Search

Intracerebral hemorrhage (ICH) is a devastating neurological disorder characterized by an exacerbation of neuroinflammation and neuronal injury, for which few effective therapies are available at present. Inhibition of excessive neuroglial activation has been reported to alleviate ICH-related brain injuries. In the present study, the anti-ICH activity and microglial mechanism of ergosta-7,9(11),22-trien-3β-ol (EK100), a bioactive ingredient from Asian medicinal herb Antrodia camphorate, were evaluated. Post-treatment of EK100 significantly attenuated neurobehavioral deficit and MRI-related brain lesion in the mice model of collagenase-induced ICH. Additionally, EK100 alleviated the inducible expression of cyclooxygenase (COX)-2 and the activity of matrix metalloproteinase (MMP)-9 in the ipsilateral brain regions. Consistently, it was shown that EK100 concentration-dependently inhibited the expression of COX-2 protein in Toll-like receptor (TLR)-4 activator lipopolysaccharide (LPS)-activated microglial BV-2 and primary microglial cells. Furthermore, the production of microglial prostaglandin E2 and reactive oxygen species were attenuated by EK100. EK100 also attenuated the induction of astrocytic MMP-9 activation. Among several signaling pathways, EK100 significantly and concentration-dependently inhibited activation of c-Jun N-terminal kinase (JNK) MAPK in LPS-activated microglial BV-2 cells. Consistently, ipsilateral JNK activation was markedly inhibited by post-ICH-treated EK100 in vivo. In conclusion, EK100 exerted the inhibitory actions on microglial JNK activation, and attenuated brain COX-2 expression, MMP-9 activation, and brain injuries in the mice ICH model. Thus, EK100 may be proposed and employed as a potential therapeutic agent for ICH.

Oxidative stress is an important factor to cause the pathogenesis of diabetic retinopathy (DR) because the retina has high vascularization and long-time light exposition. Cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes can convert arachidonic acid (AA) into eicosanoids, which are important lipid mediators to regulate DR development. COX-derived metabolites appear to be significant factors causative to oxidative stress and retinal microvascular dysfunction. Several elegant studies have unraveled the importance of LOX-derived eicosanoids, including LTs and HETEs, to oxidative stress and retinal microvascular dysfunction. The role of CYP eicosanoids in DR is yet to be explored. There is clear evidence that CYP-derived epoxyeicosatrienoic acids (EETs) have detrimental effects on the retina. Our recent study showed that the renin-angiotensin system (RAS) activation augments retinal soluble epoxide hydrolase (sEH), a crucial enzyme degrading EETs. Our findings suggest that EETs blockade can enhance the ability of RAS blockade to prevent or mitigate microvascular damage in DR. This review will focus on the critical information related the function of these eicosanoids in the retina, the interaction between eicosanoids and reactive oxygen species (ROS), and the involvement of eicosanoids in DR. We also identify potential targets for the treatment of DR.

Aims Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. Materials & methods We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC–MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. Results Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. Conclusion Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.

// Weijing Feng 1, 2, * , Kun Zhang 1, 2, * , Yu Liu 3, * , Jie Chen 1, 4, * , Qingqing Cai 5 , Yinyin Zhang 1, 2 , Mongheng Wang 6 , Jingfeng Wang 1, 2 , Hui Huang 1, 2 1 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Cardiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 2 Laboratory of RNA and Major Diseases of Brain and Heart, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 3 Department of Cardiology, the People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, China 4 Department of Radiation Oncology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, China 5 Department of Medical Oncology, Sun Yat-sen University Cancer Center, Sun Yat-sen University, Guangzhou, China 6 Department of Physiology, Georgia Regents University, Augusta, GA, USA * These authors have contributed equally to this work Correspondence to: Hui Huang, email: huanghui765@hotmail.com Keywords: vascular smooth muscle cells, osteogenic switching, vascular calcification, angiotensin II, apocynin Received: January 27, 2016 Accepted: October 19, 2016 Published: November 08, 2016 ABSTRACT Vascular calcification (VC) is a significant risk factor for cardiovascular morbidity and mortality. We recently reported that apocynin had benefits for preventing cardiovascular diseases. However, whether apocynin could attenuate VC is unknown. Here, we investigated the role of apocynin in VC and its underlying mechanisms. 163 participants with high or normal ankle–brachial index (ABI) were enrolled in this study for analyzing the demographic and biochemical data. In vitro, vascular smooth muscle cells (VSMCs) were exposed to calcification medium containing b-glycerophosphate and angiotensin II (Ang II) for 24 hours. The results showed that serum level of Ang II was significantly increased in patients with high ABI (P

Both clinical and experimental studies have demonstrated that vascular calcification (VC) is a common pathology shared in many chronic diseases such as chronic kidney disease (CKD) and diabetes. It's an independent risk factor for cardiovascular events. Since the pathogenesis of VC is complicated, current therapies have limited effects on the regression of VC. Therefore, it is urgent to investigate the potential mechanisms and find new targets for the treatment of VC. Aldosterone (Aldo), a mineralocorticoid hormone, is the metabolite of renin-angiotensin-aldosterone system (RAAS) activation, which can exert genomic and non-genomic effects on the cardiovascular system. Recent data suggests that Aldo can promote VC. Here, we summarized the roles of Aldo in the process of VC and a series of findings indicated that Aldo could act as a potentially therapeutic target for treating VC.

Arachidonic acid (AA) is metabolized by cyclooxygenase (COX) and cytochrome P450 (CYP) enzymes into eicosanoids, which are involved in cardiovascular diseases and stroke. Evidence has demonstrated the important functions of these eicosanoids in regulating cerebral vascular tone, cerebral blood flow, and autoregulation of cerebral circulation. Although COX-2 inhibitors have been suggested as potential treatments for stroke, adverse events, including an increased risk of stroke, occur following long-term use of coxibs. It is important to note that prolonged treatment with rofecoxib increased circulating levels of 20-hydroxyeicosatetraenoic acid (20-HETE), and 20-HETE blockade is a possible strategy to prevent coxib-induced stroke events. It appears that 20-HETE has detrimental effects in the brain, and that its blockade exerts cerebroprotection against ischemic stroke and subarachnoid hemorrhage (SAH). There is clear evidence that activation of EP2 and EP4 receptors exerts cerebroprotection against ischemic stroke. Several elegant studies have contributed to defining the importance of stabilizing the levels of epoxyeicosatrienoic acids (EETs), by inhibiting or deleting soluble epoxide hydrolase (sEH), in stroke research. These reports support the notion that sEH blockade is cerebroprotective against ischemic stroke and SAH. Here, we summarize recent findings implicating these eicosanoid pathways in cerebral vascular function and stroke. We also discuss the development of animal models with targeted gene deletion and specific enzymatic inhibitors in each pathway to identify potential targets for the treatment of ischemic stroke and SAH.

