Ischemic Preconditioning Provides Both Acute and Delayed Protection against Renal Ischemia and Reperfusion Injury in Mice
Jin Deok Joo*,,
Mihwa Kim*,
Vivette D. D’Agati and
H. Thomas Lee*
* Anesthesiology; Pathology, College of Physicians and Surgeons of Columbia University, New York, New York; and Department of Anesthesiology, Saint Vincent’s Hospital, The Catholic University of Korea, Paldal-gu, Suwon, Gyounggi-Do, South Korea
Address correspondence to: Dr. H. Thomas Lee, Department of Anesthesiology, Anesthesiology Research Laboratories, Columbia University, P&S Box 46 (PH-5), 630 West 168th Street, New York, NY 10032-3784. Phone: 212-305-1807; Fax: 212-305-8980; tl128{at}columbia.edu
Received for publication May 2, 2006.
Accepted for publication August 10, 2006.
Acute as well as delayed ischemic preconditioning (IPC) providesprotection against cardiac and neuronal ischemia reperfusion(IR) injury. This study determined whether delayed preconditioningoccurs in the kidney and further elucidated the mechanisms ofrenal IPC in mice. Mice were subjected to IPC (four cycles of5 min of ischemia and reperfusion) and then to 30 min of renalischemia either 15 min (acute IPC) or 24 h (delayed IPC) later.Both acute and delayed renal IPC provided powerful protectionagainst renal IR injury. Inhibition of Akt but not extracellularsignal–regulated kinase phosphorylation prevented theprotection that was afforded by acute IPC. Neither extracellularsignal–regulated kinase nor Akt inhibition prevented protectionthat was afforded by delayed renal IPC. Pretreatment with anantioxidant, N-(2-mercaptopropionyl)-glycine, to scavenge freeradicals prevented the protection that was provided by acutebut not delayed renal IPC. Inhibition of protein kinase C orpertussis toxin–sensitive G-proteins attenuated protectionfrom both acute and delayed renal IPC. Delayed renal IPC increasedinducible nitric oxide synthase (iNOS) as well as heat-shockprotein 27 synthesis, and the renal protective effects of delayedpreconditioning were attenuated by a selective inhibitor ofiNOS (l-N6[1-iminoethyl]lysine). Moreover, delayed IPC was notobserved in iNOS knockout mice. Both acute and delayed IPC wereindependent of A1 adenosine receptors (AR) as a selective A1ARantagonist failed to block preconditioning and acute and delayedpreconditioning occurred in mice that lacked A1AR. Therefore,this study demonstrated that acute or delayed IPC provides renalprotection against IR injury in mice but involves distinct signalingpathways.
Acute renal failure (ARF) results frequently from renal ischemiaand reperfusion (IR) injury and is a major contributor to morbidityand mortality during the perioperative period. We previouslydemonstrated in rats that acute renal ischemic preconditioning(IPC; brief and intermittent ischemia and reperfusion minutesbefore more prolonged ischemia) protected against IR injuryin rats via pertussis toxin–sensitive G-protein (Gi) andprotein kinase C (PKC) (1,2). In contrast to cardiac (3,4),cerebral (5), and hepatic (6) preconditioning, renal IPC invivo was not blocked by A1 adenosine receptor (AR) antagonismand was independent of K+ATP channels (1). Moreover, methacholine,morphine, or bradykinin, agents that mimic IPC in the heart,failed to protect renal function after IR injury (1). Therefore,the major signaling events of renal IPC remain unknown.
In cardiac and neuronal IR injury, acutely protective effectsof IPC dissipate over several hours but reappear 24 to 72 hlater (7,8). This phenomenon is defined as a second window ofprotection, or delayed IPC, and is proposed to be mediated byinducible nitric oxide synthase (iNOS) upregulation and enhancedrelease of NO in these organs (9). In the kidney, previous ARFand recovery protected against subsequent ARF from renal IRinjury that was induced many days later (10). However, delayedIPC with brief, intermittent periods of renal ischemia thatare not severe enough to cause ARF is incompletely characterizedand described in the kidney.
