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Heme Oxygenase 1 Up-Regulates Glomerular Decay Accelerating Factor Expression and Minimizes Complement Deposition and Injury

2016; Elsevier BV; Volume: 186; Issue: 11 Linguagem: Inglês

10.1016/j.ajpath.2016.07.009

1525-2191

Maria G. Detsika, Pu Duann, Vassilios Atsaves, Αpostolos Papalois, Elias A. Lianos,

Cannabis and Cannabinoid Research

Complement-activation controllers, including decay accelerating factor (DAF), are gaining emphasis as they minimize injury in various dysregulated complement-activation disorders, including glomerulopathies. Heme oxygenase (HO)-1 overexpression or induction has been shown to attenuate injury in complement-dependent models of glomerulonephritis. This study investigated whether up-regulation of DAF by heme oxygenase 1 (HO-1) is an underlying mechanism by using Hmox-1–deficient rats (Hmox1+/−; Hmox1−/−) or rats with HO-1 overexpression targeted to glomerular epithelial cells (GECHO-1), which are particularly vulnerable to complement-mediated injury owing to their terminally differentiated nature. Constitutively expressed DAF was decreased in glomeruli of Hmox1−/− rats and augmented in glomeruli of GECHO-1 rats. In GECHO-1 rats with anti–glomerular basement membrane antibody mediated, complement-dependent injury, complement component C3 fragment b (C3b) deposition was reduced, whereas proteinuria was diminished. In glomeruli of wild-type rats, the natural Hmox substrate, hemin, induced glomerular DAF. This effect was attenuated in glomeruli of Hmox1−/− rats and augmented in glomeruli of GECHO-1 rats. Hemin analogues differing in either metal or porphyrin ring functionalities, acting as competitive Hmox-substrate inhibitors, also increased glomerular DAF and reduced C3b deposition after spontaneous complement activation. In the presence of a DAF-blocking antibody, the reduction in C3b deposition was reversed. These observations establish HO-1 as a physiologic regulator of glomerular DAF and identify hemin analogues as inducers of functional glomerular DAF able to minimize C3b deposition. Complement-activation controllers, including decay accelerating factor (DAF), are gaining emphasis as they minimize injury in various dysregulated complement-activation disorders, including glomerulopathies. Heme oxygenase (HO)-1 overexpression or induction has been shown to attenuate injury in complement-dependent models of glomerulonephritis. This study investigated whether up-regulation of DAF by heme oxygenase 1 (HO-1) is an underlying mechanism by using Hmox-1–deficient rats (Hmox1+/−; Hmox1−/−) or rats with HO-1 overexpression targeted to glomerular epithelial cells (GECHO-1), which are particularly vulnerable to complement-mediated injury owing to their terminally differentiated nature. Constitutively expressed DAF was decreased in glomeruli of Hmox1−/− rats and augmented in glomeruli of GECHO-1 rats. In GECHO-1 rats with anti–glomerular basement membrane antibody mediated, complement-dependent injury, complement component C3 fragment b (C3b) deposition was reduced, whereas proteinuria was diminished. In glomeruli of wild-type rats, the natural Hmox substrate, hemin, induced glomerular DAF. This effect was attenuated in glomeruli of Hmox1−/− rats and augmented in glomeruli of GECHO-1 rats. Hemin analogues differing in either metal or porphyrin ring functionalities, acting as competitive Hmox-substrate inhibitors, also increased glomerular DAF and reduced C3b deposition after spontaneous complement activation. In the presence of a DAF-blocking antibody, the reduction in C3b deposition was reversed. These observations establish HO-1 as a