Endogenous estradiol contributes to vascular endothelial dysfunction in premenopausal women with type 1 diabetes.

BACKGROUND: Endogenous estrogen is cardio-protective in healthy premenopausal women. Despite this favorable action of estrogen, animal models depict a detrimental effect of estradiol on vascular function in the presence of diabetes. The present study sought to determine the role of endogenous estradiol on endothelial function in women with type 1 diabetes. METHOD: 32 women with type 1 diabetes (HbA1c = 8.6 ± 1.7%) and 25 apparently healthy women (HbA1c = 5.2 ± 0.3%) participated. Flow-mediated dilation (FMD), a bioassay of nitric-oxide bioavailability and endothelial function was performed during menses (M) and the late follicular (LF) phase of the menstrual cycle to represent low and high concentrations of estrogen, respectively. In addition, a venous blood sample was collected at each visit to determine circulating concentrations of estradiol, thiobarbituric acid reactive substances (TBARS), and nitrate/nitrite (NOx), biomarkers of oxidative stress and nitric oxide, respectively. Data were collected in (1) 9 additional women with type 1 diabetes using oral hormonal birth control (HBC) (HbA1c = 8.3 ± 2.1%) during the placebo pill week and second active pill week, and (2) a subgroup of 9 demographically matched women with type 1 diabetes not using HBC (HbA1c = 8.9 ± 2.1%). RESULTS: Overall, estradiol was significantly increased during the LF phase compared to M in both type 1 diabetes (Δestradiol = 75 ± 86 pg/mL) and controls (Δestradiol = 71 ± 76 pg/mL); however, an increase in TBARS was only observed in patients with type 1 diabetes (ΔTBARS = 3 ± 13 µM) compared to controls (ΔTBARS = 0 ± 4 µM). FMD was similar (p = 0.406) between groups at M. In addition, FMD increased significantly from M to the LF phase in controls (p = 0.024), whereas a decrease was observed in type 1 diabetes. FMD was greater (p = 0.015) in patients using HBC compared to those not on HBC, independent of menstrual cycle phase. CONCLUSION: Endogenous estradiol increases oxidative stress and contributes to endothelial dysfunction in women with diabetes. Additionally, HBC use appears to be beneficial to endothelial function in type 1 diabetes.

Sexual dimorphism in renal heme oxygenase-1 and arachidonic acid metabolizing enzymes in spontaneously hypertensive rats versus normotensive Wistar Kyoto rats.

Numerous studies have demonstrated a sexual dimorphism in blood pressure (BP) control in spontaneously hypertensive rats (SHR), however the mechanisms remain to be further elucidated. Based on the established role of arachidonic acid metabolites and heme oxygenase (HO) in BP control, we hypothesize that higher BP in male SHR is associated with differential expression in renal HO and arachidonic acid metabolizing enzymes vs. female SHR. Higher BP in male SHR coincided with significant increases in renal cortical superoxide production and thiobarbituric acid reactive substances (TBARs) levels as measures of oxidative stress compared to normotensive female WKY and female SHR. The elevations in BP and oxidative stress in male SHR were also associated with a decrease in cortical heme oxygenase-1 (HO-1) expression when compared to normotensive female WKY. Although there was no sex or strain differences in cortical expression of the epoxyeicosatrienoic acids (EETs) producing enzyme, cytochrome P450 epoxygenase (CYP2C23), in male and female SHR and WKY, SHR had greater expression of the EETs metabolizing enzyme, soluble epoxide hydrolase (sEH) vs. WKY. Cortical expression of the 20-hydroxyeicosatetraenoic acid (20-HETE) producing enzyme, cytochrome P450 hydroxylase (CYP4A), was less in female WKY and SHR compared to strain-matched males and cortical 20-HETE levels were also less in female SHR vs. male SHR. Cortical cyclooxygenase-2 (COX-2) expression was significantly greater in female SHR and WKY vs. males and cortical prostaglandin E2 (PGE2) levels in female SHR was significantly greater than male WKY. In conclusion, our data suggest that sex differences in renal oxidative stress, HO-1 and arachidonic acid metabolizing enzymes could contribute to sexual dimorphism in hypertension in young SHR.

