The Indispensability of Heme Oxygenase-1 in Protecting against Acute Heme Protein-Induced Toxicity in Vivo
2000; Elsevier BV; Volume: 156; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65024-9
ISSN1525-2191
AutoresKarl A. Nath, Jill J. Haggard, Anthony J. Croatt, Joseph P. Grande, Kenneth D. Poss, Jawed Alam,
Tópico(s)Cannabis and Cannabinoid Research
ResumoHeme oxygenase (HO) is the rate limiting enzyme in the degradation of heme, and its isozyme, HO-1, may protect against tissue injury. One posited mechanism is the degradation of heme released from destabilized heme proteins. We demonstrate that HO-1 is a critical protectant against acute heme protein-induced toxicity in vivo. In the glycerol model of heme protein toxicity—one characterized by myolysis, hemolysis, and kidney damage—HO-1 is rapidly induced in the kidney of HO-1 +/+ mice as the latter sustain mild, reversible renal insufficiency without mortality. In stark contrast, after this insult, HO-1 −/− mice exhibit fulminant, irreversible renal failure and 100% mortality; HO-1 −/− mice do not express HO-1, and evince an eightfold increment in kidney heme content as compared to HO-1 +/+ mice. We also demonstrate directly the critical dependency on HO-1 in protecting against a specific heme protein, namely, hemoglobin: doses of hemoglobin which exert no nephrotoxicity or mortality in HO-1 +/+ mice, however, precipitate rapidly developing, acute renal failure and marked mortality in HO-1 −/− mice. We conclude that the induction of HO-1 is an indispensable response in protecting against acute heme protein toxicity in vivo. Heme oxygenase (HO) is the rate limiting enzyme in the degradation of heme, and its isozyme, HO-1, may protect against tissue injury. One posited mechanism is the degradation of heme released from destabilized heme proteins. We demonstrate that HO-1 is a critical protectant against acute heme protein-induced toxicity in vivo. In the glycerol model of heme protein toxicity—one characterized by myolysis, hemolysis, and kidney damage—HO-1 is rapidly induced in the kidney of HO-1 +/+ mice as the latter sustain mild, reversible renal insufficiency without mortality. In stark contrast, after this insult, HO-1 −/− mice exhibit fulminant, irreversible renal failure and 100% mortality; HO-1 −/− mice do not express HO-1, and evince an eightfold increment in kidney heme content as compared to HO-1 +/+ mice. We also demonstrate directly the critical dependency on HO-1 in protecting against a specific heme protein, namely, hemoglobin: doses of hemoglobin which exert no nephrotoxicity or mortality in HO-1 +/+ mice, however, precipitate rapidly developing, acute renal failure and marked mortality in HO-1 −/− mice. We conclude that the induction of HO-1 is an indispensable response in protecting against acute heme protein toxicity in vivo. Heme oxygenase (HO) is the rate limiting enzyme in the degradation of heme.1Nath KA Agarwal A Vogt B Functional consequences of induction of heme oxygenase. Contemporary Issues in Nephrology. Acute Renal Failure: Emerging Concepts and Therapeutic Strategies.in: Goligorsky MS Stein J Churchill Livingstone, New York1995: 97-118Google Scholar, 2Choi AMK Alam J Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein-induced lung injury.Am J Respir Cell Mol Biol. 1996; 15: 9-19Crossref PubMed Scopus (999) Google Scholar, 3Platt JL Nath KA Heme oxygenase: protective gene or trojan horse.Nat Med. 1998; 4: 1364-1365Crossref PubMed Scopus (210) Google Scholar HO facilitates the opening of the heme ring and its conversion to biliverdin; during such conversion, iron is released from the heme prosthetic group and carbon monoxide is