Advanced oxidation protein products (AOPPs) are independent risk factor for various cardiovascular diseases. Cardiomyocyte apoptosis has been implicated as an important mechanism in cardiac remodeling in chronic kidney disease (CKD). However, whether AOPPs affect cardiomyocyte apoptosis and subsequent cardiac remodeling in CKD is still not very clear. Here, we assessed the role of AOPPs in cardiomyocyte apoptosis in CKD. H9C2 rat cardiomyoblast cells were exposed to AOPPs. Apoptotic cells were determined by TUNEL assay. The expression of apoptotic markers (cleaved caspase-3 and Bax), JNK signaling, and endoplasmic reticulum stress were explored. Serum AOPPs were measured in male Sprague-Dawley rats that underwent sham surgery and 5/6 nephrectomy, respectively. In vitro, our findings showed that AOPPs activated JNK signaling and endoplasmic reticulum stress and significantly aggravated H9C2 rat cardiomyoblast cells apoptosis. These effects were partially ameliorated by apocynin with inhibition of oxidative stress. In vivo, serum levels of AOPPs were progressively elevated with the increasing time course in CKD rats compared with sham-operated rats ( P0.05). Serum AOPP levels were positively associated with cardiomyocyte apoptosis ( R

PGE2 has an established role in renal water handling. The present study was undertaken to examine the role of microsomal prostaglandin E synthase-1 (mPGES-1) in the diuretic response to acute and chronic water loading. Compared with wild-type (+/+) controls, mPGES-1 −/− mice exhibited impaired ability to excrete an acute, but not chronic water load. In response to acute water loading, urinary PGE2 excretion in the +/+ mice increased at 2 h, in parallel with increased urine flow. In contrast, the −/− mice exhibited a delayed increase in urinary PGE2 excretion, coinciding with the stimulation of renal medullary mRNA expression of cytosolic prostaglandin E synthase but not mPGES-2. At baseline, renal aquaporin-2 (AQP2) expression in mPGES-1 −/− mice was enhanced compared with the +/+ control. In response to acute water loading, renal AQP2 expression in the +/+ mice was significantly reduced, and this reduction was blunted in the −/− mice. Despite striking changes in AQP2 protein expression, renal AQP2 mRNA in both genotypes largely remained unchanged. Overall, these data support an important role of mPGES-1 in provoking the diuretic response to acute water loading.

Prostaglandin (PG) E 2 has an established role in the regulation of vascular tone and reactivity. The present study examined the role and mechanism of microsomal PG synthase-1 (mPGES-1) in vascular response to angiotensin II (Ang II) infusion. A 7-day Ang II infusion at 0.35 mg/kg per day via osmotic minipump had no obvious effect on mean arterial blood pressure in mPGES-1 +/+ mice but induced a marked hypertensive response in mPGES-1 −/− mice, associated with a parallel increase in urinary 8-isoprostane excretion and aortic NADPH oxidase activity and mRNA expression of p47 phox , gp91 phox , and Nox1. The hypertension in mPGES-1 −/− mice was completely prevented by Tempol treatment and was fully restored on termination of the antioxidant. Apocynin induced a similar blood pressure–lowering effect as Tempol. The Ang II infusion induced mRNA expression of mPGES-1, as well as mPGES-2 and cytosolic PGE synthase in the aortas as assessed by real-time RT-PCR. Immunohistochemistry revealed remarkably enhanced immunoreactivity of mPGES-1 mostly in vascular smooth muscle cells. In cultured vascular smooth muscle cells, Ang II exerted a direct stimulatory effect on reactive oxygen species production, NADPH oxidase activity, and expression of p47 phox , gp91 phox , and Nox1 that were all inhibited by PGE 2 . The −/− mice also exhibited enhanced renal hemodynamic response to acute Ang II infusion at 150 nmol/kg per minute via a jugular vein over a period of 40 minutes. These results suggest that mPGES-1–derived PGE 2 buffers Ang II–induced vasoconstriction via inhibition of NADPH oxidase–dependent reactive oxygen species production.

OBJECTIVE—The implication of innate immunity in type 1 diabetes development has long been proposed. High-mobility group box 1 (HMGB1), an evolutionarily conserved chromosomal protein, was recently recognized to be a potent innate inflammatory mediator when released extracellularly. We sought to test the hypothesis that HMGB1 acts as an innate immune mediator implicated in type 1 diabetes pathogenesis.RESEARCH DESIGN AND METHODS—Eight- and 12-week-old NOD mice were treated with an HMGB1 neutralizing antibody once a week until 25 weeks of age and monitored for insulitis progression and diabetes onset. The underlying mechanisms of HMGB1 regulation of autoimmune response were further explored.RESULTS—During autoimmunity, HMGB1 can be passively released from damaged pancreatic β-cells and actively secreted by islet infiltrated immune cells. Extracellular HMGB1 is potent in inducing NOD dendritic cell maturation and stimulating macrophage activation. Blockade of HMGB1 significantly inhibited insulitis progression and diabetes development in both 8- and 12-week-old NOD mice. HMGB1 antibody treatment decreased the number and maturation of pancreatic lymph node (PLN) CD11c++CD11b+ dendritic cells, a subset of dendritic cells probably associated with autoantigen presentation to naïve T-cells, but increased the number for PLN CD4+Foxp3+ regulatory T-cells. Blockade of HMGB1 also decreased splenic dendritic cell allo-stimulatory capability associated with increased tolergenic CD11c+CD8a+ dendritic cells. Interestingly, the number of CD8+interferon-γ+ (Tc1) T-cells was increased in the PLNs and spleen after blockade of HMGB1, which could be associated with retarded migration of activated autoreactive T-cells into the pancreatic islets.CONCLUSIONS—Extracellular HMGB1 functions as a potent innate immune mediator contributing to insulitis progression and diabetes onset.

Previous studies have shown that the synthesis of renal cytochrome P-450 (CYP)-derived eicosanoids is downregulated in genetic or high-fat diet-induced obese rats. Experiments were designed to determine whether fenofibrate, a peroxisome proliferator-activated receptor (PPAR)-α agonist, would induce renal eicosanoid synthesis and improve endothelial function in obese Zucker rats. Administration of fenofibrate (150 mg·kg−1·day−1for 4 wk) significantly reduced plasma insulin, triglyceride, and total cholesterol levels in obese Zucker rats. CYP2C11 and CYP2C23 proteins were downregulated in renal vessels of obese Zucker rats. Consequently, renal vascular epoxygenase activity decreased by 15% in obese Zucker rats compared with lean controls. Chronic fenofibrate treatment significantly increased renal cortical and vascular CYP2C11 and CYP2C23 protein levels in obese Zucker rats, whereas it had no effect on epoxygenase protein and activity in lean Zucker rats. Renal cortical and vascular epoxygenase activities were consequently increased by 54% and 18%, respectively, in fenofibrate-treated obese rats. In addition, acetylcholine (1 μM)-induced vasodilation was significantly reduced in obese Zucker kidneys (37% ± 11%) compared with lean controls (67% ± 9%). Chronic fenofibrate administration increased afferent arteriolar responses to 1 μM of acetylcholine in obese Zucker rats (69% ± 4%). Inhibition of the epoxygenase pathway with 6-(2-propargyloxyphenyl)hexanoic acid attenuated afferent arteriolar diameter responses to acetylcholine to a greater extent in lean compared with obese Zucker rats. These results demonstrate that the PPAR-α agonist fenofibrate increased renal CYP-derived eicosanoids and restored endothelial dilator function in obese Zucker rats.

