Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes.

Overexpression of the inducible heme oxygenase (HO-1) isoform in visceral renal glomerular epithelial cells (podocytes) using in vivo transgenesis methods was shown to increase glomerular expression of the complement regulatory protein decay-accelerating factor (DAF, CD55) and reduce complement activation/deposition in a rat model of immune-mediated injury. In this preliminary study, we assessed whether constitutively expressed HO-1 regulates CD55 expression in cultured rat podocytes. We employed methods of flow cytometry, quantitative (q) RT-qPCR and post-transcriptional HO-1 gene silencing (HO-1 interfering RNA, RNAi), to assess changes in constitutive (basal) levels of podocyte HO-1 and CD55 mRNA in cultured rat podocytes. Additionally, the effect of the HO-1 inducer, heme, on HO-1 and CD55 expression was assessed. Results indicate that rat podocytes constitutively express HO-1 and DAF and that the HO-1 inducer, heme, increases both HO-1 and DAF expression. HO-1 gene silencing using RNA interference (RNAi) is feasible but the effect on constitutive CD55 transcription is inconsistent. These observations are relevant to conditions of podocyte exposure to heme that can activate the complementary cascade, as may occur in systemic or intraglomerular hemolysis.

Metalloporphyrins Reduce Proteinuria in Podocyte Immune Injury: The Role of Metal and Porphyrin Moieties.

Depending on their central metal atom, metalloporphyrins (MPs) can attenuate or exacerbate the severity of immune-mediated kidney injury, and this has been attributed to the induction or inhibition of heme oxygenase (HO) activity, particularly the inducible isoform (HO-1) of this enzyme. The role of central metal or porphyrin moieties in determining the efficacy of MPs to attenuate injury, as well as mechanisms underlying this effect, have not been assessed. Using an antibody-mediated complement-dependent model of injury directed against rat visceral glomerular epithelial cells (podocytes) and two MPs (FePPIX, CoPPIX) that induce both HO-1 expression and HO enzymatic activity in vivo but differ in their chelated metal, we assessed their efficacy in reducing albuminuria. Podocyte injury was induced using rabbit immune serum raised against the rat podocyte antigen, Fx1A, and containing an anti-Fx1A antibody that activates complement at sites of binding. FePPIX or CoPPIX were injected intraperitoneally (5 mg/kg) 24 h before administration of the anti-Fx1A serum and on days 1, 3, 6, and 10 thereafter. Upon completion of urine collection on day 14, the kidney cortex was obtained for histopathology and isolation of glomeruli, from which total protein extracts were obtained. Target proteins were analyzed by capillary-based separation and immunodetection (Western blot analysis). Both MPs had comparable efficacy in reducing albuminuria in males, but the efficacy of CoPPIX was superior in female rats. The metal-free protoporphyrin, PPIX, had minimal or no effect on urine albumin excretion. CoPPIX was also the most potent MP in inducing glomerular HO-1, reducing complement deposition, and preserving the expression of the complement regulatory protein (CRP) CD55 but not that of CD59, the expression of which was reduced by both MPs. These observations demonstrate that the metal moiety of HO-1-inducing MPs plays an important role in reducing proteinuria via mechanisms involving reduced complement deposition and independently of an effect on CRPs.

Immune-Related Functions of Heme Oxygenase-1.

Heme oxygenase (HO)-1 is a well-known cytoprotective enzyme due to its enzymatic action, which involves the catalysis of heme into anti-apoptotic and antioxidant molecules such as bilirubin, biliverdin and CO [...].

Metalloporphyrins as Tools for Deciphering the Role of Heme Oxygenase in Renal Immune Injury.

Renal immune injury is a frequent cause of end-stage renal disease, and, despite the progress made in understanding underlying pathogenetic mechanisms, current treatments to preserve renal function continue to be based mainly on systemic immunosuppression. Small molecules, naturally occurring biologic agents, show considerable promise in acting as disease modifiers and may provide novel therapeutic leads. Certain naturally occurring or synthetic Metalloporphyrins (Mps) can act as disease modifiers by increasing heme oxygenase (HO) enzymatic activity and/or synthesis of the inducible HO isoform (HO-1). Depending on the metal moiety of the Mp employed, these effects may occur in tandem or can be discordant (increased HO-1 synthesis but inhibition of enzyme activity). This review discusses effects of Mps, with varying redox-active transitional metals and cyclic porphyrin cores, on mechanisms underlying pathogenesis and outcomes of renal immune injury.

Effect of Heme Oxygenase-1 Depletion on Complement Regulatory Proteins Expression in the Rat.

