approved by localauthorities. One group of rats was treated with cerivastatin(0.5 mg/kg body wt) by gavage for 3 d before surgery. Surgerywas performed with general anesthesia with ketamine (100 mg/kgbody wt; CP-Pharma, Burgdorf, Germany) and xylazine (5 mg/kgbody wt; Rompun Bayer, Leverkusen, Germany). On the third dayof treatment, surgery was performed as described previously(10). The left renal pedicle was occluded for 45 min, duringwhich time a right nephrectomy was performed. A control groupreceived saline vehicle, by gavage, and underwent a clippingoperation and contralateral nephrectomy as described above.The third group received saline treatment and underwent a shamoperation, in which the abdomen was opened, the left renal pediclewas dissected but not clamped, and the right kidney was removed.All animals were anesthetized for 45 min and euthanized 24 hlater. Each group contained 18 animals. Blood samples were obtainedbefore surgery and at the time of euthanasia. Creatinine levelswere measured by using an automated method (Beckman analyzer;Beckman, Germany). The rats were perfused, via the aorta, with100 ml of cold phosphate-buffered saline, and their kidneyswere removed.
For measurement of inulin and p-aminohippurate (PAH) clearance,rats were anesthetized and placed on a heating table for maintenanceof body temperature (37°C). A catheter was placed in thecarotid artery for measurement of systemic BP. Catheters werealso inserted into the jugular and femoral veins and the bladder,for infusion, blood sampling, and urine collection, respectively.Inulin and PAH (in saline solution with 1% bovine serum albumin)were infused at a constant rate of 50 µl/min. After equilibriumwas reached (30 min), four consecutive urine samples (30 mineach) were collected. Blood samples were collected in the middleof each period. Inulin and PAH concentrations were measuredcolorimetrically.
For paraffin histologic assessments, kidneys were perfused withcold phosphate-buffered saline and then with cold fixative containing4% paraformaldehyde in Soerensen’s phosphate buffer. Thekidneys were fixed for an additional 4 h and then embedded inparaffin. For immunohistochemical analyses, the kidneys weresnap-frozen in isopentane (-35°C). For Western blotting,the kidneys were snap-frozen in liquid nitrogen. For morphologicevaluations, 3-µm paraffin sections were stained withtrichrome Masson-Goldner stain, by using standard procedures.
Immunohistochemical analyses were performed with the followingprimary antibodies: monoclonal mouse anti-rat ICAM-1 (1A29;Serotec, Oxford, UK), anti-rat monocytes/macrophages (ED-1;Serotec), polyclonal rabbit anti-inducible nitric oxide synthase(iNOS) (ABR, Golden, CO), rabbit anti-rat fibronectin (Paeseland Lolei, Frankfurt, Germany), and goat anti-collagen typeIV (Southern Biotechnology Associates, Birmingham, AL). Forindirect immunofluorescence assays, nonspecific binding siteswere blocked with 10% normal donkey serum (Jackson ImmunoResearch,West Grove, PA) for 30 min. The cryosections were then incubatedwith the primary antibody for 1 h. All incubations were performedin a humidified chamber at room temperature. For fluorescenceobservation of bound primary antibodies, sections were subsequentlyincubated with Cy3-conjugated secondary antibodies (JacksonImmunoResearch) for 1 h. Immunostaining for monocytes/macrophages(with ED-1) was performed by using the avidin-biotin complex-peroxidasesystem (Vector Laboratories, Burlingame, CA). The sections weretreated with 0.6% H2O2 in methanol for 10 min, to quench endogenousperoxidase activity. Subsequently, 10% goat serum was used toblock nonspecific binding sites. Endogenous biotin was quenchedby using an avidin-biotin blocking kit (Vector Laboratories).Observation was performed with an aminoethylcarbazole/H2O2 mixture.Nuclei were counterstained with Delafield’s hematoxylinsolution (Fluka, Buchs, Switzerland). Specimens were analyzedby using a Zeiss Axioplan-2 imaging microscope, with the computerprogram AxioVision 3.0 (Zeiss, Jena, Germany). Semiquantitativescoring of ED-1-positive cells was performed by using a computerizedcell-counting program (KS 300 3.0; Zeiss). Fifteen differentareas of each kidney sample were analyzed. Semiquantitativeanalysis of iNOS expression was performed by counting the numbersof glomeruli with high, medium, and low levels of expression.Scoring was performed without knowledge of the identity of theanimal group.
