Background/Aims

Studies have shown that kidney injury molecule-1 (KIM-1) is upregulated in damaged renal proximal tubules. In this study, we examined KIM-1 expression in glomerular epithelial cells in diabetic glomerulopathy.

Methods

Renal histology, immunostaining and Western blot for protein level, and real-time PCR for mRNA expression of KIM-1 and podocyte markers were evaluated in untreated or losartan-treated Zucker lean (Fa/+) and Zucker diabetic fatty (Fa/Fa) rats.

Results

The diabetic rats showed an increased glomerular expression of KIM-1. KIM-1 staining was localized primarily in the hyperplastic parietal epithelium of Bowman's capsule in the early stages of diabetes with subsequent increase in KIM-1-positive cells in the glomerular tuft in the more advanced stages. The increase in glomerular KIM-1 was associated with a decrease in podocytes in Fa/Fa rats. Antiproteinuric treatment with losartan attenuated podocytopenia and decreased renal expression of KIM-1 in treated diabetic rats. In an in vitro study, albumin overload increased KIM-1 protein in the primary cultures of rat glomerular epithelial cells.

Conclusion

These results show that glomerular KIM-1 expression was increased, in proportion to the extent of proteinuria and podocytopenia in the diabetic animals, supporting that KIM-1 could be used as a potential biomarker for glomerular injury in proteinuric kidney disease.

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid and EETs have a number of beneficial cardiovascular actions. This has led to the concept that EETs and its metabolic pathway can be therapeutically targeted for hypertension and other cardiovascular diseases. One approach has been to prevent the conversion of EETs to their inactive diols by inhibiting the soluble epoxide hydrolase (sEH) enzyme. Inhibition of sEH has been demonstrated to decrease blood pressure in certain experimental models of hypertension, decrease inflammation, and protect organs from damage associated with hypertension and other cardiovascular diseases. The development of sEH inhibitors has reached the point where they are being evaluated in humans. A second therapeutic approach has been to develop EET agonists. EET agonists have been essential for determining the structure function relationship for EETs and determining cell-signaling mechanisms by which EETs exert their cardiovascular actions. More recently, EET agonists have been administered chronically to experimental animal models of hypertension and metabolic syndrome and have been demonstrated to decrease blood pressure, improve insulin signaling, and improve vascular function. These experimental findings provide evidence for sEH inhibitors and EET agonists as a therapeutic approach for cardiovascular diseases, hypertension, and the associated end organ damage.

Obesity, hypertension and type 2 diabetes are major contributing factors to the increase in the number of patients that have chronic kidney disease. The clustering of visceral obesity and cardiovascular risk factors has been designated metabolic syndrome or cardiometabolic syndrome. Cardiometabolic syndrome is associated with a complex systemic inflammatory state that has been implicated in medically important complications including endothelial dysfunction. Inflammation, endothelial dysfunction, and insulin resistance are interrelated and have reciprocal relationships that link cardiovascular and metabolic diseases. Ultimately, cardiometabolic syndrome increases the risk for cardiovascular events and end organ damage. Although the number of patients with cardiometabolic syndrome is escalating, therapeutic approaches have not been developed that provide protection to the kidney. Eicosanoids are altered in cardiometabolic syndrome and contribute the progression of renal injury. The anti-hypertensive and anti-inflammatory actions of epoxides and soluble epoxide hydrolase inhibitors make these attractive eicosanoid therapeutic targets for chronic kidney disease in patients with cardiometabolic syndrome.

Arachidonic acid metabolites, eicosanoids, are key contributors to vascular function and improper eicosanoid regulation contributes to the progression of cardiovascular diseases. Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by epoxygenase enzymes to four regioisomers, 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET. These EETs have interesting beneficial effects like vasodilation, anti-inflammation, and anti-platelet aggregation that could combat cardiovascular diseases. There is mounting evidence that each regioisomeric EET may have unique vascular effects and that the contribution of individual EETs to vascular function differs from organ to organ. Over the past decade EET analogs and antagonists have been synthesized to determine EET structure function relationships and define the contribution of each regioisomeric EET. A number of studies have demonstrated that EET analogs induce vasodilation, lower blood pressure and decrease inflammation. EET antagonists have also been used to demonstrate that endogenous EETs contribute importantly to cardiovascular function. This review will discuss EET synthesis, regulation and physiological roles in the cardiovascular system. Next we will focus on the development of EET analogs and what has been learned about their contribution to vascular function. Finally, the development of EET antagonists and how these have been utilized to determine the cardiovascular actions of endogenous epoxides will be discussed. Overall, this review will highlight the important knowledge garnered by the development of EET analogs and their possible value in the treatment of cardiovascular diseases.