Renal cells are subjected to obligatory bursts of oxidant stressduring the reperfusion phase after each preconditioning stimulus.Accumulating evidence indicates that oxygen free radicals functionas second messengers in several cell types, including renalcells (11,12). Oxygen free radicals phosphorylate several importantcytoprotective kinases, including extracellular signal–regulatedprotein kinase mitogen-activated protein kinase (ERK MAPK) andAkt (13,14), and are involved in the upregulation of severalcytoprotective genes (15). Indeed, oxidative stress upregulatesheat-shock proteins (HSP), which are recognized molecular chaperonesto function as cytoprotective proteins (16). HSP protect otherintracellular proteins from denaturation and aggregation thatoccur in response to oxidative stress. In particular, HSP27,HSP32 (also known as heme oxygenase-1), and HSP70 have beenimplicated in mediating cytoprotection in a variety of celltypes (16,17).
In this study, we further characterized the renal protectiveeffects and probed the cellular signaling mechanisms of acuteand delayed renal IPC. We tested the following four hypothesesusing a murine model of renal IPC and IR injury: (1) Acute aswell as delayed renal IPC protects against renal IR injury inmice, (2) brief oxidative stress during the reperfusion phasesof IPC may initiate the signaling cascades of acute and/or delayedrenal IPC that protect against subsequent IR injury, (3) renalprotection with acute IPC depends on oxygen free radical–mediatedphosphorylation of preexisting cytoprotective proteins (e.g.,ERK MAPK, Akt, PKC, HSP27), and (4) delayed renal IPC protectsvia induction of cytoprotective proteins including HSP and iNOS.
Induction of Renal IPC and Renal IR Injury
All protocols were approved by the Institutional Animal Careand Use Committee of Columbia University. C57BL/6 mice (CharlesRiver, Wilmington, MA; 20 to 25 g) were anesthetized with intraperitonealpentobarbital (50 mg/kg or to effect) and placed in a heatingpad under a warming light to maintain body temperature between36 and 38°C. Additional pentobarbital was given as needed.Bilateral flank incisions were made, and after right nephrectomy,the left kidney was subjected to 30 min of ischemia. For inductionof renal IPC, mice were subjected to four cycles of 5 min ofleft renal ischemia separated by 5-min reperfusion periods.Acutely preconditioned animals were subjected to renal ischemia15 min after preconditioning. For studying delayed renal IPC,mice were subjected to renal ischemia 6 and 24 h after IPC.Some mice were subjected only to renal IPC without IR injury,and their kidney was isolated 15 min or 24 h later. The shammice for the acute IPC group were anesthetized and subjectedto right nephrectomy only, and plasma was collected 24 h later.The sham mice for the delayed IPC group were anesthetized, subjectedto midline laparotomy without left renal IPC, and allowed toawaken. Twenty-four hours later, they were re-anesthetized andsubjected to right nephrectomy only, and plasma was collected24 h later. Preliminary data showed that plasma creatinine valuesfor acute and delayed IPC sham mice were similar; therefore,the plasma creatinine values were pooled as a single sham group.
Assessment of Renal Function after IR Injury
Renal function was assessed (1) by measurement of plasma creatinine24 h after renal ischemia as described previously (1,2) and(2) by assessment of changes in renal outer medullary bloodflow (ROMBF; near the corticomedullary junction) after renalischemia as described previously (18). Previous studies demonstratedthat laser Doppler probes provide reliable measurements of relativechange in regional blood flow in the kidney (19). Needle flowprobe (480 µm diameter; Model TSD145) connected to a laserDoppler flowmeter (Biopac Systems, Goleta, CA) was used to measurethe relative changes in ROMBF after renal ischemia or afterrenal IPC plus ischemia. The flow meter generates a voltagesignal that is proportional to the blood flow velocity in approximately1 mm3 of renal tissue approximately 1 mm under the probe. Theneedle tip of the flow probe was inserted directly into thekidney tissue in the outer medullary regions (approximately1.5 mm beneath the surface of the kidney) through small holesmade in the renal capsule with a 26-G needle and was held inplace by a micromanipulator. Although the insertion of the probeis invasive, blood flow is measured in the undisturbed regionapproximately 1 mm beneath the tip of the optical fiber in theouter medullary area. Voltage output was recorded on a computerconnected to a Biopac data acquisition system and representedas blood perfusion unit. The flow data were represented as thepercentage change compared with preischemic blood perfusionunit. Zero flow was confirmed when the renal artery was completelyoccluded during renal ischemia. At the end of each experiment,the kidney was excised to confirm the position of the needleprobe tips in the outer medullary area. Mice with incorrectlyplaced probes were excluded from the study.
