Oxidative Stress and Induction of Heme Oxygenase-1 in the Kidney in Sickle Cell Disease
2001; Elsevier BV; Volume: 158; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64037-0
ISSN1525-2191
AutoresKarl A. Nath, Joseph P. Grande, Jill J. Haggard, Anthony J. Croatt, Zvonimir S. Katušić, Anna Solovey, Robert P. Hebbel,
Tópico(s)Neonatal Health and Biochemistry
ResumoChronic nephropathy is a recognized complication of sickle cell disease. Using a transgenic sickle mouse, we examined whether oxidative stress occurs in the sickle kidney, the origins and functional significance of such oxidant stress, and the expression of the oxidant-inducible, potentially protective gene, heme oxygenase-1 (HO-1); we also examined the expression of HO-1 in the kidney and in circulating endothelial cells in sickle patients. We demonstrate that this transgenic sickle mouse exhibits renal enlargement, medullary congestion, and a reduced plasma creatinine concentration. Oxidative stress is present in the kidney as indicated by increased amounts of lipid peroxidation; heme content is markedly increased in the kidney. Exacerbation of oxidative stress by inhibiting glutathione synthesis with buthionine-sulfoximine dramatically increased red blood cell sickling in the sickle kidney: in buthionine-sulfoximine-treated sickle mice, red blood cell sickling extended from the medulla into the cortical capillaries and glomeruli. HO activity is increased in the sickle mouse kidney, and is due to induction of HO-1. In the human sickle kidney, HO-1 is induced in renal tubules, interstitial cells, and in the vasculature. Expression of HO-1 is increased in circulating endothelial cells in patients with sickle cell disease. These results provide the novel demonstration that oxidative stress occurs in the sickle kidney, and that acute exacerbation of oxidative stress in the sickle mouse precipitates acute vaso-occlusive disease. Additionally, the oxidant-inducible, heme-degrading enzyme, HO-1, is induced regionally in the murine and human sickle kidney, and systemically, in circulating endothelial cells in sickle patients. Chronic nephropathy is a recognized complication of sickle cell disease. Using a transgenic sickle mouse, we examined whether oxidative stress occurs in the sickle kidney, the origins and functional significance of such oxidant stress, and the expression of the oxidant-inducible, potentially protective gene, heme oxygenase-1 (HO-1); we also examined the expression of HO-1 in the kidney and in circulating endothelial cells in sickle patients. We demonstrate that this transgenic sickle mouse exhibits renal enlargement, medullary congestion, and a reduced plasma creatinine concentration. Oxidative stress is present in the kidney as indicated by increased amounts of lipid peroxidation; heme content is markedly increased in the kidney. Exacerbation of oxidative stress by inhibiting glutathione synthesis with buthionine-sulfoximine dramatically increased red blood cell sickling in the sickle kidney: in buthionine-sulfoximine-treated sickle mice, red blood cell sickling extended from the medulla into the cortical capillaries and glomeruli. HO activity is increased in the sickle mouse kidney, and is due to induction of HO-1. In the human sickle kidney, HO-1 is induced in renal tubules, interstitial cells, and in the vasculature. Expression of HO-1 is increased in circulating endothelial cells in patients with sickle cell disease. These results provide the novel demonstration that oxidative stress occurs in the sickle kidney, and that acute exacerbation of oxidative stress in the sickle mouse precipitates acute vaso-occlusive disease. Additionally, the oxidant-inducible, heme-degrading enzyme, HO-1, is induced regionally in the murine and human sickle kidney, and systemically, in circulating endothelial cells in sickle patients. Sickle cell nephropathy is a cardinal complication of sickle cell disease, and up to 18% of patients so afflicted develop end-stage renal disease.1Falk RJ Jennette JC Sickle cell nephropathy.Adv Nephrol. 