Heme oxygenase has been implicated in the regulation of various immune responses including complement activation. Using a transgenic rat model of HO-1 depletion, the present study assessed the effect of HO-1 absence on the expression of complement regulatory proteins: decay accelerating factor (DAF), CR1-related gene/protein Y (Crry) and CD59, which act to attenuate complement activation. Protein expression was assessed by immunohistochemistry in kidney, liver, lung and spleen tissues. DAF protein was reduced in all tissues retrieved from rats lacking HO-1 (Hmox1-/-) apart from spleen tissue sections. Crry protein was also reduced, but only in Hmox1-/- kidney and liver tissue. C3b staining was augmented in the kidney and spleen from Hmox1-/- rats, suggesting that the decrease of DAF and Crry was sufficient to increase C3b deposition. The observations support an important role of HO-1 as a regulator of the complement system.

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli.

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX-) or HPX-containing (HPX+) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX- sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.

Generation of a novel decay accelerating factor (DAF) knock-out rat model using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9), genome editing.

Decay accelerating factor (DAF), a key complement activation control protein, is a 70 kDa membrane bound glycoprotein which controls extent of formation of the C3 and C5 convertases by accelerating their decay. Using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9) genome editing we generated a novel DAF deficient (Daf-/-) rat model. The present study describes the renal and extrarenal phenotype of this model and assesses renal response to complement-dependent injury induced by administration of a complement-fixing antibody (anti-Fx1A) against the glomerular epithelial cell (podocyte). Rats generated were healthy, viable and able to reproduce normally. Complete absence of DAF was documented in renal as well as extra-renal tissues at both protein and mRNA level compared to Daf+/+ rats. Renal histology in Daf-/- rats showed no differences regarding glomerular or tubulointerstitial pathology compared to Daf+/+ rats. Moreover, there was no difference in urine protein excretion (ratio of urine albumin to creatinine) or in serum creatinine and urea levels. In Daf-/- rats, proteinuria was significantly increased following binding of anti-Fx1A antibody to podocytes while increased C3b deposition was observed. The DAF knock-out rat model developed validates the role of this complement cascade regulator in immune-mediated podocyte injury. Given the increasing role of dysregulated complement activation in various forms of kidney disease and the fact that the rat is the preferred animal for renal pathophysiology studies, the rat DAF deficient model may serve as a useful tool to study the role of this complement activation regulator in complement-dependent forms of kidney injury.

Regulation of Complement Activation by Heme Oxygenase-1 (HO-1) in Kidney Injury.

Heme oxygenase is a cytoprotective enzyme with strong antioxidant and anti-apoptotic properties. Its cytoprotective role is mainly attributed to its enzymatic activity, which involves the degradation of heme to biliverdin with simultaneous release of carbon monoxide (CO). Recent studies uncovered a new cytoprotective role for heme oxygenase-1 (HO-1) by identifying a regulatory role on the complement control protein decay-accelerating factor. This is a key complement regulatory protein preventing dysregulation or overactivation of complement cascades that can cause kidney injury. Cell-specific targeting of HO-1 induction may, therefore, be a novel approach to attenuate complement-dependent forms of kidney disease.

Podocyte-targeted Heme Oxygenase (HO)-1 overexpression exacerbates age-related pathology in the rat kidney.

Although Heme Oxygenase-1 (HO-1) induction in various forms of kidney injury is protective, its role in age-related renal pathology is unknown. In the ageing kidney there is nephron loss and lesions of focal glomerulosclerosis, interstitial fibrosis, tubular atrophy and arteriolosclerosis. Underlying mechanisms include podocyte (visceral glomerular epithelial cell/GEC) injury. To assess whether HO-1 can attenuate ageing - related lesions, rats with GEC-targeted HO-1 overexpression (GECHO-1 rats) were generated using a Sleeping Beauty (SB) transposon system and extent of lesions over a 12-month period were assessed and compared to those in age-matched wild-type (WT) controls. GECHO-1 rats older than 6 months developed albuminuria that was detectable at 6 months and became significantly higher compared to age-matched WT controls at 12 months. In GECHO-1 rats, lesions of focal segmental and global glomerulosclerosis as well as tubulointerstitial lesions were prominent while podocytes were edematous with areas of foot process effacement and glomerular basement membrane thickening and wrinkling. GECHO-1 rats also developed hemoglobinuria and hemosiderinuria associated with marked tubular hemosiderin deposition and HO-1 induction, while there was depletion of splenic iron stores. Kidney injury was of sufficient magnitude to increase serum lactate dehydrogenase (LDH) and was oxidative in nature as shown by increased expression of 8-hydroxydeoxyguanosine (8-OHdg, a byproduct of oxidative DNA damage) in podocytes and tubular epithelial cells. These observations highlight a detrimental effect of podocyte-targeted HO-1 overexpression on ageing-related renal pathology and point to increased renal iron deposition as a putative underlying mechanism.