Sex differences in hypertension: lessons from spontaneously hypertensive rats (SHR).

Although numerous clinical and experimental studies have clearly identified a sexual dimorphism in blood pressure control, the mechanism(s) underlying gender differences in blood pressure remain unclear. Over the past two decades, numerous laboratories have utilized the spontaneously hypertensive rats (SHR) as an experimental model of essential hypertension to increase our understanding of the mechanisms regulating blood pressure in males and females. Previous work by our group and others have implicated that differential regulation of adrenergic receptors, the renin-angiotensin system, oxidative stress, nitric oxide bioavailability and immune cells contribute to sex differences in blood pressure control in SHR. The purpose of this review is to summarize previous findings to date regarding the mechanisms of blood pressure control in male versus female SHR.

Effect of Everolimus versus Bone Marrow-Derived Stem Cells on Glomerular Injury in a Rat Model of Glomerulonephritis: A Preventive, Predictive and Personalized Implication.

Glomerular endothelial injury and effectiveness of glomerular endothelial repair play a crucial role in the progression of glomerulonephritis. Although the potent immune suppressive everolimus is increasingly used in renal transplant patients, adverse effects of its chronic use have been reported clinically in human glomerulonephritis and experimental renal disease. Recent studies suggest that progenitor stem cells could enhance glomerular endothelial repair with minimal adverse effects. Increasing evidence supports the notion that stem cell therapy and regenerative medicine can be effectively used in pathological conditions within the predictive, preventive and personalized medicine (PPPM) paradigm. In this study, using an experimental model of glomerulonephritis, we tested whether bone marrow-derived stem cells (BMDSCs) could provide better effect over everolimus in attenuating glomerular injury and improving the repair process in a rat model of glomerulonephritis. Anti-Thy1 glomerulonephritis was induced in male Sprague Dawley rats by injection of an antibody against Thy1, which is mainly expressed on glomerular mesangial cells. Additional groups of rats were treated with the immunosuppressant everolimus daily after the injection of anti-Thy1 or injected with single bolus dose of BMDSCs after one week of injection of anti-Thy1 (n = 6-8). Nine days after injection of anti-Thy1, glomerular albumin permeability and albuminuria were significantly increased when compared to control group (p < 0.05). Compared to BMDSCs, everolimus was significantly effective in attenuating glomerular injury, nephrinuria and podocalyxin excretion levels as well as in reducing inflammatory responses and apoptosis. Our findings suggest that bolus injection of BMDSCs fails to improve glomerular injury whereas everolimus slows the progression of glomerular injury in Anti-Thy-1 induced glomerulonephritis. Thus, everolimus could be used at the early stage of glomerulonephritis, suggesting potential implications of PPPM in the treatment of progressive renal injury.

Salvianolic Acid B Slows the Progression of Breast Cancer Cell Growth via Enhancement of Apoptosis and Reduction of Oxidative Stress, Inflammation, and Angiogenesis.

