Management of oxidative stress by heme oxygenase-1 in cisplatin-induced toxicity in renal tubular cells

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, alpha-tocopherol (TOCO) and N-acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.     

Stabilization of mast cells by heme oxygenase-1: an anti-inflammatory role

This study examined the role of bilirubin in heme oxygenase (HO)-1-mediated amelioration of mast cell (MC)-elicited inflammatory responses. Pretreatment of rats with an intraperitoneal injection of hemin, an inducer of HO-1, evolved a marked induction of the enzyme in MCs. Intravital videomicroscopy revealed that hemin pretreatment attenuated compound 48/80-elicited degranulation of MCs and resultant leukocyte adhesion in venules. Superfusion with biliverdin or bilirubin, but not with carbon monoxide (CO), another product of the HO reaction, mimicked suppressive actions of the HO-1 induction on both the cell degranulation and leukocyte adhesion elicited by the stimulus, suggesting a requirement of the enzyme reaction to generate bilirubin in the inhibitory mechanisms. Such MC-desensitizing actions of bilirubin were observed in primary-cultured MCs and reproduced irrespective of the choice of stimuli, such as compound 48/80, calcium ionophore, and anti-IgE serum. Furthermore, MC-stabilizing effects of HO-1 were reproduced by the gene transfection of the enzyme into mastocytoma cell line RBL2H3. These results suggest that bilirubin generated through HO-1 serves as an anti-inflammatory substance that desensitizes MCs and ameliorates leukocyte recruitment.     

Influence of heme and heme oxygenase-1 transfection of pulmonary microvascular endothelium on oxidant generation and cGMP

Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 microM 2',7'-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 microM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 microM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 microM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 microM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide.     

Atrial natriuretic peptide elevates cGMP contents in glomeruli and in distal tubules of rat kidney

Effect of synthetic rat atrial natriuretic peptide (1-28) (ANP) on the cGMP content was studied using defined nephron segments of rat kidney. ANP elevates cGMP contents in glomeruli in a concentration and time-dependent manner. The increase of cGMP was observed in glomeruli, distal convoluted tubule (DCT) and cortical collecting tubule (CCT) (delta %; 279 +/- 35, 148 +/- 10 and 152 +/- 18, respectively), and no effect was observed in proximal convoluted (PCT) and straight tubule (PST). These results suggest that ANP may act directly on the tubular cells as well as glomeruli. In glomeruli, effects of ANP and carbamylcholine on cGMP contents were additive suggesting that these two agents may act on different receptors. Angiotensin II and norepinephrine failed to affect the ANP-induced cGMP production in the glomeruli.     

JunB and JunD regulate human heme oxygenase-1 gene expression in renal epithelial cells

Heme oxygenase-1 is a highly inducible gene, the product of which catalyzes breakdown of the prooxidant heme. The purpose of this study was to investigate the regulation of the human heme oxygenase-1 gene in renal epithelial cells. DNase I hyper-sensitivity studies identified three distal sites (HS-2, -3, and -4) corresponding to approximately -4.0, -7.2, and -9.2 kb, respectively, of the heme oxygenase-1 promoter in addition to one proximal region, HS-1, which we have shown previously to be an E box. In vivo dimethyl sulfate footprinting of the HS-2 region revealed six individual protected guanines. Two mutations within HS-2 combined with a third mutation of the proximal E box abolished hemin- and cadmium-driven heme oxygenase-1 promoter activation, suggesting that these three sites synergized for maximal heme oxygenase-1 induction. Jun proteins bound to the antioxidant response element in the HS-2 region in vitro and associated with the heme oxygenase-1 promoter in vivo. JunB and JunD contribute opposing effects; JunB activated whereas JunD repressed heme oxygenase-1 expression in human renal epithelial cells, results that were corroborated in junB(-)(/)(-) and junD(-)(/)(-) cells. We propose that heme oxygenase-1 induction is controlled by a dynamic interplay of regulatory proteins, and we provide new insights into the molecular control of the human heme oxygenase-1 gene.     

Atrial natriuretic peptide concentrations in pre-eclampsia.

The concentration of plasma immunoreactive atrial natriuretic peptide is positively associated with right atrial and pulmonary capillary wedge pressure, suggesting that blood volume and hence atrial pressure govern its release. Expansion of plasma volume is a central physiological adjustment in normal pregnancy. Conversely, pregnancies complicated by pre-eclampsia are associated with a reduction in plasma volume and central venous pressure. A study was therefore undertaken to test the hypothesis that plasma atrial natriuretic peptide concentrations are low in pre-eclampsia owing to deficient secretion. Concentrations of the peptide were measured by a specific radioimmunoassay. The mean plasma immunoreactive atrial natriuretic peptide concentration in healthy pregnant women (n = 22; third trimester) was higher (56 (1 SD 29) ng/l) than in 25 young, non-pregnant controls (37 (19) ng/l). Concentrations in patients suffering from mild pre-eclampsia (n = 9) were higher (127 (60) ng/l) than in normal pregnant women, and in patients with severe pre-eclampsia (n = 6) concentrations were higher still (392 (225) ng/l). Despite failure of plasma volume expansion and low central venous and pulmonary capillary wedge pressures in pre-eclampsia this condition is associated with greatly increased plasma concentrations of plasma immunoreactive atrial natriuretic peptide, which increase still further with the severity of the disease. These findings are clear evidence that atrial pressure may not be the principal determinant of the release of the natriuretic peptide in pre-eclampsia.     

