Genetic suppression of HO-1 exacerbates renal damage: reversed by an increase in the antiapoptotic signaling pathway.

Apoptosis has been shown to contribute to the development of acute and chronic renal failure. The antiapoptotic action of the heme oxygenase (HO) system may represent an important protective mechanism in kidney pathology. We examined whether the lack of HO-1 would influence apoptosis in clipped kidneys of two-kidney, one-clip (2K1C) rats. Five-day-old Sprague-Dawley rats were injected in the left ventricle with approximately 5 x 10(9) colony-forming units/ml of retrovirus containing rat HO-1 antisense (LSN-RHO-1-AS) or control retrovirus (LXSN). After 3 mo, a 0.25-mm U-shaped silver clip was placed around the left renal artery. Animals were killed 3 wk later. Clipping the renal artery in LSN-RHO-1-AS rats did not result in increased HO-1 expression. In contrast to LXSN animals, 2K1C LSN-RHO-1-AS rats showed increased expression of cyclooxygenase 2 (COX-2) and higher 3-nitrotyrosine (3-NT) content as well as increased expression of the proapoptotic protein Apaf-1 and caspase-3 activity. Clipping the renal artery in LXSN rats resulted in increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xl, while clipping the renal artery in LSN-RHO-1-AS rats did not change Bcl-2 levels and decreased the levels of Bcl-xl. Treatment of LSN-RHO-1-AS rats with cobalt protoporphyrin resulted in induction of renal HO-1, which was accompanied by decreases in blood pressure, COX-2, 3-NT, and caspase-3 activity, and increased expression of anti-apoptotic molecules (Bcl-2, Bcl-xl, Akt and p-Akt) in the clipped kidneys. These findings underscore the prominent role of HO-1 in counteracting apoptosis in this 2K1C renovascular hypertension model.

Up-regulation of heme oxygenase provides vascular protection in an animal model of diabetes through its antioxidant and antiapoptotic effects.

Heme oxygenase (HO) plays a critical role in the regulation of cellular oxidative stress. The effects of the reactive oxygen species scavenger ebselen and the HO inducers cobalt protoporphyrin and stannous chloride (SnCl(2)) on HO protein levels and activity, indices of oxidative stress, and the progression of diabetes were examined in the Zucker rat model of type 2 diabetes. The onset of diabetes coincided with an increase in HO-1 protein levels and a paradoxical decrease in HO activity, which was restored by administration of ebselen. Up-regulation of HO-1 expressed in the early development of diabetes produced a decrease in oxidative/nitrosative stress as manifested by decreased levels of 3-nitrotyrosine, superoxide, and cellular heme content. This was accompanied by a decrease in endothelial cell sloughing and reduced blood pressure. Increased HO activity was also associated with a significant increase in the antiapoptotic signaling molecules Bcl-xl and phosphorylation of p38-mitogen-activated protein kinase but no significant increases in Bcl-2 or BAD proteins. In conclusion, 3-nitrotyrosine, cellular heme, and superoxide, promoters of vascular damage, are reduced by HO-1 induction, thereby preserving vascular integrity and protecting cardiac function involving an increase in antiapoptotic proteins.

Carbon monoxide and biliverdin prevent endothelial cell sloughing in rats with type I diabetes.

Hyperglycemia has been linked to increased oxidative stress, a resultant endothelial cell dysfunction, and, ultimately, apoptosis. Heme oxygenases (HO-1/HO-2) and the products of their activity, biliverdin/bilirubin and carbon monoxide (CO), play a physiological role in the vascular system. The effects of heme-mediated HO-1 induction, CO, and biliverdin on urinary 8-epi-isoprostane PGF(2alpha) and endothelial cell sloughing were examined in an animal model of streptozotocin (STZ)-induced diabetes. Hyperglycemia itself did not affect HO-1 and HO-2 protein levels, but caused a net decrease in HO activity. Weekly heme administration induced HO-1 protein, as demonstrated by immunohistochemistry and Western blot analyses. Administration of biliverdin or the CO donor, CORM-3, decreased urinary 8-epi-isoprostane PGF(2alpha), P < 0.5 compared to diabetes. Hyperglycemia increased endothelial cell sloughing; 8.2 +/- 0.8 cells/ml blood in control rats vs. 48 +/- 4.8 cells/ml blood in diabetic rats (P < 0.05). Heme administration significantly increased endothelial cell sloughing in diabetic rats (98 +/- 8.1 cells/ml blood, P < 0.0007) whereas biliverdin modestly decreased endothelial cell sloughing (26 +/- 3.5 cells/ml blood, P < 0.003). Administration of CORM-3 to diabetic rats resulted in a significant decrease in endothelial cell sloughing to 21.3 +/- 2.3 (P < 0.001). Administration of SnMP to CORM-3 diabetic rats only partially reversed the protective effects of CORM-3 on endothelial cell sloughing from 21.3 +/- 2.3 to 29 +/- 2.1 cells/ml, thus confirming a direct protective of CO, in addition to the ability of CORM-3 to induce HO-1 protein. These results demonstrate that exogenously administered CO or bilirubin can prevent endothelial cell sloughing in diabetic rats, likely via a decrease in oxidative stress, and thus represents a novel approach to prophylactic vascular protection in diabetes.

Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes.

Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity.

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.

Increase in heme oxygenase-1 levels ameliorates renovascular hypertension.

BACKGROUND: The heme oxygenase system (HO-1 and HO-2) catalyzes the conversion of heme to free iron, carbon monoxide (CO), a vasodepressor, and biliverdin, which is further converted to bilirubin, an antioxidant. HO-1 induction has been shown to suppress arachidonic acid metabolism by cytochrome P450 (CYP450) monooxygenases and cyclooxygenases (COX), and to decrease blood pressure in spontaneously hypertensive rats (SHR). The Goldblatt 2K1C model is a model of renovascular hypertension in which there is increased expression of COX-2 in the macula densa and increased renin release from the juxtaglomerular apparatus of the clipped kidney. We examined whether HO-1 overexpression, as a prophylactic approach, would attenuate renovascular hypertension and evaluated potential mechanisms that may account for its effect. METHODS: 2K1C rats were treated with cobalt protoporphyrin (CoPP) or tin mesoporphyrin (SnMP) one day before surgery and weekly for three weeks thereafter. We measured systolic blood pressure, HO activity, HO-1, HO-2, COX-1 and COX-2 protein expression, heme content, and nitrotyrosine levels as indices of oxidative stress. Urinary prostaglandin excretion (PGE2), plasma renin activity (PRA), and plasma aldosterone levels were also measured. RESULTS: CoPP administration induced renal HO-1 expression by 20-fold and HO activity by 6-fold. This was associated with a reduction in heme content, nitrotyrosine levels, COX-2 expression and urinary PGE2 excretion, and attenuation of the development of hypertension in the 2K1C rats. There was no decrease in plasma renin activity; however, plasma aldosterone levels were significantly lower. In the 2K1C SnMP-treated rats, blood pressure was significantly higher than that of untreated 2K1C rats throughout the study, and the difference in the size of the smaller left clipped kidney compared to the nonclipped right kidney was significantly increased. CONCLUSION: These findings define an action of prolonged HO-1 induction to interrupt and counteract the influence of the renin-angiotensin-aldosterone system (RAAS) to increase in blood pressure in the 2K1C model of renovascular hypertension. Multiple mechanisms include a decrease in oxidative stress as indicated by the decrease in cellular heme and nitrotyrosine levels, an anti-inflammatory action as evidenced by a decrease in COX-2 and PGE2, interference with the action of angiontensin II (Ang II) as evidenced by an increase in PRA in the face of a decrease in PGE2 and aldosterone, as well as the inhibition of aldosterone synthesis.

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.

Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes.

Heme oxygenase (HO)-1 represents a key defense mechanism against oxidative injury. Hyperglycemia produces oxidative stress and various perturbations of cell physiology. The effect of streptozotocin (STZ)-induced diabetes on aortic HO activity, heme content, the number of circulating endothelial cells, and urinary 8-epi-isoprostane PGF2alpha (8-Epi) levels in control rats and rats overexpressing or underexpressing HO-1 was measured. HO activity was decreased in hyperglycemic rats. Hyperglycemia increased urinary 8-Epi, and this increase was augmented in rats underexpressing HO-1 and diminished in rats overexpressing HO-1. The number of detached endothelial cells and O2- formation increased in diabetic rats and in hyperglycemic animals underexpressing HO-1 and decreased in diabetic animals overexpressing HO-1 compared with controls. These data demonstrate that HO-1 gene transfer in hyperglycemic rats brings about a reduction in O2- production and a decrease in endothelial cell sloughing. Upregulation of HO-1 decreases oxidant production and endothelial cell damage and shedding and may attenuate vascular complications in diabetes.

