Heat Shock–Induced Protection of Renal Proximal Tubular Epithelial Cells from Cold Storage and Rewarming Injury
Declan A. Healy,
Padraig J. Daly,
Neil G. Docherty,
Madeline Murphy,
John M. Fitzpatrick and
R. William G. Watson
University College Dublin School of Medicine and Medical Science, University College Dublin Conway Institute of Biomolecular and Biomedical Research, Mater Misericordiae University Hospital, University College Dublin and Dublin Molecular Medicine Centre, Dublin, Ireland
Address correspondence to: Dr. R. William G. Watson, UCD School of Medicine and Medical Science, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. Phone: +353-1-716-6733; Fax: +353-1-716-6887; E-mail: william.watson{at}ucd.ie
Received for publication September 21, 2005.
Accepted for publication December 5, 2005.
Cold storage and reperfusion injury to transplanted kidneyscontributes to increased incidence of delayed graft functionand may have a negative impact on graft survival. This studyexamined the mechanisms by which previous heat shock protectsagainst cell death in an in vitro model of kidney storage. Coldstorage is mimicked by incubating human renal proximal tubularepithelial (HK-2) cells in University of Wisconsin solutionat 4°C with and without subsequent rewarming. Heat shockwas induced by incubation of cells at 42°C for 1 h. Alteredprotein expression was measured by Western blot, and cell viabilityand apoptosis were measured by propidium iodide DNA stainingusing flow cytometry. The specific role of heat-shock protein70 (HSP-70) was determined both by siRNA knockdown and by stableoverexpression approaches. Cold storage and rewarming-inducedcell death was associated with decreased expression of HSP-70,HSP-90, HSP-27, and Bcl-2. Previous heat shock significantlyreduced HK-2 cell death after cold storage and rewarming andwas associated with the maintenance of HSP-70, HSP-27, and Bcl-2protein levels. Blocking heat stress–induced HSP-70 withsiRNA did not significantly block the protective effect of heatstress against cold storage and rewarming cell death; however,overexpression of HSP-70 protected HK-2 cells from this stress.It is concluded that previous heat shock protects HK-2 cellsfrom cold storage and rewarming injury. siRNA inhibition ofHSP-70 induction did not block the protective effect of heatshock, indicating that HSP-70 is not essential to the heat stress–inducedprotective effect reported in this study.
Transplanted kidneys have only a limited lifespan, with posttransplantationgraft failure rates at 1 and 10 yr of approximately 10 and 50%,respectively, for kidneys from cadaveric donors and approximately5 and 30%, respectively, for kidneys from living donors (1).Graft loss can result from immunologic rejection of the graftby the recipient immune system. However, nonimmune factors,including the occurrence of delayed graft function (DGF) posttransplantation,are also believed to influence negatively graft survival (2–4).The histologic feature of transplanted kidneys with DGF is acutetubular necrosis (ATN), occurring as a result of damage to thekidney from donor, recipient, and transplant procedural factors.Kidney damage and nephron loss as a result of the transplantprocess is caused by ischemia that results from the need todisconnect temporarily the blood supply and subsequent reperfusionafter revascularization (5). Removal of the kidney from thedonor is accompanied by warm ischemic damage, whereupon a prolongedperiod of cold ischemia commences. The use of University ofWisconsin (UW) preservation solution during HLA matching andtransport is designed to help prevent these damaging processes.However, a second period of warm ischemia occurs during anastomosisto the recipient artery before unclamping (6,7) and is almostalways associated with damage caused by the reperfusion afterunclamping (8). During ischemia (both normothermic or hypothermic),ATP production is curtailed (9) and intracellular ATP concentrationsfall off rapidly, leading to deactivation of ATP-dependent sodium/potassiumpumps, loss of electrolyte gradients, and subsequent cell swellingand loss of membrane integrity. Cold storage of kidneys duringtransportation is used as a preservation technique to slow downthe deleterious effects of normothermic ischemia and reducesthe oxygen demand of the kidney to approximately only 3% ofnormothermic values (9). However, energy is still required tomaintain cellular integrity and although used to reduce therate of ischemic injury, cold storage is nevertheless damagingto kidneys and induces cell death through mitochondrial events,including mitochondrial swelling. This is associated with alterationsin the Bcl-2/Bax ratio and release of cytochrome C (10,11).Cold storage has a direct and time-dependent effect on the incidenceof DGF and graft survival (12,13).