produced; subsequently, biliverdin is reduced to bilirubin by biliverdin reductase.1Nath KA Agarwal A Vogt B Functional consequences of induction of heme oxygenase. Contemporary Issues in Nephrology. Acute Renal Failure: Emerging Concepts and Therapeutic Strategies.in: Goligorsky MS Stein J Churchill Livingstone, New York1995: 97-118Google Scholar, 2Choi AMK Alam J Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein-induced lung injury.Am J Respir Cell Mol Biol. 1996; 15: 9-19Crossref PubMed Scopus (999) Google Scholar, 3Platt JL Nath KA Heme oxygenase: protective gene or trojan horse.Nat Med. 1998; 4: 1364-1365Crossref PubMed Scopus (210) Google Scholar HO-1 is the isozyme that is induced by heme, oxidants, cytokines, glycated albumin, and other stressors, and the induction of this isozyme is associated with a protective response in some of these states.1Nath KA Agarwal A Vogt B Functional consequences of induction of heme oxygenase. Contemporary Issues in Nephrology. Acute Renal Failure: Emerging Concepts and Therapeutic Strategies.in: Goligorsky MS Stein J Churchill Livingstone, New York1995: 97-118Google Scholar, 2Choi AMK Alam J Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein-induced lung injury.Am J Respir Cell Mol Biol. 1996; 15: 9-19Crossref PubMed Scopus (999) Google Scholar, 3Platt JL Nath KA Heme oxygenase: protective gene or trojan horse.Nat Med. 1998; 4: 1364-1365Crossref PubMed Scopus (210) Google Scholar, 4Yan SD Schmidt AM Anderson GM Zhang J Brett J Zou YS Pinsky D Stern DM Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins.J Biol Chem. 1994; 269: 9889-9897Abstract Full Text PDF PubMed Google Scholar, 5Willis D Moore AR Frederick R Willoughby DA Heme oxygenase: a novel target for the modulation of the inflammatory response.Nat Med. 1996; 2: 87-90Crossref PubMed Scopus (704) Google Scholar, 6Soares MP Lin Y Anrather J Cxizmadia E Takigami K Sato K Grey ST Colvin RB Choi AM Poss KD Bach FH Expression of heme oxygenase-1 can determine cardiac xenograft survival.Nat Med. 1998; 4: 1073-1077Crossref PubMed Scopus (555) Google Scholar, 7Hancock WW Buelow R Sayegh MH Turka LA Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes.Nat Med. 1998; 4: 1392-1396Crossref PubMed Scopus (427) Google Scholar, 8Yet S-F Perrella MA Layne MD Hsieh C-M Maemura K Kobzik L Wiesel P Christou H Kourembanas S Lee M-E Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice.J Clin Invest. 1999; 103: R23-R29Crossref PubMed Scopus (355) Google Scholar, 9Otterbein LE Kolls JK Mantell LL Cook JL Alam J Choi AMK Exogenous administration of heme oxygenase-1 by gene transfer provide protection against hyperoxia-induced lung injury.J Clin Invest. 1999; 103: 1047-1054Crossref PubMed Scopus (468) Google Scholar HO-2 is the constitutive isozyme that may be involved in the regulation of vascular tone and neural function effected, in part, through the cellular effects of carbon monoxide.10Burnett AL Johns DG Kriegsfeld LJ Klein SL Calvin DC Demas GE Schramm LP Tonegawa S Nelson RJ Snyder SH Poss KD Ejaculatory abnormalities in mice with targeted disruption of the gene for heme oxygenase-2.Nat Med. 1998; 4: 84-87Crossref PubMed Scopus (102) Google Scholar, 11Christodoulides N Durante W Kroll MH Schafer AI Vascular smooth muscle cell heme oxygenases generate guanylyl cyclase-stimulatory carbon monoxide.Circulation. 