Ischemic preconditioning is an extremely powerful means to reproducibly protect from subsequent ischemia. Stress response or heat shock proteins (HSPs) are induced by many physiological stressors and are thought to be critical in this protection. Both heat stress and ischemia cause extensive cytoskeletal and mitochondrial damage and uncoupling of oxidative phosphorylation. HSPs are thought to limit injury and accelerate recovery by refolding disrupted proteins and preventing deleterious peptide interactions1.
In cultured renal epithelial cells, protection from adenosine 5'-triphosphate (ATP) depletion or heat stress is correlated with HSP72 levels2,3,4. HSP70 expression is inversely correlated with myocardial infarct size in rats5 and overexpression of HSP70 in mice protects against cardiac ischemia6,7. HSP70 induction, however, correlates poorly with protection of isolated rat proximal tubular segments from hypoxia8. The induction of HSPs prior to renal ischemia in vivo has had variable results9,10,11. The present study was designed to test the effect of heat stress at multiple time points before ischemia and of an inhibitor of HSP induction (quercetin) on the functional and histologic consequences of renal ischemia.
METHODS
Animal protocols
Studies conformed to the "Guiding Principles for Research Involving Animals and Human Beings." Male Sprague-Dawley rats (180 to 240 g) and Swiss Webster mice (20 to 25 g; Harlan Laboratories, Indianapolis, IN, USA) were placed in a 45°C incubator until rectal temperature reached 41 to 42°C for eight or four minutes, respectively. Sham hyperthermia animals were placed in a 37°C incubator for the same total time period. The quercetin group received quercetin (100 mg/kg) via gavage two hours before heat stress (8 min). Quercetin prevents the induction of HSPs of different sizes in several cell types12,13. At multiple time points after hyperthermia, animals were sacrificed for kidney removal or were subjected to ischemia. Mice were anesthetized with pentobarbital (100 mg/kg intraperitoneally) before removal of kidneys via midline incision. Cortex and medulla were separated on ice and frozen in liquid nitrogen.
Prior to ischemia14,15, rats were anesthetized with sodium pentobarbital (65 mg/kg intraperitoneally). Both renal pedicles were occluded for 30 minutes with microaneurysm clamps. All experiments were blinded. Tail vein blood samples (0.15 mL) were obtained daily. Blood urea nitrogen (BUN) and creatinine were measured by standard urease/conductivity and picric acid reactions, respectively.
Northern analysis
Renal RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's protocol. Blots were probed with 32P-labeled HSP7016, HSP8417, and HSP2218 or 28S (ATCC, Manassas, VA, USA) cDNA as previously described14. Membranes were exposed for 24 to 72 hours and read by PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA).
Light microscopy
Kidneys were removed 48-hour postischemia and were formalin fixed, paraffin embedded, sectioned at 4 microns and were stained with hematoxylin and eosin. The number of tubules with necrotic cells or debris was quantitated (0 = none, 1+ = <10%, 2+ = 10 to 25%, 3+ = 26 to 75%, 4+ => 75%)14,15 in multiple coded sections.
Myeloperoxidase activity
Myeloperoxidase (MPO) activity, an index of leukocyte infiltration19, was measured in coded specimens at 48 hours postischemia as previously described15. Activity was normalized to protein content and expressed as the percentage of sham surgery activity.
Statistics
Analysis of variance was used to compare blood urea nitrate (BUN) and MPO data. Tukey's test was used to correct for multiple comparisons. Differences in mortality and histology were evaluated using Fisher's exact test. Rats sacrificed before 48 hours were not included in mortality statistics.
RESULTS
Effect of prior heat stress on postischemic renal function
Mean BUN was lower in the rats subjected to renal ischemia six hours after hyperthermia than in the sham hyperthermia group on days 1 to 6 postischemia (P < 0.05; Figure 1). Partial protection was observed with heat stress 12 hours before ischemia with mean BUN less (P < 0.05) than that after sham hyperthermia on days 1, 2, and 6. Minimal protection was seen when renal ischemia was induced 48 hours after hyperthermia. Mean creatinine values showed a similar pattern of protection (data not shown).
