Heat Preconditioning Attenuates Renal Injury in Ischemic ARF in Rats: Role of Heat-Shock Protein 70 on NF-B–Mediated Inflammation and on Tubular Cell Injury
Sang-Kyung Jo,
Gang Jee Ko,
Chang Su Boo,
Won Yong Cho and
Hyoung Kyu Kim
Division of Nephrology, Department of Internal Medicine, Institute of Renal Disease, Korea University Hospital, Seoul, Korea
Address correspondence to: Dr. Won Yong Cho, Division of Nephrology, Department of Internal Medicine, Korea University Hospital, 126-1, 5Ka, Anam-Dong, Sungbuk-Ku, Seoul, Korea 136-705. Phone: +82-2-920-5599; Fax: +82-2-927-5344; E-mail: wonyong{at}korea.ac.kr
Received for publication October 18, 2005.
Accepted for publication August 24, 2006.
Although heat preconditioning has been known to be protectivein various types of injury, the precise molecular mechanismfor this is unclear. Recent observations that indicate thatprevious heat shock has an anti-inflammatory, antiapoptoticeffect led to this investigation of the in vivo effect of heatpreconditioning on NF-B activation and inflammation and alsoon tubular cell injury in ischemic acute renal failure (ARF).Heat preconditioning provided marked functional protection andalso reduced histologic evidence of tubular necrosis. Ischemia/reperfusion–inducedNF-B activation was suppressed by heat preconditioning witha subsequent decrease in monocyte chemoattractant protein-1expression and inflammatory cell infiltration. Heat preconditioningalso suppressed the accumulation of phosphorylated inhibitoryB (IB) with a resultant depletion of cytoplasmic IB, indicatingthat heat preconditioning blocked the activation of the IB kinasecomplex. Tubular cell apoptosis, determined by terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling staining,also was decreased by heat preconditioning, and this was accompaniedby decreased caspase 3 activation. Among several heat-shockproteins (HSP), HSP-70 was induced primarily by heat preconditioning.Inhibition of HSP-70 by quercetin almost completely reversedthe functional protection that was provided by heat preconditioning.These data provide evidence that HSP-70 affords protection viainhibition of NF-B–mediated inflammation and also inhibitionof the cell death pathway in ischemic ARF. Further elucidationof the cytoprotective mechanism of stress proteins could facilitatenew target or drug development in the treatment of ARF.
Ischemic acute renal failure (ARF) is a major clinical problemwhose mortality rate still is >30% despite advances in supportivecare. It also is associated with initial delayed graft function,which might result in poor long-term graft function (1,2). Thepathophysiologic mechanism of ischemic ARF is thought to includea complex interplay among vascular endothelial cell dysfunction,inflammation, and tubular cell damage. Inflammation is thoughtto contribute to tissue damage by releasing several mediators,such as proinflammatory cytokines, proteases, and eicosanoids(3). Tubular cell apoptosis, morphologically characterized bychromatin condensation, cell shrinkage, and plasma membraneblebbing, also is known to play an important role in renal dysfunction(4,5).
NF-B is a ubiquitous, inducible transcription factor that regulatesthe expression of numerous genes, particularly those that areinvolved in inflammatory processes (6). Several recent reportsindicated that the NF-B pathway is activated after ischemia/reperfusion(I/R) and that several proinflammatory cytokine genes are underthe transcriptional control of NF-B (7,8). Usually, NF-B residesin the cytoplasm in an inactive state bound to its inhibitor,inhibitory B (IB). After various stimuli, phosphorylation andsubsequent ubiquitination of IB leads to release and nucleartranslocation of NF-B, where it promotes its gene transcription(9).
The heat-shock response consists of the expression of a familyof highly conserved proteins that are known as heat-shock proteins(HSP), and they are well known for their cytoprotective functionin a variety of injury models. Although the protective roleof heat preconditioning or HSP is thought to be due to theirability to facilitate refolding, assembly, and stabilizationof denatured proteins, precise molecular mechanisms of sucha beneficial effect are not well established (10,11). Severalreports have indicated that the protective effect of HSP-70is partially mediated through inhibition of NF-B pathway–relatedinflammation, as well as modulation of cell necrosis or apoptosis(12–14). Heat preconditioning stabilized IB proteins andsubsequently suppressed their activation in liver I/R or myocardialinflammation (15,16). In addition, HSP-70 has been demonstratedto be protective in renal tubular cell apoptosis that is inducedby inflammatory cytokine or ATP depletion. However, the in vivoeffect of heat preconditioning on inflammation and tubular cellnecrosis or apoptosis in ischemic ARF has never been examined.The purpose of our study was to examine the effect of heat preconditioningon NF-B activation and subsequent inflammation, as well as ontubular cell necrosis, and apoptosis in ischemic ARF in rats.We found that the beneficial effect of heat preconditioningis mediated partially by its inhibitory effect on NF-B pathway–mediatedinflammation as well as by attenuation of tubular cell apoptosisand necrosis.