Breast cancer is the current leading cause of cancer death in females worldwide. Although current chemotherapeutic drugs effectively reduce the progression of breast cancer, most of these drugs have many unwanted side effects. Salvianolic acid B (Sal-B) is a bioactive compound isolated from the root of Danshen Radix with potent antioxidant and anti-inflammatory properties. Since free radicals play a key role in the initiation and progression of tumor cells growth and enhance their metastatic potential, the current study was designed to investigate the antitumor activity of Sal-B and compare it with the antitumor activity of the traditional anticancer drug, cisplatin. In vitro, Sal-B decreased the human breast cancer adenocarcinoma (MCF-7) cells proliferation in a concentration and time dependent manner. In vivo and similar to cisplatin treatment, Sal-B significantly reduced tumor volume and increased the median survival when compared to tumor positive control mice group injected with Ehrlich solid carcinoma cell line (ESC). Sal-B decreased plasma level of malondialdehyde as a marker of oxidative stress and increased plasma level of reduced glutathione (GSH) as a marker of antioxidant defense when compared to control ESC injected mice. Either Sal-B or cisplatin treatment decreased tumor tissue levels of tumor necrosis factor (TNF-α), matrix metalloproteinase-8 (MMP-8), and Cyclin D1 in ESC treated mice. Contrary to cisplatin treatment, Sal-B did not decrease tumor tissue Ki-67 protein in ESC injected mice. Immunohistochemical analysis revealed that Sal-B or cisplatin treatment increased the expression of the apoptotic markers caspase-3 and P53. Although Sal-B or cisplatin significantly reduced the expression of the angiogenic factor vascular endothelial growth factor (VEGF) in ESC injected mice, only Sal-B reduced expression level of COX-2 in ESC injected mice. Our data suggest that Sal-B exhibits antitumor features against breast cancer cells possibly via enhancing apoptosis and reducing oxidative stress, inflammation, and angiogenesis.

Influence of the selective COX-2 inhibitor celecoxib on sex differences in blood pressure and albuminuria in spontaneously hypertensive rats.

We previously reported that female spontaneously hypertensive rats (SHR) have greater cyclooxygenase-2 (COX-2) expression in the renal medulla and enhanced urinary excretion of prostaglandin (PG) E2 (PGE2) metabolites compared to male SHR. Based on the role of COX-2-derived prostanoids in the regulation of cardiovascular health, the aim of the current study was to test the hypothesis that blood pressure (BP) in female SHR is more sensitive to COX-2 inhibition than in males. Seven week old male and female SHR were implanted with telemetry transmitters for continuous BP recording. After one week of baseline BP recording, male and female SHR were randomized to receive the selective COX-2 inhibitor celecoxib (10 mg/kg/day) or vehicle for six weeks (from 9 to 14 weeks of age). Female SHR had lower BP and albuminuria compared to male SHR as well as enhanced urinary excretion of PGE metabolite (PGEM), 6-keto PGF1α and thromboxane B2, indicators of PGE2, PGI2 and TXA2, respectively. Treatment with celecoxib did not significantly alter BP or albuminuria in either female or male SHR. Celecoxib did not change PGs metabolites excretion in male SHR; however, excretion levels of PGEM and 6-keto PGF1α were reduced in female SHR. COX-2 derived PG can also induce oxidative stress. Markers of oxidative stress (thiobarbituric acid reactive substances (TBARs) and H2O2 excretion) were lesser in female SHR versus male SHR. Celecoxib treatment did not significantly change markers of oxidative stress in female SHR, however, urinary TBARs excretion was significantly reduced in male SHR after 6 weeks of treatment with celecoxib. Therefore, although celecoxib treatment appears to have distinct effects on prostanoids levels in female SHR vs. males, it is unlikely that COX-2 contributes to established sex differences in BP in SHR.

Meloxicam fails to augment the reno-protective effects of soluble epoxide hydrolase inhibition in streptozotocin-induced diabetic rats via increased 20-HETE levels.

The pro-inflammatory cyclooxygenase (COX)-derived prostaglandins and the anti-inflammatory cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) play an important role in the regulation of renal injury. The current study examined whether COX inhibition augments the reno-protective effects of increased EETs levels via inhibiting EETs degradation by soluble epoxide hydrolase (sEH) in diabetic rats. Streptozotocin (50mg/kg, i.v) was used to induce diabetes in male Sprague Dawley rats. Rats were then divided into 5 groups (n=6-8); control non diabetic, diabetic, diabetic treated with the sEH inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), diabetic treated with the COX inhibitor meloxicam and diabetic treated with meloxicam plus t-AUCB for 2 months. Glomerular albumin permeability and urinary albumin and nephrin excretion levels were significantly elevated in diabetic rats together with decreased glomerular α3 integrin and nephrin expression levels. Inhibition of sEH reduced glomerular albumin permeability, albumin and nephrin excretion levels and restored the decrease in glomerular α3 integrin and nephrin expression in diabetic rats. Meloxicam failed to reduce renal injury or even to synergize the reno-protective effects of sEH inhibition in diabetic rats. Furthermore, inhibition of sEH reduced the elevation in renal collagen deposition and urinary MCP-1 excretion levels together with a reduction in the number of renal TUNEL positive cells in diabetic vs. control rats (P<0.05). Meloxicam did not reduce renal inflammation or apoptosis in diabetic rats or even exacerbate the anti-inflammatory and anti-apoptotic effects of sEH inhibition. Renal 20-hydroxyeicosatetranoic acid (20-HETE) levels were elevated in diabetic rats and meloxicam further exacerbated this elevation. In conclusion, our study suggests that inhibition of COX failed to provide renal protection or to augment the reno-protective effects of sEH inhibition in diabetic rats, at least in part, via increased inflammatory 20-HETE levels.