Atrial natriuretic peptide and endothelin-3 target renal sodium-glucose cotransporter

Atrial natriuretic peptide (ANP) and endothelin (ET) are endogenous vasoactive factors that exert potent diuretic and natriuretic actions. We have previously shown that ANP and ET-3 act through an NO pathway to inhibit the sodium-glucose cotransporter (SGLT) in the intestine [Gonzalez Bosc LV, Elustondo PA, Ortiz MC, Vidal NA. Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine. Peptides 1997; 18: 1491-5; Gonzalez Bosc LV, Majowicz MP, Ortiz MC, Vidal NA. Effects of endothelin-3 on intestinal ion transport. Peptides 2001; 22: 2069-75.]. Here we address the role of ANP and ET-3 on SGLT activity in renal proximal tubules. In rat renal cortical brush border membranes (BBV), fluorescein isothiocianate (FITC) labeling revealed a specific 72-kD peptide that exhibits increased FITC labeling in the presence of Na+ and D-glucose. Using alpha-14C-methylglucose active uptake, rat BBV were shown to possess SGLT activity with an affinity constant (K(0.5) approximately 2.4 mM) that is consistent with the expression of the low-affinity, high-capacity SGLT2 isoform. SGLT2 activity in these preparations is dramatically inhibited by ANP and ET-3. This inhibition is independent of changes in membrane lipids and is mimicked by the cGMP analogue, 8-Br-cGMP, suggesting the involvement of cGMP/PKG pathways. These results are the first demonstration that both ANP and ET-3 inhibit rat cortical renal SGLT2 activity, and suggest a novel mechanism by which these vasoactive substances modulate hydro-saline balance at the proximal tubular nephron level.     

Paracrine effects of mesenchymal stem cells in cisplatin-induced renal injury require heme oxygenase-1

Multipotent mesenchymal stem cells (MSC) have become a popular and promising therapeutic approach in many clinical conditions. MSC are beneficial in animal models of acute kidney injury (AKI), by mediating differentiation-independent paracrine properties, and have prompted ongoing clinical trials to evaluate the safety and efficacy of MSC. Heme oxygenase-1 (HO-1) is induced in response to stress including AKI and has important anti-apoptotic, anti-inflammatory, and proangiogenic properties in these settings. We therefore examined whether HO-1 plays a role in the beneficial effects of MSC in AKI. We isolated MSC from bone marrow of age-matched HO-1+/+ and HO-1-/- mice. Our studies indicate that while differentiation of MSC into osteo- and adipocytic lineages did not differ between cells isolated from HO-1+/+ and HO-1-/- mice, MSC from HO-1-/- mice had significantly lower angiogenic potential. Moreover, HO-1-/- MSC demonstrated reduced expression and secretion of several important growth and proangiogenic factors (stromal cell-derived factor-1, vascular endothelial growth factor-A, and hepatocyte growth factor) compared with MSC derived from HO-1+/+ mice. In addition, conditioned medium of HO-1+/+ MSC rescued functional and morphological changes associated with cisplatin-induced AKI, while the HO-1-/--conditioned medium was ineffectual. Our studies indicate that HO-1 plays an important role in MSC-mediated protection. The results expand understanding of the renoprotective effects of MSC and may provide novel strategies to better utilize MSC in various disease models.     

Changes in temperature modulate heme oxygenase-1 induction by curcumin in renal epithelial cells

The stress protein heme oxygenase-1 (HO-1) plays an essential role in the prevention of transplant-associated organ injury and rejection. Prior to transplantation, organs are normally subjected to variable periods of cold storage in appropriate preservation solutions. Here, we examined whether curcumin, a phenolic plant extract which strongly induces HO-1 in many cell types, could up-regulate HO-1 protein in cultured renal epithelial cells at temperatures lower than the physiological 37 degrees C. We found that stimulation of HO-1 following incubation of cells with curcumin for 6h was dramatically reduced by decreasing the temperature from 37 to 10 degrees C. Interestingly, renal cells displayed high HO-1 expression and heme oxygenase activity when exposed to a programmed change in temperature that consisted of 3h at 37 degrees C followed by 1.5h at 20 degrees C and 1.5h at 10 degrees C. Increased HO-1 levels were observed also after incubation of cells with curcumin during the programmed change in temperature under hypoxia, another feature typical of cold storage procedures. Upon challenge with an oxidant-generating system, cells pretreated with curcumin at 37 degrees C or during the programmed change in temperature exhibited increased resistance to oxidative stress-mediated injury. These findings highlight the feasibility of modulating HO-1 expression during hypothermic storage to confer tissues a better protection to counteract the damage characteristic of organ transplantation.     