Animals and Experimental Protocol
Male Sprague-Dawley rats that weighed 150 to 200 g were purchasedfrom Orient (Orient Bio Department, Kyungki-Do, Korea) and hadfree access to water and food before manipulation. Animal carewas in accordance with the criteria established by the animalcare committee of Korea University for the care and use of laboratoryanimals in research. Rats were randomly assigned to one of fourgroups: sham (n = 6), heat preconditioning + sham (HP; n = 6),I/R (n = 6), and heat preconditioning + I/R (HP+I/R; n = 6).For heat preconditioning, rats were anesthetized with an intraperitonealinjection of 100 mg/kg ketamine and 12.5 mg/kg xylazine andplaced on a heating pad until their rectal temperature reachedand was maintained at 42 ± 0.5°C for 15 min. Sixteenhours after heat preconditioning, ischemic injury was introducedby bilateral clamping of the renal pedicles for 40 min. Theanimals were kept at a constant temperature (37°C) duringthis procedure and allowed to recover after intraperitonealinstillation of 1 ml of warmed normal saline. Sham operationwas performed in a similar manner, except for clamping of therenal pedicles. At the time of reperfusion and after 1, 6, and24 h of reperfusion, rats were killed and blood was collectedby intracardiac puncture. These time points were selected becauseNF-B activation and subsequent inflammation have been reportedto occur early after reperfusion and tubular cell damage becomesevident thereafter. Whole-kidney tissue or bluntly dissectedcortex and medulla were processed for molecular and histologicexamination. In addition, more complete time-course study ofplasma creatinine was done in both groups to determine whetherprevious heat preconditioning ameliorates or delays renal injury.For another set of experiments, heat preconditioning was introducedas described previously. Rats were killed at 1, 6, and 14 hafter heat to determine the degree of expression of variousHSP. To define the role of specific HSP in heat preconditioning–inducedrenal protection, quercetin (20 mg/kg, intraperitoneally; SigmaAldrich, St. Louis, MO) was administered 1 h before heat, andthe rats were killed at 14 h after heat and at 24 h after I/R,respectively.
Biochemical and Histologic Examination
Plasma creatinine levels were measured using a Hitachi 747 automaticanalyzer (Tokyo, Japan). For histologic examination, paraformaldehyde-fixed(4%) and paraffin-embedded kidney tissues were stained withperiodic acid-Schiff. Histologic changes in the outer medullawere evaluated semiquantitatively. Briefly, tubular damage wasestimated in 8 to 10 high-power fields (HPF; x200) per sectionby using a scoring system based on the percentage of damagedtubules per field (1 = <25%; 2 = 25 to 50%; 3 = 50 to 75%;and 4 = >75%), and the mean scores of each rat were compared.Detection of apoptotic cells in the kidney also was performedon paraffin-embedded kidney tissue sections using ApopTag Plus(Intergen, Purchase, NY), following the manufacturer’sprotocol. The number of apoptotic cells in the outer medullaand cortex was measured semiquantitatively by counting 8 to10 HPF (x200) per section, and the mean number of terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL)-positivecells was compared between groups. Inflammation was assessedby staining with Naphthol AS-D chloracetate and anti-rat macrophageantibody (ED-1). The number of esterase-positive leukocytesor ED-1–positive mononuclear cells was measured by counting8 to 10 HPF (x200) in the outer medulla, and the mean numberof positive cells was compared between groups.
Preparation of Cytoplasmic and Nuclear Protein Extracts
Cytoplasmic and nuclear protein extracts were prepared usingNE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology,Rockford, IL), following the manufacturer’s protocol,and the protein concentration was measured (BCA protein assayreagent; Pierce Biotechnology).
Electrophoretic Mobility Shift Assay
NF-B consensus oligonucleotide sequence (5'-AGTGAGGGACTTTCCCAGGC-3';Promega, Madison, WI) was end-labeled with [-32P]ATP (Bio-Rad,Hercules, CA) using T4 polynucleotide kinase (Promega), andunlabeled oligonucleotide was removed by MicroSpin G-25 columns(Amersham, Piscataway, NJ). Nuclear extracts (30 µg) wereincubated with 2 µl of 5x binding buffer (Promega). Onemicroliter of labeled probe was added to the reaction mixtureand incubated for 20 min at room temperature. HeLa cell nuclearextracts were used as a positive control; the negative controlcontained no nuclear extract. The specificity of this reactionwas confirmed by a competition assay in which 100-molar excessof unlabeled cold NF-B (specific inhibition) or activating protein1 (AP-1) oligonucleotide (nonspecific inhibition) was added10 min before the addition of the labeled probe. The protein-DNAcomplexes were separated on 4% nondenaturing polyacrylamidegel and visualized by autoradiography.
Western Blot
Samples of cytoplasmic, nuclear (30 µg), or total (30µg) protein were separated by 10% SDS-PAGE and then transferredonto a polyvinylidene difluoride membrane. Western blot analysiswas performed using primary antibodies for phosphorylated IB(phospho-IB), HSP-27, HSP-32 (Santa Cruz Biotechnology, SantaCruz, CA), p65, IB-, HSP-90 (Cell Signaling Technology, Beverly,MA), or HSP-70 (BD Transduction Laboratories, Greenland, NH).Membranes were washed three times and incubated with appropriateperoxidase-conjugated secondary antibodies. The membranes werestained with Ponceau S (Sigma-Aldrich) to ensure that equalamounts of proteins were loaded.
Quantification of Monocyte Chemoattractant Protein-1 by Real-Time Reverse Transcriptase–PCR
Total RNA from bluntly dissected kidney cortex and medulla wasisolated by using TRIZOL reagent (Life Technology, Rockville,MD) according to the manufacturer’s protocol. After precipitationby isopropyl alcohol, total RNA was purified further using RNeasyMinikit (Qiagen, Valencia, CA). One microgram of total RNA wasreverse-transcribed in a reaction volume of 50 µl thatcontained 10x reverse transcriptase buffer, 5.5 mM MgCl2, 500µM of each dNTP, 2.5 µM random hexamer, 0.4 U/µlRNase inhibitor, and 3.125 U/µl MultiScribe Reverse Transcriptaseat 25°C for 10 min, 48°C for 30 min, and 95°C for5 min (Taqman Reverse Transcription Reagents, Applied Biosystem,Foster City, CA). Subsequently, real-time PCR was run in triplicateon the iCycler system (Bio-Rad) under the amplification conditionof 40 cycles of 95°C for 15 s and 60°C for 60 s, withgene-specific primer and probe set designed by Beacon DesignerSoftware (version 2; Bio-Rad) based on sequences from GenBank,which were as follows: sense 5'-GATCTCTCTTCCTCCACCACTATG-3',antisense 5'-GAATGAGTAGCAGCAGGTGAGT-3', and probe 5'-AGGTCTCTGTCACGCTTCTGGGCC-3'.Taqman probes were labeled with 6-carboxy-fluorescein as a reporterdye and 6-carboxy-tetramethyl-rhodamine as a quencher dye. 18Sribosomal RNA (Taqman ribosomal control reagent; Applied Biosystem)was used as an internal control to normalize the data. The dynamicrange of each primer/probe set was verified by a serial dilutionof cDNA template. The abundance of monocyte chemoattractantprotein-1 (MCP-1) gene expression was normalized to that of18S and was expressed as fold differences relative to sham-operatedcontrol rats.
