Elias A. Lianos

Heme Oxygenase-2 Deficiency Contributes to Diabetes-Mediated Increase in Superoxide Anion and Renal Dysfunction

Heme oxygenase-1 (HO-1) and -2 play an important role in cytoprotection and are physiologic regulators of heme-dependent protein synthesis in renal tissues. The impact of HO-2 deletion comparing hyperglycemic HO-2 (+/+) mice and HO-2 knockout (-/-) mice was examined. Hyperglycemia was induced by streptozotocin (STZ) injection, and its effect on renal HO-1/HO-2 protein, HO activity, and creatinine levels were assessed. The effect of HO induction using systemic administration of the HO inducers heme or cobalt protoporphyrin and the effect of HO inhibition using systemic administration of the HO inhibitor tin mesoporphyrin also were assessed in STZ-treated mice. In STZ-treated HO-2 (-/-) mice, there was marked renal functional impairment as reflected by an increase in plasma creatinine, associated with acute tubular damage and microvascular pathology as compared with HO-2 (+/+). In these animals, HO activity was decreased with a concomitant increase in superoxide anion. Upregulation of HO-1 in HO-2 (-/-) mice by weekly administration of cobalt protoporphyrin prevented the increase in plasma creatinine levels and tubulointerstitial and microvascular pathology. Inhibition of HO activity by administration of tin mesoporphyrin accentuated superoxide production and increased creatinine levels in hyperglycemic HO-2 (-/-) mice. In conclusion, HO-2 deficiency enhanced STZ-induced renal dysfunction and morphologic injury and HO-1 upregulation in HO-2 (-/-) mouse rescue and prevented the morphologic damage. These observations indicate that HO activity is essential in preserving renal function and morphology in STZ-induced diabetic mice probably via mitigation of concomitant oxidative stress.

Effects of All-Trans-Retinoic Acid (atRA) on Inducible Nitric Oxide Synthase (iNOS) Activity and Transforming Growth Factor Beta-1 Production in Experimental Anti-GBM Antibody-Mediated Glomerulonephritis

Sustained high output release of Nitric oxide (NO) as result of activation of inducible nitric oxide synthase (iNOS), and increased production of the antiproliferative/profibrotic cytokine transforming growth factor-β1 (TGF-β1) are well documented in glomerulonephritis. Modulation of iNOS activity and of TGF-β1 production can therefore be viewed as anti-inflammatory strategies. The present study employed all-trans retinoic acid (atRA) which is known to have anti-inflammatory effects and to modulate expression of iNOS and TGF-β1, in order to explore its effect on iNOS enzyme activity and TGF-β1 production in anti-GBM antibody induced glomerulonephritis. Glomerulonephritis was induced in Lewis rats by injection of anti-GBM antibody. A group of nephritic rats were given daily administration of atRA for 14–16 days. Extent of proteinuria was assessed by measuring urine protein and creatinine excretion. iNOS enzyme activity was measured by calculating conversion of L[14C]arginine to L-[14C]citrulline in glomerular protein lysates. Levels of TGF-β1 in glomerular protein lysates were measured by quantitative ELISA. Levels of proliferating nuclear antigen (PCNA), TGF-β receptor II (TGFβ-RII), and fibronectin were assessed by Western blot analysis. Glomerular iNOS activity in atRA treated nephritic animals was attenuated in comparison to that in nephritic controls that were not. Glomerular expression of PCNA was also reduced. Levels of TGF-β1 were increased in glomeruli of atRA treated nephritic animals. In these animals, there was no change in glomerular levels of TGF-β receptor II (TGFβ-RII) or fibronectin, and there was no reduction in urine protein excretion. These results suggest that atRA attenuates iNOS activity and proliferation in glomeruli of nephritic animals. The failure of atRA treatment to reduce proteinuria could be due to the increase in TGF-β1 levels and to inhibition of iNOS-driven NO production.

