Renal Hemodynamic, Inflammatory, and Apoptotic Responses to Lipopolysaccharide in HO-1−/− Mice
2007; Elsevier BV; Volume: 170; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.061093
ISSN1525-2191
AutoresMichał Tracz, Julio P. Juncos, Joseph P. Grande, Anthony J. Croatt, Allan W. Ackerman, Govindarajan Rajagopalan, Keith L. Knutson, Andrew D. Badley, Matthew D. Griffin, Jawed Alam, Karl A. Nath,
Tópico(s)Neonatal Health and Biochemistry
ResumoLipopolysaccharide (LPS) induces the stress-responsive gene heme oxygenase-1 (HO-1). The present study examined the significance of HO-1 in response to LPS. In HO-1−/− mice, as compared with HO-1+/+ mice, LPS provoked a greater reduction in glomerular filtration rate and renal blood flow, increased renal cytokine expression, and increased activation of nuclear factor (NF)-κB. Conversely, HO-1-overexpressing renal epithelial cells, exposed to LPS, exhibited a blunted activation of NF-κB and less phosphorylation of its inhibitor, IκB. In HO-1−/− mice, as compared with HO-1+/+ mice, LPS provoked markedly greater elevations in serum levels of Th1 cytokines, Th2 cytokines, chemokines, and cytokines that stimulate bone marrow progenitors. The liver, a major source of serum cytokines, showed an increased activation of NF-κB in LPS-treated HO-1−/− mice. In addition, LPS provoked widespread apoptosis of immune cells in the spleen and thymus in HO-1−/− mice but not in HO-1+/+ mice. We conclude that HO-1 deficiency exhibits a heightened and dysregulated inflammatory response to LPS accompanied by greater impairment in renal hemodynamic response and widespread apoptosis of immune cells. Because polymorphisms in the HO-1 gene with diminished HO activity predispose to human disease, we speculate that our findings may be relevant to the clinical outcome in patients with sepsis syndromes. Lipopolysaccharide (LPS) induces the stress-responsive gene heme oxygenase-1 (HO-1). The present study examined the significance of HO-1 in response to LPS. In HO-1−/− mice, as compared with HO-1+/+ mice, LPS provoked a greater reduction in glomerular filtration rate and renal blood flow, increased renal cytokine expression, and increased activation of nuclear factor (NF)-κB. Conversely, HO-1-overexpressing renal epithelial cells, exposed to LPS, exhibited a blunted activation of NF-κB and less phosphorylation of its inhibitor, IκB. In HO-1−/− mice, as compared with HO-1+/+ mice, LPS provoked markedly greater elevations in serum levels of Th1 cytokines, Th2 cytokines, chemokines, and cytokines that stimulate bone marrow progenitors. The liver, a major source of serum cytokines, showed an increased activation of NF-κB in LPS-treated HO-1−/− mice. In addition, LPS provoked widespread apoptosis of immune cells in the spleen and thymus in HO-1−/− mice but not in HO-1+/+ mice. We conclude that HO-1 deficiency exhibits a heightened and dysregulated inflammatory response to LPS accompanied by greater impairment in renal hemodynamic response and widespread apoptosis of immune cells. Because polymorphisms in the HO-1 gene with diminished HO activity predispose to human disease, we speculate that our findings may be relevant to the clinical outcome in patients with sepsis syndromes. Rising in incidence, sepsis and its syndromes afflict 750,000 patients each year, ∼30% of whom die from complications of these syndromes.1Angus DC Linde-Zwirble WT Lidicker J Clermont G Carcillo J Pinsky MR Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care.Crit Care Med. 2001; 29: 1303-1310Crossref PubMed Scopus (6677) Google