Quantification of MCP-1 Protein by ELISA
Concentration of MCP-1 protein in whole-kidney tissue was measuredby ELISA (OptEIA Set Rat MCP-1; BD Biosciences, Palo Alto, CA)according to the manufacturer’s protocol. Briefly, whole-kidneytissues were homogenized in 500 µl of assay diluent (PBSwith 10% FBS [pH 7.0]) and centrifuged at 12,000 rpm for 15min at 4°C. After coating and blocking plates, MCP-1 standardsand triplicates of samples were added, followed by incubationwith biotinylated anti-rat MCP-1 with avidin–horseradishperoxidase. Absorbance was read at 450 nm, and MCP-1 tissuecontent was derived from a standard curve and expressed as pgMCP-1/mg protein.
Measurement of Caspase Activity
The activity of caspase 3 in kidney tissue was determined byfluorometric detection of free 7-amino-4-trifluoromethylcoumarineaccording to the manufacturer’s protocol (ApoAlert CaspaseFluorescence Assay Kit; BD Biosciences) by using Synergy HTMulti-Detection Microplate Reader (Biotek, Woburn, MA). Briefly,kidney tissues were homogenized in 1 ml of lysis buffer, incubatedon ice for 10 min, and centrifuged at 15,000 x g for 10 minat 4°C. Supernatants that contained 50 µg of proteinwere incubated for 1 h at 37°C in the presence of reactionbuffer, 1 mmol/L dithiothreitol, and 50 µmol/L 7-amino-4-trifluoromethylcoumarinesubstrate conjugates. Fluorescence was read at 400/505 nm (excitation/emission),and the samples were run in triplicate. The activity of caspase3 was expressed as a percentage increase compared with the sham-operatedcontrol group, and samples that were incubated with specificcaspase inhibitor served as a negative control.
Statistical Analyses
All data are presented as means ± SEM and were analyzedby an unpaired t test. P < 0.05 was considered statisticallysignificant.
Heat Preconditioning Afforded Functional and Histologic Renal Protection in Ischemic ARF in Rats
To determine the effect of heat preconditioning in I/R injury,we first evaluated biochemical and histologic renal damage.Plasma creatinine levels at 24, 48, and 96 h after I/R injurywere decreased significantly in rats with heat preconditioningcompared with those without (2.35 ± 0.39/3.35 ±0.54/2.65 ± 0.41 versus 0.63 ± 0.13/1.36 ±0.26/0.99 ± 0.13 mg/dl; P = 0.02, P < 0.01, P <0.01; Figure 1). Histologic examination showed extensive tubularinjury characterized by tubular cell necrosis, dilation of tubules,and cast formation in the outer medulla. However, kidneys frompreviously heat-treated rats showed less tubular injury thandid kidneys from those without heat preconditioning. A semiquantitativeassessment of histologic damage, demonstrating a significantbeneficial effect of heat preconditioning in I/R-induced renalinjury, is shown in Figure 2.
Figure 1. Effect of heat preconditioning (HP) on renal function in ischemic acute renal failure (ARF) in rats. Rats were placed on a heating pad for brief HP (42 ± 0.5°C, 15 min) or at room temperature and subjected to 40 min of bilateral renal pedicle clamping at 14 h after heat treatment. At 24, 48, 96, and 144 h after reperfusion, blood samples were collected and creatinine was measured. Data are means ± SEM (n = 6 rats per group). #P < 0.05 versus sham; *P < 0.05 versus ischemia/reperfusion (I/R).
Figure 2. Effect of HP on renal histology in ischemic ARF in rats. Rats were placed on a heating pad for brief HP (42 ± 0.5°C, 15 min) or at room temperature and subjected to 40 min of bilateral renal pedicle clamping at 14 h after heat treatment. Twenty-four hours after reperfusion, rats were killed and histologic renal damage was determined. (A) I/R cortex. (B) HP+I/R cortex. (C) I/R medulla. (D) HP+I/R medulla. (E) Semiquantitative assessment of renal damage in medulla (n = 6 per group). *P < 0.05 versus I/R. Magnification, x100 (periodic acid-Schiff [PAS]).
Heat Preconditioning Inhibited NF-B Activation in Ischemic ARF in Rats
To assess nuclear translocation and binding of NF-B, we performedelectrophoretic mobility shift assay. NF-B activation was alreadyevident at 1 h after reperfusion. Heat preconditioning suppressedthe I/R-induced activation of NF-B, whereas heat preconditioningitself had no effect on NF-B activity. A competition assay,using a 100-molar excess of unlabeled NF-B probe or AP-1 probeto show the specificity of reaction, was performed (Figure 3A).Addition of unlabeled NF-B probe before the addition of labeledprobe (specific inhibitor) reduced gel band intensity, but additionof unlabeled AP-1 probe (nonspecific inhibitor) had no effect.This effect of heat preconditioning on decreasing NF-B activationalso was confirmed by Western blot of p65 subunit (Figure 3B).Because activation of NF-B is known to depend largely on phosphorylationof the serine residue of IB and subsequent degradation, cytoplasmicprotein levels of IB and phosphorylated IB were examined. I/Rdecreased the abundance of IB, whereas the abundance of phospho-IBwas increased. Previous heat preconditioning suppressed thedepletion of cytoplasmic IB and the accumulation of phospho-IB(Figure 3, C and D), indicating that the effect of heat preconditioningis mediated through inhibition of IB kinase (IKK), an upstreamkinase that determines the phosphorylation of IB.