We examined the effect of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (HET0016), an inhibitor of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthesis on the omega-hydroxylation and epoxidation of arachidonic acid (AA) catalyzed by recombinant cytochrome P450 4A1 (CYP4A1), CYP4A2 and CYP4A3, and characterized the enzyme inhibitory profile of HET0016. The IC50 values of HET0016 for recombinant CYP4A1-, CYP4A2- and CYP4A3-catalyzed 20-HETE synthesis averaged 17.7 nM, 12.1 nM and 20.6 nM, respectively. The IC50 value for production of 11,12-epoxy-5,8,14-eicosatrienoic acid (11,12-EET) by CYP4A2 and 4A3 averaged 12.7 nM and 22.0 nM, respectively. The IC50 value for CYP2C11 activity was 611 nM which was much greater than that for CYP4As. The initial velocity study showed the Ki value of HET0016 for CYP4A1 was 19.5 nM and a plot of Vmax versus amount of recombinant CYP4A1 added shows HET0016 is an irreversible non-competitive inhibitor. These results indicate that HET0016 is a selective, non-competitive and irreversible inhibitor of CYP4A.

Divalent metal ions such as copper, manganese, and cobalt are essential for cell development, differentiation, function and survival. These essential metal ions are delivered into intracellular domains as cofactors for enzymes involved in neuropeptide and neurotransmitter synthesis, superoxide metabolism, and other biological functions in a target specific fashion. Altering the homeostasis of these essential metal ions is known to connect to a number of human diseases including Alzheimer disease, amyotrophic lateral sclerosis, and pain. It remains unclear how these essential metal ions are delivered to intracellular targets in mammalian cells. Here we report that rat spinal cord dorsal horn neurons express ACDP4, a member of Ancient Conserved Domain Protein family. By screening a pretransformed human fetal brain cDNA library in a yeast two-hybrid system, we have identified that ACDP4 specifically interacts with COX11, an intracellular metal ion chaperone. Ectopic expression of ACDP4 in HEK293 cells resulted in enhanced toxicity to metal ions including copper, manganese, and cobalt. The metal ion toxicity became more pronounced when ACDP4 and COX11 were co-expressed ectopically in HEK293 cells, suggesting a functional coupling between them. Our results indicate a role of ACDP4 in metal ion homeostasis and toxicity. This is the first report revealing a functional aspect of this ancient conserved domain protein family. We propose that ACDP is a family of transporter protein or chaperone proteins for delivering essential metal ions in different mammalian tissues. The expression of ACDP4 on spinal cord dorsal horn neurons may have implications in sensory neuron functions under physiological and pathological conditions.

Epoxyeicosatrienoic acids (EETs), which belong to cytochrome P-450 (CYP)-derived eicosanoids, have been implicated to vasodilate renal arterioles, inhibit sodium transport in the nephron, and regulate blood pressure in several animal models. Because pregnancy is associated with changes of blood pressure, the aim of this study was to examine whether renal EET synthesis is altered and whether EETs are involved in blood pressure regulation during pregnancy in rats. Renal microsomal epoxygenase activity increased by 47, 97, and 63% on days 6, 12, and 19 of gestation, respectively. The elevation of epoxygenase activity during pregnancy was associated with an increase in CYP2C11, CYP2C23, and CYP2J2 protein expression on days 6, 12, and 19 of gestation. Moreover, immunohistochemical analysis showed that renal tubular CYP2C11, CYP2C23, and CYP2J2 expression was significantly increased in pregnant rats on days 6, 12, and 19 of gestation. Administration of 6-(2-propargyloxyphenyl)hexanoic acid (PPOH), a selective epoxygenase inhibitor, caused a dose-dependent inhibition of microsomal expoxygenase activity without a significant effect on ω-hydroxylase activity in female rats. Interestingly, administration of PPOH (20 mg·kg−1·day−1for 4 days starting on day 15 of pregnancy) increased blood pressure by 21 mmHg and caused a significant decrease in the body weight of fetal pups (1.3 ± 0.08 g in control vs. 1.1 ± 0.06 g in PPOH). Moreover, PPOH treatment significantly decreased renal microsomal epoxygenase activity and the expression of CYP2C11, CYP2C23, and CYP2J in pregnant rats. This study demonstrates that EET synthesis in the kidney is elevated during pregnancy, and CYP2C11, 2C23, and CYP2J2 are responsible for the change of renal EET synthesis. The inhibition results demonstrate that the downregulation of renal epoxygenase activity by PPOH causes hypertension in pregnant rats. This study suggests that EETs may contribute to the control of blood pressure during pregnancy.

Apoptosis has been implicated in ischemic renal injury. Thus one strategy of renal protection is to antagonize apoptosis. However, apoptosis inhibitory approaches remain to be fully explored. Zn2+has long been implicated in apoptosis inhibition; but systematic analysis of the inhibitory effects of Zn2+is lacking. Moreover, whether Zn2+blocks renal cell apoptosis following ischemia is unknown. Here, we demonstrate that Zn2+is a potent apoptosis inhibitor in an in vitro model of renal cell ischemia. ATP depletion induced apoptosis in cultured renal tubular cells, which was accompanied by caspase activation. Zn2+at 10 μM inhibited both apoptosis and caspase activation, whereas Co2+was without effect. In ATP-depleted cells, Zn2+partially prevented Bax activation and cytochrome c release from mitochondria. In isolated cell cytosol, Zn2+blocked cytochrome c-stimulated caspase activation at low-micromolar concentrations. In addition, Zn2+could directly antagonize the enzymatic activity of purified recombinant caspases. We conclude that Zn2+is a potent inhibitor of apoptosis in renal tubular cells following ATP depletion. Zn2+blocks apoptosis at multiple steps including Bax activation, cytochrome c release, apoptosome function, and caspase activation.

The incidence of essential hypertension increases with obesity; however, the mechanisms that link obesity with hypertension are unclear. Renal cytochrome P450 (CYP)-derived eicosanoids—hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs)—have been shown to play an important role in the regulation of renal function, vascular tone, and blood pressure. The objective of this study was to examine CYP-derived eicosanoid synthesis in the different renal zones (cortex, medulla, and papilla) of rats fed a high fat diet (HF). Male Sprague-Dawley rats were fed a HF diet or regular rat chow for 10 weeks. After 10 weeks, HF rats showed significantly higher systolic blood pressure, body weight, and fat:body weight ratio. The renal ω-hydroxylase activity was decreased by 46% in cortex, 43% in medulla, and 46% in papilla of HF rats. The renal epoxygenase activity was decreased by 46% in cortex, 31% in medulla, and 56% in papilla of HF rats. Interestingly, the changes in the rate of 20-HETE and EET formation in different renal zones were consistent with the levels of expression of CYP4A and CYP2C23 proteins, respectively. Furthermore, there were no significant changes in the synthesis of these metabolites in the renal microvessels. These results demonstrate that HF diet causes the downregulation of CYP4A and CYP2C23 in renal tubules, and these proteins are responsible for renal 20-HETE and EET formation. The reduction in the synthesis of these eicosanoids may play an important role in the regulation of renal function and blood pressure in obesity-induced hypertension.

20-HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca2+-activated K+ channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low- K m arachidonic acid ω-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC50 from 0.37 ± 0.04 μM in plasmid-transfected arteries to 0.07 ± 0.01 μM in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine.