Adenosine kinase inhibition protects the kidney against streptozotocin-induced diabetes through anti-inflammatory and anti-oxidant mechanisms.

Adenosine provides anti-inflammatory effects in cardiovascular disease via the activation of adenosine A2A receptors; however, the physiological effect of adenosine could be limited due to its phosphorylation by adenosine kinase. We hypothesized that inhibition of adenosine kinase exacerbates extracellular adenosine levels to reduce renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in male C57BL/6 mice by daily injection of streptozotocin (50mg/kg/day, i.p. for 5 days). Control and diabetic mice were then treated with the adenosine kinase inhibitor ABT702 (1.5mg/kg, i.p. two times a week for 8 weeks, n=7-8/group) or the vehicle (5% DMSO). ABT702 treatment reduced blood glucose level in diabetic mice (∼20%; P<0.05). ABT702 also reduced albuminuria and markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in diabetic mice. Renal NADPH oxidase activity and urinary thiobarbituric acid reactive substances (TBARS) excretion, indices of oxidative stress, were also elevated in diabetic mice and ABT702 significantly reduced these changes. ABT702 increased renal endothelial nitric oxide synthase expression (eNOS) and nitrate/nitrite excretion levels in diabetic mice. In addition, the diabetic mice displayed an increase in renal macrophage infiltration, in association with increased renal NFκB activation. Importantly, treatment with ABT702 significantly reduced all these inflammatory parameters (P<0.05). Furthermore, ABT702 decreased glomerular permeability and inflammation and restored the decrease in glomerular occludin expression in vitro in high glucose treated human glomerular endothelial cells. Collectively, the results suggest that the reno-protective effects of ABT702 could be attributed to the reduction in renal inflammation and oxidative stress in diabetic mice.

Reno-protective effect of protocatechuic acid is independent of sex-related differences in murine model of UUO-induced kidney injury.

BACKGROUND: Obstructive nephropathy is a condition often caused by urinary tract obstruction either anatomical (e.g., tumors), mechanical (e.g., urolithiasis), or compression (e.g., pregnancy) and can progress to chronic kidney disease (CKD). Studies have shown sexual dimorphism in CKD, where males were found to have a more rapid decline in kidney function following kidney injury compared to age-matched females. Protocatechuic acid (PCA), an anti-oxidant and anti-inflammatory polyphenolic compound, has demonstrated promising effects in mitigating drug-induced kidney injuries. The current study aims to explore sexual dimorphism in kidney injury after unilateral ureteral obstruction (UUO) and assess whether PCA treatment can mitigate kidney injury in both sexes. METHODS: UUO was induced in 10-12 weeks old male and female C57BL/6J mice. Mice were categorized into four groups (n = 6-8/group); Sham, Sham plus PCA (100 mg/kg, I.P daily), UUO, and UUO plus PCA. RESULTS: After 2 weeks of induction of UUO, markers of kidney oxidative stress (TBARs), inflammation (IL-1α and IL-6), tubular injury (neutrophil gelatinase-associated lipocalin, NGAL and urinary kidney injury molecule-1, KIM-1), fibrosis (Masson's trichrome staining, collagen IV expression, MMP-2 and MMP-9) and apoptosis (TUNEL+ cells, active caspase-1 and caspase-3) were significantly elevated in both males and females relative to their sham counterparts. Males exhibited significantly greater kidney oxidative stress, inflammation, fibrosis, and apoptosis after induction of UUO when compared to females. PCA treatment significantly attenuated UUO-induced kidney injury, inflammation, fibrosis, and apoptosis in both sexes. CONCLUSION: Our findings suggest a differential gender response to UUO-induced kidney injury with males being more sensitive to UUO-induced kidney inflammation, fibrosis, and apoptosis than age-matched females. Importantly, PCA treatment reduced UUO-induced kidney injury in a sex-independent manner which might be attributed to its anti-oxidant, anti-inflammatory, anti-fibrotic, and anti-apoptotic properties.

