Alvin I. Goodman

Overexpression of the Heme Oxygenase Gene in Renal Cell Carcinoma

Heme oxygenase (HO) activity has been implicated in the regulation of renal function and cell growth in normal and disease states. Expression of HO genes has been shown to regulate important hemoprotein(s) such as cytochrome P450. In the present study, HO activity was measured in samples of human adenocarcinoma, jux-tatumor, and normal renal tissues. The samples were histologically examined to verify the malignant and normal nature. HO activity was 4-fold higher in the adenocarcinoma than in either normal or juxtatumor tissues. We designed a reverse transcriptase-polymerase chain reaction (RT-PCR) method to assess the presence of HO-1 and HO-2 mRNA in biopsy samples of various human renal tissues. Total RNA from renal samples was reverse transcribed and amplified simultaneously by PCR using specific primers for HO-1 and HO-2. Results show that both HO-1 and HO-2 mRNAs were expressed in all renal tissues examined and that HO-1 appeared to be amplified more than HO-2. Northern blot analysis revealed that HO-1 mRNA was elevated by several-fold in adenocarcinoma compared with juxtatumor or normal tissues. In contrast, no differences in HO-2 mRNA levels were observed using either RT-PCR or Northern blot. Cytochrome P450 arachidonic acid epoxygenase and ω-hydroxylase activities were markedly reduced in the tumor tissues, whereas, in the juxtatumor tissue, cytochrome P450 ω-hydroxylase activity was significantly increased. Northern blot analysis using cytochrome P450 cDNA probe 4A2 cDNA for the ω-hydroxylase gene family revealed that mRNA levels for ω-hydroxylase transcripts were significantly decreased in the adenocarcinoma compared with juxtatumor. The decrease in cytochrome P450 4All mRNA levels correlated with a decrease in the arachidonic acid ω-hydroxylation metabolite, 20-HETE. The production of 20-HETE was significantly higher in juxtatumor in agreement with ω-hydroxylase mRNA. Higher levels of HO-1 may be a contributing factor for the undetectable levels of cytochrome P450 arachidonic acid metabolites, 20-HETE, in the adenocarcinoma. Our results suggest that increased generation of mitogenic activities by ω-hydroxylase and 20-HETE in the juxtatumor may be a contributing factor in the development and growth of neoplastic tissues, and the induction of HO in the tumor tissue may be an attempt to limit oxidative injury caused by the cytochrome P450 metabolites and other oxidative stress.

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 (SnCl2) 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.

Fluoxetine in Depressed Patients on Dialysis

Objective: To test the safety and efficacy of fluoxetine in patients with renal failure on dialysis. Method: Fourteen patients with major depression and end stage renal disease on hemodialysis were randomized into two groups for an eight-week study. Subjects as well as investigators were blinded as to which subject received fluoxetine and which placebo. Patients were carefully monitored concerning adverse events, serum fluoxetine and norfluoxetine levels, and psychological measurements of degree of depression. Results: No patients discontinued treatment because of adverse events, all of which were minor. All psychological tests showed improvement in depression at the four-week and eight-weeks point, although statistical significance could only be demonstrated at the fourth week of this study. All patients in the active group had serum plasma concentrations of fluoxetine and norfluoxetine less than 250 ng/ml at eight weeks, similar to levels in patients with normal renal function in a previous open label study. Conclusions: This study confirms the relative safety of fluoxetine in depressed patients in renal failure on hemodialysis. It also suggests that fluoxetine may be efficacious in depressed patients on dialysis.

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.

Renal Artery Stent Placement for the Management of Ischemic Nephropathy

PURPOSE To evaluate the angiographic and clinical results of percutaneously implanted renal artery endoprostheses (stents) for the treatment of patients with ischemic nephropathy. MATERIALS AND METHODS During a 52-month period, 45 patients with azotemia (serum creatinine > or = 1.5 mg/dL) and atheromatous renal artery stenosis untreatable by, or recurrent after, balloon angioplasty were treated by percutaneous placement of Palmaz stents. Stent implantation was unilateral in 32 cases and bilateral in 11 cases. Clinical results were determined by measurements of serum creatinine and follow-up angiography. Clinical benefit was defined as stabilization or improvement in serum creatinine level. Angiographic patency was defined as less than 50% diameter recurrent arterial stenosis. RESULTS Stent placement was technically successful in 51 of 54 (94%) renal arteries. Technical failures were stent misdeployment requiring percutaneous stent retrieval (n = 2) and inadvertent placement distal to the desired position (n = 1). Complications included acute stent thrombosis (n = 1) and early initiation of hemodialysis (within 30 days; n = 1). There were two periprocedural deaths. With use of life-table analysis, clinical benefit was seen in 78% of patients at 6 months (n = 36), 72% at 1 year (n = 24), 62% at 2 years (n = 12), and 54% at 3 years (n = 3). In patients with clinical benefit, average creatinine level was reduced from 2.21 mg/dL +/- 0.91 before treatment to 2.05 mg/dL +/- 1.05 after treatment (P = .018). Lower initial serum creatinine level was associated with a better chance of clinical benefit (P = .05). No other variables affected outcome, including patient age, sex, diabetes, implanted stent diameter, unilateral versus bilateral stent placement, or ostial versus nonostial stent positioning. Conventional catheter angiography or spiral computed tomographic (CT) angiography performed in 19 patients (28 stents) at a mean interval of 12.5 months demonstrated primary patency in 75%. Maintained stent patency appeared to correlate with renal functional benefit. CONCLUSIONS Percutaneous renal artery stent placement for angioplasty failures or restenoses provides clinical benefit in most patients with ischemic nephropathy.