Induction of decay accelerating factor and membrane cofactor protein by resveratrol attenuates complement deposition in human coronary artery endothelial cells.

The involvement of complement activation in various forms of cardiovascular disease renders it an important factor for disease progression and therapeutic intervention. The protective effect of resveratrol against cardiovascular disease via moderate red wine consumption has been established but the exact mechanisms are still under investigation. The current study utilised human coronary artery endothelial cells (HCAECs) in order to assess the extent to which the protective effect of resveratrol, at concentrations present in red wine, can be attributed to the upregulation of complement regulatory proteins through heme-oxygenase (HO)-1 induction. Resveratrol at concentrations as low as 0.001 µΜ increased HO-1 expression as well as membrane cofactor protein (MCP, CD46) and decay-accelerating factor (DAF, CD55) expression with no-effect on CD59. Silencing of HO-1 expression by HO-1 siRNAs abrogated both DAF and MCP protein expression with no effect on CD59. Resveratrol-mediated induction of DAF and MCP reduced C3b deposition following incubation of HCAECs with 10% normal human serum or normal rat serum as a source of complement. Incubation of HCAECs, with either a DAF blocking antibody or following transfection with HO-1 siRNAs, in the presence of 10% normal rat serum increased C3b deposition, indicating that both DAF and HO-1 are required for C3b reduction. These observations support a novel mechanism for the protective effect of resveratrol against cardiovascular disease and confirm the important role of HO-1 in the regulation of the complement cascade.

Data on characterization of metalloporphyrin-mediated HO-1 and DAF induction in rat glomeruli and podocytes.

The data presented pertain to a research article titled "Heme Oxygenase 1 Up-Regulates Glomerular Decay Accelerating Factor Expression and Minimizes Complement Deposition and Injury" (Detsika et al., 2016). The present work provides additional data on induction and immunolocalization of heme oxygenase (HO)-1 (an antioxidant enzyme) and decay-accelerating factor (DAF) (a complement activation inhibitor) in isolated rat glomeruli and in glomerular epithelial cells (podocytes) in response to Iron Protoporphyrin IX (FePP, heme), and to non-iron protoporphyrins (PPs) with varying metal functionalities (ZnPP, SnPP), including a metal-devoid PP. Induction and immuno-localization of HO-1 and DAF in response to these metalloporphyrins (MP) were assessed using western blot analyses and confocal microscopy in isolated glomeruli and in cultured podocytes. These analyses identified podocytes as a major localization site of HO-1 and DAF induction in response to the aforementioned MPs. Effects of these MPs on a key glomerular structural protein, Nephrin, are also reported. The data identify MPs most and least capable of inducing DAF and reducing Nephrin expression and provide clues into expected outcomes of animal studies assessing MP efficacy in upregulating the cytoprotective proteins HO-1 and DAF.

Phenotypic characterization of a novel HO-1 depletion model in the rat.

Although the protective role of HO-1 induction in various forms of kidney disease is well established, mechanisms other than heme catabolism to biliverdin, bilirubin and carbon monoxide have recently been identified. Unraveling these mechanisms requires the generation of appropriate animal models. The present study describes the generation of a HO-1 deficient Hmox1 -/- rat model and characterizes its renal and extrarenal phenotype. Hmox1 -/- rats had growth retardation and splenomegaly compared to their Hmox1 +/+ littermates. Focal segmental glomerulosclerosis-type lesions and interstitial inflammatory infiltrates were prominent morphologic findings and were associated with increased blood urea nitrogen, serum creatinine and albuminuria. There was no increase in iron deposition in glomeruli, tubules or interstitium. Iron deposition in spleen and liver was reduced. Electron microscopic examination of glomeruli revealed edematous podocytes with scant areas of foot process effacement but otherwise well preserved processes and slit-diaphragms. Of the filtration barrier proteins examined, ß-catenin expression was markedly reduced both in glomeruli and extrarenal tissues. Since the rat is the preferred laboratory animal in experimental physiology and pathophysiology, the rat model of HO-1 deficiency may provide a novel tool for investigation of the role of this enzyme in renal function and disease.

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

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.

Glomerular Epithelial Cells-Targeted Heme Oxygenase-1 Over Expression in the Rat: Attenuation of Proteinuria in Secondary But Not Primary Injury.