Protective Effects of Heme-Oxygenase Expression in Cyclosporine A - Induced Injury

Cyclosporine A (CsA) is the immunosuppressant of first choice in allotransplantation. Its use is associated with side effects of nephrotoxicity and neurotoxicity, which are among the most prominent. This study was undertaken to explore whether expression and activity of heme oxygenase (HO), the rate-limiting enzyme in heme degradation, is altered in a rat model of CsA-induced injury. Male Sprague Dawley rats were divided into four groups and treated for 21 days. Group I (control) was injected with olive oil (vehicle), group II with CsA (15 mg/kg/day), group III with CsA and the HO inhibitor stannous mesoporphyrin (SnMP) (30 micromol/kg/day) and group IV with one dose of the HO inducer cobalt protoporphyrin (CoPP) 5 mg/100 or heme (10 mg/kg body weight), three days after onset of CsA treatment. Renal tissue was processed for light microscopy, and for HO-1 enzyme activity, assay and for Western blot analysis. In CsA-treated rats there was histological evidence of tubulointerstitial scarring. HO-1 was undetectable in CsA-treated rats compared to control while there was no change in HO-2. In animals treated with a combination of CsA and SnMP, HO-1 activity was further reduced. In animals treated with a combination of CsA and CoPP, HO-1 protein levels were partially restored. These observations indicate that downregulation of HO-1 expression by CsA could be one mechanism underlying CsA-induced toxicity. The CsA-induced decrease in HO-1 expression is partial and restorable, and attempts to preserve HO levels may attenuate CsA toxicity.

Heme Oxygenase-1 Localization in the Rat Nephron

Background/Aims: Renal tubules undergo oxidative injury in various nephropathies. It is unknown whether tubular cells possess mechanisms to attenuate this form of injury. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, may provide such a mechanism by reducing levels of free heme, a prooxidant molecule, and by limiting activity of heme-containing prooxidant enzymes. Determination of the distribution of HO-1 in the nephron may identify those segments where HO-1 can afford protection against oxidative injury. Methods: Rats were injected subcutaneously with two different inducers of HO-1: Stannous chloride and cobalt protoporphyrin. At completion of injections, frozen sections of kidneys were stained for HO-1 using a biotin-conjugated monoclonal anti-HO-1 antibody. To identify the origin of tubules staining positive for HO-1, Tetragonolobus purpureas (TP)-derived lectin and Arachnis hypogaea (AH)-derived lectin were applied to sequential sections of the kidney cortex. Results: In rats injected with either HO-1 inducer, HO-1 was immunolocalized in tubules but not in glomeruli. Staining of sequential sections with TP-derived lectin, which binds mainly to proximal tubular cells, was negative in the tubules that stained positive for HO-1. Staining of sequential sections with AH-derived lectin, which binds mainly to distal and collecting tubular cells, was positive in those tubules that were also positive for HO-1. Conclusion: In kidneys of rats injected with inducers of HO-1, distal and collecting tubular cells were identified as the main segments of the nephron that express HO-1. We suggest that the distal nephron, by expressing HO-1, may be less vulnerable to oxidative injury.