physiologic regulator of glomerular DAF and identify hemin analogues as inducers of functional glomerular DAF able to minimize C3b deposition. Dysregulated complement activation has recently gained emphasis as an underlying mechanism of various immune-mediated forms of glomerular injury, including the recently characterized complement component C3 (C3) glomerulopathy.1Pickering M.C. D'Agati V.D. Nester C.M. Smith R.J. Haas M. Appel G.B. Alpers C.E. Bajema I.M. Bedrosian C. Braun M. Doyle M. Fakhouri F. Fervenza F.C. Fogo A.B. Fremeaux-Bacchi V. Gale D.P. Goicoechea de Jorge E. Griffin G. Harris C.L. Holers V.M. Johnson S. Lavin P.J. Medjeral-Thomas N. Paul Morgan B. Nast C.C. Noel L.H. Peters D.K. Rodriguez de Cordoba S. Servais A. Sethi S. Song W.C. Tamburini P. Thurman J.M. Zavros M. Cook H.T. C3 glomerulopathy: consensus report.Kidney Int. 2013; 84: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar This putative mechanism increases the significance of complement-activation controllers, including decay accelerating factor (DAF), as potential therapeutic targets to achieve attenuation of complement-mediated injury. DAF has been shown to minimize complement activation on both human and rat glomerular epithelial cells (GECs), as its inhibition increased complement-mediated cytotoxicity after sensitization with a complement-fixing antibody.2Quigg R.J. Nicholson-Weller A. Cybulsky A.V. Badalamenti J. Salant D.J. Decay accelerating factor regulates complement activation on glomerular epithelial cells.J Immunol. 1989; 142: 877-882PubMed Google Scholar In two independent studies using DAF-deficient mice with anti–glomerular basement membrane (GBM) antibody–mediated or immune complex–mediated glomerulonephritis, severe podocyte damage with proteinuria and increased C3 deposition occurred, while no difference in the expression level of complement receptor 1–related protein Y or CD59 was observed on leukocytes or in kidney tissue, thus implicating DAF as an important defense molecule against complement-mediated glomerular injury.3Lin F. Emancipator S.N. Salant D.J. Medof M.E. Decay-accelerating factor confers protection against complement-mediated podocyte injury in acute nephrotoxic nephritis.Lab Invest. 2002; 82: 563-569Crossref PubMed Scopus (56) Google Scholar, 4Sogabe H. Nangaku M. Ishibashi Y. Wada T. Fujita T. Sun X. Miwa T. Madaio M.P. Song W.C. Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis.J Immunol. 2001; 167: 2791-2797Crossref PubMed Scopus (77) Google Scholar These findings were further confirmed in a mouse model of immune complex glomerulonephritis associated with marked C3 deposition in glomeruli. DAF-deficient mice had an increased prevalence of glomerulonephritis associated with significantly increased glomerular C3 deposition relative to wild-type (WT) controls. In contrast, disease expression in CD59-deficient mice was no different from that in WT controls.5Bao L. Haas M. Minto A.W. Quigg R.J. Decay-accelerating factor but not CD59 limits experimental immune-complex glomerulonephritis.Lab Invest. 2007; 87: 357-364Crossref PubMed Scopus (10) Google Scholar Collectively, these observations support the important role of DAF in protecting against complement-mediated glomerular injury. In the anti-GBM antibody–mediated, complement-dependent model of injury, we previously reported that heme oxygenase (Hmox)-1 induction in glomeruli after exogenous administration of the natural Hmox substrate/inducer, hemin, or GEC-targeted HO-1 overexpression (GECHO-1) in mice minimized extent of injury.6Datta P.K. Duann P. Lianos E.A. Long-term effect of heme oxygenase (HO)-1 induction in glomerular immune injury.J Lab Clin Med. 2006; 147: 150-155Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 7Duann P. Lianos E.A. GEC-targeted HO-1 expression reduces proteinuria in glomerular immune injury.Am J Physiol Renal Physiol. 