Expression of human heme oxygenase-1 in the thick ascending limb attenuates angiotensin II-mediated increase in oxidative injury.

BACKGROUND: Heme oxygenase-1 (HO-1) catalyzes the conversion of heme to bilirubin, carbon monoxide (CO), and free iron, thus controlling the level of cellular heme. The medullary thick ascending limb of the loop of Henle (TALH) is situated in a site of markedly diminished oxygen tension and, as such, is highly vulnerable to ischemic insult. We hypothesize that selective upregulation of HO-1 in TALH by gene transfer attenuates oxidative stress caused by angiotensin II (Ang II). METHODS: An adenoviral vector expressing the human HO-1 under the control of the TALH-specific promoter [Na(+)-K(+)-Cl(-) cotransporter (NKCC2 promoter)] was constructed and the cell specific expression of the recombinant adenovirus was examined using several types of cells, including endothelial, vascular smooth muscle, and TALH cells. The effects of HO-1 transduction on HO-1 expression, HO activity and the response to Ang II with respect to cyclooxygenase-2 (COX-2) up-regulation and oxidative injury [growth-stimulating hormone (GSH) levels and cell death] were determined. RESULTS: Western blot and reverse transcription-polymerase chain reaction (RT-PCR) revealed that human HO-1 was selectively expressed in primary cultured TALH cells following infection with Ad-NKCC2-HO-1. In TALH cells infected with Ad-NKCC2-HO-1, Ang II-stimulated prostaglandin E(2) (PGE(2)) levels were reduced by 40%. Ang II caused a marked decrease in GSH levels and this decrease was greatly attenuated in TALH cells transduced with Ad-NKCC2-HO-1. Moreover, Ang II-mediated DNA degradation was completely blocked by the site-specific expression of human HO-1 gene. CONCLUSION: These results indicate that TALH cell survival after exposure to oxidative stress injury may be facilitated by selective upregulation of HO-1, thusly blocking inflammation and apoptosis.

Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats.

Heme oxygenase-1 (HO-1) represents a key defense mechanism against oxidative injury. Hyperglycemia has been linked to increased oxidative stress, leading to endothelial dysfunction, delayed cell replication, and enhanced apoptosis. The effect of streptozotocin (STZ)-induced diabetes on HO activity, HO-1 promoter activity, superoxide anion (O*-2, and the number of circulating endothelial cells was measured. The expression of HO-1/HO-2 protein was unchanged, but HO activity was decreased in aortas of diabetic rats compared with control (p < 0.05). High glucose decreased HO-1 promoter activity (p < 0.05). Hyperglycemia increased O*-2 and this increase was augmented with HO-1 inhibition and diminished with HO-1 upregulation (p < 0.05). Circulating endothelial cells were significantly higher in diabetic rats and were decreased or increased with administration of the HO-1 inducer (CoPP) or inhibitor (SnMP), respectively (p<0.05). In conclusion, HO-1 upregulation in diabetic rats brings about an increase in serum bilirubin, a reduction in O*-2 production, and a decrease in endothelial cell sloughing.

Functional expression of human heme oxygenase-1 gene in renal structure of spontaneously hypertensive rats.

Heme oxygenase (HO), by catabolizing heme to bile pigments, regulates the levels and activity of cellular hemoprotein and HO activity. We examined the effect of delivery of the human HO-1 gene on cellular heme in renal tissue using a retroviral vector. We used a single intracardiac injection of the concentrated infectious viral particles in 5-day-old spontaneously hypertensive rats; 25 were transduced with empty vector and 25 were transduced with the human HO-1 gene. Functional expression of human and rat HO-1 was measured after 2 and 4 weeks. Reverse transcription polymerase chain reaction showed that human HO-1 mRNA was expressed as early as 2 weeks, with the highest levels in the kidney. Western blot analysis showed distribution of human HO-1 protein in rat kidney structures, predominantly in the thick ascending limb of the loop of Henle as well as in proximal tubules and preglomerular arterioles. These areas also demonstrated higher HO activity as measured by increased conversion of heme to bilirubin and carbon monoxide. Functional expression of the human HO-1 gene was associated with a decrease in blood pressure in 4- and 8-week-old spontaneously hypertensive rats. Compared with nontransduced rats, human HO-1 gene overexpression in transduced rats was associated with a 35% decrease in urinary 20-hydroxyeicosatetraenoic acid, a potent vasoconstrictor and an inhibitor of tubular Na(+) transport, which may be related to the decrease in blood pressure.

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.