Several other approaches have been applied to lessen the damagingeffects of cold storage, including pulsatile perfusion (14),inhibition of calcium uptake, and addition of protective agentsto the transport medium (15). A promising strategy to increasethe tolerance of organs to cold ischemia is exposure to short-termsublethal hyperthermia to induce the expression of heat-shockprotein (HSP) (16). The HSP are a family of stress proteinsthat are expressed in cells as a protective mechanism to cellulardamage caused by many different stresses (17). There is evidenceto support a role for HSP in protection against transplantation-associatedstresses in in vitro (18–20) and in vivo (21,22) modelsof kidney transplantation, although an exact mechanism of actionhas not been determined. This study describes the protectiveeffects of heat shock against cell death in an in vitro modelof kidney cold storage and rewarming and relates this to theexpression of HSP during the cold storage period. In addition,we assessed the effects of heat shock and cold storage on Bcl-2expression levels, because altered Bcl-2 expression is associatedwith cold storage–induced cell death and correlates withgraft function posttransplantation (23).
HK-2 cells were purchased from LGC Promochem (Teddington, UK).Mouse anti-human HSP-27, HSP-60, HSP-70, HSP-90, and Bcl-2 antibodieswere purchased from StressGen (Victoria, Canada). UW solutionwas supplied by Barr Laboratories (Pomona, NY). Scrambled andHSP-70–directed siRNA products were purchased from AmbionInc. (Cambridge, UK). Fugene 6 transfection reagent was purchasedfrom Roche Diagnostics (Basel, Switzerland). PIRESpuro3 vectorwas purchased from Clontech (Palo Alto, CA). DMEM/F12 cell culturemedium, l-glutamine, penicillin-streptomycin solution, and Lipofectamine2000 transfection reagent were purchased from Invitrogen LifeTechnologies (Paisley, UK). All cell culture materials werefrom Greiner Bio-One BioScience (Kremsmunster, Austria). Unlessotherwise stated, all other chemicals were from Sigma-Aldrich(Gillingham, UK).
Cell Culture
HK-2 cells were cultured in 75-cm2 culture flasks with DMEM/F12that contained 5% (vol/vol) FCS, 2 mM glutamine, 2.5 ng/ml EGF,and 1% penicillin-streptomycin. Cells were maintained at 37°Cin a humidified atmosphere of 5% CO2.
Induction of Heat Stress
HK-2 cells were heated to 42 ± 0.2°C for 60 min ina temperature-regulated incubator, followed by incubation at37°C for 2 to 3 h. These conditions have previously beenshown to result in optimal expression of HSP-70 in HK-2 cells(24).
Quantification of Apoptosis
Apoptosis was quantified as the proportion of cells with hypodiploidDNA, as assessed by propidium iodide (PI) incorporation as describedpreviously (25), and analysis using an Epics XL-MCL CoulterElite flow cytometer (Coulter, Fullerton, CA). A minimum of5000 events were recorded and analyzed. Apoptotic nuclei weredistinguished from normal nuclei by their hypodiploid DNA. Cellulardebris was excluded from analysis by raising the forward threshold.All measurements were performed under the same instrument settings.
Quantification of Necrosis
PI exclusion assays were performed to distinguish intact cellmembranes from the disrupted membranes of necrotic cells. Briefly,cells were centrifuged at 1300 rpm for 5 min and resuspendedin 300 µl of hypotonic fluorochrome solution (50 µg/mlPI in PBS) and analyzed using an Epics XL-MCL Coulter Eliteflow cytometer (Coulter). A minimum of 5000 events were recordedand analyzed. Membrane disruption was quantified as the proportionof cells with increased PI fluorescence.
RNA Extraction, cDNA Synthesis, and Reverse Transcription–PCR
Total RNA was isolated from HK-2 cells using TRIZOL reagent(Life Technologies Inc., Rockville, MD). Chromosomal DNA wasremoved from total RNA using DNase I (Life Technologies). First-strandcDNA was generated by reverse transcription using a SuperScriptFirst-Strand Synthesis System for PCR (Invitrogen Life Technologies)according to the manufacturer’s instructions. The cDNAwere used as a template in the subsequent PCR analysis. Real-timePCR TaqMan assay was used to quantify the relative gene expressionlevels of HSP-72 on a 7900HT Sequence Detection System (AppliedBiosystems, Foster City, CA). The primers and probe for HSP-72were designed using Primer Express version 2.0 (Applied Biosystems)and were supplied as a Pre-Developed Assay Reagent (PDAR kit;Applied Biosystems). Probes were labeled with 5'-FAM and with3'-TAMRA as quencher, with the exception of the ribosomal probe,which was labeled with 5'-VIC to facilitate multiplexing. Cyclingconditions were as follows: 2 min at 50°C, 10 min at 95°Cfor enzyme activation, and then 40 cycles of 15 s at 95°Cfor denaturation and 1 min at 60°C for annealing and extension.Values were normalized to 18S rRNA quantity for each sample.