Potential Roles of ERK, Akt, and A1AR in Renal IPC
All of the inhibitors of signaling intermediates in this protocolwere given 15 min before sham operation, renal ischemia, orrenal IPC. The dosages of PD98059 and wortmannin were selectedon the basis of previous in vivo studies (20–23). In addition,we performed preliminary experiments to demonstrate that thedosage and method of administration of PD98059 and wortmanninthat we used effectively blocked the phosphorylation of ERKand Akt in vivo, respectively (see Results). To test the hypothesisthat ERK MAPK and/or Akt participates in acute or delayed renalIPC, we pretreated the mice with PD98059 (an inhibitor of mitogen-activatedprotein kinase kinase (MEK1) to inhibit ERK phosphorylation,1 mg/kg, intraperitoneally) or wortmannin (an inhibitor of phosphatidylinositol-3kinase [PI3K] to inhibit Akt phosphorylation, 1 mg/kg, intraperitoneally).To further confirm that the A1AR are not involved in renal IPC(acute and delayed), we used complementary pharmacologic andgene knockout (KO) approaches. Some C57 mice were pretreatedwith 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 mg/kg, intraperitoneally),a selective A1AR antagonist (24). We also subjected mice thatlacked A1AR and their wild-type (WT) controls to acute or delayedrenal IPC and then to 30 min of renal ischemia either 15 minor 24 h later. Breeding pairs of A1AR heterozygous mice wereobtained from Dr. Jurgen Schnermann (National Institute of Diabetesand Digestive and Kidney Diseases, Bethesda, MD) to generateA1AR WT and A1AR KO mice as described previously (25).
Potential Role of Gi/o and PKC in Renal IPC
For determination of the role of Gi/o or PKC in renal IPC, micewere pretreated with pertussis toxin (25 µg/kg intraperitoneally48 h before) or with chelerythrine (5 mg/kg, intraperitoneally15 min before), respectively, before renal IPC (1).
Potential Role of Free Radical Generation in Mediating Renal IPC
Mice were pretreated with 100 mg/kg N-(2-mercaptopropionyl)-glycine(MPG; a free radical scavenger) 15 min before renal IPC andwere subjected to IR either 15 min or 24 h later. Some micealso were pretreated with MPG (8,26,27) and subjected to shamoperation or renal IR injury without preconditioning. We performedpreliminary experiments to demonstrate that the dosage and methodof MPG administration that we used effectively blocked the phosphorylationof ERK, Akt, and HSP27 in vivo (see Results).
Potential Role of iNOS in Delayed IPC
To test the hypothesis that induction of iNOS mediates the cytoprotectiveeffects of delayed renal IPC, we used complementary approachesusing a pharmacologic inhibitor of iNOS (l-N6-[1-iminoethyl]lysine [L-NIL]; 10 mg/kg, intraperitoneally; A.G. Scientific,San Diego, CA) and iNOS KO mice. C57BL/6 mice were preconditionedwith intermittent ischemia and reperfusion 24 h before renalischemia. Fifteen minutes before 30 min of renal ischemia, micewere given an injection of either vehicle (saline) or 10 mg/kgL-NIL intraperitoneally and were subjected to 30 min of renalischemia (28). C57BL/6J (iNOS +/+) and iNOS gene deletion (iNOS–/–) male mice (strain B6;129P2-Nos2tm1Lau/J; stockno. 002596) were purchased from Jackson Laboratory (Bar Harbor,ME). iNOS KO mice and their WT controls were subjected to renalIPC and then to renal ischemia 24 h later.