Figure 1.
Effect of prior heat stress on postischemic renal function. Mean blood urea nitrogen (BUN 
1 SEM) after sham hyperthermia (
) or hyperthermia 6 (
), 12 (
), or 48 (
) hours prior to ischemia is presented.
Effect of prior heat stress on renal histology after ischemia
Less severe tubular injury was also seen with prior hyperthermia. In the sham hyperthermia group, most (98%) of fields evaluated were graded 3 or 4+ (>25% tubules with evidence of necrosis), compared with 22% in the group subjected to ischemia six hours after heat stress (P < 0.01). Representative sections of outer medulla 48 hours postischemia in the sham and hyperthermia rats are shown in Figure 2 a and b, respectively.
Figure 2.
Effect of prior heat stress on renal histology after ischemia. (a) A representative section of outer medulla from a sham hyperthermia rat at 48 hours postischemia shows extensive (4+) diffuse tubular necrosis and cast formation. (b) A comparable section from a rat subjected to hyperthermia six hours prior to ischemia shows mild necrosis (2+).
Full figure and legend (109K)Mortality
The difference in mortality between the sham hyperthermia (25%) and six-hour hyperthermia (0%) rats did not reach statistical significance (P = 0.13).
Effect of prior heat stress on kidney myeloperoxidase activity postischemia
Ischemia-induced increases in renal MPO activity were attenuated with hyperthermia six hours prior to ischemia. MPO activity in the hyperthermia group (287 36% sham surgery) was less (P < 0.03) than in the sham hyperthermia rats (472 50%) 48 hours postischemia.
Effect of heat stress with or without quercetin on mRNA levels of HSP70, HSP84, and HSP22
Increased medullary HSP70 and HSP22 (
-Bcrystallin) mRNA was seen rapidly after hyperthermia and remained elevated for at least 48 hours. Increases in HSP84 mRNA began later (6 hours) following heat stress. In the four-minute hyperthermia and quercetin mice, no change in HSP70 and HSP84 mRNA levels was seen. No change in HSP22 mRNA levels was seen in the four-minute hyperthermia mice, but increases were observed at 24 and 48 hours in the quercetin group Figure 3. A similar pattern (with lower densities) is seen in cortical tissue (data not shown).
Figure 3.
Effect of heat stress with or without quercetin on mRNA levels of HSP70, HSP84, and HSP22. Representative Northern blots from renal medulla of mice subjected to hyperthermia (8 or 4 min) or quercetin prior to hyperthermia and sacrificed 0, 1, 3, 6, 24, or 48 hours after the end of the period of hyperthermia are presented. Tissue in the lanes labeled "Sham" was removed after sham hyperthermia.
Full figure and legend (31K)Effect of four minutes of heat stress or quercetin prior to hyperthermia on postischemic renal function
Mean BUN after a short heat stress was no different than that after sham hyperthermia. Mean BUN in the quercetin rats was not different from that in the sham hyperthermia rats at 24 and 48 hours after ischemia, but thereafter began to decrease Figure 4. Mean creatinine values showed a similar pattern of protection (data not shown).
Figure 4.
Effect of heat stress for four minutes or administration of quercetin prior to hyperthermia on postischemic renal function. Mean BUN ( 1 SEM) prior to and following bilateral renal ischemia in rats subjected to a short period of hyperthermia or quercetin prior to hyperthermia is presented. Ischemia was induced six hours after hyperthermia. Symbols are: (
) sham hyperthermia; (
) 4 minutes of hyperthermia; (
) quercetin; (
) 8 minutes of hyperthermia; *P < 0.05, **P < 0.01 vs. sham operated.