BACKGROUND/AIMS: Induction of heme oxygenase 1 (HO-1) in glomerular epithelial cells (GEC) in response to injury is poor and this may be a disadvantage. We, therefore, explored whether HO-1 overexpression in GEC can reduce proteinuria induced by puromycin aminonucleoside (PAN) or in anti-glomerular basement membrane (GBM) antibody (Ab)-mediated glomerulonephritis (GN). METHODS: HO-1 overexpression in GEC (GECHO-1) of Sprague-Dawley rats was achieved by targeting a FLAG-human (h) HO-1 using transposon-mediated transgenesis. Direct GEC injury was induced by a single injection of PAN. GN was induced by administration of an anti-rat GBM Ab and macrophage infiltration in glomeruli was assessed by immunohistochemistry and western blot analysis, which was also used to assess glomerular nephrin expression. RESULTS: In GECHO-1 rats, FLAG-hHO-1 transprotein was co-immunolocalized with nephrin. Baseline glomerular HO-1 protein levels were higher in GECHO-1 compared to wild type (WT) rats. Administration of either PAN or anti-GBM Ab to WT rats increased glomerular HO-1 levels. Nephrin expression markedly decreased in glomeruli of WT or GECHO-1 rats treated with PAN. In anti-GBM Ab-treated WT rats, nephrin expression also decreased. In contrast, it was preserved in anti-GBM Ab-treated GECHO-1 rats. In these, macrophage infiltration in glomeruli and the ratio of urine albumin to urine creatinine (Ualb/Ucreat) were markedly reduced. There was no difference in Ualb/Ucreat between WT and GECHO-1 rats treated with PAN. CONCLUSION: Depending on the type of injury, HO-1 overexpression in GEC may or may not reduce proteinuria. Reduced macrophage infiltration and preservation of nephrin expression are putative mechanisms underlying the protective effect of HO-1 overexpression following immune injury.

High Glucose Impairs Insulin Signaling in the Glomerulus: An In Vitro and Ex Vivo Approach.

OBJECTIVE: Chronic hyperglycaemia, as seen in type II diabetes, results in both morphological and functional impairments of podocytes in the kidney. We investigated the effects of high glucose (HG) on the insulin signaling pathway, focusing on cell survival and apoptotic markers, in immortalized human glomerular cells (HGEC; podocytes) and isolated glomeruli from healthy rats. METHODS AND FINDINGS: HGEC and isolated glomeruli were cultured for various time intervals under HG concentrations in the presence or absence of insulin. Our findings indicated that exposure of HGEC to HG led to downregulation of all insulin signaling markers tested (IR, p-IR, IRS-1, p-Akt, p-Fox01,03), as well as to increased sensitivity to apoptosis (as seen by increased PARP cleavage, Casp3 activation and DNA fragmentation). Short insulin pulse caused upregulation of insulin signaling markers (IR, p-IR, p-Akt, p-Fox01,03) in a greater extent in normoglycaemic cells compared to hyperglycaemic cells and for the case of p-Akt, in a PI3K-dependent manner. IRS-1 phosphorylation of HG-treated podocytes was negatively regulated, favoring serine versus tyrosine residues. Prolonged insulin treatment caused a significant decrease of IR levels, while alterations in glucose concentrations for various time intervals demonstrated changes of IR, p-IR and p-Akt levels, suggesting that the IR signaling pathway is regulated by glucose levels. Finally, HG exerted similar effects in isolated glomeruli. CONCLUSIONS: These results suggest that HG compromises the insulin signaling pathway in the glomerulus, promoting a proapoptotic environment, with a possible critical step for this malfunction lying at the level of IRS-1 phosphorylation; thus we herein demonstrate glomerular insulin signaling as another target for investigation for the prevention and/ or treatment of diabetic nephropathy.

Effect of Heme Oxygenase-1 Deficiency on Glomerular Proteomics.

BACKGROUND: The cytoprotective effect of heme oxygenase (HO)-1 in various forms of renal glomerular injury is established. However, little is known on the role of HO-1 in preserving glomerular structural/functional integrity in the absence of injury. The present study addressed this question in HO-1-deficient rats. METHODS: HO-1-deficient rats were generated using zinc finger nuclease-mediated HO-1 gene (Hmox1) disruption and studied. Glomeruli were isolated from HO-1-deficient (Hmox1-/-) rats and their wild type (WT) littermates for proteomic analysis. RESULTS: Glomerular lesions were characterized and differentially expressed proteins important for preserving integrity of the glomerular filtration barrier were identified. HO-1-deficient (Hmox1-/-) rats developed albuminuria with decreased glomerular filtration rate. In albuminuric rats, there were lesions resembling focal and segmental glomerulosclerosis (FSGS). Western blot analysis of the integral slit diaphragm proteins, nephrin and podocin revealed a significant decrease in nephrin, with no change in podocin. Proteomic analysis of glomerular protein lysates from Hmox1-/- and WT rats revealed differential expression of proteins previously linked with FSGS pathogenesis. Specifically, α-actinin-4, actin related protein 3, cytokeratins and novel candidates including transgelin-2 and lamins. Bioinformatic analysis predicted the upregulation of pathways implicated in platelet aggregation and fibrin clot formation. CONCLUSION: HO-1 is a putative regulator of proteins important in preserving glomerular structural stability and integrity, and in minimizing the activity of proinflammatory pathways.