We examined whether renal 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis is altered during gestation. Renal microsomal arachidonic acid ω-hydroxylase activity increased by 50 and 48% in rats on days 12 and 19 of gestation, respectively. Renal microvessel 20-HETE synthesis increased by 50 and 82% in rats on days 6 and 12 of gestation, respectively, and returned to control levels at day 19 of gestation. In contrast, 20-HETE synthesis in isolated medullary thick ascending limb was unchanged from control levels on days 6and 12 of gestation, but it increased twofold on day 19 of gestation. This increase on day 19 of gestation was associated with a twofold increase in urinary 20-HETE excretion, and it coincided with a 23-mmHg fall in blood pressure. Moreover, change in the rate of 20-HETE synthesis in microvessels was consistent with the level of expression of cytochrome P450 (CYP)4A proteins. Administration of the CYP4A inhibitor 1-aminobenzotriazole (ABT) for 2 days on day 12 of pregnancy or for 5 days starting on day 15 of pregnancy caused a transient but significant reduction in systolic blood pressure. ABT treatment also decreased urinary sodium, urinary 20-HETE, and renal and microvessel 20-HETE synthesis. This study, to our knowledge, is the first to demonstrate that 20-HETE synthesis in the kidney is altered in time- and site-specific manners during pregnancy. The localized pattern of changes suggests that there are distinct regulatory mechanisms for 20-HETE synthesis in the kidney during pregnancy.

Inner medulla collecting duct (IMCD) cells are the key part for urinary concentration. Hypotonic stress may trigger apoptosis of IMCD cells and induce renal injury. Epoxyeicosatrienoic acids (EETs) play an important role in anti-apoptosis, but their roles in hypotonic-induced apoptosis of IMCD cells are still unclear. Here we found increasing exogenous 11, 12-EET or endogenous EETs with Ad-CMV-CYP2C23-EGFP transfection decreased apoptosis of IMCD cells induced by hypotonic stress. Moreover, up-regulation of γ-ENaC induced by hypotonic stress was abolished by elevation of exogenous or endogenous EETs. Collectively, this study illustrated that EETs attenuated hypotonic-induced apoptosis of IMCD cells, and that regulation of γ-ENAC may be a possible mechanism contributing to the anti-apoptotic effect of EETs in response to hypotonic stress.

The cytochrome P-450 4A (CYP4A)-derived arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) affects renal tubular and vascular functions and has been implicated in the control of arterial pressure. We examined the effect of antisense oligonucleotide (ODN) to CYP4A1, the low K m arachidonic acid ω-hydroxylating isoform, on vascular 20-HETE synthesis, vascular reactivity, and blood pressure in the spontaneously hypertensive rat (SHR). Administration of CYP4A1 antisense ODN decreased mean arterial blood pressure from 137 ± 3 to 121 ± 4 mmHg ( P < 0.05) after 5 days of treatment, whereas treatment with scrambled antisense ODN had no effect. Treatment with CYP4A1 antisense ODN reduced the level of CYP4A-immunoreactive proteins along with 20-HETE synthesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine (EC50 0.69 ± 0.17 vs. 1.77 ± 0.40 μM). Likewise, mesenteric arterioles from antisense ODN-treated rats revealed attenuation of myogenic constrictor responses to increases of transmural pressure. The decreased vascular reactivity and myogenic responses were reversible with the addition of 20-HETE. These data suggest that CYP4A1-derived 20-HETE facilitates myogenic constrictor responses in the mesenteric microcirculation and contributes to pressor mechanisms in SHR.

20-Hydroxyeicosatetraenoic acid (HETE), the cytochrome P-450 (CYP) 4A ω-hydroxylation product of arachidonic acid, has potent biological effects on renal tubular and vascular functions and on the control of arterial pressure. We have expressed high levels of the rat CYP4A1, -4A2, -4A3, and -4A8 cDNAs, using baculovirus and Sf 9 insect cells. Arachidonic acid ω- and ω-1-hydroxylations were catalyzed by three of the CYP4A isoforms; the highest catalytic efficiency of 947 nM−1⋅ min−1for CYP4A1 was followed by 72 and 22 nM−1⋅ min−1for CYP4A2 and CYP4A3, respectively. CYP4A2 and CYP4A3 exhibited an additional arachidonate 11,12-epoxidation activity, whereas CYP4A1 operated solely as an ω-hydroxylase. CYP4A8 did not catalyze arachidonic or linoleic acid but did have a detectable lauric acid ω-hydroxylation activity. The inhibitory activity of various acetylenic and olefinic fatty acid analogs revealed differences and indicated isoform-specific inhibition. These studies suggest that CYP4A1, despite its low expression in extrahepatic tissues, may constitute the major source of 20-HETE synthesis. Moreover, the ability of CYP4A2 and -4A3 to catalyze the formation of two opposing biologically active metabolites, 20-HETE and 11,12-epoxyeicosatrienoic acid, may be of great significance to the regulation of vascular tone.

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol ⋅ kg body wt−1⋅ day−1iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.

Erectile dysfunction (ED) is estimated to affect more than 30 million American men and 152 million men worldwide. Therapeutic agents targeting the nitric oxide/cyclic GMP signaling pathway have successfully treated patients with ED; however, the efficacies of these treatments are significantly lower in specific populations such as patients with diabetes. The goal of this study was to discover and identify new endothelium-derived relaxing factors involved in the regulation of erectile function, providing alternative therapeutic targets for treatment of ED. Immunoblotting results showed that protein expressions of epoxygenases from cytochrome P450 (CYP)2B, 2C and 2J subfamilies, as well as NADPH CYP reductase were present in rat corpora cavernosa, which was confirmed by immunohistochemical analysis. Furthermore, CYP2C was localized in cavernosal endothelial cells using double immunolabeling. CYP epoxygenase activity was analyzed by reverse-phase high-pressure liquid chromatography; and the results showed that 11,12- epoxyeicosatrienoic acid (EET) was the major product metabolized by CYP epoxygenases in rat corpora cavernosa. Inhibition of EETs function by injection of an EETs antagonist into rat penis significantly decreased intracavernosal pressure-induced by electrical stimulation of the major pelvic ganglion in vivo. In conclusion, our results suggest that EETs, produced by CYP epoxygenases, in penile endothelial cells serve as vasodilators. Inhibition of this pathway attenuated erectile function, suggesting that EETs are required for normal erection.

In order to study the interaction between human cytochrome P450 2E1 (h2E1) and NADPH–P450 oxidoreductase (hOR) in a native membrane environment, we used two approaches to express both h2E1 and hOR in a baculovirus expression system. For a dual-expression system, h2E1 and hOR were coexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus carrying both h2E1 and hOR cDNAs (v-h2E1-hOR). The h2E1 cDNA was expressed under the control of the polyhedrin promoter P Polh , whereas hOR cDNA was expressed under the control of the P 10 promoter. The expressed enzymes were catalytically active in the cell membrane preparations. The estimated molar expression ratio of h2E1 to hOR in the membranes was 1:5. The apparent K m and k cat for N -nitrosodimethylamine (NDMA) demethylase activity were 145 μ M and 2.4 min −1 , respectively. When Sf9 cells were co-infected with the dual-expression virus (v-h2E1-hOR) and human cytochrome b 5 recombinant virus (v-hb 5 ), a 9-fold decrease in the K m of NDMA demethylase activity (16 μ M ) was observed in the membrane preparations, whereas the k cat was increased to 4 min −1 . In the second approach, recombinant viruses of h2E1 and hOR (v-h2E1 and v-hOR) were used to co-infect the Sf9 cells. In this double-expression system, with a fixed amount of v-h2E1, the expression of h2E1 in the Sf9 cells decreased as the amount of v-hOR increased. Western blot analysis of the membrane preparations showed that the level of hOR increased, but the level of h2E1 decreased with increasing amounts of v-hOR. A corresponding decrease in h2E1 mRNA, however, was not observed. In the presence of human cytochrome b 5 (hb 5 ), the optimal h2E1:hOR molar ratio for h2E1 catalytic activity was 1:1. In order to further investigate the hb 5 effect on h2E1-catalyzed reactions in the native membranes, we co-infected Sf9 cells with v-h2E1, v-hOR, and v-hb 5 and obtained a membrane preparation containing h2E1, hOR, and hb 5 . Stoichiometric analysis with membrane preparations from double-infection and triple-infection systems revealed that the presence of hb 5 decreased NADPH oxidation and H 2 O 2 formation by 72 and 80%, respectively, but increased product formation from NDMA 13-fold. These results suggest that hb 5 enhances the coupling between h2E1 and hOR for product formation. These studies also demonstrate that the baculovirus–insect cell system can produce high levels of expression of functional h2E1, hOR, and hb 5 for mechanistic studies.