Scholar, 2Martin GS Mannino DM Eaton S Moss M The epidemiology of sepsis in the United States from 1979 through 2000.N Engl J Med. 2003; 348: 1546-1554Crossref PubMed Scopus (4832) Google Scholar Such mortality reflects the failure of specific organ systems (such as acute renal failure) or the cumulative adverse effects of the multiple organ dysfunction syndrome. Sepsis syndromes include not only conditions in which there is documented infection, as occurs in sepsis, but also those mimetic conditions without demonstrable infection, and which are differentiated by the nature of the ongoing inflammatory response. 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One such countervailing system is heme oxygenase (HO). HO is the rate-limiting enzyme in the degradation of heme, converting heme to biliverdin and concomitantly enabling the release of iron and the generation of carbon monoxide.15Abraham N Drummond G Lutton J Kappas A The biological significance and physiological role of heme oxygenase.Cell Physiol Biochem. 1996; 6: 129-168Crossref Scopus (232) Google Scholar, 16Agarwal A Nick HS Renal response to tissue injury: lessons from heme oxygenase-1 gene ablation and expression.J Am Soc Nephrol. 2000; 11: 965-973Crossref PubMed Google Scholar, 17Dong Z Lavrovsky Y Venkatachalam MA Roy AK Heme oxygenase-1 in tissue pathology: the Yin and Yang.Am J Pathol. 2000; 156: 1485-1488Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 18Kanwar YS Heme oxygenase-1 in renal injury: conclusions of studies in humans and animal models.Kidney Int. 2001; 59: 378-379Crossref PubMed Scopus (28) Google Scholar, 19Sikorski EM Hock T Hill-Kapturczak N Agarwal A The story so far: molecular regulation of the heme oxygenase-1 gene in renal injury.Am J Physiol. 2004; 286: F425-F441Google Scholar, 20Abraham NG Kappas A Heme oxygenase and the cardiovascular-renal system.Free Radic Biol Med. 2005; 39: 1-25Crossref PubMed Scopus (322) Google Scholar, 21Kanwar YS A dynamic interplay between monocyte chemoattractant protein-1 and heme oxygenase-1: implications in renal injury.Kidney Int. 2005; 68: 896-897Crossref PubMed Scopus (8) Google Scholar, 22Nath KA Heme oxygenase-1: a provenance for cytoprotective pathways in the kidney and other tissues.Kidney Int. 2006; 70: 432-443Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar HO exists in two isoforms, HO-1 being the inducible isoform elicited by a wide array of stimuli including ischemia, hypoxia, oxidant stress, LPS, cytokines, and irradiation; HO-2 represents the constitutive isoform. In several of these conditions, induction of HO-1 confers protective effects by virtue of its vasorelaxant, anti-inflammatory, and anti-apoptotic actions.15Abraham N Drummond G Lutton J Kappas A The biological significance and physiological role of heme oxygenase.Cell Physiol Biochem. 1996; 6: 129-168Crossref Scopus (232) Google Scholar, 16Agarwal A Nick HS Renal response to tissue injury: lessons from heme oxygenase-1 gene ablation and expression.J Am Soc Nephrol. 2000; 11: 965-973Crossref PubMed Google Scholar, 17Dong Z Lavrovsky Y Venkatachalam MA Roy AK Heme oxygenase-1 in tissue pathology: the Yin and Yang.Am J Pathol. 2000; 156: 1485-1488Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 18Kanwar YS Heme oxygenase-1 in renal injury: conclusions of studies in humans and animal models.Kidney Int. 2001; 59: 378-379Crossref PubMed Scopus (28) Google Scholar, 19Sikorski EM Hock T Hill-Kapturczak N Agarwal A The story so far: molecular regulation of the heme oxygenase-1 gene in renal injury.Am J Physiol. 