1994; 23: 133-147PubMed Google Scholar, 2Saborio P Scheinman JI Sickle cell nephropathy.J Am Soc Nephrol. 1999; 10: 187-192PubMed Google Scholar, 3Pham T-TT Pham P-CT Wilinson AH Lew SQ Renal abnormalities in sickle cell disease.Kidney Int. 2000; 57: 1-8Crossref PubMed Scopus (170) Google Scholar, 4Powars DR Elliot-Mills DD Chan L Niland J Hiti AL Opas LM Johnson C Chronic renal failure in sickle cell disease: risk factors, clinical course, and mortality.Ann Intern Med. 1991; 115: 614-620Crossref PubMed Scopus (262) Google Scholar This latter outcome increases mortality by ∼50%, reduces patient survival to 4 years after its onset, and is associated with a median patient age of 27 years at the time of death.4Powars DR Elliot-Mills DD Chan L Niland J Hiti AL Opas LM Johnson C Chronic renal failure in sickle cell disease: risk factors, clinical course, and mortality.Ann Intern Med. 1991; 115: 614-620Crossref PubMed Scopus (262) Google Scholar Chronic renal insufficiency and end-stage renal failure are thus critical contributors to morbidity and mortality associated with sickle cell disease.1Falk RJ Jennette JC Sickle cell nephropathy.Adv Nephrol. 1994; 23: 133-147PubMed Google Scholar, 2Saborio P Scheinman JI Sickle cell nephropathy.J Am Soc Nephrol. 1999; 10: 187-192PubMed Google Scholar, 3Pham T-TT Pham P-CT Wilinson AH Lew SQ Renal abnormalities in sickle cell disease.Kidney Int. 2000; 57: 1-8Crossref PubMed Scopus (170) Google Scholar, 4Powars DR Elliot-Mills DD Chan L Niland J Hiti AL Opas LM Johnson C Chronic renal failure in sickle cell disease: risk factors, clinical course, and mortality.Ann Intern Med. 1991; 115: 614-620Crossref PubMed Scopus (262) Google Scholar Renal involvement in sickle cell disease characteristically includes renal hypertrophy in the setting of prominent hemodynamic and vascular alterations.1Falk RJ Jennette JC Sickle cell nephropathy.Adv Nephrol. 1994; 23: 133-147PubMed Google Scholar, 2Saborio P Scheinman JI Sickle cell nephropathy.J Am Soc Nephrol. 1999; 10: 187-192PubMed Google Scholar, 3Pham T-TT Pham P-CT Wilinson AH Lew SQ Renal abnormalities in sickle cell disease.Kidney Int. 2000; 57: 1-8Crossref PubMed Scopus (170) Google Scholar Such hemodynamic and vascular processes impose a spectrum of effects on renal perfusion at the whole-organ and regional levels within the kidney: at one end of this spectrum, and in the cortical circulation, renal plasma flow rates and glomerular filtration rates (GFRs) are increased, whereas at the other, perfusion of the medulla is diminished because of vaso-occlusive disease. Such alterations are incriminated in the pathogenesis of specific phenotypic features of sickle cell nephropathy: increased glomerular perfusion and filtration impose hemodynamically-mediated damage to the glomerular compartment, whereas medullary ischemia impairs concentrating ability and other aspects of tubular function, induces histological tubulointerstitial disease, and may provoke frank papillary infarction.1Falk RJ Jennette JC Sickle cell nephropathy.Adv Nephrol. 1994; 23: 133-147PubMed Google Scholar, 2Saborio P Scheinman JI Sickle cell nephropathy.J Am Soc Nephrol. 1999; 10: 187-192PubMed Google Scholar, 3Pham T-TT Pham P-CT Wilinson AH Lew SQ Renal abnormalities in sickle cell disease.Kidney Int. 2000; 57: 1-8Crossref PubMed Scopus (170) Google Scholar Such vascular alterations—hyperemia in the case of the cortical circulation and ischemia in the case of the medullary circulation—may set in train inflammatory and fibrogenic processes that lead to progressive loss of renal function. Additionally, the kidney in sickle patients may be prone to intrinsic glomerulopathic processes manifested as focal segmental glomerulosclerosis and membranoproliferative glomerulonephritis.1Falk RJ Jennette JC Sickle cell nephropathy.Adv Nephrol. 1994; 23: 133-147PubMed Google Scholar, 2Saborio P Scheinman JI Sickle cell nephropathy.J Am Soc Nephrol. 