Female SHR have greater blood pressure sensitivity and renal T cell infiltration following chronic NOS inhibition than males.

Nitric oxide is a critical regulator of blood pressure (BP) and inflammation, and female spontaneously hypertensive rats (SHR) have higher renal nitric oxide bioavailability than males. We hypothesize that female SHR will have a greater rise in BP and renal T cell infiltration in response to nitric oxide synthase (NOS) inhibition than males. Both male and female SHR displayed a dose-dependent increase in BP to the nonspecific NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME: 2, 5, and 7 mg·kg(-1)·day(-1) for 4 days each); however, females exhibited a greater increase in BP than males. Treatment of male and female SHR with 7 mg·kg(-1)·day(-1) L-NAME for 2 wk significantly increased BP in both sexes; however, prior exposure to L-NAME only increased BP sensitivity to chronic NOS inhibition in females. L-NAME-induced hypertension increased renal T cell infiltration and indices of renal injury in both sexes, yet female SHR exhibited greater increases in Th17 cells and greater decreases in regulatory T cells than males. Chronic L-NAME was also associated with larger increases in renal cortical adhesion molecule expression in female SHR. The use of triple therapy to block L-NAME-mediated increases in BP attenuated L-NAME-induced increases in renal T cell counts and normalized adhesion molecule expression in SHR, suggesting that L-NAME-induced increases in renal T cells were dependent on both increases in BP and NOS inhibition. Our data suggest that NOS is critical in the ability of SHR, females in particular, to maintain BP and limit a pro-inflammatory renal T cell profile.

Role of haem oxygenase in the renoprotective effects of soluble epoxide hydrolase inhibition in diabetic spontaneously hypertensive rats.

We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-ß (transforming growth factor ß) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.

Acute nitric oxide synthase inhibition induces greater increases in blood pressure in female versus male Wistar Kyoto rats.

Nitric oxide (NO) contributes to blood pressure (BP) regulation via its vasodilatory and anti-inflammatory properties. We and others previously reported sex differences in BP in normotensive and hypertensive rat models where females have lower BP than age-matched males. As females are known to have greater NO bioavailability than age-matched males, the current study was designed to test the hypothesis that anesthetized female normotensive Wistar Kyoto rats (WKY) are more responsive to acute NOS inhibition-induced increases in BP compared to male WKY. Twelve-week-old male and female WKY were randomized to infusion of the nonspecific NOS inhibitor NG -nitro-L-arginine methyl ester (L-NAME, 1 mg/kg/min) or selective NOS1 inhibition with vinyl-L-NIO (VNIO, 0.5 mg/kg/min) for 60 min. Mean arterial BP, glomerular filtration rate (GFR), urine volume, and electrolyte excretion were assessed before, and during L-NAME or VNIO infusion. L-NAME and VNIO significantly increased BP in both sexes; however, the increase in BP with L-NAME infusion was greater in females versus males compared to baseline BP values. Acute infusion of neither L-NAME nor VNIO for 60 min altered GFR in either sex. However, urine volume, sodium, chloride and potassium excretion levels increased comparably in male and female WKY with L-NAME and VNIO infusion. Our findings suggest sex differences in BP responses to acute non-isoform-specific NOS inhibition in WKY, with females being more responsive to L-NAME-induced elevations in BP relative to male WKY. However, sex differences in the BP response did not coincide with sex differences in renal hemodynamic responses to acute NOS inhibition.