Western Blot Analysis
Cell lysates that contained 40 µg of protein were loadedonto 12% SDS polyacrylamide gradient gels. Resolved proteinswere electrophoretically transferred to Immobilon-P (Millipore,Bedford, MA) membranes. Membranes were incubated in blockingbuffer (5% BSA in PBS plus 0.1% Tween) for 1 h at room temperatureand then incubated in primary antibody (1:1000 HSP-70, 1:1000HSP-27, 1:100 HSP-90, 1:1000 Bcl-2) for an additional 1 h atroom temperature. After washing for 5 min x5 in PBS-0.1% Tween,membranes were incubated for 1 h at room temperature in horseradishperoxidase–conjugated anti-mouse IgG at 1:5000 dilution(Transduction Laboratories, Lexington, KY). Blots were washedfor an additional 5 min x5 in PBS-Tween and developed usingan ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK) enhancedchemiluminescence substrate system.
siRNA Inhibition of HSP-70 Production
Incorporation of siRNA targeted against HSP-70 was achievedusing Lipofectamine 2000 according to the manufacturer’sinstructions. Initial experiments used fluorescence siRNA (siGLO)to optimize transfection efficiency. The cells were transfectedfor 4 h before replacement of the transfection medium with DMEM/F12medium.
Generation of Stable Cell Line Overexpressing HSP-70
The HSP-70 gene was supplied by the group of A. Catapano (Universityof Milano, Milan, Italy). The gene was subcloned into the pIRESpuro3bicistronic vector using BamH1 and Not1 restriction enzymes.Cells were grown in 75-cm2 flasks to 70% confluence before transfection.Cells were transfected with 6 µg of plasmid DNA in thepresence of Fugene 6 reagent for 24 h at 37°C (ratio ofFugene 6:DNA was 6:1). Stable transfectants were selected with1.0 µg/ml puromycin for approximately 10 d followed bycontinued growth in the presence of 0.5 µg/ml puromycin.
Statistical Analyses
All values are expressed as mean ± SD unless specified.Statistical analysis among three or fewer groups was determinedusing a t test (SPSS Inc., Chicago, IL). ANOVA was used to determinestatistical differences among four or more groups. Further analysiswas performed using the Student-Newman-Keuls post hoc test.P < 0.05 was considered statistically significant.
Effect of Cold Storage and Rewarming on HK-2 Apoptosis and Membrane Integrity
Previous studies in our laboratory demonstrated that cells thatare stored in medium at 4°C or UW solution at 37°C undergosignificant apoptosis and necrosis within 24 h (unpublisheddata). However, when HK-2 cells were stored in UW solution at4°C, there was no significant (P = 0.09) increase in apoptosis(Figure 1A) up to 24 h, but there was a significant increase(P = 0.008) in cell necrosis (Figure 1B). Rewarming of the cellsafter 8 or 16 h of cold storage significantly increased apoptosis(P = 0.02) as well as necrosis (P = 0.002) at 24 h (Figure 1A).Furthermore, 16 h of cold storage followed by 8 h of rewarmingresulted in increased necrosis (P = 0.03) compared with 8 hof cold storage followed by 16 h of rewarming (Figure 1B). Theseresults indicate that reperfusion of renal tubular epithelialcells after cold storage was associated with a significant increasein apoptosis in the presence of ongoing membrane disruptioncompared with control cells or those that were cold-stored for24 h without a rewarming period.