1999; 10: 187-192PubMed Google Scholar, 3Pham T-TT Pham P-CT Wilinson AH Lew SQ Renal abnormalities in sickle cell disease.Kidney Int. 2000; 57: 1-8Crossref PubMed Scopus (170) Google Scholar Exploration of the mechanisms by which renal and other complications of sickle cell disease arise has been greatly facilitated by the recent availability of transgenic mice expressing sickle hemoglobin.5Fabry ME Nagel RL Pachnis A Suzuka SM Costantini F High expression of human β S- and α-globins in transgenic mice. I. Hemoglobin composition and hematological consequences.Proc Natl Acad Sci USA. 1992; 89: 12150-12154Crossref PubMed Scopus (96) Google Scholar, 6Fabry ME Costantini FD Pachnis A Suzuka SM Bank N Aynedjian HS Factor S Nagel RL High expression of human βs and α-genes in transgenic mice. II. Red cell abnormalities, organ damage, and the effect of hypoxia.Proc Natl Acad Sci USA. 1992; 89: 12155-12159Crossref PubMed Scopus (85) Google Scholar, 7Nagel RL A knockout of a transgenic mouse: animal models of sickle cell anemia.N Engl J Med. 1998; 339: 194-195Crossref PubMed Scopus (23) Google Scholar The use of these models has provided insights into mechanisms underlying sickling in vivo and the mechanisms contributing to end-organ damage.5Fabry ME Nagel RL Pachnis A Suzuka SM Costantini F High expression of human β S- and α-globins in transgenic mice. I. Hemoglobin composition and hematological consequences.Proc Natl Acad Sci USA. 1992; 89: 12150-12154Crossref PubMed Scopus (96) Google Scholar, 6Fabry ME Costantini FD Pachnis A Suzuka SM Bank N Aynedjian HS Factor S Nagel RL High expression of human βs and α-genes in transgenic mice. II. Red cell abnormalities, organ damage, and the effect of hypoxia.Proc Natl Acad Sci USA. 1992; 89: 12155-12159Crossref PubMed Scopus (85) Google Scholar, 7Nagel RL A knockout of a transgenic mouse: animal models of sickle cell anemia.N Engl J Med. 1998; 339: 194-195Crossref PubMed Scopus (23) Google Scholar These latter studies, for example, have revealed up-regulation of eNOS and iNOS in the kidneys of such mice,8Bank N Aynedjian HS Qiu J-H Osei SY Ahima RS Fabry ME Nagel RL Renal nitric oxide synthases in transgenic sickle cell mice.Kidney Int. 1996; 50: 184-189Crossref PubMed Scopus (77) Google Scholar, 9Bank N Kiroycheva M Ahmed F Anthony GM Fabry ME Nagel RL Singhal PC Peroxynitrite formation and apoptosis in transgenic sickle cell mouse kidneys.Kidney Int. 1998; 54: 1520-1528Crossref PubMed Scopus (46) Google Scholar and have raised the possibility that such alterations may contribute to the development of renal complications of sickle cell disease. The present studies represent characterization of changes in the kidney in a more recently described transgenic sickle mouse.10Fabry ME Sengupta A Suzuka SM Costantini F Rubin EM Hofrichter J Christoph G Manci E Culberson D Factor SM Nagel RL A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity.Blood. 1995; 86: 2419-2428Crossref PubMed Google Scholar This characterization includes, and focuses on, alterations in renal redox in this model. The rationale for examining renal redox resides in the recognition that the kidney in sickle cell disease is exposed to copious amounts of sickle hemoglobin. Because sickle hemoglobin is an unstable heme protein that may undergo scission to its heme and globin moieties,11Hebbel RP The sickle erythrocyte in double jeopardy: autooxidation and iron decompartmentalization.Semin Hematol. 1990; 27: 51-69PubMed Google Scholar, 12Hebbel RP Membrane-associated iron.in: Embury SH Hebbel RP Mohandas N Steinberg MH Sickle Cell Disease: Basic Principles and Clinical Practice, ch 12. Raven Press, Ltd., New York1994: 163-172Google Scholar we reasoned that the kidney would be exposed to large amounts of heme, the latter representing a lipophilic pro-oxidant.11Hebbel RP The sickle erythrocyte in double jeopardy: autooxidation and iron decompartmentalization.Semin Hematol. 