For Western blotting, the frozen kidneys were pulverized inliquid nitrogen and resuspended in 2 ml of lysis buffer (20mM Tris buffer, pH 7.5, containing 10 mM glycerophosphate, 2mM pyrophosphate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonylfluoride, 1 g/ml leupeptin, 1 mM dithiothreitol, and 1 mM ethylenediaminetetraacetate).Homogenates were sonicated with three 20-s bursts, on ice, andcentrifuged at 500 x g for 1 min, to remove cell debris. Aliquotsof the supernatants were stored at -80°C. Protein amountswere measured by using the Lowry assay. Seventy micrograms ofprotein from each sample were suspended in loading buffer, separatedon a 10% polyacrylamide gel, and electrophoretically transferredto a nitrocellulose membrane. Membranes were blocked with 5%skim milk/1% bovine serum albumin for 1 h at room temperature.A primary antibody against phosphorylated extracellular activatedkinase-1/2 (ERK1/2) [p42/44 mitogen-activated protein (MAP)kinase; NE Biolabs, UK] was applied, with gentle rocking, overnightat 4°C. After three 10-min washing steps with TBST buffer(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% Tween 20), incubationwith horseradish peroxidase-conjugated goat anti-rabbit secondaryantibody (Dianova, Hamburg, Germany) was performed for 1 h atroom temperature. After three additional TBST washes, the membranewas incubated with Renaissance reagent (NEN Life Science, Zaventem,Belgium), according to the instructions provided by the manufacturer,and then exposed to x-ray film (Kodak, Rochester, NY). Quantificationwas performed with relative density measurements (Scion Image,Frederick, MD).
Tissue preparation for electrophoretic mobility shift assayswas performed as described previously (16). Nuclear extracts(5 µg) were incubated in binding reaction medium with0.5 ng of [32P]dATP-end-labeled oligonucleotide, containingthe NF-B binding site from the MHC enhancer (H2K, 5'-GATCCAGGGCTGGGGATTCCCCATCTCCACAGG-3').For AP-1, double-stranded oligonucleotides containing the consensussequence for AP-1 (5'-CGCTTGATGACTCAGCCGGAA-3') (Santa CruzBiochemicals, Santa Cruz, CA) were radiolabeled with -32P withthe use of T4 polynucleotide kinase, by standard methods, andwere purified on a column. The DNA-protein complexes were analyzedon 5% polyacrylamide gels, dried, autoradiographed. In competitionassays, 50 ng of unlabeled H2K or AP-1 oligonucleotides wereused.
Data are presented as mean ± SEM. The data were comparedby using ANOVA and the Scheffé test, as appropriate.Significant differences were accepted at P < 0.05. Data analysiswas performed by using StatView software (SAS Institute, Cary,NC) for Macintosh computers. For GFR (inulin) and PAH measurements,normal distributions were analyzed by using the Kolmogorov-Smirnovtest, and statistical significance was calculated by using thet test for independent groups. SPSS 10.0 (SPSS, Chicago, IL)software was used.
As demonstrated in Figure 1, 45 min of ischemia and subsequentreperfusion caused severe loss of renal function, as reflectedin a 7.5-fold elevation in creatinine levels (P < 0.005),compared with the sham-operated group. Renal function decreaseswere ameliorated by statin treatment, which reduced the elevationby 40%. Statin-treated rats demonstrated threefold creatininelevel increases 24 h after surgery, compared with sham-operatedanimals (P < 0.005). Right nephrectomy alone (sham operation)did not increase creatinine concentrations. To substantiatethe effects of statin treatment on renal function after ischemia,we analyzed GFR (inulin clearance) and PAH clearance. Theseresults are presented in Figure 2; GFR and PAH clearance insham-operated animals were 0.95 ± 0.09 ml/min and 4.4± 0.33 ml/min (n = 13), respectively. Ischemia (45 min)reduced these values to 0.06 ± 0.02 ml/min and 0.07 ±0.02 ml/min (n = 9, P < 0.001), respectively. Pretreatmentwith a statin significantly ameliorated the decreases in bothGFR and PAH clearance (0.21 ± 0.03 ml/min and 0.36 ±0.09 ml/min, respectively; n = 11, P < 0.001).