Renal Cortical NOS Activity Assay
We measured NOS activity in the renal cortices (including corticomedullaryjunction) of mice that were subjected to sham operation or toIPC 24 h before with a commercially available kit (Calbiochem,EMD Biosciences, San Diego, CA). The assay is based on the enzymaticconversion of nitrate to nitrite by nitrate reductase, followedby the spectrophotometric quantification of nitrite levels usingthe Griess reagent.
Histologic Detection of Necrosis
Morphologic assessment was performed by an experienced renalpathologist who was unaware of the treatment that each animalhad received. A grading scale of 0 to 4, as outlined by Jablonskiet al. (29), was used to assess the degree of renal tubularnecrosis in the outer medullary area after renal IR injury asdescribed previously (1,2).
Renal Cortical Protein Preparation and Immunoblot Analyses
For determination of the signaling pathways of acute and delayedrenal IPC, kidneys were isolated 15 min or 24 h after renalIPC without being subjected to ischemic injury. Mouse kidneycortical tissues (including corticomedullary junction) weredissected on ice and immediately placed in ice-cold RIPA buffer(150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, and 1% Triton-X [pH7.4]) and homogenized for 10 s on ice. The samples were centrifugedfor 30 min at 50,000 x g. The supernatant was collected andused for immunoblotting as described previously (30,31).
We measured the phosphorylation of ERK MAPK, Akt (protein kinaseB), and phospho-HSP27 by immunoblotting. Phospho-ERK, phospho-Akt,or phospho-HSP27 blots were stripped and reprobed for totalERK, Akt, or HSP27. We also measured expression of HSP70, HSP32(heme oxygenase-1), and iNOS protein expression after renalIPC. The primary antibodies for phospho-ERK1/2, total ERK1/2,HSP70, and HSP32 were from Santa Cruz Biotechnologies (SantaCruz, CA). Antibodies for phospho-Akt, phospho-HSP27, totalAkt, and total HSP27 were from Cell Signaling Technologies (Danvers,MA). The iNOS antibody was from BD Biosciences Pharmingen (SanJose, CA). The antibody for the inducible form of HSP70 wasfrom Stressgen Biotechnologies (San Diego, CA). The secondaryantibody (goat anti-rabbit or anti-mouse IgG conjugated to horseradishperoxidase at 1:5000 dilution) was detected with ECL immunoblottingdetection reagents (Amersham, Piscataway, NJ), with subsequentexposure to a CCD camera coupled to a UVP Bio-imaging System(Upland, CA). The band intensities of the immunoblots were withinthe linear range of exposure for all experiments.
Statistical Analyses
The data were analyzed with t test when means between two groupswere compared or with one-way (e.g., plasma creatinine) or two-way(e.g., renal medullary blood flow) ANOVA plus Tukey post hocmultiple comparison test to compare mean values across multipletreatment groups. The ordinal values of the Jablonski scalewere analyzed by the Kruskal-Wallis nonparametric test withDunn posttest comparison among groups. In all cases, P <0.05 was taken to indicate significance. All data are expressedas mean ± SEM.
Renal Protective Effects of Acute and Delayed Renal IPC
As expected, 30 min of renal ischemia and 24 h of reperfusionresulted in significant rises in serum creatinine (2.6 ±0.2 mg/dl; n = 11; P < 0.001 versus sham; Table 1) comparedwith the sham-operated mice (0.4 ± 0.1 mg/dl; n = 6).Acute IPC (four cycles of 5 min of renal ischemia and 5 minof reperfusion, 15 min before 30 min of renal ischemia) significantlyimproved renal function (creatinine = 1.1 ± 0.2 mg/dl;n = 10; P < 0.001 versus IR) after 30 min of renal ischemiaand 24 h of reperfusion compared with mice that were subjectedto IR injury alone.