Cytochrome P450 2E1 (P450 2E1) catalyzes the biotransformation of many low-molecular-weight compounds including industrial solvents, indoor pollutants, alcohol, and drugs. In order to understand the nature of the P450 2E1 active site, we studied the competitive inhibition of the P450 2E1-catalyzed N -nitrosodimethylamine demethylation by alcohols and carboxylic acids with different alkyl chain lengths. Using microsomes from acetone-treated rats as the enzyme source of P450 2E1, the K i value for each compound was measured. With primary alcohols and secondary alcohols, the K i decreased with the increase in the carbon chain length until the carbon number reached 6 or 7; the free energy increment of binding was 0.28 kcal/mol CH 2 group. Similar inhibitory effects were also observed with human P450 2E1 heterologously expressed in Hep G2 cells. These results suggest that both rat and human P450 2E1 contain a pocket with hydrophobicity that serves as a binding site for low-molecular-weight substrates. Among a series of carboxylic acids and ω-hydroxycarboxylic acids investigated, dodecanoic acid was the strongest inhibitor ( K i = 22 μM), and 12-hydroxydodecanoic acid had the lowest K i (320 μM) within the series of ω-hydroxycarboxylic acids. The free energy increment of binding for carboxylic acids was 0.35 kcal/mol CH2 group. 1,10-Decanedicarboxylic acid, which contains a carboxylic group at each end, showed a K i > 20 mM. We suggest that for optimal interaction of a carboxylic acid moiety with the active site of P450 2E1, the hydrocarbon end of the molecule binds to the substrate binding site, leaving the carboxylic acid group outside of a proposed substrate access channel. The length of optimal substrates such as dodecanoic acid may reflect the length of the substrate access channel and the size of the active site. We estimate that the distance from the opening of the access channel to the oxygenation site is about 15 A. Based on the structural features of P450 2E1 substrates and competitive inhibitors, we propose a conceptual model to illustrate the binding of these molecules in the active site of P450 2E1.

Studies suggest that soluble epoxide hydrolase (sEH) inhibition reduces end-organ damage in cardiovascular diseases. We hypothesize that sEH gene ( Ephx2) knockout (KO) improves endothelial function and reduces renal injury in streptozotocin-induced diabetes. After 6 wk of diabetes, afferent arteriolar relaxation to acetylcholine was impaired in diabetic wild-type (WT) mice, as the maximum relaxation was 72% of baseline diameter in the WT but only 31% in the diabetic mice. Ephx2 KO improved afferent arteriolar relaxation to acetylcholine in diabetes as maximum relaxation was 58%. Urinary monocyte chemoattractant protein-1 (MCP-1) excretion significantly increased in diabetic WT mice compared with control (868 ± 195 vs. 31.5 ± 7 pg/day), and this increase was attenuated in diabetic Ephx2 KO mice (420 ± 98 pg/day). The renal phospho-IKK-to-IKK ratio and nuclear factor-κB were significantly decreased, and hemeoxygenase-1 (HO-1) expression increased in diabetic Ephx2 KO compared with diabetic WT mice. Renal NADPH oxidase and urinary thiobarbituric acid reactive substances excretion were reduced in diabetic Ephx2 KO compared with diabetic WT mice. Albuminuria was also elevated in diabetic WT mice compared with control (170 ± 43 vs. 37 ± 13 μg/day), and Ephx2 KO reduced this elevation (50 ± 15 μg/day). Inhibition of sEH using trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB) also reduced renal inflammation and injury in diabetic WT mice. Furthermore, inhibition of HO with stannous mesoporphyrin negated the reno-protective effects of tAUCB or Ephx2 KO during diabetes. These data demonstrate that Ephx2 KO improves endothelial function and reduces renal injury during diabetes. Additionally, our data also suggest that activation of HO-1 contributes to improved renal injury in diabetic Ephx2 KO mice.

Background— Despite intensive investigation, how DNA methylation influences endothelial function remains poorly understood. We used methyl-CpG–binding domain protein 2 (MBD2), an interpreter for DNA methylome–encoded information, to dissect the impact of DNA methylation on endothelial function in both physiological and pathophysiological states. Methods and Results— Human umbilical vein endothelial cells under normal conditions express moderate levels of MBD2, but knockdown of MBD2 by siRNA significantly enhanced angiogenesis and provided protection against H 2 O 2 -induced apoptosis. Remarkably, Mbd2 −/− mice were protected against hind-limb ischemia evidenced by the significant improvement in perfusion recovery, along with increased capillary and arteriole formation. Loss of MBD2 activated endothelial survival and proangiogenic signals downstream of vascular endothelial growth factor signaling characterized by an increase in endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor 2 expression, along with enhanced extracellular signal-regulated kinase 1/2 activation and BCL-2 expression. Mechanistic studies confirmed the methylation of CpG elements in the eNOS and vascular endothelial growth factor receptor 2 promoter. MBD2 binds to these methylated CpG elements and suppresses eNOS promoter activity. On ischemic insult, key endothelial genes such as eNOS and vascular endothelial growth factor receptor 2 undergo a DNA methylation turnover, and MBD2 interprets the changes of DNA methylation to suppress their expressions. Moreover, MBD2 modulation of eNOS expression is likely confined to endothelial cells because nonendothelial cells such as splenocytes fail to express eNOS after loss of MBD2. Conclusions— We provided direct evidence supporting that DNA methylation regulates endothelial function, which forms the molecular basis for understanding how environmental insults (epigenetic factor) affect the genome to modify disease susceptibility. Because MBD2 itself does not affect the methylation of DNA and is dispensable for normal physiology in mice, it could be a viable epigenetic target for modulating endothelial function in disease states.

Arachidonic acid (AA) is metabolized by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes into eicosanoids, which are involved in diverse diseases, including type 1 and type 2 diabetes. During the last thirty years, evidence has been accumulated that suggests important functions for eicosanoids in the control of pancreatic β-cell function and destruction. AA metabolites of the COX pathway, especially prostaglandin E2 (PGE2), appear to be significant factors to β-cell dysfunction and destruction, participating in the pathogenesis of diabetes and its complications. Several elegant studies have contributed to the sorting out of the importance of 12-LOX eicosanoids in cytokine-mediated inflammation in pancreatic β cells. The role of CYP eicosanoids in diabetes is yet to be explored. A recent publication has demonstrated that stabilizing the levels of epoxyeicosatrienoic acids (EETs), CYP eicosanoids, by inhibiting or deleting soluble epoxide hydrolase (sEH) improves β-cell function and reduces β-cell apoptosis in diabetes. In this review we summarize recent findings implicating these eicosanoid pathways in diabetes and its complications. We also discuss the development of animal models with targeted gene deletion and specific enzymatic inhibitors in each pathway to identify potential targets for the treatment of diabetes and its complications.