1990; 27: 51-69PubMed Google Scholar, 12Hebbel RP Membrane-associated iron.in: Embury SH Hebbel RP Mohandas N Steinberg MH Sickle Cell Disease: Basic Principles and Clinical Practice, ch 12. Raven Press, Ltd., New York1994: 163-172Google Scholar In our examination of the involvement of oxidative stress in sickle cell nephropathy, we examined indices of oxidative stress along with heme content of the kidney. We also examined expression of the redox sensitive enzyme, HO-1, as an index of oxidant stress.13Platt JL Nath KA Heme oxygenase: protective gene or trojan horse.Nat Med. 1998; 4: 1364-1365Crossref PubMed Scopus (210) Google Scholar, 14Dong Z Lavrovsky Y Venkatachalam MA Roy AK Heme oxygenase-1 in tissue pathology.Am J Pathol. 2000; 156: 1485-1488Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 15Agarwal 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 Heme oxygenase (HO) is the rate limiting enzyme in the degradation of heme, converting heme to biliverdin in the course of which iron is released and carbon monoxide is emitted. HO consists of three isozymes, HO-1 representing the isozyme that is induced by oxidative stress, heme, and other stressors; HO-1 thus provides a relevant and sensitive index by which to assess alterations in cellular redox. This model is homozygous for deletion of mouse β-globin, and contains transgenes for human βS- and βS-antilles-globins linked to the transgene for human α-globin.10Fabry ME Sengupta A Suzuka SM Costantini F Rubin EM Hofrichter J Christoph G Manci E Culberson D Factor SM Nagel RL A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity.Blood. 1995; 86: 2419-2428Crossref PubMed Google Scholar Studies were conducted in aged-matched control and sickle mice comprising similar numbers of male and female mice. Although the various studies of the kidney, in aggregate, involved mice that ranged in age from 0.5 to 1.5 years, each study was undertaken in similarly aged control and transgenic mice, each group comprising similar numbers of male and female mice. Renal function was assessed by the concentration of plasma creatinine, the latter determined on plasma derived from tail-vein blood samples and using a Beckman Creatinine Analyzer II (Beckman Instruments, Inc., Fullerton, CA).16Nath KA Haggard JJ Croatt AJ Grande JP Poss KD Alam J The indispensability of heme oxygenase-1 (HO-1) in protecting against heme protein-induced toxicity in vivo.Am J Pathol. 2000; 156: 1527-1535Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar Urinary protein concentration was determined by the Coomassie method. Lipid peroxidation was assessed by the thiobarbituric acid reactive substance assay, as previously described.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 Heme content of whole kidney homogenate, and of various cellular fractions, was determined by the pyridine hemochromogen method, as previously described.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 As described in detail in our previous study,18Nath 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 HO activity was measured by the method of Pimstone and colleagues,19Pimstone NR Engel P Tenhunen R Seitz PT Marver HS Schmid R Inducible heme oxygenase in the kidney: a model for the homeostatic control of hemoglobin catabolism.J Clin Invest. 1971; 50: 2042-2050Crossref PubMed Scopus (92) Google Scholar catalase by the method of Aebi and colleagues,20Aebi H Catalase.in: Bergmeyer HU Methods of Enzymatic Analysis. vol 2. Academic Press, Inc., New York1974: 673-684Google Scholar and glutathione peroxidase by the method of Lawrence and Burk.21Lawrence RA Burk RF Glutathione peroxidase activity in selenium deficient rat liver.Biochem Biophys Res Commun. 1976; 95: 351-358Google Scholar RNA from kidneys was extracted using the Trizol method (Life Technologies, Inc., Gaithersburg, MD). Twenty micrograms of total RNA from each sample were separated on an agarose gel and transferred to a nylon membrane.16Nath KA Haggard JJ Croatt AJ Grande JP Poss KD Alam J The indispensability of heme oxygenase-1 (HO-1) in protecting against heme protein-induced toxicity in vivo.Am J Pathol. 