Table 1. Plasma creatinine values in mice subjected to Sham, IR, acute IPC+IR, and delayed IPC+IRa
When the mice were subjected to renal ischemia 6 h after renalIPC, renal protective effects of IPC were lost (creatinine =3.0 ± 0.3 mg/dl; n = 4; P = 0.38 versus IR, P < 0.001versus acute IPC+IR). However, when the mice were subjectedto renal ischemia 24 h after renal IPC, serum creatinine wassignificantly lower (0.7 ± 0.1 mg/dl; n = 8; P < 0.001versus IR).
Acute and Delayed Renal IPC Improves ROMBF in Renal IR Injury
Thirty minutes of renal ischemia and 60 min of reperfusion ledto a significant reduction in ROMBF (61± 4% of preischemicvalue; n = 7; P < 0.001 versus preischemic value). Both acute(15 min before renal IR; 84± 4% of preischemic value;n = 6; P < 0.01 versus IR group) and delayed renal IPC (24h before renal IR; 100± 8% of preischemic value; n =10; P < 0.001 versus IR group) led to better preserved ROMBFafter IR injury (with 30 min of renal ischemia and reperfusion;Figure 1). It is interesting that approximately 20% increasesin ROMBF were observed immediately after postischemic reperfusionin mice that were subjected to a preconditioning stimulus 24h earlier (P = 0.04 versus IR group).
Figure 1. Changes in renal outer medullary blood flow measured with laser Doppler flux (blood perfusion unit [BPU]) in mice. Mice were subjected to 30 min of renal ischemia and 60 min of reperfusion (IR; n = 7). Some mice were subjected to ischemic preconditioning (IPC) and then to 30 min of renal ischemia 15 min later (acute IPC+IR; n = 6) or 24 h later (delayed IPC+IR; n = 10). Changes in renal medullary blood compared with preischemic values (% preischemic BPU) are represented. *P < 0.05 versus IR; #P < 0.01 versus IR; $P < 0.001 versus IR. Renal medullary blood flow values between acute IPC+IR and delayed IPC+IR groups were not statistically different at all reperfusion time points.
Signaling Pathways of Acute Renal IPC: Role of Akt, PKC, Gi/o, and Free Radical Generation
Acute renal IPC resulted in rapid phosphorylation of ERK MAPKand Akt as well as phosphorylation of HSP27 (Figure 2). TotalERK, Akt, and HSP27 expression did not change with acute renalIPC. Free radical scavenger (MPG), inhibitor of Akt phosphorylation(wortmannin), PKC inhibitor (chelerythrine), or Gi/o inhibitor(pertussis toxin) prevented the renal protection that was inducedwith acute IPC (Table 1). In contrast, inhibitors of ERK phosphorylation(PD98059) and the A1AR (DPCPX) failed to prevent the protectiveeffects of acute renal IPC against IR injury. Inhibitors alonehad no additional effects on renal function after IR injury(Table 1). Acute and delayed IPC occurred in A1 AR KO mice (Table 1).
Figure 2. (A) Representative immunoblots for phosphorylated extracellular signal–regulated protein kinase (pERK) and total ERK, pAkt and total Akt, phosphorylated heat-shock protein 27 (pHSP27) and total HSP27, and inducible nitric oxide synthase (iNOS) from renal cortices of mice subjected to sham operation (Sham; n = 6), acute IPC (Acute IPC; n = 5), or delayed IPC (Delayed IPC; n = 6). Delayed IPC indicates samples collected from mice 24 h after being subjected to IPC. (B) Densitometric quantifications of relative band intensities. #P < 0.05 versus Sham; *P < 0.01 versus Sham. Error bars = 1 SEM.
Signaling Pathways of Delayed Renal IPC: HSP27 and iNOS Upregulation
Twenty-four hours after renal ischemic preconditioning, proteinexpression of HSP27 increased (Figure 2) without affecting HSP70(data not shown) or HSP32 (data not shown) expression. We thenquestioned whether induction of iNOS and increased NO releasecontribute to the cytoprotection with delayed renal IPC. Figure 2shows that 24 h after renal IPC, iNOS protein expression increased.Inhibition of iNOS function with L-NIL prevented the protectiveeffects of delayed renal IPC (Table 1). In addition, mice thatlacked iNOS failed to demonstrate delayed renal protection,indicating that induction of iNOS is critical for the mechanismof delayed renal protection (Table 1). Inhibition of Gi/o orPKC prevented renal protection with delayed IPC (Table 1). Inhibitionof ERK phosphorylation, Akt phosphorylation, free radical generation,or A1AR had no effect on renal protection with delayed renalIPC (Table 1).