Figure 1. The effect of cold storage and recovery on HK-2 apoptosis and viability. Cells were grown in 24-well plates to 80% confluence. After varying periods of cold storage (4°C) in University of Wisconsin (UW) solution, the cells were incubated under normal cell culture conditions in warm (37°C) DMEM/F12 for varying times as indicated. (A) Apoptosis was determined as the percentage of cells with hypodiploid DNA on propidium iodide (PI) staining using flow cytometry. (B) The percentage of cells with disrupted membranes was assessed by PI exclusion using flow cytometry (n = 6). Statistical analysis was carried out using ANOVA, using Student-Newman-Keuls post hoc test. *P = 0.02 versus control; **P < 0.05 versus all other data points at 24 h.
Effect of Cold Storage and Rewarming on HK-2 Intracellular Stress Protein and Bcl-2 Expression Levels
Short-term cold storage (4 h) did not significantly alter theexpression of HSP-70, HSP-90, HSP-7, or Bcl-2 (Figure 2). After8 h of cold storage, intracellular HSP-90 levels were lowerthan control levels, with no change in levels of any other proteinsexamined (Figure 2). However, upon extension of the cold storageperiod to 16 h, there was a clear decrease in the levels ofintracellular HSP-70, HSP-90, HSP-27, and Bcl-2 compared withcontrol levels (Figure 2). After 16 h of cold storage, it wasfound that subsequent rewarming of the HK-2 cells at 37°Cfor 6 h led to a significant increase in the expression of allHSP and Bcl-2 to levels similar to or above both those of controlcells and those that had been cold-stored alone (Figure 2).
Figure 2. The effect of cold storage and rewarming on protein expression in HK-2 cells. Cells were grown in 24-well plates to 80% confluence and cold-stored (4°C) in UW solution with or without a subsequent reperfusion period under normal cell culture conditions at 37°C. Total cellular protein was extracted at the times indicated for Western blot analysis. Blots shown are a representative of at least three independent experiments.
Effect of Previous Heat Shock on Cold Storage and Rewarming-Induced HK-2 Cell Death
HK-2 cells were either cultured at 37°C (control) or heatshocked (42°C for 1 h) and allowed to recover for 2 h at37°C. The cells then were cold-stored for 16 h, at whichpoint they were either harvested for analysis of cell deathor rewarmed with DMEM/F12 at 37°C for 6 h and then analyzedfor cell death. Previous heat shock significantly reduced therate of apoptosis (Figure 3A) and necrosis (Figure 3B) aftercold storage and rewarming compared with non–heat-shockedcells.
Figure 3. The effect of previous heat shock on cold storage and recovery-induced HK-2 cell death. Cells were grown in 24-well plates to 80% confluence. Cells were treated with or without heat shock at 42°C for 1 h and allowed to recover for 2 h at 37°C. Cells then were cold-stored (4°C) in UW solution for 18 h and returned to optimum culture conditions for 6 h. (A) Apoptosis then was determined as the percentage of cells with hypodiploid DNA on PI staining using flow cytometry. *P < 0.05 versus unshocked cells. (B) The percentage of cells with disrupted membranes was assessed by PI incorporation using flow cytometry. *P < 0.001 versus unshocked cells. Each data point represents n = 9 from three independent experiments.
Effect of Previous Heat Shock on Cold Storage and Rewarming-Induced Changes in HK-2 Protein Expression
Transient heat shock of HK-2 cells was shown previously to induceincreased HSP-70 (24). In this study, we confirmed that heatshock for 1 h at 42°C followed by 2 to 3 h of recovery at37°C results in increased expression of HSP-70 with no changein the expression of HSP-27 or HSP-90 compared with controllevels (Figure 4, A through C, lane 1 versus 2 for each protein).In addition, we report that heat shock also results in an upregulationof Bcl-2 (Figure 4D, lane 1 versus 2).
Figure 4. The effect of previous heat shock on protein expression levels in HK-2 cells after cold storage and rewarming. Cells were grown in 24-well plates to 80% confluence and either maintained at 37°C or subjected to heat-shock treatment at 42°C for 1 h followed by recovery for 2 h at 37°C. Cells then were cold-stored (4°C) in UW solution with or without a subsequent rewarming period under normal cell culture conditions at 37°C. Cells were harvested, and protein was extracted for Western blot analysis. Western blots shown are representative of the results of at least three independent experiments.