Figure 1. Serum creatinine levels before () and 24 h after () ischemia-reperfusion injury. Ischemia-reperfusion injury (IR) and treatment with saline vehicle resulted in significant creatinine level elevation after 24 h. Treatment with a statin before ischemia-reperfusion injury (IR + statin) dramatically reduced the impairment of kidney function. A right nephrectomy without ischemia of the contralateral kidney (sham OP) caused mild creatinine elevation after 24 h.
Figure 2. Inulin clearance (A) and PAH clearance (B) after 45 min of ischemia without treatment (IR) (n = 9) or with statin pretreatment (IR + statin) (n = 11), compared with sham-operated control animals (sham OP) (n = 13). Ischemia induced major decreases in both inulin clearance and PAH clearance (P < 0.001, compared with control animals). Pretreatment with a statin significantly increased GFR (*P < 0.001, compared with ischemia-reperfusion) and PAH clearance (*P < 0.05, compared with ischemia-reperfusion).
We next studied the effects of statins on ischemia-induced tissuedamage. Representative examples of these experiments are presentedin Figure 3. Ischemia-reperfusion injury caused severe tissuedamage in the S3 segment of proximal tubules. The outer medullarystripe exhibited loss of the brush border and detachment ofepithelial cells from the basement membrane. This effect leftnaked basement membranes (Figure 3D, arrow) and caused tubularobstruction (Figure 3D, arrowhead). In kidneys of statin-treatedanimals, the tubular necrosis was markedly reduced. Most tubuleswere intact and demonstrated normal brush borders (Figure 3, B and E).Kidneys from sham-operated animals are presented inFigure 3, C and F.
Figure 3. Morphologic damage in the S3 segment of proximal tubules in the outer medullary stripe. (A and D) Ischemia-reperfusion (IR) caused severe damage, with loss of the brush border (arrow), detachment of epithelial cells from the basement membrane, and tubular obstruction (arrowhead). (B and E) Statin pretreatment (IR + statin) markedly reduced the extent of acute tubular necrosis. (C and F) Control animals after right nephrectomy without ischemia (sham OP) exhibited almost no abnormalities in the contralateral kidney. V marks veins, and the bar marks the outer medullary stripe. Paraffin sections, Masson-Goldner trichrome stain. Magnification, x50 in A to C; x200 in D to F.
Because monocyte/macrophage infiltration is an important stepin tissue damage, we examined the effects of statins on leukocyteinfiltration after ischemia-reperfusion. Representative immunohistologicspecimens stained with ED-1 are presented in Figure 4, A to C.Ischemia-reperfusion injury produced marked perivascularand interstitial infiltration with ED-1-positive monocytes/macrophagesat 24 h (Figure 4A). Statin treatment before ischemia substantiallyblocked the infiltration of monocytes/macrophages (Figure 4B).Kidneys from sham-operated animals demonstrated only solitaryED-1-positive cells (Figure 4C). Figure 4, lower, presents asemiquantitative analysis of these findings. We observed analmost 50% decrease in monocyte/macrophage infiltration withstatin treatment (P < 0.005).
Figure 4. Perivascular infiltration by ED-1-positive monocytes and macrophages. (A) Ischemia-reperfusion (IR) caused severe infiltration of ED-1-positive cells. (B) The statin (IR + statin) reduced the influx of monocytes/macrophages into renal tissue. (C) The sham operation (sham OP) resulted in solitary infiltrating cells. (Lower) Semiquantitative scoring of ED-1-positive cells was performed. Results are depicted as mean ± SEM for each group. *P < 0.005. Magnifications, x200 in A through C.
We next addressed the effects of statins on adhesion moleculeexpression. Postischemic changes were reflected by the upregulationof adhesion molecules. Representative immunohistologic specimensstained for ICAM-1 are presented in Figure 5. Figure 5, A to C,demonstrates representative glomeruli, and Figure 5, D to F,presents representative arteries. Ischemia-reperfusion injuryinduced massive upregulation of ICAM-1 in the glomeruli andcortical peritubular interstitium (Figure 5A). The adhesionmolecule ICAM-1 was dramatically upregulated in the intima andin the perivascular interstitium of arteries after ischemia-reperfusioninjury (Figure 5D). Statins blocked ICAM-1 upregulation in theglomeruli and cortical peritubular interstitium (Figure 5B),as well as perivascular and endothelial upregulation of ICAM-1expression (Figure 5E). Figure 5, C and F, presents controlsections from the corresponding areas in kidneys of sham-operatedrats, with low ICAM-1 expression.