Figure 3. Effect of HP on NF-B activation in ischemic ARF in rats. Rats were placed on a heating pad for brief HP (42 ± 0.5°C, 15 min) or at room temperature and subjected to 40 min of bilateral renal pedicle clamping at 14 h after heat treatment. Rats were killed at 1 or 6 h after reperfusion. (A) Electrophoretic mobility shift assay (EMSA) for NF-B. (B) Western blot of nuclear p65 protein. (C) Western blot of cytoplasmic IB-. (D) Western blot of cytoplasmic phosphorylated inhibitory B (p-IB). For EMSA, HeLa cell nuclear extracts were used as positive control and a free probe was used as negative control. One hundred-molar excess of unlabeled NF-B probe and unlabeled activating protein 1 (AP-1) probe served as a specific inhibitor or nonspecific inhibitor, respectively.
Heat Preconditioning Suppressed Expression of MCP-1 in Ischemic ARF in Rats
Because several chemokines and cytokines are known to be underthe transcriptional control of NF-B, we examined the mRNA expressionof MCP-1 in bluntly dissected cortex and medulla using real-timequantitative reverse transcriptase–PCR. At 6 h after reperfusion,MCP-1 mRNA expression increased in medulla compared with cortex,and heat preconditioning significantly reduced their expressions(Figure 4A). MCP-1 protein from whole-kidney tissue as measuredby ELISA also showed marked reduction by heat preconditioning(Figure 4B).
Figure 4. Effect of HP on monocyte chemoattractant protein-1 (MCP-1) expression in ischemic ARF in rats. MCP-1 mRNA and protein expression was measured at 6 h after reperfusion by real-time quantitative reverse transcriptase–PCR (RT-PCR) and ELISA. In real-time RT-PCR, the gene expression level was normalized to that of 18S and expressed as fold differences relative to sham-operated rats. (A) MCP-1 mRNA. (B) MCP-1 protein. Data are means ± SEM (n = 6 rats per group). #P < 0.05 versus sham; *P < 0.05 versus I/R.
Heat Preconditioning Reduced Inflammation in Ischemic ARF in Rats
Inflammatory cell recruitment after I/R is facilitated by variousmediators such as chemokines, cytokines, and adhesion moleculesand is thought to contribute to renal injury. At 24 h afterreperfusion, numerous ED-1–and esterase-positive leukocyteswere found in the medullary interstitium, but in cortex, veryfew inflammatory cells were found in both groups. Heat preconditioningsignificantly reduced the infiltration of inflammatory cellsin medulla (Figures 5 and 6).
Figure 5. Effect of HP on anti-rat macrophage antibody (ED-1) leukocyte infiltration in ischemic ARF in rats. Eight to 10 high-power fields (HPF; x200) per section were counted, and mean numbers of ED-1–positive leukocytes were compared. (A) Sham cortex. (B) I/R cortex. (C) HP+I/R cortex. (D) Sham medulla. (E) I/R medulla. (F) HP+I/R medulla. (G) Mean numbers of ED-1–positive leukocytes in medulla. Data are means ± SEM (n = 6 rats per group). #P < 0.05 versus sham; *P < 0.05 versus I/R. Magnification, x400 (ED-1 staining, 24 h).
Figure 6. Effect of HP on esterase-positive leukocyte infiltration in ischemic ARF in rats. Eight to 10 HPF (x200) were counted, and mean numbers of esterase-positive leukocytes were compared. (A) Sham cortex. (B) I/R cortex. (C) HP+I/R cortex. (D) Sham medulla. (E) I/R medulla. (F) HP+I/R medulla. (G) Mean numbers of esterase-positive leukocytes in medulla. Data are means ± SEM (n = 6 rats per group). #P < 0.05 versus sham; *P < 0.05 versus I/R. Magnification, x400 (naphthol AS-D chloracetate esterase staining, 24 h).
Heat Preconditioning Reduced Caspase Activation and Apoptosis
Caspases are cysteine proteases that are responsible for apoptosis.Because the in vivo effect of heat preconditioning on apoptosisin ischemic ARF has never been examined, we measured the activationof caspase 3, an executioner caspase, and also apoptosis. Heatpreconditioning reduced the activation of caspase 3 at 6 h afterI/R injury predominantly in cortex (Figure 7), and this wasaccompanied by reduced TUNEL-positive cells that also were foundmainly in cortical distal tubules (Figure 8). These findingssuggest that heat preconditioning decreases caspase 3–mediatedapoptosis in ischemic ARF.
Figure 7. Effect of HP on caspase activation in ischemic ARF in rats. Caspase 3 activities were measured in cortex and medulla at 6 h after reperfusion and were expressed as percentage increase compared with sham group rats. Samples in which specific caspase inhibitors were added served as negative control for the reactions. Data are means ± SEM (n = 6 rats per group). #P < 0.05 versus sham; *P < 0.05 versus I/R.