Figure 3 shows that the dosage and the method of administrationof PD98059 and wortmannin that we used effectively blocked thephosphorylation of ERK and Akt in vivo, respectively. It alsoshows that the dosage and the method of MPG administration thatwe used effectively blocked the phosphorylation of ERK, Akt,and HSP27 in vivo.
Figure 3. Representative immunoblots illustrating specificity of pharmacologic inhibitors. Mice were pretreated with PD98059 (PD; an inhibitor of mitogen-activated protein kinase kinase [MEK1], 1 mg/kg), N-(2-mercaptopropionyl)-glycine (MPG; 100 mg/kg), or wortmannin (Wort; an inhibitor of phosphatidylinositol-3 kinase [PI3K], 1 mg/kg), and their effects on the phosphorylation status of ERK (A), HSP27 (B), and Akt (C) were measured. Representative of four experiments.
Consistent with the finding that IPC induces iNOS protein expressionand that inhibition or genetic deletion of iNOS prevented renalprotection with delayed IPC, IPC significantly increased theNOS activity in renal cortices of mice that were subjected toIPC 24 h before (162 ± 7% of sham; n = 6; P < 0.001versus sham group; Figure 4).
Figure 4. NOS activity in renal cortices of mice subjected to sham operation (Sham; n = 6) or IPC 24 h before (Delayed IPC; n = 6). *P < 0.001 versus Sham. Error bars = 1 SEM.
Assessment of Renal Necrosis with Jablonski Grading Scores
In Figure 5, the renal protective effects of acute and delayedIPC are supported further by representative histologic slides.Thirty minutes of renal ischemia followed by 24 h of reperfusion(IR) resulted in significant renal injury as evidenced by severetubular necrosis, medullary congestion and hemorrhage, and developmentof proteinaceous casts in the outer medullary area. Acute anddelayed renal IPC preserved near-normal morphology. The Jablonskiscale histology grading scores in the outer medullary area areshown in Figure 6. Thirty minutes of renal ischemia and 24 hof reperfusion resulted in severe acute tubular necrosis (grade= 3.0 ± 0.5; n = 7; P < 0.001 versus sham grade =0.3 ± 0.3, n = 5). Acute ischemic preconditioning (grade= 1.1 ± 0.3; n = 6; P < 0.001 versus IR) and delayedischemic preconditioning (grade = 1.4 ± 0.3; n = 6; P< 0.001 versus IR) groups showed significantly improved necrosisscores compared with IR alone.
Figure 5. Representative hematoxylin and eosin staining photomicrographs of the outer medulla of kidneys of sham-operated mice (Sham; A) and mice subjected to IR (B), acute IPC+IR (C), and delayed IPC+IR (D). Severe tubular dilation, tubular swelling and necrosis, medullary luminal congestion, and hemorrhage are present in the kidneys of mice subjected to IR (arrows), but these changes are drastically attenuated in mice subjected to IR injury after preconditioning. Magnification, x200.
Figure 6. Jablonski grading scale scores of outer medullary area for the histologic appearance of acute tubular necrosis in sham-operated mice (Sham; n = 5) and mice subjected to IR (n = 7), acute IPC+IR (n = 6), and Delayed IPC+IR (n = 6). *P < 0.001 versus Sham; #P < 0.001 versus IR. Error bars = 1 SEM.
The major findings of this study are that (1) acute renal IPCprotects against renal IR injury in the murine kidney; (2) protectionby acute renal IPC disappears but recurs 24 h after initialpreconditioning (delayed IPC); (3) only the inhibition of Aktphosphorylation prevented renal protection with acute IPC; (4)delayed renal IPC leads to upregulation of HSP27 and iNOS; (5)pharmacologic inhibition or genetic deletion of iNOS leads toa significant attenuation of renal protection with delayed IPC;(6) Gi/o and PKC activation are involved in both acute and delayedIPC; and (7) as we observed in rat kidney in vivo, acute ordelayed renal IPC is independent of the A1AR activation.