2000; 156: 1527-1535Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar Membranes were hybridized overnight with32P-labeled mouse HO-1 and mouse HO-2 cDNA probes.22Haugen EN Croatt AJ Nath KA Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro.Kidney Int. 2000; 58: 144-152Crossref PubMed Scopus (147) Google Scholar Autoradiograms were standardized, as previously described,22Haugen EN Croatt AJ Nath KA Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro.Kidney Int. 2000; 58: 144-152Crossref PubMed Scopus (147) 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. Kidney sections, derived at autopsy, were studied for the expression of HO-1 by immunoperoxidase. The patient was female, 47 years old, had a history of modest severity (average of three pain crises per year), and died of a massive thromboembolism during an acute painful episode. Normal kidney tissue from a nephrectomized specimen served as a control. Kidney sections were stained for HO-1 using a monoclonal HO-1 antibody (OSA-111; Stressgen, Victoria, BC, Canada) as the primary antibody, a polyclonal goat anti-mouse IgG as the secondary antibody (SAB-100, Stressgen), and diaminobenzidine as substrate for localization, as previously described in detail.16Nath KA Haggard JJ Croatt AJ Grande JP Poss KD Alam J The indispensability of heme oxygenase-1 (HO-1) in protecting against heme protein-induced toxicity in vivo.Am J Pathol. 2000; 156: 1527-1535Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 22Haugen EN Croatt AJ Nath KA Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro.Kidney Int. 2000; 58: 144-152Crossref PubMed Scopus (147) Google Scholar CECs were evaluated for expression of HO-1 using the methods we have previously described in detail.23Solovey A Lin Y Browne P Choong S Wayner E Hebbel RP Circulating activated endothelial cells in sickle cell anemia.N Engl J Med. 1997; 337: 1584-1590Crossref PubMed Scopus (555) Google Scholar, 24Solovey A Gui L Key NS Hebbel RP Tissue factor expression by endothelial cells in sickle cell anemia.J Clin Invest. 1998; 101: 1899-1904Crossref PubMed Scopus (184) Google Scholar Briefly, using fresh whole blood anticoagulated with ethylenediaminetetraacetic acid, we used immunomagnetic beads (Dynal, Oslo, Norway) coated with the anti-endothelial cell monoclonal antibody, P1H12, to prepare a CEC-enriched population of cells from the buffy coat.23Solovey A Lin Y Browne P Choong S Wayner E Hebbel RP Circulating activated endothelial cells in sickle cell anemia.N Engl J Med. 1997; 337: 1584-1590Crossref PubMed Scopus (555) Google Scholar, 24Solovey A Gui L Key NS Hebbel RP Tissue factor expression by endothelial cells in sickle cell anemia.J Clin Invest. 1998; 101: 1899-1904Crossref PubMed Scopus (184) Google Scholar After transfer to slides, cells were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100. Cells were stained for HO-1 using a primary polyclonal antibody (SPA-895, Stressgen), followed by a rhodamide-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa). A negative control was provided by use of an irrelevant primary antibody. Cells were scored as being “negative” if they showed no increase in staining higher than that of the negative control sample done in parallel. Cells were scored as being “low positive” if their staining intensity exceeded that of the negative control but was still less than that of the red autofluorescence of the immunomagnetic beads in the preparation (Figure 9, top left). Cells were scored as being “high positive” if their staining was brighter than the bead autofluorescence (Figure 9, top right). Results are expressed as percentage of CECs being negative, low positive, or high positive. Sufficient sample was evaluated for each donor so that at least 10 CECs were screened per normal donor and at least 20 CECs were screened per sickle donor; this difference is accounted for by the much smaller number of CECs per ml of blood in normal donors.23Solovey A Lin Y Browne P Choong S Wayner E Hebbel RP Circulating activated endothelial cells in sickle cell anemia.N Engl J Med. 1997; 337: 1584-1590Crossref PubMed Scopus (555) Google Scholar These studies were conducted in four healthy patients and in five patients with sickle cell disease. Buthionine-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase (the first step in glutathione synthesis), was administered every 12 hours for 3 days to control and sickle mice (5 mmol/kg i.p.).25Minchinton AI Rojas A Smith KA Soranson JA Shrieve DC Jones NR Bremner JC Glutathione depletion in tissues after administration of buthionine sulphoximine.Int J Radiat Oncol Biol Phys. 1984; 10: 1261-1264Abstract Full Text PDF PubMed Scopus (49) Google Scholar The kidneys were then harvested for the assessment of thiol content and histological studies. Thiol content was determined by the method based on the reduction of the Ellman reagent, 5,5′-dithiobis (2-nitrobenzoic acid), as previously described.26Nath KA Enright H Nutter L Fischereder MF Zou JN Hebbel RP Effect of pyruvate on oxidant injury to isolated and cellular DNA.Kidney Int. 1994; 45: 166-176Crossref PubMed Google Scholar Results are presented as means ± SEM. For statistical analyses, the Student’s t-test or the Mann-Whitney test was used as appropriate. Results are considered significant for P < 0.05. The transgenic sickle mouse demonstrates increased kidney weight and increased body weight as compared to control mice: both the absolute kidney weight as well as the kidney weight factored for body weight were increased in sickle mice (Table 1). The concentration of plasma creatinine was significantly lower in sickle mice, and in light of the greater body weight in sickle mice, this likely reflects a higher GFR in the transgenic sickle mouse. Neither the urinary protein concentration, nor the ratio of urinary concentration of protein/urinary concentration of creatinine, was altered in sickle mice (Table 1).Table 1Characteristics of Control and Sickle Mice at 6 to 8 Months of AgeControlSickleP valueBody weight (g)23.7 ± 0.928.1 ± 2.0<0.05Hematocrit (%)49 ± 148 ± 2NSKidney weight (g)0.16 ± 0.010.23 ± 0.02<0.01Kidney weight/body weight ×10−36.8 ± 0.37.8 ± 0.4<0.05Plasma creatinine (mg/dl)0.34 ± 0.020.20 ± 0.01<0.0001Urinary protein concentration (mg/dl)73.7 ± 10.368.1 ± 9.7NSUrinary protein concentration/urinary creatinine concentration2.4 ± 0.31.7 ± 0.3NSThe control group comprised 12 to 14 mice; the sickle group comprised 9 to 10 mice.NS, not significant. Open table in a new tab The control group comprised 12 to 14 mice; the sickle group comprised 9 to 10 mice. NS, not significant. Histological assessment of the kidney in the sickle mouse demonstrated congestion of the medullary vessels with spontaneous sickling of RBCs (Figure 1). Sickle mice studied for up to 1.5 years of age did not develop progressive renal disease; in particular, there was no significant segmental or global glomerulosclerosis, interstitial fibrosis, atrophy of cortical or medullary tubules other than that associated with aging in control, aged-matched, mice. We determined lipid peroxidation by the thiobarbituric acid-reactive substance assay and demonstrate a significant increase in the kidney in the sickle mouse (Figure 2). The content of heme—the latter representing a strong pro-oxidant species—in the sickle kidney was uniformly increased in all cellular compartments including whole-kidney homogenates, and in cytosolic, mitochondrial, and microsomal fractions of the kidney (Figure 3).Figure 3Heme content in whole kidney homogenate and subcellular fractions in control mice (open column) and sickle mice (striped column). In all studies, n= 8 in control mice and n = 10 in sickle mice. *, P < 0.05, control versus sickle mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because heme content was increased in the sickle kidney in conjunction with increased oxidative stress, we assessed, by measuring HO activity, whether heme-degrading activity was altered in the sickle kidney. Such enzyme activity was increased in kidney microsomes prepared from sickle mice (181 ± 9 versus 236 ± 10 pmol/hour/mg protein; P < 0.05; n = 8 in control and sickle mice). As HO is an antioxidant enzyme, we measured other antioxidant enzyme activity to determine whether such increase in HO activity was part of a more generalized antioxidant response. In this regard, catalase activity