Figure 5. Representative immunohistochemical evaluations of intercellular adhesion molecule-1 (ICAM-1) expression in glomeruli (A to C) and in the perivascular interstitium (D to F). (A and D) Ischemia-reperfusion injury (IR) induced massive upregulation of ICAM-1 in the glomeruli and the cortical peritubular interstitium (A), as well as in the arterial endothelium and the perivascular interstitium (D). (B and E) The statin (IR + statin) partially blocked ICAM-1 upregulation after ischemia-reperfusion injury. (C and F) Sham-operated animals (sham OP) demonstrated only faint ICAM-1 expression. Magnification, x250.
Next, we analyzed the effects of statins on the upregulationof iNOS expression in glomeruli. Representative immunostainingsfor iNOS are presented in Figure 6, A to C. Ischemia-reperfusioninjury caused significant elevation of iNOS expression in theglomeruli of the corticomedullary junction (Figure 6A); thestaining pattern was mainly focal. The statin blocked iNOS upregulationin the glomeruli (Figure 6B). Sham-operated animals demonstratedlow levels of iNOS expression, comparable to that of statin-treatedanimals (Figure 6C). Figure 6, lower, presents the semiquantitativeanalysis of iNOS expression; iNOS expression was defined ashigh, medium, or low. Ischemia-reperfusion yielded a significantlygreater number of glomeruli with high levels of iNOS expression,compared with the statin-treated and sham-operated animals (P< 0.005).
Figure 6. Representative immunohistochemical evaluations of inducible nitric oxide synthase (iNOS) expression in glomeruli. (A) Ischemia-reperfusion injury (IR) induced iNOS expression in glomeruli. (B) The statin (IR + statin) inhibited iNOS upregulation to low iNOS expression levels. (C) Comparable levels were noted for sham-operated control animals (sham OP). (Lower) Semiquantitative analysis of iNOS expression was performed. iNOS expression was defined as high (red), medium (blue), or low (yellow). Ischemia-reperfusion resulted in a significantly greater number of glomeruli with high levels of iNOS expression, compared with statin-treated and sham-operated animals (P < 0.05). Magnification, x200 in A through C.
We then examined fibronectin expression. Fibronectin is a multifunctionalprotein that is upregulated in the early phase of ischemia-reperfusioninjury and remains at high levels for several weeks. Representativeimmunostaining for fibronectin in the outer medullary stripeis presented in Figure 7, A to C. We observed that tubulointerstitialfibronectin was upregulated in ischemia-reperfusion injury (Figure 7A).Statin treatment completely prevented the marked expressionof fibronectin (Figure 7B). Control animals demonstrated similarstaining patterns, compared with statin-treated animals (Figure 7C).Collagen IV is a marker for matrix accumulation in thekidney. After ischemia-reperfusion injury, there was a broadeningof the peritubular interstitium, with increased expression ofcollagen IV. Representative staining for collagen IV is presentedin Figure 7, D to F. Ischemia-reperfusion injury led to markedupregulation of collagen IV expression in the periglomerularand peritubular interstitium (Figure 7D). We observed that thestatin partially prevented matrix expansion (Figure 7E), comparedwith sham-operated control animals (Figure 7F).
Figure 7. Representative immunohistochemical evaluations of fibronectin expression (A to C) and collagen IV expression (D to F). (A) Ischemia-reperfusion injury (IR) caused matrix accumulation in the peritubular interstitium, with increased expression of fibronectin. (B) The statin (IR + statin) partially prevented the matrix expansion. (C) Sham-operated animal (sham OP). (D) Ischemia-reperfusion injury caused matrix accumulation in the periglomerular and peritubular interstitium, with markedly elevated collagen IV expression. (E) The statin partially prevented the matrix expansion. (F) Sham-operated animal. Magnification, x200 in A through F.
We next assessed possible intracellular statin-related effects.MAP kinases are involved in inflammatory processes and can beupregulated by ischemia. Therefore, we studied the activationof the MAP kinase ERK1/2. The p42/44 antibody directed againstthe phosphorylated form of ERK1/2 detected activation. RepresentativeWestern blots are presented in Figure 8. Figure 8A presentsthe Western blot results; each lane corresponds to results forone animal. Figure 8B presents the densitometric analysis results.Ischemia-reperfusion injury caused marked activation of ERK1/2(Figure 8A, left). We observed that the statin was effectivein decreasing ERK1/2 activation after ischemia-reperfusion injury(Figure 8A, middle). The sham operation caused only slight activationof ERK1/2 (Figure 8A, right).