2009; 297: F629-F638Crossref PubMed Scopus (21) Google Scholar However, the underlying mechanisms were not explored. It was recently reported that, in cultured endothelial cells derived from Hmox-1–deficient mice, DAF expression was reduced.8Kinderlerer A.R. Pombo Gregoire I. Hamdulay S.S. Ali F. Steinberg R. Silva G. Ali N. Wang B. Haskard D.O. Soares M.P. Mason J.C. Heme oxygenase-1 expression enhances vascular endothelial resistance to complement-mediated injury through induction of decay-accelerating factor: a role for increased bilirubin and ferritin.Blood. 2009; 113: 1598-1607Crossref PubMed Scopus (78) Google Scholar This finding points to heme oxygenase 1 (HO-1) as a putative regulator of DAF and raises the question of whether the protective effect of HO-1 in complement-dependent forms of glomerular injury involves DAF up-regulation. To address this question, we generated two novel rat models: Hmox-1–depleted rats, and GECHO-1 rats, which are the only cell type expressing DAF in the rat nephron.9Bao L. Spiller O.B. St John P.L. Haas M. Hack B.K. Ren G. Cunningham P.N. Doshi M. Abrahamson D.R. Morgan B.P. Quigg R.J. Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes.Kidney Int. 2002; 62: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Anti–Hmox-1 polyclonal antibody was purchased from Enzo Life Sciences (New York, NY). Anti-DAF antibody clone RDII-24 was a kind gift from Prof P. Morgan (University of Cardiff, Cardiff, UK), and clone RDIII-7 was purchased from Hycult (Uden, the Netherlands). Anti-rat C3/C3 fragment b (C3b) antibody was purchased from Hycult; anti–β-actin antibody, from Sigma-Aldrich (St. Louis, MO); and anti–glyceraldehyde-3-phosphate dehydrogenase antibody, from Cell Signaling Technology (Danvers, MA). Hemin, cobalt protoporphyrin, zinc protoporphyrin, protoporphyrin IX (PPIX), cycloheximide (CHX), and phosphatidylinositol-specific phospholipase C were purchased from Sigma-Aldrich. Tin protoporphyrin and tin mesoporphyrin (SnMP) were purchased from Tocris Bioscience (Bristol, UK). A deglycosylation kit was purchased from CalBiochem (EMD Millipore, Billerica, MA). Adult male Sprague-Dawley rats, 300 g in body weight, were used in this study. Animals were reared in accordance with the European Union Directive for the care and use of laboratory animals, and all procedures were approved by the Hellenic Veterinary Administration and the ethics committee of Evangelismos Hospital (Paphos, Cyprus). For the generation of transgenic rats, all procedures described were conducted according to the NIH's Guide for the Care and Use of Laboratory Animals.10Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar Zinc finger nuclease (ZFN) constructs targeting the rat Hmox-1 sequence 5′-GGTGGCCCACGCATATACCCGCTACCTGGGTGACCTCTCAG-3′ within exon 3 (Table 1) were designed and assembled in a ready-to-inject mRNA format by Sigma-Aldrich. The underlined sequences are recognized by the right and left ZFNs, respectively (on opposite strands), and are separated by the lowercase 7-bp spacer sequences, where the nuclease domains interact to cause a double-strand break. mRNA at a concentration of 2 ng/μL was introduced by male pronuclear microinjection into Sprague-Dawley rat embryos, and surviving founder generation pups were screened using a CEL-I nuclease assay as previously described.11Geurts A.M. Cost G.J. Remy S. Cui X. Tesson L. Usal C. Menoret S. Jacob H.J. Anegon I. Buelow R. Generation of gene-specific