DISCUSSION
Acute renal failure is a common clinical problem with increasing incidence20 and an unacceptably high mortality21. Exogenous agents protective in animal models have not shown a significant clinical benefit in recent trials22,23. In the present study, we examined the role of endogenous protective mechanisms in renal ischemia.
In many organs, an ischemic insult results in protection from subsequent prolonged ischemia. HSPs are thought to be critical in this protection1,24. After renal ischemia, increases in HSP72 occur quickly25,26. The ubiquity and conservation of the heat shock response suggest that it is a fundamental mechanism of cellular defense. HSPs are molecular chaperones that transiently bind nascent polypeptides and facilitate correct folding and assembly. Following injury, HSPs may refold denatured proteins and restore function, limit detrimental peptide interactions, translocate proteins to the correct intracellular location, and degrade irreparably damaged proteins and toxic metabolites. Heat shock and ischemia both cause extensive damage to the cytoskeleton, mitochondrial swelling, and uncoupling of oxidative phosphorylation. After such stresses, general protein synthesis is inhibited, but HSPs are efficiently translated and synthesized and can rapidly reach 15 to 25% of intracellular protein content1,24.
The effect of increases in HSP expression on ischemic renal injury in vivo has been variable9,10,11. The role of HSPs in the kidney may depend on the exact nature and timing of the initial and subsequent insults1. Chatson et al found protection with 8 to 11 but not 12 to 15 minutes of hyperthermia prior to renal ischemia9. Joannidis et al also did not demonstrate protection with 15 minutes of hyperthermia11. Santos et al found increased resistance to heat stress, ATP depletion, and cyclosporine, but increased sensitivity to cadmium chloride in inner medulla collecting duct cells after hyperosmolar induction of HSP70, OSP94, and HSP110 mRNA (abstract; Santos et al, J Am Soc Nephrol 8:130A, 1997). In the present model, the pattern of renal expression of HSPs suggests that the induction of HSP84 is not critical to the protection observed.
The protection observed was dependent on the timing of ischemia after heat stress. Partial protection was seen with ischemia 12 hours after hyperthermia, but minimal or no protection was seen with ischemia 48 hours after heat shock Figure 1. A lack of protection at 48 hours was also seen by Joannidis et al11. In a lung transplant model, Hiratsuka et al found protection with hyperthermia 6 but not 12 hours before organ harvest27. We also found no protection with a short period of hyperthermia Figure 4 that did not increase HSP mRNA levels Figure 3. Protection was also dependent on the induction of HSPs and was not seen in the short hyperthermia group. These and the results of Chen et al (abstract, J Am Soc Nephrol 10:630A, 1999) showing protection with tunicamycin (which up-regulates endoplasmic HSPs) suggest that heat stress is not necessary for protection. The possibility that the increase in HSP22 mRNA 24 and 48 hours after quercetin/heat shock is associated with the improvement in renal function in this group on days 3 and 4 is intriguing.
The induction of HSPs may afford protection from subsequent insults by decreasing leukocyte infiltration of postischemic tissue27. Javadpour et al found fewer pulmonary neutrophils and less MPO activity after aortic occlusion in rats with prior induction of HSPs via hyperthermia28 or pharmacologic means29. They also showed that hyperthermia prevents the decrease in leukocyte rolling velocity seen with mesenteric ischemia/reperfusion30. Decreased MPO activity in transplanted lung tissue with prior hyperthermia has been demonstrated27. Prior hyperthermia also decreases intestinal neutrophil infiltration and mucosal injury after intestinal ischemia31. In the present study, we found decreased renal MPO activity with heat stress prior to ischemia.
These studies demonstrate a role for endogenous protective mechanisms in renal ischemia. Pharmacological strategies to increase stress protein expression have potential merit to prevent ischemic injury to the kidney and other organs. However, these and other studies illustrate that protection is dependent on many factors, including the specific HSPs induced and the nature and timing of the insults studied.