Figure 8. Western blots of phosphorylated extracellular activated kinase-1/2 (ERK1/2) (42/44 kD). Each lane represents 70 µg of protein from a single kidney. (A) Ischemia-reperfusion (IR) led to marked phosphorylation of ERK1/2. The statin (IR + statin) decreased the ischemia-reperfusion injury-induced activation of ERK1/2. Kidneys from sham-operated rats (sham OP) demonstrated only slight ERK1/2 phosphorylation. (B) The results of densitometric analysis of phospho-ERK1/2 intensities are depicted as mean values.
In Figure 9, we demonstrate activation of the DNA-binding nuclearfactor NF-B in electrophoretic mobility shift assays. Two lanesare representative of the group. NF-B DNA-binding activity wasupregulated in the kidney after ischemia-reperfusion injury.The sham operation resulted in slight NF-B activation. The statinblocked NF-B activation in a time-dependent manner. Three daysof statin treatment before ischemia were more effective in blockingNF-B activation, compared with 2 d of treatment. Similar effectson AP-1 activity were observed (data not shown).
Figure 9. Activation of DNA-binding nuclear factors by ischemia-reperfusion injury. (Lanes 1 and 2) Ischemia-reperfusion injury (IR) caused strong upregulation of nuclear factor-B (NF-B) DNA-binding activity. (Lanes 3 and 4) Sham-operated animals (sham). (Lanes 5 to 8) Statin pretreatment before ischemia-reperfusion injury (IR + statin) could partially prevent the upregulation of NF-B DNA-binding activity. Three-day treatment (lanes 7 and 8) was more effective than 2-d treatment (lanes 5 and 6).
We investigated the effects of cerivastatin on acute ischemia-reperfusioninjury. We observed that 3-d treatment significantly amelioratedthe decrease in renal function with postischemic acute tubularnecrosis and considerably limited the structural damage afterischemia. Histologically, we observed that statin treatmentreduced damage in the S3 segment of proximal tubules in theouter medullary stripe, the area that is most susceptible tohypoperfusion and hypoxia. Untreated animals demonstrated typicalchanges in the S3 segment, namely loss of the brush border,destruction of epithelial cells, naked basement membranes, andtubular obstruction. Statin treatment clearly reduced the morphologicdamage. Inflammatory reactions attributable to ischemia-reperfusioninjury are characterized by leukocyte infiltration. In postischemicARF, the majority of infiltrating cells are ED-1-positive monocytesand macrophages (10). We observed that the statin reduced ED-1-positivecell infiltration by >50%, compared with no treatment. Inflammatorycell infiltration in ischemia-reperfusion injury is accompaniedby significant upregulation of adhesion molecules (17). We observedthat activation of the adhesion cascade, as reflected by upregulationof ICAM-1 on the endothelium of arteries and in the perivascularspace, was inhibited by the statin. The upregulation of ICAM-1expression in glomeruli, the periglomerular area, and the peritubularinterstitium was also prevented. Other strategies to preventICAM-1 expression after ischemia-reperfusion injury during ratrenal transplantation have been demonstrated to reduce chronicgraft dysfunction (11). The statins might also be beneficialin such situations.
Enhanced NO production, presumably attributable to macrophage-typeiNOS, participates in hypoxic/ischemic proximal tubular injury(18). Experiments in gene-disrupted mice demonstrated that iNOSblockade attenuates renal ischemia-reperfusion injury (19).We observed that statin treatment prevented increased iNOS expressionin glomeruli. This statin-related effect could lead to a decreasein free radical oxygen levels during ischemia and reperfusion.In other models of renal disease, inhibition of oxidative systemswas demonstrated to have marked beneficial effects and to preventtissue damage (20).
Fibronectin is a major glycoprotein in plasma and in the extracellularmatrix. Fibronectin plays a role as a chemoattractant for severalcell types and generates a scaffold to which other matrix componentscan attach. In response to ischemia-reperfusion injury, fibronectinis markedly upregulated in the renal interstitium (21). We observedthat cerivastatin effectively blocked the increased expressionof fibronectin in the tubulointerstitium of the outer medullarystripe. We also examined collagen IV expression, because abnormalcollagen turnover is a major histologic feature in many formsof renal disease. Collagen IV expression is also predictiveof final outcomes. In ischemia-reperfusion injury, increaseddeposition of collagen IV occurs in the renal interstitium,Bowman’s capsule, and tubular basement membranes (22).We observed that the increased collagen IV deposition in theperitubular interstitium after ischemia-reperfusion injury wasdecreased with statin treatment.