Figure 8. Effect of HP on apoptosis in ischemic ARF in rats. Rats were placed on a heating pad for brief HP (42 ± 0.5°C, 15 min) or at room temperature and subjected to bilateral renal pedicle clamping for 40 min. Detection of apoptosis was done at 24 h. (A) Sham. (B) HP 14 h. (C) I/R. (D) HP+I/R. (E) Mean numbers of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL)-positive apoptotic cells. Data are means ± SEM (n = 6 rats per group). #P < 0.05 versus sham; *P < 0.05 versus I/R. Magnification, x200 (TUNEL).
HSP-70–Mediated Protective Effect of Heat Preconditioning in Ischemic ARF in Rats
Although heat preconditioning afforded functional and histologicrenal protection in this study, the role of individual HSP wasnot clear. In addition, it is possible that this previous thermalstress might have other, non–HSP-related, nonspecificeffects. Therefore, we examined the expression of various HSP.After heat treatment, HSP-70 expression markedly increased,compared with HSP-27, HSP-32, or HSP-90 (Figure 9A). Previousadministration of quercetin, a known inhibitor of HSP-70, completelyreversed the functional protection that was induced by heatpreconditioning (Figure 9B), and this also was accompanied byreversal of p65 subunit downregulation by heat preconditioning(Figure 9C). These data suggest that HSP-70 is responsible forthe protective effect of heat preconditioning in this study.
Figure 9. Effect of quercetin (Q) on expression of heat-shock proteins (HSP), renal function, and NF-B activation in ischemic ARF in rats. Q was administered 1 h before HP, and rats were killed at 1, 6, and 14 h after HP or 24 h after I/R. (A) Western blot of various HSP after heat-shock treatment or Q+heat-shock treatment. (B) Plasma creatinine 24 h after I/R. Data are means ± SEM (n = 6 rats per group). #P < 0.05 versus sham; *P < 0.05 versus I/R; ##P < 0.05 versus HP+I/R. (C) Western blot of nuclear p65 protein 1 h after I/R.
HSP-70 Induction by I/R Was Blunted in Heat-Preconditioned Animals
To identify the effect of induced HSP by heat preconditioningon subsequent HSP-70 induction after I/R, we performed Westernblot analysis of HSP-70 at 0, 1, and 6 h after I/R. HSP-70 inductionwas markedly blunted in heat-preconditioned rats at 1 and 6h compared with normothermic rats (Figure 10).
Figure 10. HSP-70 induction after I/R. Rats were placed on a heating pad for brief HP (42 ± 0.5°C, 15 min) or at room temperature and subjected to bilateral renal pedicle clamping for 40 min. Rats were killed at 0, 1, and 6 h after I/R.
In this study, we have demonstrated that heat preconditioningafforded functional and histologic renal protection in ischemicARF in rats, and this was accompanied by suppression of NF-Bactivation and subsequent inflammation, as well as tubular cellnecrosis and apoptosis. We also found that the beneficial effectof heat preconditioning seems to be mediated primarily throughHSP-70 induction.
In the kidney, I/R induces inflammatory mediators such as chemokines,cytokines, and adhesion molecules (17). These mediators arethought to initiate the inflammatory cascade that leads to leukocyterecruitment and microcirculatory compromise with resultant renaldysfunction. Because these mediators, such as TNF- and MCP-1,are known to have B-binding motifs in their promoter regions,their transcriptions are thought to be under the control ofNF-B (9,18). Therefore, NF-B may be a potential therapeutictarget in ischemic ARF.
HSP are known to confer protection against diverse forms ofcellular injury by having a capacity to bind to misfolded ordenatured proteins and thereby prevent their irreversible denaturation(10,11). Although the beneficial effect of heat preconditioningin ischemic injury has been reported in several organs, includingthe kidney, the precise mechanism of protection remains unclear.Recently, several in vitro and in vivo studies suggested thatone of the protective mechanisms of heat preconditioning, orHSP, is their inhibitory effect on NF-B–mediated inflammation(19,20). However, the in vivo effect of heat preconditioningon inflammation in the kidney has never been demonstrated. Therefore,we first examined the effect of heat preconditioning on NF-Bactivation and inflammation. Heat preconditioning suppressedI/R-induced NF-B activation in the kidney at 1 h after reperfusion.These findings again were confirmed by Western blot analysisof p65 subunit of NF-B complexes. Suppression of NF-B activationby heat preconditioning at 1 h was accompanied by a subsequentdecrease in MCP-1 mRNA and protein expression at 6 h and alsoby decreased leukocyte recruitment at 24 h. We divided kidneytissues into cortex and medulla by blunt dissection and foundthat MCP-1 expression increased significantly in medulla, wheresubsequent inflammation and tubular necrosis were predominant.These results are consistent with other studies (21,22) andsupport the possibility that heat preconditioning reduces thetranscriptional activation of proinflammatory mediators andsubsequent inflammation by blocking the activation of the NF-Bpathway in ischemic ARF. However, there is a limitation of usingan activation assay rather than nuclear transcription reporterassay because activation of NF-B does not necessarily lead toincreased transcription. Although we cannot provide direct evidencein this study, NF-B–dependent production of MCP-1 or othercytokines has been demonstrated in several in vitro and in vivostudies (23,24). Cao et al. (23) demonstrated that inhibitionof NF-B by in vivo transfection of decoy oligodeoxynucleotidesnot only prevented NF-B activation but also decreased MCP-1gene expression with decreased monocyte/macrophage infiltration.The role of MCP-1 in I/R injury also has been studied extensively,and it is thought to play an important role in leukocyte recruitmentwith further compromise of the outer medullary reflow and facilitationof tubular cell damage (17,25,26). However, the effect of NF-Bon facilitating cell survival or inhibiting apoptosis in othercell types also has been reported (27,28). The major pathwayfor NF-B activation depends on the activation of IKK complexes,which leads to the phosphorylation of the serine residue ofcytoplasmic IB and degradation by the ubiquitin-proteasome system.However, other mechanisms that are not dependent on phosphorylationand degradation of IB also have been reported (29). To evaluatefurther the inhibitory effect of heat preconditioning on NF-Bpathway, we examined IB and phospho-IB protein levels that recognizethe phosphorylation of serine residue of IB, using cytoplasmicprotein extracts. We found that the suppression of NF-B activationby previous heat preconditioning was secondary to decreasedphosphorylation and resultant stabilization of cytoplasmic IB.This finding might suggest that the beneficial effect of previousheat preconditioning is mediated by inhibition of IKK activation,an upstream kinase of IB that results in inhibition of NF-Bnuclear translocation. This is in agreement with several otherstudies (20). However, according to several recent reports,inhibition of IB degradation by increased phosphatase activityafter heat shock also could be a possible mechanism (30).