We examined the effect of previous heat shock on the alteredexpression of HSP and Bcl-2 described in Figure 2. We foundthat the expression levels of HSP-70, HSP-27, and Bcl-2 in heat-shockedcells were maintained above those in unshocked cells that weresubjected to similar cold storage periods (Figure 4, A, C, andD, lane 5 versus 6). It is interesting that the levels of theseproteins in heat-shocked cells that were stored in the coldfor 16 h did not fall below control levels (Figure 4, A, C,and D, lane 1 versus 6). Previous heat shock did not maintainHSP-90 levels above those of unshocked cells during cold storage(Figure 4B). Levels of HSP-70 and HSP-90 expression were upregulatedupon rewarming to a greater extent in heat-shocked comparedwith unshocked cells (Figure 4, A and B, lane 7 versus 8), withno differences seen in postwarming HSP-27 and Bcl-2 levels (Figure 4,C and D, lane 7 versus 8).
Effect of siRNA Directed against HSP-70 on the Protective Effect of Heat-Shock against Cold Storage and Rewarming Injury
To determine whether the specific increase in HSP-70 expressionwas involved in the heat shock–induced protective response,we used siRNA directed against HSP-70. HSP-70–directedsiRNA (5 nM) led to a significant decrease in heat shock–inducedlevels of HSP-70 mRNA (Figure 5A) and protein in HK-2 cells(Figure 5B). In contrast, the same concentration of scrambledsiRNA had no effect (Figure 5, A and B). Despite blocking theincrease in HSP-70 after heat shock with siRNA, there was nosignificant reversal of the protective effect of heat shockagainst cold storage and rewarming-induced apoptosis (Figure 5C)or necrosis (Figure 5D) of HK-2 cells.
Figure 5. The effects of targeted siRNA to heat-shock protein (HSP-70) on HSP-70 expression and cold storage and rewarming injury in HK-2 cells. Cells were grown in 24-well plates to 80% confluence and were divided into control, transfection control, or transfected with either 5 nM siRNA directed against HSP-70 or 5 nM scrambled siRNA for 4 h. Cells then were either maintained at 37°C (unshocked) or subjected to heat shock at 42°C for 1 h followed by recovery for 2 h at 37°C. Cells were divided into two, and one set was harvested at this point for RNA and protein analysis (A and B). The remaining cells were maintained in normal cell culture conditions for an additional 24 h followed by cold storage (4°C) in UW solution for 16 h with a subsequent 6-h rewarming period at 37°C and then harvested for analysis of cell death (C and D). (A) Heat-shocked samples were harvested and RNA extracted for reverse transcription–PCR analysis of HSP-70 mRNA content. Expression levels are expressed relative to those in control (untransfected) cells. **P < 0.01 versus transfection control and scrambled siRNA. (B) Samples were harvested, and protein was extracted for Western blot analysis. (C) Percentage of apoptosis was assessed by cells with hypodiploid DNA on PI staining using flow cytometry. *P < 0.05 versus unshocked. (D) Cells were harvested for analysis of membrane disruption, which was assessed by PI incorporation using flow cytometry (n = 6); *P < 0.05 versus unshocked.
Effect of Overexpression of HSP-70 on Cold Storage and Rewarming Injury in HK-2 Cells
We next wanted to determine whether overexpression of HSP-70could protect HK-2 cells from cold storage and reperfusion injury.We generated a HK-2–derived cell line stably overexpressingHSP-70 using the bicistronic pIRESpuro-3 vector. Stably transfectedHK-2 cells expressed higher levels of HSP-70 than control ormock-transfected cells (Figure 6A). Transfected cells seemedto grow normally, exhibited basal rates of apoptosis and necrosissimilar to control cells (Figure 6, B and C), and maintainedtheir responsiveness to heat shock as indicated by the factthat levels of HSP-70 were higher in heat-shocked transfectedcells compared with unshocked transfected cells (data not shown).After 16 h of cold storage and 6 h of rewarming, apoptotic rateswere lower for the HSP-70–overexpressing cells comparedwith control cells or mock-transfected cells (Figure 6B), whereasthere was no change in the rate of necrosis observed (Figure 6C).
Figure 6. The effect of HSP-70 overexpression on cold storage and rewarming injury in HK-2 cells. Control cells or those that were stably transfected with empty pIRESpuro3 vector (mock) or vector that contained the human HSP-70 gene were grown in 24-well plates to 80% confluence. Cells were either maintained at 37°C under normal culture conditions (untreated) or cold-stored (4°C) in UW solution (16 h) followed by 6 h of rewarming in normal culture conditions at 37°C. (A) Total cellular protein was extracted for Western blot analysis of HSP-70. (B) Cells were harvested for analysis of apoptosis, which was determined as the percentage of cells with hypodiploid DNA on PI staining using flow cytometry. (C) Cells were harvested for analysis of membrane disruption, which was assessed by PI incorporation using flow cytometry. Each data point represents n = 6 from three independent experiments. *P < 0.05 versus untreated cells; #P < 0.05 versus control and mock-transfected cells.