We focused on MAP kinase ERK1/2 activation because, in our earlierangiotensin II-induced glomerulosclerosis model, we observedthat these signaling molecules were probably involved in renalinjury (14). Many extracellular signals, including oxidativestress, are transduced to the nucleus via activation of theMAP kinase cascade (5). Statins can directly interfere withintracellular signaling mechanisms (23). The most likely mechanisminvolves statin inhibition of G protein prenylation, via reductionsof farnesylation and geranylgeranylation. In vivo studies havedemonstrated that these statin-related effects on intracellularsignaling mechanisms can be overcome with the addition of eithermevalonate or farnesol (24,25). Small G proteins of the Rasfamily regulate MAP kinase activation. Ras binds to Raf, thusactivating MEK, which in turn phosphorylates ERK1/2 (26). Asin our previous in vitro studies, we observed that cerivastatindownregulated the ischemia-induced activation of ERK1/2 (14).We have now extended these observations to the in vivo settingand to a different model, namely postischemic ARF.
NF-B and AP-1 are redox-sensitive transcription factors thatare activated by many stimuli, including atherogenic events(16). NF-B and AP-1 regulate the expression of genes encodinginflammatory adhesion molecules, cytokines, and chemokines,leading to inflammation (27). We demonstrated that statin treatmentpartially prevented the upregulation of the DNA-binding activityof NF-B and AP-1 in ischemia-reperfusion injury. NF-B belongsto a family of transcription factors that are recognized bythe B enhancer element. Numerous proinflammatory genes havebinding sites for NF-B, and the products of these genes areintegral parts of cellular activation and inflammatory responsesystems. There are close relationships between NF-B and mediatorsof cell activation (28). We and others previously demonstratedthat the DNA-binding activity of NF-B and AP-1 could be blockedby statin treatment in rat models (15). Direct inhibition ofthis transcription factor prevents tissue damage in other formsof renal disease as well (29).
We do not think that our findings are related to a reductionin LDL cholesterol levels. We did not measure cholesterol levelsin this study, because in our earlier study we demonstratedthat treatment of Sprague-Dawley rats with cerivastatin (0.5mg/kg per d for 3 wk) did not decrease cholesterol levels (15).We performed our study from clinical interest, because prophylactictreatment with a statin drug for 3 d would clearly be welcomefor patients at risk for developing ARF. Elective transplantrecipients, patients electively undergoing major surgery, andperhaps patients undergoing radiologic interventions might becandidates. We can only speculate regarding the clinical relevanceof our findings. We selected cerivastatin because this drugpenetrates nonhepatocyte cells, in which it also inhibits hydroxy-3-methylglutarylcoenzyme A reductase (30). Other lipid-soluble statins alsoexhibit this property (31). Whether our findings are characteristicof relatively lipid-soluble statins only or can be extrapolatedto hydrophilic statins cannot be determined on the basis ofour data. Our speculations regarding intracellular mechanismsalso require further testing. However, the data we report hereand those from other studies (32,33) support the concept thatstatins have novel pleiotropic effects on intracellular signaling,independent of circulating cholesterol levels.
In conclusion, we demonstrated that a statin ameliorated postischemicARF. Inflammatory mechanisms were significantly affected, supportingthe hypothesis that the statin exerted direct anti-inflammatoryeffects in vivo. Cell infiltration, ICAM-1 and iNOS upregulation,matrix molecule expression, MAP kinase ERK1/2 activation, andtranscription factor activation were all reduced. Our resultsindicate that certain statins may be useful in the preventionof ARF.
Acknowledgments
We thank Kerstin Bankes, Yvonne Nikolai, Michaela Beese, PetraBerkefeld, and Karin Dressler for technical assistance. Thisstudy received support from the Bayer Corporation. Drs. Muller,Elger, Luft, and Haller were supported by grants-in-aid fromthe Deutsche Forschungsgemeinschaft. This study was also supportedby the Klinisch-Pharmakologischer Verbund, Berlin-Brandenburg.