Renal tubular cells that are lethally injured after ischemicor toxic insults can die by necrosis or apoptosis (4,31). Contraryto apoptosis, which is a well-coordinated process, necrosisusually results from overwhelming cellular ATP depletion thatis beyond their capability to repair and has been known to bean unregulated event. However, recent studies suggest that necrosisalso is controlled by a specific program that involves manysignaling cascades such as reactive oxygen species and stresskinases (14). In our study using 40-min bilateral ischemia,extensive tubular necrosis in outer medulla was observed at24 h after I/R, and heat preconditioning significantly decreasedtubular necrosis. The mechanisms of heat preconditioning orHSP-mediated cell protection from necrosis is not well understood.The direct inhibitory effect of HSP-72 on c-JUN N-terminal kinasepathways that mediate cell necrosis in addition to their functionas molecular chaperones has been suggested by Yaglom et al.(14), and it is possible that induced HSP-70 in our study inhibitedc-JUN N-terminal kinase–mediated tubular cell death. Apoptosisis characterized by cell shrinkage, nuclear chromatin condensation,and activation of a family of cysteine proteases called caspase.HSP-70 was reported previously to decrease apoptosis throughinhibition of the mitochondrial apoptotic pathway (32–34).The in vivo effect of HSP-70 on the mitochondrial apoptoticpathway also was demonstrated by Lee et al. (35), who observedmore cytochrome c release into the cytosol with a larger infarctvolume in HSP-70 knockout mice in the cerebral ischemia model.Although the inhibitory effect of previous heat stress on caspase3 activation and apoptosis in ATP-depleted tubular cells hasbeen reported (36), the in vivo effect of heat preconditioningon various caspase activation has never been assessed. Therefore,we measured caspase activation after heat preconditioning orI/R. Caspase 3 activity increased in cortex at 6 h after I/R,and heat preconditioning decreased the activations significantly.Tubular cell apoptosis, determined by TUNEL staining, was observedpredominantly in cortical distal tubules and also decreasedmarkedly by heat preconditioning. However, caspase 8 or 9 activity,which represents the activation of membrane death receptor pathwayor mitochondrial pathway, did not match with those of caspase3 (data not shown), indicating that the effect of heat preconditioningis independent of activation of caspase 8 or 9 in I/R-inducedARF. The precise biochemical pathways by which heat preconditioningdecreases apoptosis is not clear in this study. Recent studiessuggested that HSP-70 precipitates Bcl2, a widely known antiapoptoticprotein, and this interaction between HSP-70 and Bcl2 proteinmay afford cytoprotection by restoring Bcl2 function, a rolethat is compatible with the chaperone function of this stressprotein (37,38).
These above findings suggest that the effect of heat preconditioningmight be mediated via inhibition of NF-B–mediated inflammationand inhibition of cell death pathways, but there still are uncertaintiesas to the specificity of individual HSP in these circumstancesor the possible nonspecific effects of other HSP molecules.We therefore performed Western blot analysis of various HSP,including HSP-27, HSP-32 (heme oxygenase-1), HSP-70, and HSP-90.In contrast to HSP-27, -32, and -90, which showed a minimalincrease after heat stress, HSP-70 protein increased significantlyafter 1 h and reached a maximum level at 14 h. Quercetin administration,a widely known inhibitor of HSP-70, decreased its expressionmarkedly, and this was accompanied not only by complete reversalof the functional protection that was provided from heat preconditioningat 24 h after I/R but also by reversal of suppression of thep65 subunit of NF-B. This observation strongly suggests thatthe beneficial effect of heat preconditioning is mediated primarilyby HSP-70 induction. HSP-70 induction is correlated with theinhibition of NF-B. Although the above data can provide evidenceof the importance of HSP-70 in renal protection, we furtherexamined the alterations in HSP-70 expression after I/R. Itis interesting that we found that HSP-70 expression after I/Rwas blunted significantly in previously heat-preconditionedrats. The precise mechanism is not clear, but Basile et al.(39) reported that in ischemia-resistant Brown-Norway rats,which have a higher level of constitutive HSP, the inductionof HSP-25 and HSP-72 by I/R was blunted and their kidneys werefunctionally protected from I/R. This finding is comparablewith our results and further supports that induced HSP beforeinjury is critical in providing protection.
We have shown that heat preconditioning resulted in attenuationof renal injury. This protective effect was accompanied by inhibitionof NF-B activation with subsequent decrease in inflammationand also decrease in tubular cell necrosis and apoptosis. HSP-70is thought to be responsible for this beneficial effect. A betterunderstanding of the role of stress proteins in modifying proinflammatory,proapoptotic mediators could facilitate new drug developmentfor reducing renal injury in ischemic ARF and other diseases.
Acknowledgments
We thank Mr. H.W. Kim, Mrs. Y.S. Ko, and Mrs. K.H. Chang fortheir excellent technical support.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Star RA: Treatment of acute renal failure.