In vitro cold storage of human renal tubular epithelial HK-2cells in UW solution was used as a model of kidney storage.HK-2 cells were used because tubular epithelial cells bear thebrunt of ischemia and reperfusion injury and undergo ATN, leadingto the pathology of DGF and acute renal failure. In our model,we subjected cells to 16 h of cold storage, which is comparableto cold storage times experienced by cadaveric kidneys (1).We found that cell death that was caused by cold storage wasalmost exclusively necrotic in nature, but rewarming inducedboth apoptosis and necrosis. Similar findings were shown inprimary human renal tubular epithelial cells, in which coldstorage induced necrosis and rewarming induced significant apoptosis(26). The degree of cell death was significantly greater aftera period of at least 8 h of cold storage followed by rewarmingcompared with cold storage alone, indicating that cold storagemay prime kidney cells for cell death during rewarming. An extrapolationbetween our in vitro cell death findings and the situation inthe intact kidney is made difficult by a number of factors.First, the rapid and efficient clearance of apoptotic cellsmakes apoptosis extremely difficult to detect in tissue sections,and the incidence of apoptosis may generally be underestimatedbecause of this phenomenon. Also, there is a scarcity of knowledgeregarding the relative contributions of cold storage alone versuscold storage with subsequent reperfusion on cell death in humankidneys. Only two studies have compared the effects of coldstorage with and without subsequent reperfusion on cell deathin human kidney biopsies (27,28), and further research is requiredto determine the contribution of cold storage to kidney damagebefore transplantation.
All organisms respond to stresses by the preferential productionof stress proteins. However, little is known about whether humanor animal cells can mount a stress response, including productionof stress proteins, during cold storage. It was reported previouslythat cold storage induces increased HSP-70 expression in humanumbilical vein endothelial cells (29) and human neutrophils(30), whereas other studies have demonstrated no effect of coldon HSP expression in a rat model (31). In this study, we examinedthe effects of prolonged cold storage on stress protein expression.In addition, because the cold storage and rewarming injury inthis study is likely to proceed via mitochondrial-derived increasesin oxidative stress (10,11), we also examined Bcl-2 expression,which has been correlated with cadaveric graft survival (23).Only one previous study examined the effect of cold storagein isolation on Bcl-2 expression, with no effect being reported(11). We report that whereas a short period of cold storage(4 h) does not alter the expression levels of any of the HSP(HSP-70, HSP-90, or HSP-27) or Bcl-2 (Figure 2), 16 h significantlydecreased their expression compared with controls (Figure 2).We had expected that the stress of cold storage would induceincreased expression of stress proteins such as HSP-70, andthe decreased expression seen may be due to a number of possibilities,such as the enhanced degradation or decreased synthesis of HSPprotein as a result of altered transcriptional or translationalactivity in HK-2 cells at low temperatures. The timing of thechanges in protein expression caused by cold storage describedabove may explain the pattern of cell death induced by the stressesseen in Figure 1. For instance, 4 h of cold storage does notalter the expression levels of any of the HSP or Bcl-2, explainingthe lack of cell death in Figure 1. However, on increasing thetime of cold storage there is a loss of HSP and Bcl-2 with acorresponding sensitivity to rewarming-induced apoptosis, despitethe increased expression of these proteins upon rewarming (Figure 2).The increased expression of such pro-survival proteins duringrewarming may represent an inadequate and somewhat belated attemptby the cells to respond to the cold storage and reperfusioninsult. It is clear that there is a lack of understanding ofthe effects of cold storage on cell stress responses. Furtherstudies are required to elucidate why pro-survival proteinssuch as HSP and Bcl-2 are lost during cold storage so that thesechanges may be manipulated for therapeutic intervention to improveorgan graft survival.
