Renoprotective potency of amifostine in rat renal ischaemia-reperfusion.

PURPOSE: Kidneys from haemodynamically unstable donors may suffer from renal ischaemia-reperfusion (RIR) injury. RIR is associated with reactive oxygen species production that induces inflammation and activates the arachidonic acid (AA) pathway which converts AA into prostaglandin E(2). Amifostine was investigated for its renoprotective potential in RIR. MATERIALS AND METHODS: The effect of amifostine (25 mg/kg = 910 mg/m(2)) on the COX pathway, enzymatic antioxidant activity, the lipid peroxidation marker MDA, serum creatinine and apoptosis was determined in rats. Kidneys were subjected to 45 min of ischaemia and 1 or 24 h of reperfusion. Control groups (sham: coeliotomy, no ischaemia; r1: 45 min ischaemia/1 h reperfusion; r2: 45 min ischaemia/24 h reperfusion) were administered physiological saline intraperitoneally, and treated groups (E1: 45 min ischaemia/1 h reperfusion; E2: 45 min ischaemia/24 h reperfusion) received amifostine 30 min before reperfusion. RESULTS: Serum creatinine increased in non-treated control rats: r1 vs sham (1.6-fold; P <0.007), r2 vs sham (2-fold; P <0.007). Amifostine decreased serum creatinine levels in treated rats: E1 vs r1 (8%; P <0.0025), E2 vs r2 (44%; P <0.0025). Amifostine reduced acute tubular necrosis (25%) 24 h after reperfusion: E1 vs r1 (P <0.004), E2 vs r2 (P <0.03) and reduced COX-2 and microsomal prostaglandin E synthase expression: E1 vs r1 (P <0.03), E2 vs r2 (P <0.02). Amifostine decreased MDA (P <0.04) and reduced caspase-3 expression but did not alter enzymatic antioxidant activity after RIR. CONCLUSIONS: Amifostine decreased the degree and severity of tubular damage after reperfusion, probably by scavenging oxygen free radicals and attenuating the cytotoxic effects of inflammatory infiltrates and apoptosis.

Renoprotective potency of heme oxygenase-1 induction in rat renal ischemia-reperfusion.

PURPOSE: Renal injury caused by ischemia-reperfusion (IR) can lead to acute renal failure or delayed graft function. Renal ischemia-reperfusion (RIR) induces inflammatory disorders via activation of arachidonic acid metabolism into prostaglandin E(2) (PGE(2)). Two inducible enzymes, COX-2 and microsomal prostaglandin E synthase (mPGES), regulate PGE(2) production. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated during cellular stress. Overexpression of HO-1 is beneficial in transplantation models including antigen-independent IR injury, acute and chronic allograft rejection. MATERIALS AND METHODS: We investigated the effect of HO-1 induction on the COX pathway, antioxidant enzyme activities, malondialdehyde (MDA) levels, and apoptosis in rat kidneys subjected to 45 min ischemia and 1 h or 24 h reperfusion. Rats were injected intraperitoneally with either: 50 mg/kg hemin (HO-1 inducer groups: H1, H2); 50 micromol/kg ZnPP (HO-1 inhibitor groups: Hz1, Hz2); or 0.9% saline (control groups: r1, r2). Sham animals (Sh) did not undergo RIR. RESULTS: Serum creatinine increased significantly after RIR (r vs Sh; p <0.05). Hemin treatment induced a significant decrease in serum creatinine after RIR (H vs r; p <0.05) whereas ZnPP treatment significantly increased serum creatinine levels (Sh vs Hz; p <0.05). Hemin reduced the severity of acute tubular necrosis and significantly reduced COX-2 and mPGES expression (p <0.05). Hemin did not alter depleted antioxidant enzyme activity but did decrease levels of MDA (p <0.05). Hemin also reduced caspase-3 expression. CONCLUSIONS: HO-1 decreased the degree and severity of tubular damage after IR, probably by attenuating the cytotoxic effects of inflammatory infiltrates and apoptosis.

Heme oxygenase-1 prevents airway mucus hypersecretion induced by cigarette smoke in rodents and humans.

We investigated the role of heme oxygenase-1 (HO-1), a powerful anti-inflammatory and anti-oxidant enzyme, in modulating cigarette smoke (CS)-induced mucus secretion. In both rats and mice, 5-day CS exposure increased HO-1 expression and activity, mucus secretion, MUCIN 5AC (MUC5AC) gene and protein expression, and local inflammation, along with up-regulation of dual oxidase 1 gene expression and both the activity and phosphorylation of the epidermal growth factor receptor, which is involved in MUC5AC induction. Pharmacological induction of HO-1 prevented these actions and inhibition of HO-1 expression by a specific siRNA potentiated them. In French participants to the European Community Respiratory Health Survey II (n = 210, 30 to 53 years of age, 50% males) exposed to CS, a significant increase in the percentage of participants with chronic sputum was observed in those harboring at least one allele with a long (GT)(n) in the HO-1 promoter gene (>33 repeats), which is associated with a low level of HO-1 protein expression, compared with those with a short number of (GT)n repeats (21.7% versus 8.6%, P = 0.047). No such results were observed in those who had never smoked (n = 297). We conclude that HO-1 has a significant protective effect against airway mucus hypersecretion in animals and humans exposed to CS.