Kidney Int 54
: 1817
–1831, 1998[CrossRef][Medline]
Bonventre JV, Weinberg JM: Recent advances in the pathophysiology of ischemic acute renal failure.
J Am Soc Nephrol 14
: 2199
–2210, 2003[Free Full Text]
Okusa MD: The inflammatory cascade in acute ischemic renal failure.
Nephron 90
: 133
–138, 2002[CrossRef][Medline]
Lieberthal W, Koh JS, Levine JS: Necrosis and apoptosis in acute renal failure.
Semin Nephrol 18
: 505
–518, 1998[Medline]
Bonegio R, Lieberthal W: Role of apoptosis in the pathogenesis of acute renal failure.
Curr Opin Nephrol Hypertens 11
: 301
–308, 2002[CrossRef][Medline]
Barnes PJ, Karin M: Nuclear factor-kappaB: A pivotal transcription factor in chronic inflammatory diseases.
N Engl J Med 336
: 1066
–1071, 1997[Free Full Text]
Ishiyama T, Dharmarajan S, Hayama M, Moriya H, Grapperhaus K, Patterson GA: Inhibition of nuclear factor kappaB by IkappaB superrepressor gene transfer ameliorates ischemia-reperfusion injury after experimental lung transplantation.
J Thorac Cardiovasc Surg 130
: 194
–201, 2005[Abstract/Free Full Text]
Nurmi A, Lindsberg PJ, Koistinaho M, Zhang W, Juettler E, Karjalainen-Lindsberg ML, Weih F, Frank N, Schwaninger M, Koistinaho J: Nuclear factor-kappaB contributes to infarction after permanent focal ischemia.
Stroke 35
: 987
–991, 2004[Abstract/Free Full Text]
Chen F, Castranova V, Shi X: New insights into the role of nuclear factor-kappaB in cell growth regulation.
Am J Pathol 159
: 387
–397, 2001[Abstract/Free Full Text]
Agashe VR, Hartl FU: Roles of molecular chaperones in cytoplasmic protein folding.
Semin Cell Dev Biol 11
: 15
–25, 2000[CrossRef][Medline]
Hartl FU, Martin J: Molecular chaperones in cellular protein folding.
Curr Opin Struct Biol 5
: 92
–102, 1995[CrossRef][Medline]
Malhotra V, Wong HR: Interactions between the heat shock response and the nuclear factor-kappaB signaling pathway.
Crit Care Med 30[Suppl]
: S89
–S95, 2002[CrossRef][Medline]
Yaglom JA, Ekhterae D, Gabai VL, Sherman MY: Regulation of necrosis of H9c2 myogenic cells upon transient energy deprivation. Rapid deenergization of mitochondria precedes necrosis and is controlled by reactive oxygen species, stress kinase JNK, HSP72 and ARC.
J Biol Chem 278
: 50483
–50496, 2003[Abstract/Free Full Text]
Uchinami H, Yamamoto Y, Kume M, Yonezawa K, Ishikawa Y, Taura K, Nakajima A, Hata K, Yamaoka Y: Effect of heat shock preconditioning on NF-kappaB/I-kappa pathway during I/R injury of the rat liver.
Am J Physiol Gastrointest Liver Physiol 282
: G962
–G971, 2002[Abstract/Free Full Text]
Chen Y, Arrigo AP, Currie RW: Heat shock treatment suppresses angiotensin II-induced activation of NF-kappaB pathway and heart inflammation: A role for IKK depletion by heat shock?
Am J Physiol Heart Circ Physiol 287
: H1104
–H1114, 2004[Abstract/Free Full Text]
Furuichi K, Wada T, Yokoyama H, Kobayashi KI: Role of cytokines and chemokines in renal ischemia-reperfusion injury.
Drug News Perspect 15
: 477
–482, 2002[CrossRef][Medline]
Lee JI, Burckart GJ: Nuclear factor kappa B: Important transcription factor and therapeutic target.
J Clin Pharmacol 38
: 981
–993, 1998[Abstract/Free Full Text]
Meldrum KK, Burnett AL, Meng X, Misseri R, Shaw MB, Gearhart JP, Meldrum DR: Liposomal delivery of heat shock protein 72 into renal tubular cells blocks nuclear factor-kappaB activation, tumor necrosis factor-alpha production, and subsequent ischemia-induced apoptosis.
Circ Res 92
: 293
–299, 2003[Abstract/Free Full Text]
Yoo CG, Lee S, Lee CT, Kim YW, Han SK, Shim YS: Anti-inflammatory effect of heat shock protein induction is related to stabilization of I kappa B alpha through preventing I kappa B kinase activation in respiratory epithelial cells.
J Immunol 164
: 5416
–5423, 2000[Abstract/Free Full Text]
Uchinami H, Yamamoto Y, Kume M, Yonezawa K, Ishikawa Y, Taura K, Nakajima A, Hata K, Yamaoka Y: Effect of heat shock preconditioning on NF-kappaB/I-kappaB pathway during I/R injury of the rat liver.
Am J Physiol Gastrointest Liver Physiol 282
: G962
–G971, 2002[Abstract/Free Full Text]
Chen Y, Arrigo AP, Currie RW: Heat shock treatment suppresses angiotensin II-induced activation of NF-kappaB pathway and heart inflammation: A role for IKK depletion by heat shock?
Am J Physiol Heart Circ Physiol 287
: H1104
–H1114, 2004[Abstract/Free Full Text]
Cao CC, Ding XQ, Ou ZL, Liu CF, Li P, Wang L, Zhu CF: In vivo transfection of NF-kappaB decoy oligodeoxynucleotides attenuate renal ischemia/reperfusion injury in rats.