Dragun D, Hoff U, Park JK, Qun Y, Schneider W, Luft FC, Haller H: Ischemia-reperfusion injury in renal transplantation is independent of the immunologic background. Kidney Int 58: 2166–2177, 2000[CrossRef][Medline]
Cosentino F: Drugs for the prevention and treatment of acute renal failure. Cleve Clin J Med 62: 248–253, 1995[Medline]
Dishart MK, Kellum JA: An evaluation of pharmacological strategies for the prevention and treatment of acute renal failure. Drugs 59: 79–91, 2000[CrossRef][Medline]
Liu ZX, Nickel CH, Cantley LG: HGF promotes adhesion of ATP-depleted renal tubular epithelial cells in a MAPK-dependent manner. Am J Physiol 281: F62–F70, 2001
Edelstein CL, Alkhunaizi AA, Schrier RW: The role of calcium in the pathogenesis of acute renal failure. Ren Fail 19: 199–207, 1997[Medline]
Zager RA, Burkhart KM, Johnson A: Sphingomyelinase and membrane sphingomyelin content: Determinants of proximal tubule cell susceptibility to injury. J Am Soc Nephrol 11: 894–902, 2000[Abstract/Free Full Text]
Zuk A, Bonventre J, Brown D, Matlin KS: Polarity, integrin, and extracellular matrix dynamics in the postischemic rat kidney. Am J Physiol 275: C711–C731, 1998
Ysebaert DK, De Greef KE, Vercauteren SR, Ghielli M, Verpooten GA, Eyskens EJ, De Broe ME: Identification and kinetics of leukocytes after severe ischaemia/reperfusion renal injury. Nephrol Dial Transplant 15: 1562–1574, 2000[Abstract/Free Full Text]
Dragun D, Tullius SG, Park JK, Maasch C, Lukitsch I, Lippoldt A, Gross V, Luft FC, Haller H: ICAM-1 antisense oligodesoxynucleotides prevent reperfusion injury and enhance immediate graft function in renal transplantation. Kidney Int 54: 590–602, 1998[CrossRef][Medline]
Kelly KJ, Williams WW Jr, Colvin RB, Meehan SM, Springer TA, Gutierrez-Ramos JC, Bonventre JV: Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury. J Clin Invest 97: 1056–1063, 1996[Medline]
Crisby M, Nordin-Fredriksson G, Prediman KS, Yano J, Zhu J, Nilsson J: Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteases, and cell death in human carotid plaques. Circulation 103: 926–933, 2001[Abstract/Free Full Text]
Park JK, Muller DN, Mervaala EM, Dechend R, Fiebeler A, Schmidt F, Bieringer M, Schafer O, Lindschau C, Schneider W, Ganten D, Luft FC, Haller H: Cerivastatin prevents angiotensin II-induced renal injury independent of blood pressure- and cholesterol-lowering effects. Kidney Int 58: 1420–1430, 2000[CrossRef][Medline]
Dechend R, Fiebler A, Lindschau C, Bischoff H, Muller D, Park JK, Dietz R, Haller H, Luft FC: Modulating angiotensin II-induced inflammation by HMG Co-A reductase inhibition. Am J Hypertens 14: 55S–61S, 2001[CrossRef][Medline]
Muller DN, Dechend R, Mervaala EM, Park JK, Schmidt F, Fiebeler A, Theuer J, Breu V, Ganten D, Haller H, Luft FC: NF-B inhibition ameliorates angiotensin II-induced inflammatory damage in rats. Hypertension 35: 193–201, 2000[Abstract/Free Full Text]
Linas SL, Whittenburg D, Parsons PE, Repine JE: Ischemia increases neutrophil retention and worsens acute renal failure: Role of oxygen metabolites and ICAM 1. Kidney Int 48: 1584–1591, 1995[Medline]
Liang M, Knox FG: Production and functional roles of nitric oxide in the proximal tubule. Am J Physiol 278: R1117–R1124, 2000
Ling H, Edelstein C, Gengaro P, Meng X, Lucia S, Knotek M, Wangsiripaisan A, Shi Y, Schrier R: Attenuation of renal ischemia-reperfusion injury in inducible nitric oxide synthase knockout mice. Am J Physiol 277: F383–F390, 1999
Scheuer H, Gwinner W, Hohbach J, Grone EF, Brandes RP, Malle E, Olbricht CJ, Walli AK, Grone HJ: Oxidant stress in hyperlipidemia-induced renal damage. Am J Physiol 278: F63–F74, 2000
Zuk A, Bonventre JV, Matlin KS: Expression of fibronectin splice variants in the postischemic rat kidney. Am J Physiol 280: F1037–F1053, 2001