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.

Fluoxetine in depressed patients with renal failure and in depressed patients with normal kidney function

Nine depressed patients with normal kidney function and seven depressed patients with renal failure undergoing hemodialysis were treated with open-label fluoxetine 20 mg/day in an 8-week study. The study was designed to evaluate the pharmacokinetics of fluoxetine during repeated administration and to acquire preliminary data regarding the effectiveness of this antidepressant in a population undergoing hemodialysis. Six patients in each group completed the study. Of these, five patients undergoing hemodialysis and five patients with normal renal function experienced moderate to marked improvement in their depression. Side effects were equal and minor in both groups, indicating that fluoxetine is safe in patients with renal impairment. The mean +/- standard deviation steady-state plasma concentrations of the sum of fluoxetine plus its metabolite norfluoxetine for patients completing 8 weeks (N = 6, both groups) were comparable for the patients undergoing hemodialysis (253 +/- 61 ng/ml) and those with normal kidney function (218 +/- 122 ng/ml; t = 1.5, df = 70, p > 0.13). These data suggest that the efficacy of fluoxetine in patients with renal failure undergoing hemodialysis is comparable to that in patients with normal kidney function. These data further suggest that renal failure and the process of hemodialysis do not materially alter the pharmacokinetics of fluoxetine or its major metabolite norfluoxetine.

Role of human heme oxygenase‐1 in attenuating TNF‐α‐mediated inflammation injury in endothelial cells

Heme oxygenase (HO) is the rate‐limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme‐HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF‐α (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO‐1 gene exhibited a several‐fold increase in HO‐1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE2 and 6‐keto PGF1α levels. We also assessed the effect of retrovirus‐mediated HO‐1 gene transfer in the sense and antisense orientation on HO‐1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO‐1 sense and were greatly suppressed in cells transduced with HO‐1 antisense as compared to control sham‐transduced cells (P < 0.05). The percentage of the G1‐phase in cells transduced with HO‐1 significantly increased (41.4% ± 9.1) compared with control endothelial cells (34.8% ± 4.9). We measured COX activity by determining the levels of PGI2 and PGE2. The levels of PGI2 decreased in cells transduced with HO‐1 sense and increased in cells transduced with HO‐1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO‐1 sense and a marked increase in the HO‐1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF‐α‐induced decrease in the proportion of G1‐phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO‐1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO‐1. These results demonstrate that overexpressing HO‐1 mitigated the TNF‐α‐mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity. J. Cell. Biochem. 87: 377–385, 2002.

Heme Oxygenase-1 Gene Expression Attenuates Angiotensin II-Mediated DNA Damage in Endothelial Cells

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression using a retroviral vector on attenuation of angiotensin II (Ang II)-mediated oxidative stress. Comet and glutathione (GSH) levels were used as indicators of the levels of oxidative stress. Comet assay was performed to evaluate damage on DNA, whereas GSH levels were measured to determine the unbalance of redox potential. Pretreatments with inducers, such as heme 10 μM, SnCl2 10 μM, and inhibitors, such as tin-mesoporphyrin 10 μM was followed by treatment with Ang II 200 ng/ml. Pretreatment with heme or SnCl2 provoked significant reductions (P < 0.01) of tail moment in the comet assay. Opposite effects were evident by pretreatment for 16 hr with tin-mesoporphyrin. A decrease in tail moment levels was found in human endothelial cells transduced with the human HO-1 gene. The addition of Ang II (200 ng/ml) to human dermal microvessel endothelial cel1-1 for 16 hr resulted in a significant (P < 0.05) reduction of GSH contents control endothelial cells but not in endothelial cells transduced with HO-1 gene. The results presented indicated that stimulation or overexpression of HO-1 attenuated DNA damages caused by exposures of Ang II.

Properties of human kidney heme oxygenase: Inhibition by synthetic heme analogues and metalloporphyrins

Heme oxygenase activities in human kidney microsomes were found to be from 0.238 to 0.620 nmol of bilirubin/mg/hr (mean 0.375, SD 0.134), which represent approximately 30% of activities determined for human adult liver. There was interindividual variation in heme oxygenase activity of a 2-5-fold difference. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from human kidney was identified on Western blots by its reaction with the anti-heme oxygenase liver antibody similar to the purified enzyme protein. Thus, a homology exists between human hepatic and kidney heme oxygenase. The enzyme activity was sensitive to inhibition by metalloporphyrins, such as tin-protoporphyrin IX and, to a lesser degree, by zinc and cobalt protoporphyrin IX. In a study of different synthetic heme analogues for in vitro inhibition of heme oxygenase, we found that replacement of iron by zinc in deuteroporphyrin IX 2,4 bis glycol dramatically potentiated the inhibition of heme oxygenase activity. This finding demonstrated that zinc deuteroporphyrin IX 2,4 bis glycol is a most potent inhibitor of heme oxygenase activity.