Induction of hemeoxygenase-1 reduces glomerular injury and apoptosis in diabetic spontaneously hypertensive rats.

Induction of hemeoxygenase-1 (HO-1) lowers blood pressure and reduces organ damage in hypertensive animal models; however, a potential protective role for HO-1 induction against diabetic-induced glomerular injury remains unclear. We hypothesize that HO-1 induction will protect against diabetes-induced glomerular injury by maintaining glomerular integrity and inhibiting renal apoptosis, inflammation, and oxidative stress. Diabetes was induced with streptozotocin in spontaneously hypertensive rats (SHR) as a model where the coexistence of hypertension and diabetes aggravates the progression of diabetic renal injury. Control and diabetic SHR were randomized to receive vehicle or the HO-1 inducer cobalt protoporphyrin (CoPP). Glomerular albumin permeability was significantly greater in diabetic SHR compared with control, consistent with an increase in apoptosis and decreased glomerular nephrin and α(3)ß(1)-integrin protein expression in diabetic SHR. CoPP significantly reduced albumin permeability and apoptosis and restored nephrin and α(3)ß(1)-integrin protein expression levels in diabetic SHR. Glomerular injury in diabetic SHR was also associated with increases in NF-κB-induced inflammation and oxidative stress relative to vehicle-treated SHR, and CoPP significantly blunted diabetes-induced increases in glomerular inflammation and oxidative stress in diabetic SHR. These effects were specific to exogenous stimulation of HO-1, since incubation with the HO inhibitor stannous mesoporphyrin alone did not alter glomerular inflammatory markers or oxidative stress yet was able to prevent CoPP-mediated decreases in these parameters. These data suggest that induction of HO-1 reduces diabetic induced-glomerular injury and apoptosis and these effects are associated with decreased NF-κB-induced inflammation and oxidative stress.

Reno-protective mechanisms of epoxyeicosatrienoic acids in cardiovascular disease.

Cardiovascular disease (CVD) is the leading cause of mortality worldwide, and it is well known that end-stage renal disease (ESRD) is a profound consequence of the progression of CVD. Present treatments only slow CVD progression to ESRD, and it is imperative that new therapeutic strategies are developed to prevent the incidence of ESRD. Because epoxyeicosatrienoic acids (EETs) have been shown to elicit reno-protective effects in hypertensive animal models, the current review will focus on addressing the reno-protective mechanisms of EETs in CVD. The cytochrome P-450 epoxygenase catalyzes the oxidation of arachidonic acid to EETs. EETs have been identified as endothelium-derived hyperpolarizing factors (EDHFs) with vasodilatory, anti-inflammatory, antihypertensive, and antiplatelet aggregation properties. EETs also have profound effects on vascular migration and proliferation and promote angiogenesis. The progression of CVD has been linked to decreased EETs levels, leading to the concept that EETs should be therapeutically targeted to prevent end-organ damage associated with CVD. However, EETs are quickly degraded by the enzyme soluble epoxide hydrolase (sEH) to their less active diols, dihydroxyeicosatrienoic acids (DHETs). As such, one way to increase EETs level is to inhibit their degradation to DHETs by using sEH inhibitors. Inhibition of sEH has been shown to effectively reduce blood pressure and organ damage in experimental models of CVD. Another approach to target EETs is to develop EET analogs with improved solubility and resistance to auto-oxidation and metabolism by sEH. For example, stable ether EET analogs dilate afferent arterioles and lower blood pressure in hypertensive rodent animal models. EET agonists also improve insulin signaling and vascular function in animal models of metabolic syndrome.

Oxidative stress contributes to sex differences in angiotensin II-mediated hypertension in spontaneously hypertensive rats.