Autophagy, Innate Immunity and Tissue Repair in Acute Kidney Injury.

Kidney is a vital organ with high energy demands to actively maintain plasma hemodynamics, electrolytes and water homeostasis. Among the nephron segments, the renal tubular epithelium is endowed with high mitochondria density for their function in active transport. Acute kidney injury (AKI) is an important clinical syndrome and a global public health issue with high mortality rate and socioeconomic burden due to lack of effective therapy. AKI results in acute cell death and necrosis of renal tubule epithelial cells accompanied with leakage of tubular fluid and inflammation. The inflammatory immune response triggered by the tubular cell death, mitochondrial damage, associative oxidative stress, and the release of many tissue damage factors have been identified as key elements driving the pathophysiology of AKI. Autophagy, the cellular mechanism that removes damaged organelles via lysosome-mediated degradation, had been proposed to be renoprotective. An in-depth understanding of the intricate interplay between autophagy and innate immune response, and their roles in AKI pathology could lead to novel therapies in AKI. This review addresses the current pathophysiology of AKI in aspects of mitochondrial dysfunction, innate immunity, and molecular mechanisms of autophagy. Recent advances in renal tissue regeneration and potential therapeutic interventions are also discussed.

Presence of an HO-1 expression threshold in renal glomeruli.

This article reports data describing HO-1 expression patterns of heme oxygenase (HO)-1 in isolated rat glomeruli and in cultured glomerular epithelial cells (GEC) in response to its natural substrate heme. Qualitative and quantitative data are presented to support presence of a HO-1 expression threshold in glomeruli but not in GEC. Interpretation of our data and further insight into HO-1 expression pattern in glomeruli may be found in 'HO-1 expression control in the rat glomerulus' [1].

HO-1 expression control in the rat glomerulus.

The differential localization of HO-1 in renal cells under conditions of injury, and the demonstration that exaggerated HO-1 expression can have detrimental rather than beneficial effects, raises the question of whether HO-1 expression in these cells is subject to control. The present study identifies a unique HO-1 expression pattern in the renal glomerulus indicative of presence of HO-1 expression control following prolonged HO-1 induction. HO-1 and HO-2 expression in response to the natural HO substrate/inducer Fe(++) protoporphyrin (PP) IX (hemin) was assessed in normal rat glomeruli. Following 18 h incubations with hemin (0-200 µM), HO-1 expression increased in a concentration-dependent manner and via a hemopexin (HPX) independent mechanism with no effect on HO-2. In incubations with higher hemin concentrations (400 µM), likely to be encountered in hemolytic disorders, HO-1 expression, decreased. This was preceded by a prolonged and sustained increase in HO-1 protein and was independent of the Fe(++) moiety as incubations with Cobalt protoporphyrin (CoPP) resulted in an identical expression pattern. The decrease of HO-1 protein could not be accounted for by proteasomal degradation since it was not reversed in co-incubations with hemin and the proteasome inhibitor, MG132, at concentrations sufficient to increase HO-1 glomerular content when used alone. Moreover, in the presence of MG132, a decrease of HO-1 expression also occurred at 100 and 200 µM hemin. The effect of MG132 was mimicked by two additional mechanistically different approaches which also raised HO-1 content: a) co-incubations of hemin with ZnPP which increased HO-1 protein when used alone, and b) glomerular HO-1 over expression achieved by SB transposon mediated transgenesis. In contrast, the decrease in HO-1 levels observed at high hemin concentrations was reversed in co-incubations with hemin and SnPP, which reduced HO-1 content when used alone. Expression of NF-E2 related factor 2 (Nrf2) protein, which mediates HO-1 induction in response to hemin, had a similar expression pattern with that of HO-1 protein indicating involvement of Nrf2 in the response of HO-1 to hemin. The above observations indicate presence of a HO-1 expression control mechanism in the glomerulus that may serve to protect it against potentially detrimental effects of exaggerated HO-1 expression.