Atrial natriuretic peptide in kidney of renal disease patients and healthy persons

Regulation of renal excretion of atrial natriuretic peptide (ANP) was studied in kidney disease patients and healthy kidney donors. The measured ANP concentration in the patient's plasma did not correlate with their creatinine clearance (Ccr), while the fractional excretion of ANP (FEANP) significantly correlated with Ccr. FEANP in healthy persons is less than 1%. In the healthy donors of kidneys for transplantation, approximately 80% of the plasma ANP from the renal artery appeared in the renal vein. From these results, this high recovery of ANP in the veins does not appear to be adequately explained by its degradation in the renal arterioles and nephrons. The FEANP from kidney disease patients significantly correlated with FENa, FEK and FEP, but not with FECa and FEMg. The manner of ANP handling in the nephron may possibly differ from that of Ca or Mg.     

Protective role of heme oxygenase-1 in Listeria monocytogenes-induced abortion

It is well-known fact that various pathogens, including bacteria, virus, and protozoa, induce abortion in humans and animals. However the mechanisms of infectious abortion are little known. In this study, we demonstrated that Listeria monocytogenes infection in trophoblast giant cells decreased heme oxygenase (HO)-1 and B-cell lymphoma-extra large (Bcl-XL) expression, and that their overexpression inhibited cell death induced by the infection. Furthermore, HO-1 and Bcl-XL expression levels were also decreased by L. monocytogenes in pregnant mice. Treatment with cobalt protoporphyrin, which is known to induce HO-1, inhibited infectious abortion. Taken together, our study indicates that L. monocytogenes infection decreases HO-1 and Bcl-XL expression and induces cell death in placenta, leading to infectious abortion.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Atrial natriuretic peptide hormonal system in plants.

To determine if atrial natriuretic peptides are present in plants as well as animals, where they are important for water and sodium metabolism, the leaves and stems of the Florida Beauty (Dracena godseffiana) were examined. The N-terminus consisting of amino acids (a.a.) 1-98 (i.e., pro ANF 1-98), the mid portion of the N-terminus (a.a. 31-67; pro ANF 31-67), and C-terminus (a.a. 99-126; ANF) of the 126 a.a. atrial natriuretic factor (ANF) prohormone were all present in the leaves and stems of this plant. The concentrations of pro ANF 1-98, pro ANF 31-67 and ANF-like peptides of 120 +/- 20, 123 +/- 21, and 129 +/- 20 ng/g of plant tissue in leaves and 109 +/- 20, 96 +/- 21, and 124 +/- 18 ng/g of tissue, respectively, in the stems were lower (P less than 0.05) than their concentrations in rat (Rattus norvegicus) heart atria of 196 +/- 40, 192 +/- 28, and 189 +/- 15 ng/g of tissue respectively, but higher (P less than 0.001) than their respective concentrations of 4.3 +/- 1.4, 4.1 +/- 1.2, and 3.9 +/- 1 ng/g of rat heart ventricular tissue. We conclude that the atrial natriuretic peptide-like hormonal system is present in the plant kingdom as well as in the animal kingdom.     

Atrial natriuretic peptide concentration and natriuretic hormone activity in plasma of patients with chronic renal failure

To elucidate further the possible role of atrial natriuretic peptide (ANP) and hypothetical natriuretic hormone (NH) in volume and BP regulation in chronic renal failure (CRF) we measured plasma ANP, digitalis-like substances (DLS) and Na+-K+-ATPase activity (using 86Rb influx into RBC) in 9 patients with CRF before and after hemodialysis. Volume expansion between consecutive dialyses led in all patients to the elevation of plasma ANP (83.4 +/- 14.2 pmol/l) reaching in some overhydrated subjects and/or patients with concomitant cardiac insufficiency concentration greater than 150 pmol/l. Reduced 86Rb influx into RBC before hemodialysis (37.7 +/- 4.9% of controls) was accompanied by higher DLS concentrations (201 +/- 32 pmol/l). Ultrafiltration during hemodialysis with ECFV reduction lowered both ANP and DLS concentrations to 28.1 +/- 9.4 pmol/l and to 151 +/- 23 pmol/l, respectively, and abolished partly the inhibition of Na+-K+-ATPase activity (64.9 +/- 7.6% of controls). These changes corresponded to the degree of ECFV alteration. Our results suggest that both natriuretic principles are activated during ECFV expansion in CRF, probably as a corrective mechanism, with a tendency to normalize when ECFV is reduced during hemodialysis.     

血红素加氧酶-1在中枢神经系统疾病中的作用

血红素加氧酶-1(heme oxygenase-1,HO-1)是一种应激蛋白,可将血红素降解为胆绿素、游离铁和一氧化碳。目前,国内外普遍认为HO-1在神经系统损伤后的应激反应中有至关重要的作用,其表达量与神经元凋亡和神经变性密切相关。本文对近年来HO-1在神经系统损伤性疾病中的现有研究结果进行了总结分析,旨在为相关研究的发展提供新的思路和方向。