1995; 91: 2306-2309Crossref PubMed Scopus (235) Google Scholar One posited, but primarily unsubstantiated, mechanism invoked for the cytoprotective effects of HO-1 in the setting of tissue injury centers on the degradation of heme released from intracellular heme proteins that are destabilized pari passu as cells are injured.1Nath KA Agarwal A Vogt B Functional consequences of induction of heme oxygenase. Contemporary Issues in Nephrology. Acute Renal Failure: Emerging Concepts and Therapeutic Strategies.in: Goligorsky MS Stein J Churchill Livingstone, New York1995: 97-118Google Scholar, 2Choi AMK Alam J Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein-induced lung injury.Am J Respir Cell Mol Biol. 1996; 15: 9-19Crossref PubMed Scopus (999) Google Scholar, 3Platt JL Nath KA Heme oxygenase: protective gene or trojan horse.Nat Med. 1998; 4: 1364-1365Crossref PubMed Scopus (210) Google Scholar The heme prosthetic group is widespread in cells as it is found in proteins that store or carry oxygen, cytochromes, and numerous enzymes involved in diverse aspects of cellular metabolism. Destabilization of heme proteins in injured cells may lead to the disengagement of heme from its linked protein moiety. The linkage of heme with a given protein moiety not only enables the functional activity of the specific heme protein, but also restrains free heme from exerting injurious effects. A large body of literature confirms the cytotoxicity of free heme,12Hebbel RP Eaton JW Pathobiology of heme interaction with the erythrocyte membrane.Semin Hematol. 1989; 26: 136-149PubMed Google Scholar, 13Balla G Vercellotti GM Muller-Eberhard U Eaton J Jacob HS Exposure of endothelial cells to free heme potentiates damage mediated by granulocytes and toxic oxygen species.Lab Invest. 1991; 64: 648-655PubMed Google Scholar, 14Muller-Eberhard U Fraig M Bioactivity of heme and its containment.Am J Hematol. 1993; 42: 59-62Crossref PubMed Scopus (67) Google Scholar, 15Eaton JW Hemoglobin-based blood substitutes: a dream-like trade of blood and guile?.J Lab Clin Med. 1996; 127: 416-417Abstract Full Text PDF PubMed Scopus (10) Google Scholar and indeed, there are clinical observations attesting to the marked nephrotoxicity of heme when the latter—used to induce remission in patients with acute intermittent porphyria—is inadvertently administered in inordinate doses.16Jeelani Dhar G Bossenmaier I Cardinal R Petryka ZJ Watson CJ Transitory renal failure following rapid administration of a relatively large amount of hematin in a patient with acute intermittent porphyria in clinical remission.Acta Med Scand. 1978; 203: 437-443Crossref PubMed Scopus (65) Google Scholar Heme is a lipophilic prooxidant that can impair lipid bilayers and organelles such as mitochondria and nuclei; heme can also destabilize the cytoskeleton; finally, heme can impair a number of enzymes.12Hebbel RP Eaton JW Pathobiology of heme interaction with the erythrocyte membrane.Semin Hematol. 1989; 26: 136-149PubMed Google Scholar, 13Balla G Vercellotti GM Muller-Eberhard U Eaton J Jacob HS Exposure of endothelial cells to free heme potentiates damage mediated by granulocytes and toxic oxygen species.Lab Invest. 1991; 64: 648-655PubMed Google Scholar, 14Muller-Eberhard U Fraig M Bioactivity of heme and its containment.Am J Hematol. 1993; 42: 59-62Crossref PubMed Scopus (67) Google Scholar, 15Eaton JW Hemoglobin-based blood substitutes: a dream-like trade of blood and guile?.J Lab Clin Med. 1996; 127: 416-417Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 16Jeelani Dhar G Bossenmaier I Cardinal R Petryka ZJ Watson CJ Transitory renal failure following rapid administration of a relatively large amount of hematin in a patient with acute intermittent porphyria in clinical remission.Acta Med Scand. 