2004; 286: F425-F441Google Scholar, 20Abraham NG Kappas A Heme oxygenase and the cardiovascular-renal system.Free Radic Biol Med. 2005; 39: 1-25Crossref PubMed Scopus (322) Google Scholar, 21Kanwar YS A dynamic interplay between monocyte chemoattractant protein-1 and heme oxygenase-1: implications in renal injury.Kidney Int. 2005; 68: 896-897Crossref PubMed Scopus (8) Google Scholar, 22Nath KA Heme oxygenase-1: a provenance for cytoprotective pathways in the kidney and other tissues.Kidney Int. 2006; 70: 432-443Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar The present study evaluated the functional significance of HO-1 in determining the response to LPS in vivo by adopting an approach based on HO-1−/− mice. In this study, attention was focused on the kidney for the following reasons: this organ is commonly impaired in sepsis, exhibiting a well-defined alteration in its hemodynamic profile7Schor N Acute renal failure and the sepsis syndrome.Kidney Int. 2002; 61: 764-776Crossref PubMed Scopus (62) Google Scholar; the occurrence of acute renal failure in patients with sepsis substantially increases mortality23Chertow GM Soroko SH Paganini EP Cho KC Himmelfarb J Ikizler TA Mehta RL Mortality after acute renal failure: models for prognostic stratification and risk adjustment.Kidney Int. 2006; 70: 1120-1126Crossref PubMed Scopus (236) Google Scholar; even relatively mild renal insufficiency can adversely affect mortality in critically ill patients24Chertow GM Burdick E Honour M Bonventre JV Bates DW Acute kidney injury, mortality, length of stay, and costs in hospitalized patients.J Am Soc Nephrol. 2005; 16: 3365-3370Crossref PubMed Scopus (2526) Google Scholar; and, as recently demonstrated in relevant disease models, renal insults of diverse origin, even when relatively mild, can markedly exaggerate the systemic inflammatory response elicited by LPS.10Zager RA Johnson AC Hanson SY Lund S Ischemic proximal tubular injury primes mice to endotoxin-induced TNF-alpha generation and systemic release.Am J Physiol. 2005; 289: F289-F297Crossref PubMed Scopus (69) Google Scholar, 11Zager RA Johnson AC Hanson SY Lund S Acute nephrotoxic and obstructive injury primes the kidney to endotoxin-driven cytokine/chemokine production.Kidney Int. 2006; 69: 1181-1188Crossref PubMed Scopus (51) Google Scholar, 12Zager RA Johnson AC Lund S Hanson S Acute renal failure: determinants and characteristics of the injury-induced hyperinflammatory response.Am J Physiol. 2006; 291: F546-F556Crossref PubMed Scopus (8) Google Scholar In addition to examining the renal and systemic responses to LPS in HO-1−/− mice, this study analyzed organs enriched in immune cells, in view of the increasing recognition that critical participants in the sepsis syndrome include altered immunity, in general, and apoptosis of lymphocytes, in particular.25Weaver JG Rouse MS Steckelberg JM Badley AD Improved survival in experimental sepsis with an orally administered inhibitor of apoptosis.FASEB J. 2004; 18: 1185-1191Crossref PubMed Scopus (69) Google Scholar, 26Hotchkiss RS Schmieg Jr, RE Swanson PE Freeman BD Tinsley KW Cobb JP Karl IE Buchman TG Rapid onset of intestinal epithelial and lymphocyte apoptotic cell death in patients with trauma and shock.Crit Care Med. 2000; 28: 3207-3217Crossref PubMed Scopus (155) Google Scholar, 27Hotchkiss RS Osmon SB Chang KC Wagner TH Coopersmith CM Karl IE Accelerated lymphocyte death in sepsis occurs by both the death receptor and mitochondrial pathways.J Immunol. 