Forbes JM, Leaker B, Hewitson TD, Becker GJ, Jones CL, Gailit J, Colflesh D, Rabiner I, Simone J, Goligorsky MS: Macrophage and myofibroblast involvement in ischemic acute renal failure is attenuated by endothelin receptor antagonists. Kidney Int 55: 198–208, 1999[CrossRef][Medline]
Davignon J, Mabile L: Mechanisms of action of statins and their pleiotropic effects (in French). Ann Endocrinol (Paris) 62: 101–112, 2001[Medline]
Guijarro C, Blanco-Colio LM, Ortego M, Alonso C, Ortiz A, Plaza JJ, Diaz C, Hernández G, Egido J: 3-Hydroxy-3-methylglutaryl coenzyme A reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle cells in culture. Circ Res 83: 490–500, 1998[Abstract/Free Full Text]
Bellosta S, Ferri N, Bernini F, Paoletti R, Corsini A: Non-lipid-related effects of statins. Ann Med 32: 164–176, 2000[Medline]
di Mari JF, Davis R, Safirstein RL: MAPK activation determines renal epithelial cell survival during oxidative injury. Am J Physiol 277: F195–F203, 1999
Guijarro C, Kim Y, Kasiske BL, Massy ZA, O’Donnel MP, Kashtane CE, Keane WF: Central role of transcription factor nuclear factor B in mesangial cell production of chemokines. Contrib Nephrol 120: 210–218, 1997[Medline]
Massy ZA, Guijarro C, O’Donnell MP, Kim Y, Kashtan CE, Egido J, Kasiske BL, Keane WF: The central role of nuclear factor-B in mesangial cell activation. Kidney Int Suppl 71: S76–S79, 1999[CrossRef][Medline]
Vos IH, Govers R, Grone HJ, Kleij L, Schurink M, De Wegner RA, Goldschmeding R, Rabelink TJ: NFB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts. FASEB J 14: 815–822, 2000[Abstract/Free Full Text]
Christians U, Jacobsen W, Floren LC: Metabolism and drug interactions of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in transplant patients: Are the statins mechanistically similar? Pharmacol Ther 80: 1–34, 1998[CrossRef][Medline]
Joukhadar C, Klein N, Prinz M, Schrolnberger C, Vukovich T, Wolzt M, Schmetterer L, Dorner GT: Similar effects of atorvastatin, simvastatin and pravastatin on thrombogenic and inflammatory parameters in patients with hypercholesterolemia. Thromb Haemost 85: 47–51, 2001[Medline]
Faggiotto A, Paoletti R: State-of-the-art lecture: Statins and blockers of the renin-angiotensin system: Vascular protection beyond their primary mode of action. Hypertension 34: 987–996, 1999[Abstract/Free Full Text]
Faggiotto A, Paoletti R: Do pleiotropic effects of statins beyond lipid alterations exist in vivo? What are they and how do they differ between statins? Curr Atheroscler Rep 2: 20–25, 2000[Medline]
Received for publication August 28, 2001.
Accepted for publication May 14, 2002.
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Abstract
Introduction
B and activator protein-1 were activated. Statin treatment reduced this activation toward levels observed in sham-operated rats. The data suggest that hydroxy-3-methylglutaryl coenzyme A reductase inhibition protects renal tissue from the effects of ischemia-reperfusion injury and thus reduces the severity of ARF. The chain of events may involve anti-inflammatory effects, with inhibition of mitogen-activated protein kinase activation and the redox-sensitive transcription factors nuclear factor-
-32P with the use of T4 polynucleotide kinase, by standard methods, and were purified on a column. The DNA-protein complexes were analyzed on 5% polyacrylamide gels, dried, autoradiographed. In competition assays, 50 ng of unlabeled H2K or AP-1 oligonucleotides were used. 
) and 24 h after (
) ischemia-reperfusion injury. Ischemia-reperfusion injury (IR) and treatment with saline vehicle resulted in significant creatinine level elevation after 24 h. Treatment with a statin before ischemia-reperfusion injury (IR + statin) dramatically reduced the impairment of kidney function. A right nephrectomy without ischemia of the contralateral kidney (sham OP) caused mild creatinine elevation after 24 h.