Kidney Int 65
: 834
–845, 2004[CrossRef][Medline]
Sung FL, Zhu TY, Au-Yeung KK, Siow YL, O K: Enhanced MCP-1 expression during ischemia/reperfusion injury is mediated by oxidative stress, NF-kappaB.
Kidney Int 62
: 1160
–1170, 2002[CrossRef][Medline]
Rice JC, Spence JS, Yetman DL, Safirstein RL: Monocyte chemoattractant protein-1 expression correlates with monocyte infiltration in the post-ischemic kidney.
Ren Fail 24
: 703
–723, 2002[CrossRef][Medline]
Yamada T, Murase N, Maeda T, Ye Q, Sakamoto T, Terakura M, Starzl TE, Todo S: Protective effect of TNF-alpha and IL-1 beta inhibitor FR167653 on ischemia-reperfusion injury in rat small intestinal transplantation.
Transplant Proc 30
: 2638
, 1998[CrossRef][Medline]
Chaisson ML, Brooling JT, Ladiges W, Tsai S, Fausto N: Hepatocyte-specific inhibition of NF-kappaB leads to apoptosis after TNF treatment, but not after partial hepatectomy.
J Clin Invest 110
: 193
–202, 2002[CrossRef][Medline]
Yang H, Bocchetta M, Kroczynska B, Elmishad AG, Chen Y, Liu Z, Bubici C, Mossman BT, Pass HI, Testa JR, Franzoso G, Carbone M: TNF-alpha inhibits asbestos-induced cytotoxicity via a NF-kappaB-dependent pathway, a possible mechanism for asbestos-induced oncogenesis.
Proc Natl Acad Sci U S A 103
: 10392
–10402, 2006
Zwacka RM, Zhang Y, Zhou W, Halldorson J, Engelhardt JF: Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor kappaB independently of IkappaB degradation.
Hepatology 28
: 1022
–1030, 1998[CrossRef][Medline]
Grossman BJ, Shanley TP, Odoms K, Dunsmore KE, Denenberg AG, Wong HR: Temporal and mechanistic effects of heat shock on LPS-mediated degradation of IkappaBalpha in macrophages.
Inflammation 26
: 129
–137, 2002[CrossRef][Medline]
Lieberthal W, Menza SA, Levine JS: Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells.
Am J Physiol 274
: F315
–F327, 1998[Medline]
Saleh A, Srinivasula SM, Balkir L, Robbins PD, Alnemri ES: Negative regulation of the Apaf-1 apoptosome by Hsp70.
Nat Cell Biol 2
: 476
–483, 2000[CrossRef][Medline]
Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, Tailor P, Morimoto RI, Cohen GM, Green DR: Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.
Nat Cell Biol 2
: 469
–475, 2000[CrossRef][Medline]
Steel R, Doherty JP, Buzzard K, Clemons N, Hawkins CJ, Anderson RL: Hsp72 inhibits apoptosis upstream of the mitochondria and not through interactions with Apaf-1.
J Biol Chem 279
: 51490
–51499, 2004[Abstract/Free Full Text]
Lee SH, Kwon HM, Kim YJ, Lee KM, Kim M, Yoon BW: Effects of hsp70.1 gene knockout on the mitochondrial apoptotic pathway after focal cerebral ischemia.
Stroke 35
: 2195
–2199, 2004[Abstract/Free Full Text]
Wang Y, Knowlton AA, Christensen TG, Shih T, Borkan SC: Prior heat stress inhibits apoptosis in adenosine triphosphate-depleted renal tubular cells.
Kidney Int 55
: 2224
–2235, 1999[CrossRef][Medline]
Seidberg NA, Clark RS, Zhang X, Lai Y, Chen M, Graham SH, Kochanek PM, Watkins SC, Marion DW: Alterations in inducible 72-kDa heat shock protein and the chaperone cofactor BAG-1 in human brain after head injury.
J Neurochem 84
: 514
–521, 2003[CrossRef][Medline]
Townsend PA, Cutress RI, Sharp A, Brimmell M, Packham G: BAG-1: A multifunctional regulator of cell growth and survival.
Biochim Biophys Acta 1603
: 83
–98, 2003[Medline]
Basile DP, Donohoe D, Cao X, Van Why SK: Resistance to ischemic acute renal failure in the Brown Norway rat: A new model to study cytoprotection.
Kidney Int 65
: 2201
–2211, 2004[CrossRef][Medline]
This article has been cited by other articles:
Y. Yokomaku, T. Sugimoto, S. Kume, S.-i. Araki, K. Isshiki, M. Chin-Kanasaki, M. Sakaguchi, N. Nitta, M. Haneda, D. Koya, et al. Asialoerythropoietin Prevents Contrast-Induced Nephropathy
J. Am. Soc. Nephrol.,
February 1, 2008;
19(2):
321 - 328.
[Abstract][Full Text][PDF]
B–Mediated Inflammation and on Tubular Cell Injury
Top
Abstract
Introduction
B activation and inflammation and also on tubular cell injury in ischemic acute renal failure (ARF). Heat preconditioning provided marked functional protection and also reduced histologic evidence of tubular necrosis. Ischemia/reperfusion–induced NF-
(I
-32P]ATP (Bio-Rad, Hercules, CA) using T4 polynucleotide kinase (Promega), and unlabeled oligonucleotide was removed by MicroSpin G-25 columns (Amersham, Piscataway, NJ). Nuclear extracts (30 µg) were incubated with 2 µl of 5x binding buffer (Promega). One microliter of labeled probe was added to the reaction mixture and incubated for 20 min at room temperature. HeLa cell nuclear extracts were used as a positive control; the negative control contained no nuclear extract. The specificity of this reaction was confirmed by a competition assay in which 100-molar excess of unlabeled cold NF-