1978; 203: 437-443Crossref PubMed Scopus (65) Google Scholar, 17Nath KA Grande JP Croatt AJ Likely S Hebbel RP Enright H Intracellular targets in heme protein-induced renal injury.Kidney Int. 1998; 53: 100-111Crossref PubMed Scopus (97) Google Scholar In injured cells, the recruitment of HO-1 may degrade heme released from intracellular heme proteins, thereby safeguarding against this mechanism of cytotoxicity. Besides the removal of heme, the induction of HO-1 procures potentially cytoprotectant molecules such as ferritin, bilirubin, and carbon monoxide. Ferritin provides a storage site for iron,18Balla G Jacob HS Balla J Rosenberg ME Nath KA Apple F Eaton JW Vercellotti GM Ferritin: a cytoprotective antioxidant stratagem of endothelium.J Biol Chem. 1992; 267: 18148-18153Abstract Full Text PDF PubMed Google Scholar whereas bilirubin is a metabolite with recognized antioxidant properties.19Stocker R Induction of haem oxygenase as a defense against oxidative stress.Free Radic Res Commun. 1990; 9: 101-112Crossref PubMed Scopus (303) Google Scholar, 20Llesuy SF Tomaro ML Heme oxygenase and oxidative stress. Evidence of involvement of bilirubin as physiological protector against oxidative damage.Biochim Biophys Acta. 1994; 1223: 9-14Crossref PubMed Scopus (286) Google Scholar Carbon monoxide is a critical intracellular signaling molecule in neural tissue;10Burnett AL Johns DG Kriegsfeld LJ Klein SL Calvin DC Demas GE Schramm LP Tonegawa S Nelson RJ Snyder SH Poss KD Ejaculatory abnormalities in mice with targeted disruption of the gene for heme oxygenase-2.Nat Med. 1998; 4: 84-87Crossref PubMed Scopus (102) Google Scholar additionally, carbon monoxide possesses vasodilatory, anti-inflammatory, and cytoprotectant properties.10Burnett AL Johns DG Kriegsfeld LJ Klein SL Calvin DC Demas GE Schramm LP Tonegawa S Nelson RJ Snyder SH Poss KD Ejaculatory abnormalities in mice with targeted disruption of the gene for heme oxygenase-2.Nat Med. 1998; 4: 84-87Crossref PubMed Scopus (102) Google Scholar, 11Christodoulides N Durante W Kroll MH Schafer AI Vascular smooth muscle cell heme oxygenases generate guanylyl cyclase-stimulatory carbon monoxide.Circulation. 1995; 91: 2306-2309Crossref PubMed Scopus (235) Google Scholar, 21Morita T Kourembanas S Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide.J Clin Invest. 1995; 96: 2676-2682Crossref PubMed Scopus (325) Google Scholar, 22Otterbein LE Mantell LL Choi AM Carbon monoxide provides protection against hyperoxic lung injury.Am J Physiol. 1999; 276: L688-L694PubMed Google Scholar Thus, the salutary effects afforded by the induction of HO-1 may reside in the recruitment of biochemically diverse cytoprotective substances, in addition to the reduction in an intracellular toxicant. Much of the literature demonstrating a protective effect of induced HO-1, however, relies on pharmacological approaches which may exert effects besides those involving HO-1;23Meffert MK Haley JE Schuman EM Schulman H Madison DV Inhibition of hippocampal heme oxygenase, nitric oxide synthase, and long-term potentiation by metalloporphyrins.Neuron. 1994; 13: 1233-1255Abstract Full Text PDF Scopus (149) Google Scholar, 24Grundemar L Ny L Pitfalls using metalloporphyrins in carbon monoxide research.Trends Pharmacol Sci. 1997; 18: 193-195Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 25Serfass L Burstyn JN Effect of heme oxygenase inhibitors on soluble guanylyl cyclase activity.Arch Biochem Biophys. 1998; 359: 8-16Crossref PubMed Scopus (50) Google Scholar the availability of genetically engineered mice deficient in HO-1 provides an opportunity to examine the protective effects of induced HO-1 without the confounding actions of such pharmacological manipulations.6Soares MP Lin Y Anrather J Cxizmadia E Takigami K Sato K Grey ST Colvin RB Choi AM Poss KD Bach FH Expression of heme oxygenase-1 can determine cardiac xenograft survival.Nat Med. 1998; 4: 1073-1077Crossref PubMed Scopus (555) Google Scholar, 8Yet S-F Perrella MA Layne MD Hsieh C-M Maemura K Kobzik L Wiesel P Christou H Kourembanas S Lee M-E Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice.J Clin Invest. 