2005; 174: 5110-5118PubMed Google Scholar, 28Hotchkiss RS Coopersmith CM Karl IE Prevention of lymphocyte apoptosis—a potential treatment of sepsis?.Clin Infect Dis. 2005; 41: S465-S469Crossref PubMed Scopus (122) Google Scholar Homozygous HO-1-null mutant mice, used in this and previous studies by our laboratory,29Nath KA Haggard JJ Croatt AJ Grande JP Poss KD Alam J The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo.Am J Pathol. 2000; 156: 1527-1535Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 30Nath KA Vercellotti GM Grande JP Miyoshi H Paya CV Manivel JC Haggard JJ Croatt AJ Payne WD Alam J Heme protein-induced chronic renal inflammation: suppressive effect of induced heme oxygenase-1.Kidney Int. 2001; 59: 106-117Crossref PubMed Scopus (193) Google Scholar, 31Pittock ST Norby SM Grande JP Croatt AJ Bren GD Badley AD Caplice NM Griffin MD Nath KA MCP-1 is up-regulated in unstressed and stressed HO-1 knockout mice: pathophysiologic correlates.Kidney Int. 2005; 68: 611-622Crossref PubMed Scopus (89) Google Scholar were generated by targeted disruption of the HO-1 gene as described by Poss and Tonegawa.32Poss KD Tonegawa S Heme oxygenase 1 is required for mammalian iron reutilization.Proc Natl Acad Sci USA. 1997; 94: 10919-10924Crossref PubMed Scopus (866) 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 from tail samples. HO-1+/+ mice were used as controls. For all experiments, HO-1+/+ and HO-1−/− mice were age-matched, and mice from 10 to 28 weeks were used. For studies of hemodynamic and other responses to LPS, LPS (Escherichia coli, serotype 0127:B8, 1 mg/kg, catalog no. L3129, lot 123K4143; Sigma/Aldrich, St. Louis, MO) was administered intravenously to HO-1+/+ and HO-1−/− mice. At 24 hours after administration of LPS, mice were sacrificed for the harvest of tissues and plasma or subjected to renal hemodynamic measurements as described below. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of the Mayo Clinic. These studies were performed as described in detail in our previous investigations.33Juncos JP Grande JP Murali N Croatt AJ Juncos LA Hebbel RP Katusic ZS Nath KA Anomalous renal effects of tin protoporphyrin in a murine model of sickle cell disease.Am J Pathol. 2006; 169: 21-31Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Mice were anesthetized with sodium pentobarbital (60 mg/kg body weight, i.p.) and placed on a temperature-regulated table to maintain body temperature at 37°C. During these studies, a solution of 0.9% saline containing 2.25% bovine serum albumin and 0.75% fluorescein isothiocyanate-inulin (FITC-I; Sigma-Aldrich) was infused, and glomerular filtration rate (GFR) was determined by the clearance of inulin.34Lorenz JN Gruenstein E A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin.Am J Physiol. 1999; 276: F172-F177PubMed Google Scholar Mean arterial pressure was continuously monitored throughout the experiment. Renal blood flow (RBF) was determined by an electromagnetic flow probe and a small animal blood flow meter (T206; Transonic Systems Inc., Ithaca, NY).35Traynor TR Schnermann J Renin-angiotensin system dependence of renal hemodynamics in mice.J Am Soc Nephrol. 