Disulfiram, widely used in avoidance therapy for alcohol abuse, has been shown to have protective effects against chemically induced toxicity and carcinogenesis. The purpose of this work was to elucidate the biochemical mechanisms of this protective action by examining its effects on cytochrome P450IIE1 and other related microsomal enzyme activities. When a dose of disulfiram was given intragastrically to rats, a very rapid decrease of N -nitrosodimethylamine (NDMA) demethylase activity, possibly due to the inactivation of P450IIE1, was seen. The loss of P450IIE1 protein from the microsomal membrane was observed at 18 hr after receiving disulfiram, but not within the first 5 hr after the treatment. P450IIB1, on the other hand, was induced markedly between 15 and 72 hr after the disulfiram treatment. The treatment, however, caused only moderate changes in some other P450 isozymes. Carbon disulfide, a putative metabolite of disulfiram, produced similar effects on P450IIE1, but with shorter duration. Carbon disulfide, however, did not induce P450IIB1. Diethyldithiocarbamate, a reductive product of disulfiram, was an inhibitor of P450IIE1 activity in vitro , and upon preincubation with microsomes, it produced an NADPH-dependent inactivation of NDMA demethylase activity. The results suggest that this or other metabolites of disulfiram are inhibitors of P450IIE1 and are responsible for the inactivation of P450IIE1 in vivo . Hepatotoxicity of NDMA or CCl 4 in rats was blocked by pretreatment with disulfiram. The present work demonstrates that P450IIE1 was inhibited and inactivated by disulfiram, and this mechanism can account for many of the reported inhibitory actions of disulfiram against chemically induced toxicity and carcinogenesis.

Nitroalkene derivatives of linoleic acid (nitrolinoleic acid; LNO2) and nitro-oleic acid (OA-NO2) are endogenous lipid products with potent anti-inflammatory properties. The present study was undertaken to evaluate the therapeutic potential of OA-NO2in a mouse model of renal ischemia-reperfusion (I/R) injury. B6129SF2/J mice were subjected to bilateral renal ischemia for 30 min, followed by 24 h of reperfusion. Fifty minutes after ischemia, mice received intraperitoneal (ip) injections of OA-NO2(500 μg/kg; I/R OA-NO2), vehicle for OA-NO2(i.e., 0.8 ml/kg ethanol; I/R veh), or oleic acid (500 μg/kg; I/R OA) every 6 h during the 24-h recovery period. A sham-operated group was not subjected to ischemia and received 0.8 ml/kg ethanol ip every 6 h during the 24-h recovery period (sham veh). While plasma urea and creatinine were elevated ( P < 0.05) in I/R veh vs. sham veh mice, the severity was less ( P < 0.05) in I/R OA-NO2animals. Indices of histological damage, polymorphonucleocyte infiltration, together with expression of intracellular adhesion molecule-1, interleukin-1β, and tumor necrosis factor-α, p47phox, and gp91phoxwere greater in I/R veh vs. sham veh mice, but were attenuated ( P < 0.05) in I/R OA-NO2animals. Because indices of renal dysfunction were similar between I/R veh and I/R OA mice ( P > 0.05), but less ( P < 0.05) in I/R OA-NO2animals compared with both groups, protection from bilateral renal ischemia is afforded by the nitrated but not free form of oleic acid. Together, delayed administration of nitrated fatty acid OA-NO2attenuates renal I/R injury in the mouse likely via inhibition of the inflammatory response.

Here we performed studies to demonstrate SUMO4 maturation process. Unlike other SUMO proteins, cells under physiological condition mediate a rapid degradation for SUMO4. However, when cells under stressed condition, SUMO4 can be matured by the stress-induced endogenous hydrolase and be able to covalently conjugate to its substrate proteins. Furthermore, we failed to obtain evidence supporting a role for proline-90 unique to SUMO4 in its activation and functionality. Both wild-type SUMO4 and SUMO4-P90Q can be hydrolyzed by the stressed RAW264.7 cell lysates, and no significant functional difference between SUMO4, SUMO4-P90Q, and SUMO4-GG (matured form) was observed as determined by luciferase assay. However, the C-terminal di-glycine motif, a prerequisite for sumoylation, is necessary for SUMO4 to exert its functional activity. These data not only confirmed our previous published data, but also provided additional evidence suggesting a role for SUMO4 sumoylation in the regulation of intracellular stress.

Experiments were performed to establish the pharmacological profile of purinoceptors and to identify the signal transduction pathways responsible for increases in intracellular calcium concentration ([Ca2+]i) for cultured mouse mesangial cells. Mouse mesangial cells were loaded with fura 2 and examined using fluorescent spectrophotometry. Basal [Ca2+]iaveraged 102 ± 2 nM ( n = 346). One hundred micromolar concentrations of ATP, ADP, 2′,3′-(benzoyl-4-benzoyl)-ATP (BzATP), ATP-γ-S, and UTP in normal Ca2+medium evoked peak increases in [Ca2+]iof 866 ± 111, 236 ± 18, 316 ± 26, 427 ± 37, and 808 ± 73 nM, respectively. UDP or 2-methylthio-ATP (2MeSATP) failed to elicit significant increases in [Ca2+]i, whereas identical concentrations of adenosine, AMP, and α,β-methylene ATP (α,β-MeATP) had no detectable effect on [Ca2+]i. Removal of Ca2+from the extracellular medium had no significant effect on the peak increase in [Ca2+]iinduced by ATP, ADP, BzATP, ATP-γ-S, or UTP compared with normal Ca2+; however, Ca2+-free conditions did accelerate the rate of decline in [Ca2+]iin cells treated with ATP and UTP. [Ca2+]iwas unaffected by membrane depolarization with 143 mM KCl. Western blot analysis for P2 receptors revealed expression of P2X2, P2X4, P2X7, P2Y2, and P2Y4receptors. No evidence of P2X1and P2X3receptor expression was detected, whereas RT-PCR analysis reveals mRNA expression for P2X1, P2X2, P2X3, P2X4, P2X7, P2Y2, and P2Y4receptors. These data indicate that receptor-specific P2 receptor activation increases [Ca2+]iby stimulating calcium influx from the extracellular medium and through mobilization of Ca2+from intracellular stores in cultured mouse mesangial cells.