1999; 103: R23-R29Crossref PubMed Scopus (355) Google Scholar Moreover, very few studies have examined whether the potential cytoprotective effects of HO-1 are derived from restraining the tissue buildup of heme; and these studies do not include heme protein-induced toxicity. We thus examined the requirement for HO-1 in protecting against heme proteins—a possible intracellular toxicant in injured tissue, and one common to insults in which HO-1 is induced—using mice deficient in HO-1 (HO-1 −/−) or wild-type mice (HO-1 +/+). We used a long-established, in vivo model of heme protein-induced tissue injury, the glycerol model, in which we examined the relative sensitivity of these mice to such injury. The intramuscular injection of hypertonic glycerol induces myolysis and hemolysis thereby exposing tissues, especially the kidney, to large amounts of myoglobin and hemoglobin;26Zager RA Burkhart KM Conrad DS Gmur DJ Iron, heme oxygenase, and glutathione: effects on myohemoglobinuric proximal tubular injury.Kidney Int. 1995; 48: 1624-1634Crossref PubMed Scopus (123) Google Scholar, 27Zager RA Rhabdomyolysis and myohemoglobinuric acute renal failure.Kidney Int. 1996; 49: 314-326Crossref PubMed Scopus (501) Google Scholar acute renal failure dominates this disease model and the analogous clinical syndromes, such as those caused by crush injuries and a host of nontraumatic and medical disorders.27Zager RA Rhabdomyolysis and myohemoglobinuric acute renal failure.Kidney Int. 1996; 49: 314-326Crossref PubMed Scopus (501) Google Scholar Using this model of heme protein-induced renal injury, as well as the direct examination of the nephrotoxicity of a specific heme protein (hemoglobin), we provide compelling evidence for the indispensability of HO-1 in protecting against heme protein-mediated injury in vivo. Homozygous HO-1 null mutants were generated by targeted disruption of the HO-1 gene as described by Poss and Tonegawa.28Poss KD Tonegawa S Heme oxygenase 1 is required for mammalian iron reutilization.Proc Natl Acad Sci USA. 1997; 94: 10919-10924Crossref PubMed Scopus (844) Google Scholar Colonies of mice were maintained by breeding HO-1 −/− males with HO-1 +/− females. Offspring were genotyped at the time of weaning by using polymerase chain reaction to amplify the wild-type and mutant alleles of genomic DNA obtained from tail samples. HO-1 +/+ mice (wild type. were used as controls. The characteristics of HO-1 −/− mice, described previously,28Poss KD Tonegawa S Heme oxygenase 1 is required for mammalian iron reutilization.Proc Natl Acad Sci USA. 1997; 94: 10919-10924Crossref PubMed Scopus (844) Google Scholar include slower rate of somatic growth as compared to HO-1 +/+ mice; and thus to ensure that HO-1 +/+ and HO-1 −/− mice received comparably severe glycerol-induced exposure to heme proteins, HO-1 −/− mice were matched in body weight to HO-1 +/+ mice, the mean age of the HO-1 −/− mice being 30 weeks whereas the mean age of the HO-1 +/+ mice was 20 weeks. Groups of HO-1 +/+ and HO-1 −/− mice comprised similar numbers of male and female mice. Such groups were used in the study of the relative effects of the glycerol model of heme protein toxicity and the relative effects of a specific heme protein, namely, hemoglobin. In additional studies of the nephrotoxic actions of hemoglobin in HO-1 +/+ and HO-1 −/− mice, we examined the effects of hemoglobin in HO-1 +/+ and HO-1 −/− mice that were much younger (age, 7 to 10 weeks. than those