1999; 10: S184-S188PubMed Google Scholar Renal vascular resistance (RVR) was determined as the ratio of mean arterial pressure:RBF. Renal plasma flow (RPF) was calculated by RBF × (100 − hematocrit)/100. Filtration fraction was calculated as GFR/RPF. For analysis of gene expression, total RNA was extracted from mouse tissues using the Trizol method (Invitrogen, Carlsbad, CA), further purified with the RNeasy mini kit (Qiagen, Valencia, CA) and subsequently used in reverse transcription reactions (Transcriptor First Strand cDNA synthesis kit; Roche Applied Science, Indianapolis, IN) according to each manufacturer's protocol. The resulting cDNA was used in quantitative real-time PCR reactions performed as described in detail by our previous publications and analyzed on an ABI Prism 7900HT (Applied Biosystems, Foster City, CA).31Pittock ST Norby SM Grande JP Croatt AJ Bren GD Badley AD Caplice NM Griffin MD Nath KA MCP-1 is up-regulated in unstressed and stressed HO-1 knockout mice: pathophysiologic correlates.Kidney Int. 2005; 68: 611-622Crossref PubMed Scopus (89) Google Scholar, 36Pedraza-Chaverri J Murali NS Croatt AJ Alam J Grande JP Nath KA Proteinuria as a determinant of renal expression of heme oxygenase-1: studies in models of glomerular and tubular proteinuria in the rat.Am J Physiol. 2006; 290: F196-F204Google Scholar Probe and primer sets used for quantification were designed with Primer Express software (Applied Biosystems) and are detailed in Table 1. In addition, a probe and primer set for the analysis of endothelin-1 (ET-1) gene expression was purchased from Applied Biosystems (TaqMan gene expression assay, stock no. Mm00438656) and was used in our standard procedure.Table 1Primers and Probes Used for Quantitative Real-Time RT-PCRGenePrimer/probeSequenceIL-6Forward5′-CCAGAAACCGCTATGAAGTTCCT-3′Reverse5′-CACCAGCATCAGTCCCAAGA-3′TaqMan5′-(FAM)TCTGCAAGAGACTTCCATCCAGTTGCC(TAMRA)-3′IL-10Forward5′-AGCAGCCTTGCAGAAAAGAGA-3′Reverse5′-AGTAAGAGCAGGCAGCATAGCA-3′TaqMan5′-(FAM)-CCATCATGCCTGGCTCAGCA(TAMRA)-3′MCP-1Forward5′-GGCTCAGCCAGATGCAGTTAA-3′Reverse5′-CCTACTCATTGGGATCATCTTGCT-3′TaqMan5′-(FAM)-CCCCACTCACCTGCTGCTACTCATTCAC(TAMRA)-3′RANTESForward5′-GCAAGTGCTCCAATCTTGCA-3′Reverse5′-CTTCTCTGGGTTGGCACACA-3′TaqMan5′-(FAM)-CGTGTTTGTCACTCGAAGGAACCGC(TAMRA)-3′TNFForward5′-TCTCTTCAAGGGACAAGGCTG-3′Reverse5′-ATAGCAAATCGGCTGACGGT-3′TaqMan5′-(FAM)-CCCGACTACGTGCTCCTCACCCA(TAMRA)-3′18SForward5′-TGTCTCAAAGATTAAGCCATGCAT-3′Reverse5′-AACCATAACTGATTTAATGAGCCATTC-3′TaqMan5′-(FAM) TACGCACGGCCGGTACAGTGAAACT(TAMRA)-3′ Open table in a new tab Nuclear extracts were prepared from cells in culture and mouse tissues, and EMSA was performed for the assessment of activation of NF-κB as described in our previous studies.30Nath KA Vercellotti GM Grande JP Miyoshi H Paya CV Manivel JC Haggard JJ Croatt AJ Payne WD Alam J Heme protein-induced chronic renal inflammation: suppressive effect of induced heme oxygenase-1.Kidney Int. 2001; 59: 106-117Crossref PubMed Scopus (193) Google Scholar, 31Pittock ST Norby SM Grande JP Croatt AJ Bren GD Badley AD Caplice NM Griffin MD Nath KA MCP-1 is up-regulated in unstressed and stressed HO-1 knockout mice: pathophysiologic correlates.Kidney Int. 2005; 68: 611-622Crossref PubMed Scopus (89) Google Scholar, 37Murali NS Ackerman AW Croatt AJ Cheng J Grande JP Sutor SL Bram RJ Bren GD Badley AD Alam J Nath KA Renal upregulation of HO-1 reduces albumin-driven MCP-1 production: implications for chronic kidney disease.Am J Physiol. 