Differential Nephron HO-1 Expression following Glomerular Epithelial Cell Injury

Background: In proteinuria of glomerular origin there is upregulation of heme-oxygenase (HO), the rate-limiting enzyme of heme degradation, in the nephron in a segment-specific manner. To better characterize this phenomenon, we employed a model of proteinuria resulting from disruption of the glomerular capillary permeability barrier to protein by administration of the glomerular epithelial cell toxin puromycin aminonucleoside (PAN) to rats. In this model, we assessed nephron distribution of the expression of the inducible HO isoform, HO-1, and the role of free radicals in modulating HO-1 expression. Methods: Rats were injected with either vehicle (dimethyl sulfoxide) or PAN or the spin trap free radical stabilizer α-phenyl-N-tert butyl nitrone (PBN), or with both PAN and PBN. Ten days following the PAN injection, urine protein, creatinine, nitric oxide (NO) and malonyldialdehyde (MDA) were measured. Kidney sections and protein lysates were assessed for changes in HO-1 expression by immunohistochemistry and Western blot analysis. Results: In control animals (DMSO or PBN alone) there was no proteinuria and very weak or absent HO-1 staining in nephron segments. PAN treatment induced proteinuria and increased urine MDA excretion. In these animals, there was a robust HO-1 expression mainly in tubules and in glomerular parietal but not visceral epithelial cells. Unilateral ureteral obstruction to interrupt glomerular filtration in animals treated with PAN abrogated tubular HO-1 expression in the kidney ipsilateral to the obstruction. Administration of PBN to PAN-treated animals reduced proteinuria and MDA excretion while it markedly augmented tubular HO-1 expression. This augmentation was prominent in tubular cells of the inner cortex/outer medulla. Conclusions: These observations indicate that upregulation of nephron HO-1 following disruption of the glomerular permeability barrier occurs at sites downstream of this barrier and is mediated by a filtered HO-1 inducer(s). Scavenging of free radicals potentiates the effect of this inducer and unmasks nephron segments most and least capable of upregulating HO-1.

Regulation of human P2X1 promoter activity by β helix–loop–helix factors in smooth muscle cells

We isolated and characterized genomic clones of the human P2X1 receptor (hP2X1) gene in an effort to understand its tissue specific expression. The hP2X1 gene contains 12 exons spanning 20 kb, with exon sizes ranging from 59 to 143 bp. A 385 bp upstream fragment promoted hP2X1 gene expression in smooth muscle (A7R5 and primary trachealis) and fibroblast (NIH3T3) cell lines, and mutation of a consensus E box sequence (CACCTG) within this fragment (-340 to -345) did not alter basal promoter activity. However, co-transfected bHLH factors regulated activity of the 385 bp minimal P2X1 promoter in a tissue-specific manner. E12 expression inhibited and ITF2b augmented activity in A7R5 cells, but had no effect in NIH3T3 cells. ITF2a, Myo-D, and Id1 proteins had no effect on either cell line, but co-expression of ITF2a blocked E12 inhibition in A7R5 cells, while ITF2b failed to reverse the inhibition. Northern analysis of A7R5 RNA identified high levels of E12 and ITF2b transcripts, and gel shift assays using A7R5 and NIH3T3 nuclear extracts indicated the formation of a protein-DNA complex with an oligonucleotide corresponding to -330 and -348, which was abolished by base substitutions within the E box motif. Our results identify a critical E box response element in the hP2X1 promoter that binds bHLH factors and demonstrate smooth muscle specific transcriptional regulation by E proteins.

Mechanisms of HO-1 mediated attenuation of renal immune injury: a gene profiling study.

Using a mouse model of immune injury directed against the renal glomerular vasculature and resembling human forms of glomerulonephritis (GN), we assessed the effect of targeted expression of the cytoprotective enzyme heme oxygenase (HO)-1. A human (h) HO-1 complementary DNAN (cDNA) sequence was targeted to glomerular epithelial cells (GECs) using a GEC-specific murine nephrin promoter. Injury by administration of antibody against the glomerular basement membrane (anti-GBM) to transgenic (TG) mice with GEC-targeted hHO-1 was attenuated compared with wild-type (WT) controls. To explore changes in the expression of genes that could mediate this salutary effect, we performed gene expression profiling using a microarray analysis of RNA isolated from the renal cortex of WT or TG mice with or without anti-GBM antibody-induced injury. Significant increases in expression were detected in 9 major histocompatibility complex (MHC)-class II genes, 2 interferon-γ (IFN-γ)-inducible guanosine triphosphate (GTP)ases, and 3 genes of the ubiquitin-proteasome system. The increase in MHC-class II and proteasome gene expression in TG mice with injury was validated by real-time polymerase chain reaction (PCR) or Western blot analysis. The observations point to novel mechanisms underlying the cytoprotective effect of HO-1 in renal immune injury.

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.