previously used (age, 20 to 30 weeks). In these additional studies using HO-1 +/+ and HO-1 −/− mice in the age range 7 to 10 weeks, mean body weights of these mice were not significantly different (21.5 ± 1.5 g versus 22.7 ± 1.7 g, respectively; P = ns); the toxicity of a specific heme protein was thus further studied in younger mice similar in age and body weight. HO-1 +/+ and HO-1 −/− mice, deprived of water overnight for 16 hours but allowed free access to rodent chow, were anesthetized with ether, and then injected with 50% glycerol in water, 7.5 ml/kg, half of the dose injected into each anterior thigh muscle.26Zager RA Burkhart KM Conrad DS Gmur DJ Iron, heme oxygenase, and glutathione: effects on myohemoglobinuric proximal tubular injury.Kidney Int. 1995; 48: 1624-1634Crossref PubMed Scopus (123) Google Scholar, 29Nath KA Balla G Vercellotti GM Balla J Jacob HS Levitt MD Rosenberg ME Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.J Clin Invest. 1992; 90: 267-270Crossref PubMed Scopus (592) Google Scholar In initial studies, the renal effects of intramuscular injections of 50% glycerol at two doses, 7.5 ml/kg and 10 ml/kg, were examined in C57BL/6 mice. HO-1 +/+ and HO-1 −/− mice were deprived of water for 16 hours but allowed free access to rodent chow. Mice were given two tail vein injections of mouse hemoglobin (Sigma Chemical Co., St. Louis, MO), each consisting of 90 mg/100 g mouse hemoglobin, and administered 1 hour apart. Renal function was assessed by plasma creatinine concentrations. the latter were determined on plasma derived from tail vein blood samples and using a Beckman Creatinine Analyzer II (Beckman Instruments, Inc., Fullerton, CA).29Nath KA Balla G Vercellotti GM Balla J Jacob HS Levitt MD Rosenberg ME Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.J Clin Invest. 1992; 90: 267-270Crossref PubMed Scopus (592) Google Scholar Plasma hemoglobin concentrations were determined by the method of Winterbourn.30Winterbourn CC Reactions of superoxide with hemoglobin.in: Greenwald RA Handbook of Methods for Oxygen Radical Research. CRC, Boca Raton1985: 137-141Google Scholar Heme content of whole kidney homogenates was determined by the pyridine hemochromogen method.31Paul KG Theorell H Akeson A The molar light absorption of pyridine ferroprotoporphyrin (pyridine haemochromogen).Acta Chem Scand. 1953; 7: 1284-1287Crossref Google Scholar, 32Vogt BA Shanley TP Croatt A Alam J Johnson KJ Nath KA Glomerular inflammation induces resistance to tubular injury in the rat: a novel form of acquired heme oxygenase-dependent resistance to renal injury.J Clin Invest. 1996; 98: 2139-2145Crossref PubMed Scopus (115) Google Scholar Plasma lactate dehydrogenase (LDH) activity was measured by determining the rate of formation of NADH.17Nath KA Grande JP Croatt AJ Likely S Hebbel RP Enright H Intracellular targets in heme protein-induced renal injury.Kidney Int. 1998; 53: 100-111Crossref PubMed Scopus (97) Google Scholar Plasma creatine kinase activity was measured by a colorimetric method using a Sigma Diagnostics Creatine Phosphokinase kit (Sigma Chemical Co.). RNA from kidneys was extracted using the Trizol method (Life Technologies, Inc., Gaithersburg, MD). Ten μg of total RNA from each sample were separated on an agarose gel and transferred to a nylon membrane. Membranes were hybridized overnight with a 32P-labeled mouse HO-1 cDNA probe. Autoradiograms were standardized, as previously described,33Agarwal A Balla J Alam J Croatt AJ Nath KA Induction of heme oxygenase in toxic renal injury: a protective role in cisplatin nephrotoxicity in the rat.Kidney Int. 1995; 48: 1298-1307Crossref PubMed Scopus (228) Google Scholar by