Preconditioning of cells or organs with exposure to short-termsublethal hyperthermia induces the expression of HSP and mayprove to be a promising strategy to increase the tolerance oforgans to cold ischemia and reperfusion injury. We were interestedto determine whether such preconditioning heat stress couldprotect HK-2 cells from cold storage and rewarming-induced celldeath, particularly because we have already demonstrated thatsuch cell death is associated with decreased expression of HSPduring the cold storage period. We demonstrated that previousheat shock significantly reduced cold storage and rewarming-inducedapoptotic and necrotic cell death in HK-2 cells. The heat-shockeffect was associated with increased expression of HSP-70 withoutaffecting the levels of HSP-27 or HSP-90, as previously reportedby Power et al. (24). It is interesting that heat-shocked HK-2cells that then were subjected to subsequent cold storage werefound to maintain their HSP-70 and HSP-27 content far abovelevels in nonshocked cells that were subjected to 16 h of coldstorage. Upon rewarming, HSP-70 and HSP-90 expression was significantlyincreased in heat-shocked cells to a level greater than thatseen for nonshocked cells. To our knowledge, this is the firststudy to report the maintenance of HSP expression levels duringcold storage and rewarming by previous heat shock and helpsto explain the protective effects of heat shock reported inthis study. HSP-70, HSP-27, and HSP-90 all are known to promotecell survival through various mechanisms within cells, includingstress kinase inhibition (32,33), protection of cells from DNAdamage (34,35), modulation of mitochondrial reactive oxygenspecies production (36), and inhibition of caspase activation(37,38). Thus, maintenance of HSP levels during cold storagemay help cells to cope better with reperfusion-associated increasesin oxidative stress, mitochondrial disruption, and activationof caspase pathways.
As already described, Bcl-2 levels were dramatically decreasedafter 16 h of cold storage compared with control levels. Itis interesting that previous heat shock was associated withan increased Bcl-2 expression. Bcl-2 upregulation after heatshock may be linked to increased HSP levels, because HSP-90has been shown to upregulate Bcl-2 expression after vascularendothelial growth factor addition (39). HSP-70 has also beenshown to activate Bcl-2 via a direct link made by a nucleotideexchange factor for HSP-70 known as BAG (40). We speculate thatheat shock–induced upregulation and maintenance of Bcl-2levels during cold storage may contribute to cell survival byblocking the mitochondrial membrane disruption that is likelyto be central to cold storage and reperfusion injury in HK-2cells.
We next focused specifically on whether HSP-70 plays a directrole in the heat-shock effect observed. We generated HK-2 cellsthat stably overexpress HSP-70 and found them to be more resistantto cold storage and rewarming-induced apoptosis than controlHK-2 cells. This finding indicates that HSP-70 alone in theabsence of a stress response is sufficient to confer resistanceagainst cold storage and rewarming injury in HK-2 cells andis in agreement with other HSP-70 overexpression studies. Forexample, addition of recombinant HSP-70 has been shown to inhibitcytochrome C/dATP-mediated caspase activation (41) and to blockcell death via reduced p38 kinase activation (42). In addition,an overexpression study in human lung adenocarcinoma cells demonstrateda specific role for HSP-70 in blocking hyperoxia-mediated lipidperoxidation and cell death (43). Despite the ability of HSP-70overexpression to protect HK-2 cells, blocking of HSP-70 upregulationwith siRNA did not inhibit the protective effect of heat shock.In agreement with our finding, it was reported previously thatantisense inhibition of HSP-70 failed to abolish the heat shock–inducedprotection of neuroblastoma cells from apoptosis (44). We speculatethat the heat-shock protective effect that was seen in thisstudy is a multifactorial response to the complex insult ofcold storage and rewarming and that a degree of redundancy mayexist such that blocking induction of a specific protectivefactor such as HSP-70 will not blunt the overall protectiveeffect. Further research is required to understand fully thestress response mechanisms of organ preconditioning for transplantation.
Our findings indicate that cold storage of HK-2 cells predisposesthem to further injury and death after rewarming. This injuryis associated with decreased expression of stress proteins (HSP-70,HSP-90, and HSP-27) and Bcl-2 during the cold storage period.Such injury can be partially blocked by previous heat shockassociated with maintenance of HSP and Bcl-2 expression levelsduring cold storage. HSP-70 overexpression is sufficient toconfer protection against cold storage and rewarming injury.However, the upregulation of HSP-70 is not the only factor involvedin heat shock–induced protection against such injury.
Acknowledgments
This work was supported by a Punchestown Kidney Research Fundgrant to D.A.H., a Wellcome Trust grant to M.M., and in partby the Health Research Board.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