mutated rats using zinc-finger nucleases.Methods Mol Biol. 2010; 597: 211-225Crossref PubMed Scopus (89) Google Scholar A total of 149 fertilized oocytes were microinjected, and 78 surviving embryos were kept for implantation into the oviducts of pseudopregnant female Sprague-Dawley rats and allowed to go to term. Nineteen pups were born, among which 18 could be screened for ZFN-mediated disruption of the Hmox-1–coding sequence. Screening to verify disruption of the ZFN target site was performed by standard PCR amplification of DNA prepared from rat tissue (ear or tail). PCR primers sequences were: forward, 5′-ATGCCCCACTCTACTTCCCT-3′; reverse, 5′-TTCATGCGAGCACGATAGAG-3′.Table 1Target DNA Sequences of the Hmox1 Gene ORF (Exon 3) to Which the Designed ZFN Pairs BindZNF pair #PositionSequence#063507-35155′-GGCCTCCTTGTACCATATCTATACGGCCCTGGAAGAG-3′#073514-35215′-CTCCTTGTACCATATCTATACGGCCCTGGAAGAGGAG-3′#083580-35875′-TGCCCCACTCTACTTCCCTGAGGAGCTGCACCGAAGG-3′#093583-35895′-CCACTCTACTTCCCTGAGGAGCTGCACCGAAGGGCTGCC-3′#103590-35975′-TACTTCCCTGAGGAGCTGCACCGAAGGGCTGCCCTAG-3′#113628-36345′-CAGGACATGGCCTTCTGGTATGGGCCCCACTGGCAGGA-3′#123638-36445′-CCTTCTGGTATGGGCCCCACTGGCAGGAGGCC-3′#133744-37525′-GGTGGCCCACGCATATACCCGCTACCTGGGTGACCTCTCAG-3′#143750-37565′-CCACGCATATACCCGCTACCTGGGTGACCTCTCAG-3′#153754-37625′-CGCATATACCCGCTACCTGGGTGACCTCTCAGGGGGTC-3′#163756-37625′-ATATACCCGCTACCTGGGTGACCTCTCAGGGGGTC-3′Underlined sequence: sequence targeted for deletion. ZNF pair 13 was the most active pair.ORF, open reading frame; ZFN, zinc finger nucleases. Open table in a new tab Underlined sequence: sequence targeted for deletion. ZNF pair 13 was the most active pair. ORF, open reading frame; ZFN, zinc finger nucleases. Two founders were identified (Supplemental Figure S1), one of which was mosaic and harbored Hmox-1 mutant alleles 1 and 2. Hmox-1 mutant allele 1 was a 16-bp frameshift deletion in the Hmox-1 exon 3 coding sequence, overlapping the ZFN target site. Hmox-1 mutant allele 2 was a 21-bp in-frame deletion associated with a 3-bp insertion "ggg," resulting in a net loss of 18 bp and maintaining the open reading frame of Hmox-1. A litter of pups was obtained, suggesting that the disruption of Hmox-1 in the Sprague-Dawley rat can be tolerated. The second founder was apparently heterozygous for a single mutation, Hmox-1 mutant allele 3, a 10-bp frameshift deletion, in Hmox-1 exon 3. While rats with biallelic Hmox-1 gene disruption were short-lived and developed various anomalies, hemizygotes survived to breeding age and were apparently healthy and reproductively fit. The WT allele was 340 bp; the mutant allele 1, 324 bp; the mutant allele 2, 322 bp; and the mutant allele 3, 330 bp. PCR products were resolved on a 10% TRIS/Borate/EDTA polyacrylamide high-resolution electrophoresis gel. Sequence analysis was further applied to confirm the mutations. Real-time PCR primers and probe sequences for total tissue RNA were the following: forward, 5′-GGCCCACGCATATACCCGC-3′; reverse, 5′-AGCCAGGCCTTCCCCAGAG-3′; double-dye oligonucleotide probe, FAM-AATGTTGAGCAGGAAGGCGGTCT-BHQ1 (fluorescein amidite, black hole quencher 1). All rats were genotyped after rat tissue (tail or ear) DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen, Manchester, UK) and the following PCR amplification primers: WT, 5′-GGGGTTTTGACAGCTGGGC-3′ (forward) and 5′-CCCAGGTAGCGGGTATATGC-3′ (reverse); mutant, 5′-GGTGGCCCACGCATACTGG-3′ (forward) and 5′-GTCAGCATCCGAAGCCTGG-3′ (reverse). After an initial denaturation step (95°C for 5 minutes), samples were subjected to 30 cycles of denaturation at 95°C (1 minute), annealing (60°C for 1 minute), and extension (72°C for 1 minute). A