factoring the optical density of the message for HO-1 with the optical density of the 18S rRNA, the latter obtained on a negative of the ethidium bromide-stained nylon membrane. Western analysis was performed using a polyclonal HO-1 antibody (SPA-895, Stressgen, Victoria, BC, Canada).33Agarwal A Balla J Alam J Croatt AJ Nath KA Induction of heme oxygenase in toxic renal injury: a protective role in cisplatin nephrotoxicity in the rat.Kidney Int. 1995; 48: 1298-1307Crossref PubMed Scopus (228) Google Scholar For immunohistochemical localization, tissue sections were fixed in 10. formalin and embedded in paraffin. Staining for HO-1 was performed using a polyclonal antibody (SPA-895, Stressgen) as the primary antibody, a horseradish peroxidase conjugated secondary antibody (R14745, Transduction Laboratories, Lexington, KY) and diaminobenzidine as substrate for localization. Results are expressed as means ± SEM, and are considered statistically significant for P < 0.05. For comparison between unpaired groups, the Student's t-test or the Mann-Whitney test was used as appropriate. Because the glycerol model in the mouse is not characterized as it is in the rat, we first examined the renal response to this insult in C57BL/6 mice. As in the rat, markers of renal injury (creatinine), muscle injury (creatine kinase), and red cell injury (hemoglobin), are all reversibly elevated in mice subjected to heme protein-induced renal injury (see below); and, as shown in Figure 1, the kidney mounts a robust response consisting of the induction of HO-1 mRNA. We proceeded to impose the glycerol model in HO-1 +/+ and HO-1 −/− mice, and to determine the severity of renal injury. Before this, we assessed the severity of myolysis and hemolysis by plasma creatine kinase and plasma hemoglobin, respectively. Injection of hypertonic glycerol elevated plasma creatine kinase concentrations in HO-1 +/+ and HO-1 −/− mice to values that were not significantly different at 6 hours (257 ± 119 versus 82 ± 26 Sigma Units, P = ns, n = 8 in each group) and 24 hours (8 ± 4 versus 20 ± 7 Sigma Units, P = ns, n = 6 and n = 5, respectively); no significant difference was observed in this index before the administration of glycerol (13 ± 7 versus 4 ± 2 Sigma Units, P = ns, n = 6 in each group) in HO-1 +/+ and HO-1 −/− mice. Plasma hemoglobin levels in HO-1 +/+ and HO-1 −/− mice at 6 hours (75 ± 15 versus 142 ± 39 μmol/l, P = ns, n = 8 in each group) and 24 hours (28 ± 5 versus 46 ± 17 μmol/L, P = ns, n = 6 and n = 5, respectively) were not significantly different; nor were there significant differences in this index before glycerol (17 ± 1 versus 25 ± 4 μmol/L, P = ns, n = 6 in each group). Thus, HO-1 +/+ and HO-1 −/− mice exhibit comparable degrees of muscle injury and hemolysis. After glycerol, however, alterations in renal function were fundamentally different in HO-1 +/+ and HO-1 −/− mice (Figure 2). Although plasma creatinines were identical before the administration of glycerol, the plasma creatinine was markedly increased in HO-1 −/− mice by days 2 and 3 after the administration of hypertonic glycerol, findings that demonstrate greater deterioration in renal function in HO-1 −/− mice (Figure 2). Worsened renal histological injury accompanied this greater impairment in renal function, as demonstrated by widespread necrosis of tubular epithelial cells and tubular cast formation in the kidney in HO-1 −/− mice on day 2 after glycerol (Figure 3, right panel); such histological features were uncommon in HO-1 +/+ mice in that only isolated tubules showed evidence of such tubular necrosis (Figure 3, left panel); no gross renal histological abnormalities were present in either group