2007; 292: F837-F844Google Scholar In brief, 5.0 μg of each extract were added to a binding reaction containing the following components: 20 mmol/L HEPES, pH 8.0, 100 mmol/L NaCl, 1.0 mmol/L MgCl2, 0.05 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol, 3% glycerol, 0.2 μg/μl poly(dI+dC), and 50,000 cpm of [32P]γATP-labeled NF-κB probe. A consensus oligonucleotide containing DNA binding sites for NF-κB transcription factors (catalog no. E3291; Promega, Madison, WI) was used. Bound and free probe were separated on a 5% TBE nondenaturing polyacrylamide gel and visualized by autoradiography. A concentrated anti-p65 antibody (catalog no. sc-109; Santa Cruz Biotechnology, Santa Cruz, CA) was used for supershift analysis of selected extracts. Serum samples were collected using BD Microtainer serum separators (BD Biosciences, Franklin Lakes, NJ) and analyzed for cytokine levels using a multiplex bead suspension array system (Bio-Plex Mouse Cytokine 23-Plex Panel, catalog no. 171-F11241; Bio-Rad Laboratories, Hercules, CA), according to the manufacturer's protocol. Western analysis was performed on proteins extracted from mouse tissues as described previously.31Pittock ST Norby SM Grande JP Croatt AJ Bren GD Badley AD Caplice NM Griffin MD Nath KA MCP-1 is up-regulated in unstressed and stressed HO-1 knockout mice: pathophysiologic correlates.Kidney Int. 2005; 68: 611-622Crossref PubMed Scopus (89) Google Scholar In brief, lysate proteins (20 to 100 μg) were separated on 10% Tris-HCl gels and transferred to polyvinylidene difluoride membranes. Blots were incubated with primary antibodies to caspase-3, pIκBα, or IκBα (catalog nos. 9665, 9246, and 9242, respectively; Cell Signaling Technology, Danvers, MA) overnight at 4°C. This was followed by incubation with goat anti-mouse or anti-rabbit IgG secondary antibodies and visualization using a chemiluminescence method (Amersham Pharmacia, Piscataway, NJ). Equal protein loading was confirmed by immunoblotting for β-actin (catalog no. 612657; BD Transduction Laboratories, San Diego, CA). Rat proximal tubular epithelial cells, engineered to overexpress HO-1 by stable transfection of NRK-52E cells (American Type Culture Collection, Manassas, VA) as described in detail in our previous study,37Murali NS Ackerman AW Croatt AJ Cheng J Grande JP Sutor SL Bram RJ Bren GD Badley AD Alam J Nath KA Renal upregulation of HO-1 reduces albumin-driven MCP-1 production: implications for chronic kidney disease.Am J Physiol. 2007; 292: F837-F844Google Scholar were used in the present study to test the effect of HO-1 overexpression on responses to LPS treatment. Control cells (containing an empty cassette) and HO-1-overexpressing cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum, 20 mmol/L HEPES, 1 mmol/L sodium pyruvate, 100 μg/ml G418 sulfate, and 100 U/ml penicillin/100 μg/ml streptomycin.37Murali NS Ackerman AW Croatt AJ Cheng J Grande JP Sutor SL Bram RJ Bren GD Badley AD Alam J Nath KA Renal upregulation of HO-1 reduces albumin-driven MCP-1 production: implications for chronic kidney disease.Am J Physiol. 2007; 292: F837-F844Google Scholar For NF-κB EMSA studies, cells were washed twice with phosphate-buffered saline (PBS) and incubated for 2 hours with serum-free DMEM or serum-free DMEM containing various concentrations of LPS (1.0 to 0.01 μg/ml). EMSA was performed as described above. For the pIκBα/IκBα studies, cells were incubated in serum-free DMEM or serum-free DMEM containing 1.0 μg/ml LPS for 5 to 30 minutes. Cells were then washed once with cold PBS and lysed in complete, prechilled RIPA buffer for Western analysis. As previously used by our laboratory, an Apoptag Plus peroxidase in situ apoptosis kit (Chemicon, Temecula, CA) was used to assess apoptosis in mouse tissues.36Pedraza-Chaverri J Murali NS Croatt AJ Alam J Grande JP Nath KA Proteinuria as a determinant of renal expression of heme oxygenase-1: studies in models of glomerular and tubular proteinuria in the rat.Am J Physiol. 