IPC in the heart and the brain occurs in a biphasic manner;acute protective effects of IPC wanes over several hours, butthe protective effect reappears 12 to 24 h later (delayed preconditioning,or the second window of protection [7,8,32]). Although we demonstratedin rats that IPC produced renal protection when ischemia isinduced acutely (minutes) after a preconditioning protocol (1,2),it was not known whether delayed renal IPC occurs as has beenobserved in cardiac and neuronal tissues. Our study demonstratesfor the first time not only that mouse kidney can be protectedwith acute renal IPC but also that delayed renal IPC protectsthe murine kidney against renal IR injury, as illustrated byreduced plasma creatinine and renal necrosis scores. We demonstratein this study that the acute or delayed renal IPC shares common(PKC and Gi/o) as well as several distinct signaling pathways(free radical generation and Akt phosphorylation for acute preconditioningand iNOS induction for delayed preconditioning)
A significant component of ischemic reperfusion injury occursduring the reperfusion phase as a result of increased oxidativestress and the generation of free radicals. However, althoughhigh concentrations of oxygen free radicals induce tissue injuryduring the reperfusion period after prolonged ischemia, moderateoxidative stress is considered to be an important prerequisiteof IPC in cardiac (33), cerebral (34,35), and endothelial (36)cells. A growing body of evidence indicates that oxygen freeradicals are important second messengers in several cell types,including renal cells (11,12). Reactive oxygen species are attractivesignaling candidates to account for preconditioning in the kidneybecause renal cells are subjected to obligatory bursts of oxidantstress during the reperfusion phase after each preconditioningstimulus. Therefore, we hypothesized that exposure to moderateoxidant stress may initiate cytoprotective signaling to defendagainst subsequent and more severe free radical–mediatedinjury in renal tubule cells. Our data support this hypothesisin that the free radical scavenger MPG prevented acute renalIPC. However, that MPG failed to prevent the delayed preconditioningsuggests separate signaling mechanisms of renal protection foracute and delayed IPC.
In this study, we demonstrate upregulation of iNOS with concomitantincrease in NOS activity in the renal cortices of mice thatwere subjected to delayed IPC. Genetic deletion of iNOS or pharmacologicinhibition of iNOS with L-NIL prevented the renal protectiveeffects of delayed renal IPC. Therefore, iNOS induction playsa key role in mediating renal protection with delayed IPC. Upregulationof NO with iNOS induction has been implicated in cardiac protectionby preconditioning (9), and inhibition of iNOS prevents theprotective effects of delayed IPC in the heart as well as inthe kidney (28,37). However, the role of NO in renal IR injuryhad been controversial as some studies demonstrate protectiveeffects whereas others show detrimental effects against renalIR injury (38,39). NO downregulates inflammatory reactions,which are important contributors to renal IR injury (40). NOregulates neutrophil recruitment by inhibiting the expressionof adhesion molecules in the vascular endothelium, resultingin increased blood flow to ischemic regions (41). Therefore,NO’s vasodilative effects, inhibition of platelet plugformation, and reduction of the inflammatory response can producebeneficial effects in renal IR injury. Goligorsky et al. (42,43)proposed that the cellular effects of NO depend on its concentration,site of release, and duration of action. Low levels of NO maybe protective, but higher levels may be detrimental. It is increasinglyclear that NO produces distinct renal physiologic effects dependingon its concentration reached in the kidney.
It is interesting that approximately 20% increases in outermedullary blood flow were observed immediately after postischemicreperfusion in mice that were subjected to a preconditioningstimulus 24 h earlier (Figure 1). Increased outer medullaryperfusion persisted for 5 to 10 min and was unique to this experimentalgroup. This fundamental physiologic difference after delayedIPC could be mechanistically important because increased postischemicouter medullary perfusion may be due to the induction of iNOSprotein synthesis and function, suggesting a significant hemodynamiccomponent to the protective effect(s) of the delayed IPC protocol.