final extension step (72°C for 1 minute) was performed. Hmox1 knockout animals (homozygotes, Hmox1−/−) were generated through breeding of the Hmox1+/− rats. Cross-breeding of the Hmox1+/− rats did not yield the expected mendelian ratio. This finding is in agreement with those described earlier in mice lacking Hmox1.12Poss K.D. Tonegawa S. Heme oxygenase 1 is required for mammalian iron reutilization.Proc Natl Acad Sci U S A. 1997; 94: 10919-10924Crossref PubMed Scopus (862) Google Scholar Hmox1−/− rats had growth retardation compared with their WT littermates, and most died at 6 months of age, without any previous signs of abnormality. They otherwise appeared healthy with respect to fur color and motility. As previously described,13Detsika M.G. Duann P. Lianos E.A. HO-1 expression control in the rat glomerulus.Biochem Biophys Res Commun. 2015; 460: 786-792Crossref PubMed Scopus (8) Google Scholar GECHO-1 rats were generated using a Sleeping Beauty (SB) transposon vector, SB human HO-1, harboring a FLAG-tagged human HO-1 sequence under the control of a murine nephrin promoter7Duann P. Lianos E.A. GEC-targeted HO-1 expression reduces proteinuria in glomerular immune injury.Am J Physiol Renal Physiol. 2009; 297: F629-F638Crossref PubMed Scopus (21) Google Scholar and the SB transposon system methodology, as reported by Katter et al.14Katter K. Geurts A.M. Hoffmann O. Mates L. Landa V. Hiripi L. Moreno C. Lazar J. Bashir S. Zidek V. Popova E. Jerchow B. Becker K. Devaraj A. Walter I. Grzybowksi M. Corbett M. Filho A.R. Hodges M.R. Bader M. Ivics Z. Jacob H.J. Pravenec M. Bosze Z. Rulicke T. Izsvak Z. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.FASEB J. 2013; 27: 930-941Crossref PubMed Scopus (74) Google Scholar All rats were genotyped after rat tissue (tail or ear) DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen) and the following PCR amplification primer pairs: SB1, 5′-GAGGGAAGAGAGAAGGGCGAGT-3′ (forward) and 5′-CCTTGTTGCGCTCAATCTCCT-3′ (reverse); SB2, 5′-CGACAGCATGCCCCAGGATT-3′ (forward) and 5′-CTCTGGGAGTCTCCACGGGG-3′ (reverse). After an initial denaturation step (95°C for 5 minutes), samples were subjected to 30 cycles of denaturation at 95°C (1 minute), annealing (60°C for 1 minute), and extension (72°C for 1 minute). A final extension step (72°C for 1 minute) was performed. The model of anti-GBM antibody–mediated, complement-dependent glomerulonephritis was induced as we previously reported.6Datta P.K. Duann P. Lianos E.A. Long-term effect of heme oxygenase (HO)-1 induction in glomerular immune injury.J Lab Clin Med. 2006; 147: 150-155Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Briefly, rats were immunized with 500 μg i.p. of rabbit IgG emulsified in complete Freund's adjuvant (Sigma-Aldrich) 1 week before a single subnephritogenic i.v. injection of 1 mL of rabbit anti-rat GBM serum. Control rats were preimmunized with rabbit IgG in complete Freund's adjuvant and subsequently injected with nonimmune rabbit serum. Urine was collected using metabolic cages 4 days before preimmunization with rabbit IgG and 7 days after the administration of the anti-GBM antibody. Urinary albumin was measured using an enzyme-linked immunosorbent assay method (Nephrat kit; Exocell, Philadelphia, PA). Urinary and serum creatinine concentrations were measured by the Jaffe colorimetric method. Albuminuria was expressed as the ratio of urinary albumin to urinary creatinine. Glomeruli were isolated from the kidneys of WT or transgenic animals by an established differential sieving method.15Wiggins J.E. Patel S.R. Shedden K.A. Goyal M. Wharram B.L. Martini S. Kretzler M. Wiggins R.C. NFkappaB promotes inflammation, coagulation, and fibrosis in the aging glomerulus.J Am Soc Nephrol. 