NADPH oxidase has been implicated in ANG II-induced oxidative stress and hypertension in males; however, the contribution of oxidative stress to ANG II hypertension in females is unknown. In the present study, we tested the hypothesis that greater antioxidant capacity in female spontaneously hypertensive rats (SHR) blunts ANG II-induced oxidative stress and hypertension relative to males. Whole body and renal cortical oxidative stress levels were assessed in female and male SHR left untreated or following 2 wk of chronic ANG II infusion. Chronic ANG II infusion increased NADPH oxidase enzymatic activity in the renal cortex of both sexes; however, this increase only reached significance in female SHR. In contrast, male SHR demonstrated a greater increase in all measurements of reactive oxygen species production in response to chronic ANG II infusion. ANG II infusion increased plasma superoxide dismutase activity only in female SHR (76 ± 9 vs. 190 ± 7 Units·ml(-1)·mg(-1), P < 0.05); however, cortical antioxidant capacity was unchanged by ANG II in either sex. To assess the functional implication of alterations in NADPH enzymatic activity and oxidative stress levels following ANG II infusion, additional experiments assessed the ability of the in vivo antioxidant apocynin to modulate ANG II hypertension. Apocynin significantly blunted ANG II hypertension in male SHR (174 ± 2 vs. 151 ± 1 mmHg, P < 0.05), with no effect in females (160 ± 11 vs. 163 ± 10 mmHg). These data suggest that ANG II hypertension in male SHR is more dependent on increases in oxidative stress than in female SHR.

Hyperhomocysteinemia dysregulates plasma levels of polyunsaturated fatty acids-derived eicosanoids.

Hyperhomocysteinemia (HHcy) contributes to the incidence of many cardiovascular diseases (CVD). Our group have previously established crucial roles of eicosanoids and homocysteine in the incidence of vascular injury in diabetic retinopathy and renal injury. Using cystathionine-ß-synthase heterozygous mice (cßs+/-) as a model of HHcy, the current study was designed to determine the impact of homocysteine on circulating levels of lipid mediators derived from polyunsaturated fatty acids (PUFA). Plasma samples were isolated from wild-type (WT) and cßs+/- mice for the assessment of eicosanoids levels using LC/MS. Plasma 12/15-lipoxygenase (12/15-LOX) activity significantly decreased in cßs+/- vs. WT control mice. LOX-derived metabolites from both omega-3 and omega-6 PUFA were also reduced in cßs+/- mice compared to WT control (P < 0.05). Contrary to LOX metabolites, cytochrome P450 (CYP) metabolites from omega-3 and omega-6 PUFA were significantly elevated in cßs+/- mice compared to WT control. Epoxyeicosatrienoic acids (EETs) are epoxides derived from arachidonic acid (AA) metabolism by CYP with anti-inflammatory properties and are known to limit vascular injury, however their physiological role is limited by their rapid degradation by soluble epoxide hydrolase (sEH) to their corresponding diols (DiHETrEs). In cßs+/- mice, a significant decrease in the plasma EETs bioavailability was obvious as evident by the decrease in EETs/ DiHETrEs ratio relative to WT control mice. Cyclooxygenase (COX) metabolites were also significantly decreased in cßs+/- vs. WT control mice. These data suggest that HHcy impacts eicosanoids metabolism through decreasing LOX and COX metabolic activities while increasing CYP metabolic activity. The increase in AA metabolism by CYP was also associated with increase in sEH activity and decrease in EETs bioavailability. Dysregulation of eicosanoids metabolism could be a contributing factor to the incidence and progression of HHcy-induced CVD.

Deletion of soluble epoxide hydrolase gene improves renal endothelial function and reduces renal inflammation and injury in streptozotocin-induced type 1 diabetes.

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.

Obesity is the major contributor to vascular dysfunction and inflammation in high-fat diet hypertensive rats.