We show that acute renal IPC is associated with rapid phosphorylationof the cytoprotective kinases ERK and Akt. However, we determinedthat inhibition of PI3K Akt pathway but not the MEK1 ERK MAPKpathway blocked the renal protective effects of acute renalIPC. Therefore, although ERK activation was observed, it isnot responsible for the renal protective effects of acute renalIPC. The serine/threonine kinase Akt is an important componentof cell survival pathways in many cell types (44,45). In particular,Akt has diverse functions to counteract apoptosis, includinginhibition of cytochrome c release from mitochondria and phosphorylationof several proapoptotic factors (e.g., BAD, caspase 9, glycogensynthase kinase 3) (46,47). Akt also can increase the activityof HSP27 in certain cell types (48–50).
We demonstrate in this study that renal IPC produced increasedphosphorylation of HSP27 as well as total HSP27 protein expression24 h after renal IPC. HSP27 is a widely known cytoprotectiveHSP (51). Both phosphorylated and nonphosphorylated forms ofHSP27 can reduce cellular injury against diverse forms of stress,including renal injury. HSP27 stabilizes actin cytoskeletonto preserve the renal architectural integrity after IR injury.HSP27 also prevents activation of several caspases and inhibitsthe release of cytochrome c from mitochondria. We previouslyshowed that brief oxidant stress before renal tubular necrosisin vitro led to increased HSP27 activation (52).
Our study of murine renal IPC shares several important similaritieswith cardiac IPC. Similar to cardiac preconditioning, we demonstratedthat acute renal IPC is mediated, at least in part, by activationof the PI3K Akt pathway and not through the ERK MAPK pathway(53). Phosphorylation and upregulation of HSP27 to mediate cardiacpreconditioning has been suggested (54). Moreover, we demonstratean important role for iNOS induction in mediating delayed renalischemic preconditioning as demonstrated in delayed cardiacIPC (55).
However, there are some mechanistic differences between renaland cardiac preconditioning as well. Unlike the findings incardiac IPC (56), the renal A1AR do not mediate renal IPC, asa selective inhibitor of A1AR (DPCPX) failed to block the protectiveeffects of renal IPC in mice (in our study) and rats (2). Moreover,our data demonstrating that the A1AR KO mice can benefit fromboth acute and delayed renal IPC conclusively rules out a rolefor the A1AR in renal IPC. In contrast, cardiac IPC failed tooccur in A1AR KO mice (56).
Park et al. demonstrated that previous ARF induced significantprotection when the kidney is subjected to another 30 min ofischemia several weeks later (10). They also implicated iNOSand HSP27 induction as mechanisms of their renal "preconditioning."Our study has several distinct differences with the studiesby Park et al (10). In their study, 30 min of renal ischemiainduced preconditioning that protected renal function againstanother 30 min of renal ischemia that was induced 1 to 12 wklater. This "delayed IPC" was associated with increased synthesisof HSP27 as well as iNOS. Similar to our study, inhibition orgenetic deletion of iNOS prevented their preconditioning-induceddelayed protection. However, they reported that a single 15-minepisode of renal ischemia was not sufficient to induce iNOSand renal protection failed to occur. However, in our study,four cycles of 5 min of ischemia interspersed with 5 min ofreperfusion indeed produced upregulation of iNOS and renal protection.
We demonstrate in this study that renal IPC can occur in miceand that there are acute and delayed phases of renal protectionwith renal IPC. Acute IPC is mediated via Gi/o, PKC and Aktphosphorylation, whereas delayed IPC involves Gi/o, PKC, andiNOS upregulation. Clinical manipulation of the signaling pathwaysof IPC that mediate protection may lead to therapeutic improvementsto prevent or reduce the incidence of acute renal failure duringthe perioperative period.
Acknowledgments
This work was supported by National Institutes of Health grantRO1 DK-58547 (H.T.L.); by the Department of Anesthesiology,Columbia University; and by Saint Vincent's Hospital, The CatholicUniversity of Korea (J.D.J.).
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Abstract
Introduction
Akt pathway but not the MEK1 