ETB receptor polymorphism is associated with airway obstruction.

BACKGROUND: Endothelin-1 (EDN1) has been involved in the development of airway obstruction and inflammation in asthma. Several polymorphisms have been identified among the genes encoding for preproET1, an inactive precursor of ET-1, and for ETA (EDNRA) and ETB (EDNRB), the two receptors for EDN1. In the present work, we hypothesised that molecular variation in these genes could be a major determinant of the degree of bronchial obstruction. The purpose of this study was to investigate whether the genetic polymorphisms of preproET-1, EDNRA and EDNRB genes were associated with the degree of airway obstruction, assessed by FEV1. METHODS: Polymorphisms of preproET-1, EDNRA and EDNRB were first studied in a population of adult asthmatic patients. Results were confirmed in a large population of adults from the general population from the ECRHS II study. RESULTS: In our population of adult asthmatic patients, the EDNRB-30G>A (Leu277Leu) polymorphism (GG genotype) is strongly associated with a low FEV1 and with a higher percentage of patients with FEV1 < 80% of predicted value. No relationship was found between pulmonary function and EDNRA-1363C>T (His323His) or preproET-1-595G>T (Lys198Asp) polymorphism. In the adult population from the ECRHS II, we found a similar association between GG genotype and a low FEV1 or a higher percentage of subjects with FEV1 < 80% predicted, especially in the subgroups of asthmatics subjects (OR = 4.31 (95%CI 1.03 - 18.04)) and smokers (OR = 7.42 (95%CI 1.69 - 32.6)). CONCLUSION: the EDNRB-30G>A polymorphism could be a determinant of airway obstruction in humans with predisposing factors such as tobacco smoke exposure or asthma.

Bilirubin decreases nos2 expression via inhibition of NAD(P)H oxidase: implications for protection against endotoxic shock in rats.

We investigated a possible beneficial role for bilirubin, one of the products of heme degradation by the cytoprotective enzyme heme oxygenase-1 in counteracting Escherichia coli endotoxin-mediated toxicity. Homozygous jaundice Gunn rats, which display high plasma bilirubin levels due to deficiency of glucuronyl transferase activity, and Sprague-Dawley rats subjected to sustained exogenous bilirubin administration were more resistant to endotoxin (LPS)-induced hypotension and death compared with nonhyperbilirubinemic rats. LPS-stimulated production of nitric oxide (NO) was significantly decreased in hyperbilirubinemic rats compared with normal animals; this effect was associated with reduction of inducible NO synthase (NOS2) expression in renal, myocardial, and aortic tissues. Furthermore, NOS2 protein expression and activity were reduced in murine macrophages stimulated with LPS and preincubated with bilirubin at concentrations similar to that found in the serum of hyperbilirubinemic animals. This effect was secondary to inhibition of NAD(P)H oxidase since 1) inhibition of NAD(P)H oxidase attenuated NOS2 induction by LPS, 2) bilirubin decreased NAD(P)H oxidase activity in vivo and in vitro, and 3) down-regulation of NOS2 by bilirubin was reversed by addition of NAD(P)H. These findings indicate that bilirubin can act as an effective agent to reduce mortality and counteract hypotension elicited by endotoxin through mechanisms involving a decreased NOS2 induction secondary to inhibition of NAD(P)H oxidase.

Heme oxygenase attenuates allergen-induced airway inflammation and hyperreactivity in guinea pigs.

Heme oxygenase (HO), the heme-degrading enzyme, has shown anti-inflammatory effects in several models of pulmonary diseases. HO is induced in airways during asthma; however, its functional role is unclear. Therefore, we evaluated the role of HO on airway inflammation [evaluated by bronchoalveolar lavage (BAL) cellularity and BAL levels of eotaxin, PGE(2), and proteins], mucus secretion (evaluated by analysis of MUC5AC gene expression and periodic acid-Schiff staining), oxidative stress (evaluated by quantification of 4-hydroxynonenal adducts and carbonylated protein levels in lung homogenates), and airway responsiveness to histamine in ovalbumin (OVA)-sensitized and multiple aerosol OVA or saline-challenged guinea pigs (6 challenges, once daily, OVA group and control group, respectively). Airway inflammation, mucus secretion, oxidative stress, and responsiveness were significantly increased in the OVA group compared with the control group. HO upregulation by repeated administrations of hemin (50 mg/kg i.p.) significantly decreased airway responsiveness in control animals and airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These effects were reversed by the concomitant administration of the HO inhibitor tin protoporphyrin-IX (50 micromol/kg i.p.). Repeated administrations of tin protoporphyrin-IX alone significantly increased airway responsiveness in control animals but did not modify airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These results suggest that upregulation of the HO pathway has a significant protective effect against airway inflammation, mucus hypersecretion, oxidative stress, and hyperresponsiveness in a model of allergic asthma in guinea pigs.

Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation.

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.

Heme oxygenase modulates oxidant-signaled airway smooth muscle contractility: role of bilirubin.

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3',5'-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.