2010; 21: 587-597Crossref PubMed Scopus (75) Google Scholar Glomeruli were incubated with defined concentrations of the following metalloporphyrins (MPs): hemin, cobalt protoporphyrin, zinc protoporphyrin, tin protoporphyrin, SnMP, and PPIX. MPs were dissolved in dimethyl sulfoxide. Negative controls consisted of glomeruli incubated with MP vehicle (dimethyl sulfoxide) only. Protein extracts were prepared using lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris pH 8.0, and 1% Triton X-100 containing a protease-inhibitors cocktail), and concentration was determined by the Bradford assay (BioRad Laboratories, Hercules, CA). Glomerular RNA was extracted using an established TRIzol-based method. Protein lysates were resolved by SDS-PAGE, transferred onto polyvinyledinedifluoride membrane, and probed with primary antibodies overnight. Secondary antibodies were purchased from Cell Signaling Technology, and ECL reagent, from Santa Cruz Biotechnology (Dallas, TX). Equal loading was determined by probing for β-actin or glyceraldehyde-3-phosphate dehydrogenase. RT-PCR reactions were performed using a TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Waltham, MA). Reactions were performed in a PTC-200 PCR cycler (MJ Research, Bio-Rad Laboratories, Hercules, CA) with a Chromo4 detector system (Bio-Rad) under the following conditions: 25°C for 10 minutes, 48°C for 30 minutes, and 95°C for 5 minutes. Each reaction consisted of 2 μL of primer-probe assay mix (Integrated DNA Technologies, Coralville, IA), 10 μL of Master Mix (Applied Biosystems), and 8 μL of cDNA. Reactions were performed in triplicate, and results were analyzed by the ΔΔCT method. Kidney cortical sections obtained after nephrectomy were fixed in formalin 30%. Sections were stained with periodic acid-Schiff following established methodology. Glomerular damage was quantified as previously described by assessing glomerular size and cellularity.4Sogabe H. Nangaku M. Ishibashi Y. Wada T. Fujita T. Sun X. Miwa T. Madaio M.P. Song W.C. Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis.J Immunol. 2001; 167: 2791-2797Crossref PubMed Scopus (77) Google Scholar Immunofluorescence for C3b deposition or rabbit IgG deposition in glomeruli was performed on cryosections using a mouse anti-rat C3/C3b primary antibody and a goat anti-mouse secondary antibody labeled with Alexa Fluor 568 or a goat anti-rabbit secondary labeled with Alexa Fluor 488, respectively (Thermo Fisher Scientific, Renfrew, UK). Sections incubated in the absence of primary antibody (secondary antibody only) served as negative controls. In immunohistochemistry detection of DAF in kidney cortical sections, an anti-DAF antibody (clone RDIII-7) and standard techniques were used. Values are expressed either as means ± SEM or as means ± SD. Statistical analysis was performed with either t-test, where applicable, or analysis of variance for more than two group comparisons. When significant, post hoc analysis was performed, with either a Turkey test or the least significant difference test. A P value <0.05 was chosen as statistically significant. Binding specificity of the anti-DAF antibody used was verified by immunohistochemistry analysis. Figure 1A demonstrates antibody binding exclusively in glomeruli and GECs, confirming the findings from a previous report of restricted DAF expression to GECs in the rat nephron.9Bao L. Spiller O.B. St John P.L. Haas M. Hack B.K. Ren G. Cunningham P.N. Doshi M. Abrahamson D.R. Morgan B.P. Quigg R.J. Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes.Kidney Int. 2002; 62: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar To confirm that DAF protein is preserved in its glycosylated, glycosylphosphatidylinositol (gpi)-anchored form, after glomerular isolation, glomeruli were treated with phosphatidylinositol-specific phospholipase C at various concentrations for 90 minutes at 37°C. Phosphatidylinositol-specific phospholipase C treatment resulted in a loss of DAF protein of approximately 90% (Figure 1B). To assess the glycosylation state of gpi-anchored DAF, glomerular protein lysates were subjected to enzymatic deglycosylation using deglycosylation enzymes alone or in combination. The presence of a heavily glycosylated DAF protein was demonstrated. Neuraminidase treatment resulted in a band of approximately 52 kDa (Figure 1C). Treatment with O-glycosidase combined with either neuraminidase or N-glycosidase F further reduced DAF molecular weight to approximately 47 kDa (Figure 1B). The addition of two more enzymes [β-(1-4)-galactosidase and β-N-acetylglucosaminidase] to the previous enzyme mix resulted in a band of approximately 43 kDa (Figure 1C), representing the fully deglycosylated, gpi-anchored glomerular DAF protein. The regulatory effect of HO-1 on glomerular DAF was initially assessed in the Hmox-1–deficient rats. As shown in Figure 2A, Hmox-1 protein in the glomeruli isolated from the Hmox1−/− rats was completely absent, whereas in the glomeruli of the Hmox1+/− animals, there was a significant reduction in Hmox-1 protein compared with that in the WT animals. Constitutive DAF expression was significantly reduced at both the mRNA and protein levels in the glomeruli of both the Hmox1−/− rats (Figure 2, B and C) and the Hmox1+/− rats (Figure 2, A and B). A significant increase in constitutive DAF expression was observed in the glomeruli isolated from the GECHO-1 rats compared to those from the WT rats, supporting the regulation of DAF by HO-1 (Figure 2D). We next examined whether complement deposition and extent of injury (proteinuria) in the GECHO-1 rats are reduced in a model of anti-GBM antibody–mediated, complement-dependent glomerular injury. At the time point chosen for the studies performed (day 7), proteinuria was associated with minimal inflammatory cell infiltration in the glomeruli, segmental areas of glomerular hypercellularity, and no crescent formation or scaring (Figure 3). Quantification of injury by the assessment of changes in glomerular size and cellularity (nuclear count) revealed no differences in glomerular size between any two groups of animals, and a statistically significant increase in nuclear count in the glomeruli of the WT rats treated with anti-GBM compared with those in all of the other groups (Table 2). In the GECHO-1 rats treated with anti-GBM antibody, albuminuria (ratio of urinary albumin to urinary creatinine) was no different from that in the GECHO-1 or WT controls treated with nonimmune rabbit serum (Figure 4A), but was significantly increased compared with that in the anti-GBM–treated WT rats. No differences in serum creatinine values were observed between any two groups of animals (Table 3). As shown in Figure 4B, a marked reduction in glomerular C3b deposition was observed in the glomeruli of the GECHO-1 rats treated with anti-GBM antibody compared with that in the antibody-treated WT controls. This observation was corroborated by immunofluorescence staining for C3b deposition (Figure 4C). Staining for rabbit IgG ruled out differences in the extent of anti-GBM antibody deposition in the GBM (Figure 4C).Table 2Quantification of Glomerular Damage in WT and GECHO-1 Animals Treated with Nonimmune Rabbit Serum (Control) or Anti-GBM AntibodyGlomerular characteristicsWT controlWT anti-GBMGECHO-1 controlGECHO-1 anti-GBMGlomerular area, μm29944 ± 126511,410 ± 14939509 ± 790.39746 ± 607.8Nuclear count42.32 ± 4.01180.82 ± 9.744∗∗∗,††48.02 ± 2.2654.62 ± 4.466Data are expressed as means ± SD. n = 4. Statistical analyses were performed by analysis of variance and post hoc analysis by a Turkey test.