was unchanged in the sickle kidney (0.30 ± 0.02 versus 0.33 ± 0.02, k/mg protein; P = ns; n = 8 in control group and n = 10 in sickle mice) whereas glutathione peroxidase was reduced in the sickle kidney (589 ± 24 versus 509 ± 26, nmol/min/mg protein; P < 0.05; n = 8 in control group and n = 10 in sickle mice). Thus, the increase in HO activity does not seem to be part of a more widespread induction of antioxidant systems. Increased HO activity may reflect induction of one of the HO isoforms. We thus assessed the expression of HO-1 and HO-2 by Northern analysis. HO-1 mRNA was increased some fivefold in the sickle kidney (Figure 4), whereas mRNA expression for HO-2, the constitutively expressed isoform, was unaltered in the sickle kidney (mean standardized densitometric values: 7.91 ± 1.38 versus 8.40 ± 1.22; P = ns;n = 4 in control and sickle mice). Thus, induction of HO-1 accompanies the presence of oxidative stress in the sickle kidney. To determine the functional significance of oxidative stress in the sickle kidney, we depleted thiol content of the kidney in transgenic sickle mice by administering BSO, an agent that inhibits glutathione synthesis and promotes a pro-oxidant state. That BSO, so administered, did achieve reduction in kidney thiol content is shown in Figure 5: kidney thiol content was significantly reduced in both groups, ∼50% reduction in control mice and 38% reduction in sickle mice. Such administration of BSO did not have any apparent histological effect in the kidney in control mice (data not shown); however, such administration of BSO markedly exacerbated RBC sickling in the kidney of the sickle mouse (Figure 6, Figure 7). Medullary congestion was worsened in sickle mice treated with BSO as compared with sickle mice treated with vehicle (Figure 6). Additionally, and strikingly, BSO-treated sickle mice—but not vehicle-treated sickle mice—exhibited extension of vascular congestion and RBC sickling from the medulla (where they are normally exhibited, and restricted to, in this unstressed transgenic sickle kidney) into cortical capillaries, cortical arterioles, and glomeruli (Figure 7). Thus, acute oxidative stress in the kidney, as induced by reduction in kidney thiol content by BSO, exacerbates the sickling process that occurs in the kidney.Figure 6Vascular congestion and RBC sickling in the renal medulla in sickle mice treated with vehicle (left) or BSO (right). Original magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Vascular congestion and RBC sickling in the renal cortex in sickle mice treated with vehicle (left) or BSO (right). Thewhite arrows point out arterioles and capillaries. BSO-treated sickle mice demonstrate vascular congestion and RBC sickling in glomeruli, arterioles, and capillaries in the renal cortex (right), findings not observed in glomeruli, arterioles, and capillaries in vehicle-treated sickle mice (left). Original magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether HO-1 is induced in the kidney in patients with sickle cell disease, we examined the expression of HO-1 in the kidney obtained from a sickle patient who died after a massive pulmonary embolus. The healthy human kidney does not express HO-1 as shown in Figure 8A. In contrast, the sickle kidney exhibits diffuse and widespread brown staining, that was HO-1 antibody-specific, in proximal and distal renal tubules (Figure 8, Figure 9), in interstitial cells (Figure 8, Figure 9), and in endothelial cells and in the smooth muscle cells in the media of small arteries (Figure 10). Focal glomerular staining within isolated cells was also observed (data not shown); however, the nature of these cells—infiltrating mononuclear cells or mesangial cells—demonstrating such staining awaits further investigation.Figure 10Immunoperoxidase staining for HO-1 in the vasculature of the kidney of a patient with sickle cell disease. This figure shows positive staining for HO-1 in the endothelium and the smooth muscle cells of the media of a small artery. Original magnifi
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