References
| 1. | VanWhy SK & Siegel NJ. Heat shock proteins in renal injury and recovery. Curr Opin Nephrol Hypertens 1998; 7: 407–412 10.1002/(sici)1521-3919(19980701)7:4<407::aid-mats407>3.0.co;2-d. | PubMed | ChemPort | |
| 2. | Borkan SC, Emami A & Schwartz JH. Heat stress protein-associated cytoprotection of inner medullary collecting duct cells from rat kidney. Am J Physiol 1993; 265: F333–F341. | PubMed | ISI | ChemPort | |
| 3. | Nissim I, Hardy M & Pleasure J et al. A mechanism of glycine and alanine cytoprotective action: Stimulation of stress-induced HSP70 mRNA. Kidney Int 1992; 42: 775–782. | PubMed | ISI | ChemPort | |
| 4. | Wang YH & Borkan SC. Prior heat stress enhances survival of renal epithelial cells after ATP depletion. Am J Physiol 1996; 270: F1057–F1065. | PubMed | ISI | ChemPort | |
| 5. | Hutter M, Sievers R, Barbosa V & Wolfe C. Heat-shock protein induction in rat hearts: A direct correlation between the amount of heat-shock protein induced and the degree of myocardial protection. Circulation 1994; 89: 355–360. | PubMed | ISI | ChemPort | |
| 6. | Plumier J, Ross B & Currie R et al. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest 1995; 95: 1854–1860. | PubMed | ISI | ChemPort | |
| 7. | Marber M, Mestril R & Chi S et al. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 1995; 95: 1446–1456. | PubMed | ISI | ChemPort | |
| 8. | Zager R, Iwata M, Burkhart K & Schimpf B. Post-ischemic acute renal failure protects proximal tubules from O2 deprivation injury, possibly by inducing uremia. Kidney Int 1994; 45: 1760–1768. | PubMed | ISI | ChemPort | |
| 9. | Chatson G, Perdrizet G & Anderson C et al. Heat shock protects kidneys against warm ischemic injury. Curr Surg 1990; 47: 420–423. | PubMed | ChemPort | |
| 10. | Perdrizet G, Kaneko H & Buckley T et al. Heat shock protects pig kidneys against warm ischemic injury. Transplant Proc 1990; 22: 460–461. | PubMed | ISI | ChemPort | |
| 11. | Joannidis M, Cantley L & Spokes K et al. Induction of heat-shock proteins does not prevent renal tubular injury following ischemia. Kidney Int 1995; 47: 1752–1759. | PubMed | ISI | ChemPort | |
| 12. | Chen H-C, Guh J-Y, Tsai J-H & Lai Y-H. Induction of heat stock protein 70 protects mesangial cells against oxidative injury. Kidney Int 1999; 56: 1270–1273 10.1046/j.1523-1755.1999.00693.x. | Article | PubMed | ISI | ChemPort | |
| 13. | Loktionova S, Ilyinskaya O & Kabakov A. Early and delayed tolerance to simulated ischemia in heat-preconditioned endothelial cells: A role for HSP27. Am J Physiol 1998; 275: H2147–H2158. | PubMed | ISI | ChemPort | |
| 14. | Kelly KJ, Williams WW, Jr & Colvin RB et al. Intercellular adhesion molecule-1 deficient mice are protected against ischemic renal injury. J Clin Invest 1996; 97: 1056–1063. | PubMed | ISI | ChemPort | |
| 15. | Kelly KJ, Williams WW, Colvin RB & Bonventre JV. Antibody to intercellular adhesion molecule-1 protects the kidney against ischemic injury. Proc Natl Acad Sci USA 1994; 91: 812–816. | PubMed | ChemPort | |
| 16. | Wu B, Hunt C & Morimoto R. Structure and expression of the human gene encoding major heat shock protein HSP70. Mol Cell Biol 1985; 5: 330–341. | PubMed | ISI | ChemPort | |
| 17. | Hoffmann T & Hovemann B. Heat-shock proteins, hsp84 and hsp86, of mice and men: Two related genes encode formerly identified tumour-specific transplantation antigens. Gene 1988; 74: 491–501. | Article | PubMed | ISI | ChemPort | |