Obesity and hypertension are the two major risk factors that contribute to the progression of end-stage renal disease. To examine whether hypertension further exacerbates oxidative stress and vascular dysfunction and inflammation in obese rats, four groups of male Sprague-Dawley rats were fed either a normal (7% fat) or high-fat (36% fat) diet for 6 weeks and osmotic pumps were implanted to deliver ANG (angiotensin II) or vehicle for an additional 4 weeks.Treatment with the high-fat diet did not alter ANG-induced hypertension compared with the normal diet (174 +/- 6 compared with 170 +/- 5 mmHg respectively). Treatment with the high-fat diet increased body weight gain and plasma leptin levels and induced insulin resistance in normotensive and ANG-induced hypertensive rats. Plasma TBARS (thiobarbituric acid-reacting substances), a measure of oxidative stress, were elevated in high-fat diet-fed rats compared with controls (11.2 +/-1 compared with 8.4 +/- nmol/ml respectively) and was increased further in ANG-induced hypertensive rats fed a high-fat diet (18.8 +/-2.2 nmol/ml). Urinary nitrite excretion was also decreased in rats fed a high-fat diet without or with ANG infusion compared with controls. Afferent arteriolar relaxation to acetylcholine was impaired in rats fed the high-fat diet without or with ANG infusion. Renal cortical TNF-alpha(tumour necrosis factor-alpha), COX-2(cyclo-oxygenase-2) and phospho-IKK (inhibitor of nuclear factor k B kinase) expression increased in high-fat diet-fed rats compared with normal diet-fed rats. The increases in phospho-IKK and COX-2 expression were elevated further in ANG-induced hypertensive rats fed the high-fat diet.These results suggest that ANG-induced hypertension exacerbates oxidative stress and renal inflammation without further impairment in vascular dysfunction in high-fat diet-induced obesity.

Induction of hemeoxygenase-1 attenuates the hypertension and renal inflammation in spontaneously hypertensive rats.

The reno-protective mechanisms of hemeoxygenase-1 (HO-1) induction in hypertension remain unclear. We hypothesize that induction of HO-1 will decrease blood pressure and proteinuria with a marked decrease in oxidative stress and inflammation in spontaneously hypertensive rats (SHR). Male Wistar Kyoto (WKY) and SHR were injected with the HO-1 inducer cobalt protoporphyrin (CoPP, 1.5 mg/kgs.c. twice weekly) which resulted in an increase in renal HO-1 expression after 2 weeks. CoPP reduced mean arterial pressure (133±2 mmHg vs. 144±4 mmHg, p<0.05) and proteinuria (14±1 mg/day vs. 24±2 mg/day, p<0.05) in SHR as compared to baseline values, with no effect in WKY. Renal cortical superoxide (O(2)(-)) production and urinary 8-isoprostane excretion were higher in SHR compared to WKY (O(2)(-): 11±1 CPM/µg vs. 6±1 CPM/µg protein, p<0.05; 8-iso: 7±1 ng/day vs. 3±0.8 ng/day, p<0.05) and CoPP attenuated oxidative stress levels only in SHR (O(2)(-): 5±1 CPM/µg, p<0.05; 8-isoprostane: 4±0.7 ng/day) without an overall effect on antioxidant defense enzymes expression and activities. SHR showed a marked elevation in plasma C-reactive protein (CRP) and urinary monocyte chemoattractant protein-1 (MCP-1) excretion compared with WKY and HO-1 induction reduced the CRP and MCP-1 levels in SHR. Cortical COX2 expression and urinary thromboxane B(2) (TXB(2)) excretion were also significantly elevated in SHR compared to WKY and levels were reduced with induction of HO-1. Inhibition of HO with stannous mesoporphyrin further increased blood pressure and proteinuria in SHR and blocked the ability of CoPP to reduce blood pressure and proteinuria in SHR. These data demonstrate that induction of HO-1 slows the progression of hypertension and proteinuria in SHR and these changes were associated with reduced renal oxidative stress and inflammation.

Correction: Curcumin attenuates iron accumulation and oxidative stress in the liver and spleen of chronic iron-overloaded rats.

[This corrects the article DOI: 10.1371/journal.pone.0134156.].