>> In this study we examined the expression of cytochrome P450 (CYP) 2C and CYP2J isoforms in renal proximal tubules and microvessels isolated from rats at different stages of pregnancy. We also selectively inhibited epoxyeicosatrienoic acid (EET) production by the administration of N-methanesulfonyl-6-(2proparyloxyphenyl)hexanamide (MSPPOH 20 mg/kg/day iv) to rats during Days 14–17 of gestation and to age-matched virgin rats and determined the consequent effects on renal function. Western blot analysis showed that CYP2C11, CYP2C23, and CYP2J2 expression was significantly increased in the renal microvessels of pregnant rats on Day 12 of gestation. In the proximal tubules, CYP2C23 expression was significantly increased throughout pregnancy, while the expression of CYP2C11 was increased in early and late pregnancy and the expression of CYP2J2 was increased in middle and late pregnancy. MSPPOH treatment significantly increased pregnant rats’ mean arterial pressure, renal vascular resistance, and sodium balance but significantly decreased renal blood flow, glomerular filtration rate, and urinary sodium excretion, as well as fetal pups’ body weight and length. In contrast, MSPPOH treatment had no effect on renal hemodynamics or urinary sodium excretion in age-matched virgin rats. In pregnant rats, MSPPOH treatment also caused selective inhibition of renal cortical EET production and significantly decreased the expression of CYP2C11, CYP2C23, and CYP2J2 in the renal cortex, renal microvessels, and proximal tubules. These results suggest that upregulation of renal vascular and tubular EETs contributes to the control of blood pressure and renal function during pregnancy. Exp Biol Med 231:1744–1752, 2006

Hypoxia that is caused by vascular defects or disruption is commonly associated with renal diseases. During cisplatin nephrotoxicity, hypoxic regions are identified in the outer medulla and the renal cortex. However, the regulation of cisplatin injury by hypoxia is unclear. Previous work has demonstrated the cytoprotective effects of hypoxia against apoptotic injury. This study further examines the cytoprotective mechanisms in models of cisplatin-induced tubular cell apoptosis. In cultured renal tubular cells, 20 microM cisplatin induced approximately 60% apoptosis within 16 h. The rate of apoptosis was suppressed to < 20%, when the incubation was conducted under hypoxia (2% O2). Mitochondrial events of apoptosis, namely Bax accumulation and cytochrome c release, also were ameliorated. During cisplatin treatment, cell ATP was maintained in both normoxic and hypoxic cells. Hypoxic incubation lowered extracellular pH, but prevention of the pH decrease did not restore cisplatin-induced apoptosis. The cytoprotective effects of hypoxia also were independent of hypoxia-inducible factor 1 (HIF-1). Cobalt, as hypoxia, activated HIF-1 yet did not suppress cisplatin-induced apoptosis. Moreover, hypoxia suppressed cisplatin-induced apoptosis in HIF-1-deficient mouse embryonic stem cells and renal proximal tubular cells. Conversely, mitochondrial inhibitors, particularly inhibitors of respiration complex III (antimycin A and myxothiazol), mimicked hypoxia in apoptosis suppression. The effects of hypoxia and mitochondrial inhibitors were not additive. It is interesting that both hypoxia and complex III inhibitors ameliorated cisplatin-induced p53 activation. Therefore, the cytoprotective effects of hypoxia are independent of changes in cell ATP, pH, or HIF but may involve mitochondrial inhibition and the suppression of p53.

The substrates of SUMO4, a novel member for the SUMO gene family, were characterized in HEK293 cells cultured under serum starvation by proteomic analysis. We identified 90 SUMO4 substrates including anti-stress proteins such as antioxidant enzymes and molecular chaperones or co-chaperones. The substrates also include proteins involved in the regulation of DNA repair and synthesis, RNA processing, protein degradation, and glucose metabolism. Several SUMO4-associated transcription factors were characterized by Western blot analyses. AP-1 was selected for in vitro conjugation assays to confirm SUMO4 sumoylation of these transcription factors. Further functional analyses of the transcription factors suggested that SUMO4 sumoylation represses AP-1 and AP-2alpha transcriptional activity, but enhances GR DNA binding capacity. These results demonstrate that SUMO4 sumoylation may play an important role in the regulation of intracellular stress.

Tubular cell apoptosis is involved in ischemic renal failure, but the underlying mechanism is unclear. Bid, a proapoptotic Bcl-2 family protein, may regulate the intrinsic as well as the extrinsic pathway of apoptosis. In vivo, Bid is most abundantly expressed in the kidneys. However, the role played by Bid in renal pathophysiology is unknown. Our recent work demonstrated Bid activation during renal ischemia-reperfusion. The current study has determined the role of Bid in ischemic renal injury and renal failure using Bid-deficient mice. In wild-type C57BL/6 mice, Bid was proteolytically processed into active forms during renal ischemia-reperfusion, which subsequently targeted mitochondria. This was accompanied by the development of tissue damage and severe renal failure, showing serum creatinine of 3.0 mg/dl after 48 h of reperfusion. The same ischemic insult induced acute renal failure in Bid-deficient mice, which was nonetheless less severe than the wild-type, showing 1.3 mg/dl serum creatinine. In addition, Bid deficiency attenuated tubular disruption, tubular cell apoptosis, and caspase-3 activation during 48 h of reperfusion. Compared with wild-type, animal death following renal ischemia was delayed in Bid-deficient mice. Collectively, the results suggest a role for Bid in ischemic renal injury and renal failure.

Cytochrome P-450–4A1 (CYP4A1) is an ω-hydroxylase that catalyzes the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to determine the vasomotor consequences of vascular overexpression of CYP4A1. Isolated rat gracilis muscle arterioles transfected ex vivo with an expression plasmid containing CYP4A1 cDNA expressed more CYP4A protein than vessels transfected with the control plasmid. In arterioles pressurized to 80 mmHg, the internal diameter of vessels transfected with CYP4A1 cDNA (55 ± 3 μm) was surpassed ( P < 0.05) by that of vessels transfected with control plasmid (97 ± 4 μm). Treatment with a CYP4A inhibitor ( N-methylsulfonyl-12,12-dibromododec-11-enamide; DDMS) or with an antagonist of 20-HETE actions [20-hydroxyeicosa-6( Z),15( Z)-dienoic acid; 20-HEDE] elicited robust dilation of arterioles transfected with CYP4A1 cDNA, whereas the treatment had little or no effect in vessels transfected with control plasmid. Examination of the intraluminal pressure-internal diameter relationship revealed that pressure increments over the range of 40–100 mmHg elicited a more intense ( P < 0.05) myogenic constrictor response in arterioles transfected with CYP4A1 cDNA than in those with control plasmid. Arterioles transfected with CYP4A1 cDNA also displayed enhanced sensitivity to the constrictor action of phenylephrine. Treatment with DDMS or 20-HEDE greatly attenuated the constrictor responsiveness to both constrictor stimuli in vessels overexpressing CYP4A1, whereas the treatment had much less effect in control vessels. These data suggest that CYP4A1 overexpression promotes constriction of gracilis muscle arterioles by intensifying the responsiveness of vascular smooth muscle to constrictor stimuli. This effect of CYP4A1 overexpression appears to be mediated by a CYP4A1 product.