| 18. | Klemenz R, Froehli E & Aoyama A et al. AlphaB crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH 3T3 fibroblasts. Mol Cell Biol 1991; 11: 803–812. | PubMed | ISI | ChemPort | |
| 19. | Bradley PP, Priebat DA, Christensen RD & Rothstein G. Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982; 78: 206–209. | Article | PubMed | ISI | ChemPort | |
| 20. | Chertow G. On the design and analysis of multicenter trials in acute renal failure. Am J Kidney Dis 1997; 30 Suppl 4: S96–S101. | PubMed | ISI | ChemPort | |
| 21. | Kelly KJ & Molitoris BA. Acute renal failure in the new millennium: Time to consider combination therapy. Semin Nephrol 2000; 20: 4–19. | PubMed | ISI | ChemPort | |
| 22. | Allegren RL, Marbury TC & Rahman SN et al. Anaritide in acute tubular necrosis. N Engl J Med 1997; 336: 828–834. | PubMed | |
| 23. | Hirschberg R, Kopple J & Lipsett P et al. Multicenter clinical trial of recombinant human insulin-like growth factor I in patients with acute renal failure. Kidney Int 1999; 55: 2423–2432 10.1046/j.1523-1755.1999.00463.x. | Article | PubMed | ISI | ChemPort | |
| 24. | Gething M-J & Sambrook J. Protein folding in the cell. Nature 1992; 355: 33–45. | Article | PubMed | ISI | ChemPort | |
| 25. | Van Why SK, Hildebrandt F & Ardito T et al. Induction and intracellular localization of HSP-72 after renal ischemia. Am J Physiol 1992; 263: F769–F775. | PubMed | ChemPort | |
| 26. | Emami A, Schwartz JH & Borkan SC. Transient ischemia or heat stress induces a cytoprotectant protein in rat kidney. Am J Physiol 1991; 260: F479–F485. | PubMed | ISI | ChemPort | |
| 27. | Hiratsuka M, Yano M & Mora B et al. Heat shock pretreatment protects pulmonary isografts from subsequent ischemia-reperfusion injury. J Heart Lung Transplant 1998; 17: 1238–1246. | PubMed | ISI | ChemPort | |
| 28. | Javadpour M, Kelly C & Chen G et al. Thermotolerance induces heat shock protein 72 expression and protects against ischaemia-reperfusion lung injury. Br J Surg 1998; 85: 943–946. | Article | PubMed | ISI | ChemPort | |
| 29. | Javadpour M, Kelly C, Chen G & Bouchier-Hayes D. Herbimycin-A attenuates ischaemia-reperfusion induced pulmonary neutrophil infiltration. Eur J Vasc Endovasc Surg 1998; 16: 377–382. | Article | PubMed | ISI | ChemPort | |
| 30. | Chen G, Kelly C & Stokes K et al. Induction of heat shock protein 72 kDa expression is associated with attenuation of ischaemia-reperfusion induced microvascular injury. J Surg Res 1997; 69: 435–439 10.1006/jsre.1997.5059. | Article | PubMed | ISI | ChemPort | |
| 31. | Stojadinovic A, Kiang J & Smallridge R et al. Induction of heat-shock protein 72 protects against ischemia/reperfusion in rat small intestine. Gastroenterology 1995; 109: 505–515. | Article | PubMed | ISI | ChemPort | |
Acknowledgments
This work was supported in part by National Institutes of Health grant DK02364. Portions were published in abstract form (J Am Soc Nephrol 9:579A, 1998, and 10:633A, 1999) and were presented at the American Society of Nephrology meeting in October, 1999. We thank Drs. Z. Wang and M. Soleimani for assistance with molecular analyses, R. Stites for technical assistance, and Drs. J.V. Bonventre and J.H. Galla for helpful discussions.