2006; 290: F196-F204Google Scholar This method detects apoptosis-associated DNA fragmentation by labeling of 3′-OH termini with digoxigenin nucleotides using terminal deoxynucleotidyl transferase. Data are expressed as mean ± SEM. Data for HO-1−/− and HO-1+/+ mice for a given condition were compared using Student's t-test for parametric data and the Mann-Whitney test for nonparametric data. Results were considered significant for P < 0.05. Renal hemodynamics in response to LPS and vehicle were assessed in HO-1+/+ and HO-1−/− mice and are shown in Figure 1. HO-1−/− mice, as compared with HO-1+/+ mice, subjected to LPS, demonstrated a significantly lower GFR and RBF, the latter attributable to increased RVR, and a tendency toward a lower filtration fraction (Figure 1). Mean arterial pressure was not significantly different between the HO-1+/+ and HO-1−/− mice treated with vehicle (100 ± 1 versus 100 ± 2 mm Hg, P = NS) or LPS (98 ± 1 versus 105 ± 6 mm Hg, P = NS). LPS induced minor histological changes in the kidney in both groups, with vascular congestion appearing somewhat more prominent in LPS-treated HO-1−/− mice (data not shown). Despite these minor differences in histological appearance, the kidneys in HO-1−/− mice exhibited a markedly exaggerated cytokine response to LPS. As shown in Figure 2, mRNA expression for interleukin (IL)-6, tumor necrosis factor (TNF), RANTES, IL-10, MCP-1, and ET-1 was markedly increased in HO-1−/− mice as compared with HO-1+/+ mice after the administration of LPS. To determine the basis for such proinflammatory changes, studies were undertaken in the kidney to assess activation of the proinflammatory transcription factor NF-κB. NF-κB is involved in the expression of several of the cytokines exuberantly manifested in the kidney of LPS-treated HO-1−/− mice (eg, IL-6, TNF, RANTES, MCP-1). As shown by EMSA (Figure 3), activation of NF-κB in HO-1−/− mice still persisted 24 hours after the administration of LPS, whereas there was little evidence of such activation in HO-1+/+ mice after LPS. To determine a possible mechanism whereby activation of NF-κB can be influenced by HO-1, we used a complementary approach in which HO-1-overexpressing cells were exposed to LPS. As shown in Figure 4A, HO-1-overexpressing cells, as compared with control cells, exhibited a blunted activation of NF-κB in response to LPS. Because activation of NF-κB occurs after its inhibitory protein, IκB, undergoes phosphorylation and degradation, the expression of pIκB was evaluated by Western analysis. HO-1-overexpressing cells in response to LPS exhibit less expression of pIκB (Figure 4B), thereby providing a possible mechanism whereby HO-1 can suppress activation of NF-κB; namely, inhibition of phosphorylation of IκB. To evaluate the systemic inflammatory response to LPS, serum concentrations of a panel of cytokines were determined, and these data are summarized in Figure 5, Figure 6, Figure 7, 8, and 9. The concentrations of these serum cytokines were not significantly different in vehicle-treated HO-1+/+ and HO-1−/− mice. However, in response to LPS, HO-1−/− mice as compared with HO-1+/+ mice exhibited significantly greater amounts of Th1 cytokines (IL-1, IL-2, IL-6, IL-12, TNF, interferon-γ; Figure 5, Figure 6), chemokines (MCP-1, KC, RANTES, MIP-1β; Figure 7), Th2 cytokines and chemokines (IL-4, IL-5, IL-9, IL-10, eotaxin; Figure 6, Figure 8), and cytokine stimulators of bone marrow progenitor cells (IL-3 and GM-CSF; Figure 9).Figure 6Serum Th1 cytokine levels (IL-2, IL-6, and IL-12 p40), and Th2 cytokine level (IL-10) in HO-1+/+ and HO-1−/− mice treated with veh
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