∗∗∗P < 0.001 versus WT control; ††P < 0.01 versus GECHO-1 treated with anti-GBM.GBM, glomerular basement membrane; GECHO-1, heme oxygenase 1 overexpression targeted to glomerular epithelial cells; WT, wild-type. Open table in a new tab Figure 4Heme oxygenase (HO)-1 overexpression reduces complement component C3 fragment b (C3b) deposition and proteinuria after anti–glomerular basement membrane (GBM) treatment. Glomeruli were isolated from wild-type (WT) rats, rats with glomerular epithelial cell–targeted heme oxygenase 1 overexpression (GECHO-1) treated with anti-GBM, or WT and GECHO-1 controls that were treated with nonimmune rabbit serum. A: Urinary albumin (Ualb) excretion factored by urinary creatinine excretion (Uc) in WT or GECHO-1 rats treated with nonimmune rabbit serum (controls) and in WT or GECHO-1 rats treated with anti-GBM antibody. B: Total glomerular protein lysates were analyzed by Western blot analysis for C3b and decay accelerating factor (DAF) protein levels. Representative Western blot from three independent experiments. C: Immunofluorescence staining for C3b or rabbit IgG in glomeruli of WT rats treated with nonimmune rabbit serum or anti-GBM antibody and glomeruli of GECHO-1 rats treated with anti-GBM antibody. Data are expressed as means ± SEM. n = 4 per group. ∗P < 0.05 versus WT control; †P < 0.05 versus WT treated with anti-GBM antibody. Scale bars = 50 μm (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 3Serum Creatinine Values in WT or GECHO-1 Rats Treated with Nonimmune Rabbit Serum (Control) or Anti-GBM SerumWT controlWT anti-GBMGECHO-1 controlGECHO-1 anti-GBMSerum creatinine, mg/dL0.4167 ± 0.18040.5556 ± 0.24060.3125 ± 0.2250.3125 ± 0.4253Data are expressed as means ± SD. n = 4.GBM, glomerular basement membrane; GECHO-1, heme oxygenase 1 overexpression targeted to glomerular epithelial cells; WT, wild-type. Open table in a new tab Data are expressed as means ± SD. n = 4. Statistical analyses were performed by analysis of variance and post hoc analysis by a Turkey test. ∗∗∗P < 0.001 versus WT control; ††P < 0.01 versus GECHO-1 treated with anti-GBM. GBM, glomerular basement membrane; GECHO-1, heme oxygenase 1 overexpression targeted to glomerular epithelial cells; WT, wild-type. Data are expressed as means ± SD. n = 4. GBM, glomerular basement membrane; GECHO-1, heme oxygenase 1 overexpression targeted to glomerular epithelial cells; WT, wild-type. The observation that Hmox-1 overexpression up-regulated DAF raised the question of whether DAF up-regulation also occurs in response to Hmox-1 induction. We initially examined the effect of the natural Hmox substrate/inducer, hemin. The hemin concentrations chosen (50 to 400 μmol/L) reflect those likely attained within the glomerular milieu in hematuric forms of glomerular injury owing to release of heme from circulating red blood cells undergoing membrane damage while passing through injured glomeruli.16Glagov S. Zarins C. Giddens D.P. Ku D.N. Hemodynamics and atherosclerosis. Insights and perspectives gained from studies of human arteries.Arch Pathol Lab Med. 1988; 112: 1018-1031PubMed Google Scholar Hemin (50 to 400 μmol/L) increased glomerular DAF protein levels in a concentration-dependent manner (Figure 5A). A statistically significant increase in DAF protein was observed at concentrations of 200 and 400 μmol