Bid is a proapoptotic Bcl-2 family protein, which on activation translocates to mitochondria and induces damage to the organelles. Activation of Bid depends on its proteolytic processing into truncated forms of tBid. Bid is highly expressed in the kidneys; however, little is known about its role in renal pathophysiology. In this study, we initially examined Bid activation in cultured rat kidney proximal tubular cells following ATP depletion. The cells were depleted of ATP by azide incubation in the absence of metabolic substrates and then returned to normal culture medium for recovery. Typical apoptosis developed during recovery of ATP-depleted cells. This was accompanied by Bid cleavage, releasing tBid of 15 and 13 kDa. Bid cleavage was abolished in cells overexpressing Bcl-2, an antiapoptotic gene. It was also suppressed by caspase inhibitors. Peptide inhibitors of caspase-9 were more effective in blocking Bid cleavage compared with inhibitors of caspase-8 and caspase-3. Provision of glucose, a glycolytic substrate, during azide incubation inhibited Bid cleavage as well, indicating that Bid cleavage was initiated by ATP depletion. Consistently, Bid cleavage was also induced following ATP depletion by hypoxia or mitochondrial uncoupling. Of significance, cleaved Bid translocated to mitochondria, suggesting a role for Bid in the development of mitochondrial defects in ATP-depleted cells. Finally, Bid cleavage was induced during renal ischemia-reperfusion in the rat. Together, these results provide the first evidence for Bid activation in kidney cells following ATP depletion in vitro and renal ischemia in vivo.

20-Hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor and mediator of the myogenic response, is a major arachidonic acid metabolite in the microvasculature of the rat kidney formed primarily by the cytochrome P-450 (CYP) 4A isoforms, CYP4A1, CYP4A2, and CYP4A3. We examined CYP4A isoform expression and 20-HETE synthesis in microdissected interlobar, arcuate, and interlobular arteries; mRNA for all CYP4A isoforms was identified by RT-PCR. Western blot analysis indicated that the levels of CYP4A2/4A3-immunoreactive protein increased with decreased arterial diameter, whereas those of CYP4A1-immunoreactive protein remained unchanged. 20-HETE synthesis was the highest in the interlobular arteries (17 ± 1.62 nmol · mg−1· h−1) and, like CYP4A2/4A3-immunoreactive protein, decreased with increasing vessel diameter (4.5 ± 1.21, 2.65 ± 0.58, and 0.81 ± 0.14 nmol · mg−1· h−1in the arcuate, interlobar, and segmental arteries, respectively). 20-HETE synthesis in the renal artery and the abdominal aorta was undetectable. The observed decreased immunoreactivity of NADPH-cytochrome P-450 ( c) oxidoreductase with increased arterial diameter provided a possible explanation for the decreased capacity to generate 20-HETE in the large arteries. The increase in CYP4A isoform expression and 20-HETE synthesis with decreasing diameter along the preglomerular arteries and the potent biological activity of 20-HETE underscore the significance of 20-HETE as a modulator of renal hemodynamics.

P450-dependent arachidonic acid (AA) metabolites regulate arterial tone by modulating calcium-activated (BK) potassium channels in vascular smooth muscle cells (VSMC). Because eicosapentaenoic acid (EPA) has been reported to improve vascular function, we tested the hypothesis that P450-dependent epoxygenation of EPA produces alternative vasoactive compounds. We synthesized the 5 regioisomeric epoxyeicosattrienoic acids (EETeTr) and examined them for effects on K + currents in rat cerebral artery VSMCs with the patch-clamp technique. 11(R),12(S)-epoxyeicosatrienoic acid (50 nmol/L) was used for comparison and stimulated K + currents 6-fold at +60 mV. However, 17(R),18(S)-EETeTr elicited a more than 14-fold increase. 17(S),18(R)-EET and the remaining four regioisomers were inactive. The effect of 17(R),18(S)-EETeTr was blocked by tetraethylammonium but not by 4-aminopyridine. VSMCs expressed P450s 4A1 and 4A3. Recombinant P450 4A1 hydroxylated EPA at C-19 and C-20 and epoxygenated the 17,18-double bond, yielding the R, S- and S, R-enantiomers in a ratio of 64:36. We conclude that 17(R),18(S)-EETeTr represents a novel, potent activator of BK potassium channels. Furthermore, this metabolite can be directly produced in VSMCs. We suggest that 17(R),18(S)-EETeTr may function as an important hyperpolarizing factor, particularly with EPA-rich diets.

20-hydroxyeicosatetraenoic acid (20-HETE), the ω-hydroxylation product of arachidonic acid (AA), is the principal metabolite formed in tubular and vascular structures of the rat renal cortex and outer medulla. 20-HETE has potent biological activities and has been shown to contribute to the regulation of renal function and to the control of arterial pressure. In the renal tubules it inhibits sodium reabsorption, while in the renal microcirculation it is a vasoconstrictor and a regulator of the myogenic response. The ω-hydroxylation of fatty acids, including AA, is catalyzed by enzymes of the cytochrome P450 (CYP) 4A family. In the rat, four isoforms have been identified: CYP4A1, 4A2, 4A3, and 4A8. Our studies indicated that despite the high homology, these isoforms display distinct catalytic properties including differences in kinetic parameters, product profile and inhibitor sensitivity. While the constitutive level of expression of CYP4A1 is low, its recombinant form is the low Km AA-ω-hydroxylase and thus, by far, the most efficient 20-HETE synthesizing enzyme. Whereas CYP4A1 is solely an AA whydroxylase, CYP4A2 and CYP4A3 also catalyze AA 11,12-epoxidation. Systemic administration of CYP4A antisense oligodeoxynucleotides revealed that CYP4A1 contributes significantly to the renal tubular and vascular production of 20-HETE. Furthermore, these isoforms demonstrate unique intrarenal localization. Using molecular and pharmacological probes we demonstrated a unique CYP4A isoform-specific localization within the renal microvasculature. Transfection of CYP4A1 cDNA to renal microvessels resulted in enhanced 20-HETE synthesis and increased reactivity to phenylephrine. Thus, 20-HETE of vascular origin serves as a stimulatory regulator of vascular responses to constrictor agonists.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), the omega-hydroxylation product of arachidonic acid, is the major metabolite produced in the kidney. It has potent biological effects on renal tubular and vascular functions and on the long-term control of arterial pressure. The synthesis of 20-HETE is catalyzed by enzymes of the CYP4A family, among which CYP4A2 is the most abundant isozyme expressed in the kidneys of rats. We have cloned and sequenced the CYP4A2 cDNA from the kidney of Lewis-Wistar rats and directed its expression using baculovirus and Sf9 insect cells. A high level of expression of CYP4A2 was evident by Northern, Western, and spectral analyses revealing a P450 content of 0.3 nmol/mg microsomal protein. To study CYP4A2-catalyzed arachidonic acid omega-hydroxylation, Sf9 cells were coinfected with CYP4A2 and NADPH cytochrome P450 oxidoreductase (OR) recombinant viruses. CYP4A2/OR membranes metabolized lauric acid at a high rate (7 and 5.5 nmol/min/nmol P450 in the presence and absence of b5, respectively). However, arachidonic acid omega-hydroxylase activity was barely detectable. When purified OR was added to the membranes expressing CYP4A2 protein, a concentration-dependent production of 20-HETE was observed. Maximal synthesis of 20-HETE of 0.89 nmol/min/nmol P450 was achieved at OR:CYP4A2 ratio of 14:1. The omega-hydroxylation of arachidonic acid was dependent on the presence of b5. Furthermore, increasing OR concentrations yielded additional arachidonic acid metabolite identified by GC/MS as 11,12-EET. Microsomes prepared from isolated renal microvessels selectively expressed CYP4A2 protein and readily metabolized arachidonic acid to two major metabolites, 20-HETE and 11,12-DHET, the hydrolytic metabolite of 11, 12-EET. It is suggested that CYP4A2 functions as the renal microvessel arachidonate omega-hydroxylase and that it can also catalyze the 11,12-epoxidation of arachidonic acid.