not subjected to glycerol (data not shown). In these mice in which renal histological studies were undertaken, worsened renal function was again observed in the HO-1 −/− mice as compared to HO-1 +/+ mice subjected to glycerol: serum creatinine measurements on day 2 after glycerol were 0.9 ± 0.3 versus 2.7 ± 0.1 mg/dl, P < 0.005, n = 6 in each group. Accompanying these differences in kidney function was biochemical evidence of marked cellular injury, as reflected by plasma levels of LDH (Figure 4a). LDH is a ubiquitous cytosolic enzyme, and its elevation in plasma indicates cellular injury. Plasma LDH profiles in HO-1 +/+ and HO-1 −/− mice were different: in HO-1 +/+ mice, plasma LDH levels acutely rose by 6 hours but normalized by 48 hours; in contrast, HO-1−/− mice demonstrate a progressive and sustained rise in LDH, and by 48 hours, these levels were increased 30-fold greater than levels in HO-1 +/+ mice, indicating massive tissue injury. Interestingly, HO-1−/− mice evinced minimally elevated LDH levels even in the basal state, perhaps reflecting the endogenous cytoprotective effects of HO-1 in unstressed mice. The marked cellular injury in HO-1 −/− mice, in conjunction with more severe renal failure, after exposure to heme proteins, increased mortality in the HO-1−/− mice; by 6 days, seven out of the eight animals in the HO-1 −/− group were dead, and by 15 days, all HO-1 −/− mice were dead; no deaths occurred in the HO-1 +/+ mice in response to the same dose of glycerol (Figure 4b). The induction of myolysis and hemolysis by hypertonic glycerol promptly and prominently induced HO-1 mRNA and protein in the kidney in HO-1 +/+ mice; no such induction occurred in the HO-1 −/− mice (Figure 5, a and b). The lack of induction of HO-1 in the HO-1 −/− mice enhanced the accumulation of heme which occurs in this model: the increment in kidney heme content in HO-1 −/− mice was increased some eightfold above that which occurred in HO-1 +/+ mice (Figure 5c). The site of induction of HO-1 protein in the kidney in HO-1 +/+ mice after the administration of glycerol localized, primarily, to the proximal tubule (Figure 6).Figure 6Immunoperoxidase staining for HO-1 in HO-1 +/+ mice, before and 6 hours after glycerol (7.5 ml/kg). In the basal state the kidneys of HO-1 +/+ mice do not express HO-1 (left panel). In response to glycerol prominent induction of HO-1 was observed in the proximal tubules of HO-1 +/+ mice (right panel). There was no evidence of HO-1 staining in HO-1 −/− mice either in the basal state or 6 hours after glycerol (data not shown). Original magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether similar sensitivities would occur in HO-1 −/− mice when heme proteins are directly administered, we developed a model of heme protein-induced toxicity in which mouse hemoglobin was administered intravenously. In HO-1 +/+ mice such administration of mouse hemoglobin was associated with no elevation in plasma creatinine and no mortality (Figure 7, a and b). In stark contrast, hemoglobin so administered to HO-1 −/− mice led to acute renal failure and 75% mortality (Figure 7, a and b). Thus, heme proteins per se are uniquely toxic in mice rendered deficient in HO-1. After we established the remarkable sensitivity of HO-1 −/− mice to specific heme proteins, we proceeded to examine this effect in mice that were younger (7 to 10 weeks) than those previously used (20 to 30 weeks). In this age range of 7 to 10 weeks, HO-1−/− mice exhibit body weights that are comparable to HO-1 +/+ mice, and do not display inflammatory and other histological changes that subsequently emerge in much older
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