Fatty acid amides are a new class of signaling lipids that have been implicated in diverse physiological and pathological conditions. Oleamide is a fatty acid amide that induces vasorelaxation. Here, we investigated the mechanisms behind the vasorelaxation effect of oleamide in rat mesenteric resistance arteries. Oleamide-induced concentration dependent (0.01 μM–10μM) vasorelaxation in mesenteric resistance arteries. This relaxation was unaffected by the presence of the fatty acid amide hydrolase (FAAH) inhibitors. The cannabinoid type 1 (CB1) receptor antagonist, AM251 and the non-CB1/CB2 cannabinoid receptor antagonist, O-1918, attenuated the oleamide vasodilatory response, however the cannabinoid CB2 receptor antagonist, AM630, did not affect the vascular response. Moreover, inhibition of the transient receptor potential vanilloid (TRPV) 1 receptor with capsazepine shifted the oleamide-induced vasorelaxation response to the right. In agreement with the vascular functional data, the cannabinoid CB1 and TRPV1 receptor proteins were expressed in mesenteric resistance arteries but cannabinoid CB2 receptors and the FAAH enzyme were not. In endothelium-denuded arteries, the oleamide-mediated vasorelaxation was attenuated and cannabinoid CB1 or non-CB1/CB2 cannabinoid receptor blockade did not further reduce the dilatory response whereas TRPV1 antagonism further decreased the response. These findings indicate that cannabinoid receptors on the endothelium and endothelium-independent TRPV1 receptors contribute to the oleamide vasodilatory response. Taken together, these results demonstrate that the oleamide-induced vasorelaxation is mediated, in part, by cannabinoid CB1 receptors, non-CB1/CB2 cannabinoid receptors, and TRPV1 receptors in rat mesenteric resistance arteries. These mechanisms are overlapping in respect to oleamide-induced mesenteric resistance artery dilation.

The present study was designed to determine if chemokine receptor 2b (CCR2b) contributes to the development of renal injury in salt-sensitive angiotensin II (ANG) hypertension. Rats were infused with ANG and fed a high-salt diet (HS) for 14 days. Rats were divided into four groups: HS, HS administered the CCR2b antagonist, RS102895, ANG/HS hypertensive, and ANG/HS hypertensive administered RS102895. CCR2b inhibition slowed the progression of blood pressure elevation during the first week of ANG/HS hypertension; however, it did not alter blood pressure in the HS group. At two weeks, arterial pressure was not significantly different between ANG/HS and ANG/HS hypertensive rats administered RS102895. Renal cortical NFκB activity increased in ANG/HS hypertension compared to HS group (0.11 ± 0.006 vs. 0.08 ± 0.003 ng activated NFκB/μg protein) and RS102895 treatment lowered NFκB activity in ANG/HS hypertension (0.08 0.005 ng activated NFκB/μg protein). Renal TNF-alpha and ICAM-1 expression increased and Cyp2c23 expression decreased in ANG/HS hypertension compared to HS group and CCR2b inhibition reduced TNF-alpha and ICAM-1 and increased Cyp2c23 expression. Histological immunostaining revealed increased renal monocyte and macrophage infiltration in ANG/HS hypertensive rats with decreased infiltration in rats receiving RS102895 treatment. Albuminuria and cortical collagen staining also increased in ANG/HS hypertensive rats and RS102895 treatment lowered these effects. Afferent arteriolar autoregulatory responses to increasing renal perfusion pressure were blunted in ANG/HS hypertension and RS102895 treatment improved this response. These data suggest that CCR2b inhibition protects the kidney in hypertension by reducing inflammation and delaying the progression of hypertension.

Recent studies have shown that the renal cytochrome P-450 metabolites of arachidonic acid: the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE), and the vasodilator epoxyeicosatrienoic acids (EETs) play an important role in the pathophysiology of angiotensin II (ANG II)-dependent forms of hypertension and the associated target organ damage. The present studies were performed in Ren-2 renin transgenic rats (TGR) to evaluate the effects of chronic selective inhibition of 20-HETE formation or elevation of the level of EETs, alone or in combination, on the course of hypertension and hypertension-associated end-organ damage. Both young (30 days of age) prehypertensive TGR and adult (190 days of age) TGR with established hypertension were examined. Normotensive Hannover Sprague-Dawley (HanSD) rats served as controls. The rats were treated with N-methylsulfonyl-12,12-dibromododec-11-enamide to inhibit 20-HETE formation and/or with N-cyclohexyl-N-dodecyl urea to inhibit soluble epoxide hydrolase and prevent degradation of EETs. Inhibition in TGR rats of 20-HETE formation combined with enhanced bioavailability of EETs attenuated the development of hypertension, cardiac hypertrophy, proteinuria, glomerular hypertrophy and sclerosis as well as renal tubulointerstitial injury. This was also associated with an attenuation of the responsiveness of the systemic and renal vascular beds to ANG II without modifying their responses to norepinephrine. Our data suggest that altered production and/or action of 20-HETE and EETs plays a permissive role in the development of hypertension and hypertension-associated end-organ damage in this model of ANG II-dependent hypertension. This information provides a basis for a search of new therapeutic approaches to the treatment of hypertension.

Objective

The contribution of cytochrome P-450 (CYP) metabolites of arachidonic acid: epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) in the regulation of nonclipped kidney function in two-kidney, one-clip (2K1C) Goldblatt hypertensive rats during the phases of initial and stable hypertension (7 or 27 days after clipping, respectively) were investigated.

Methods

Male Hannover-Sprague Dawley rats had the right renal artery clipped or had a sham-operation. Urinary excretion of EETs, their inactive metabolites (DHETEs), and 20-HETE were measured. Intrarenal CYP protein expression and the activities of epoxygenase, ω-hydroxylase and soluble epoxide hydrolase (sEH) were also determined.

The responses of renal hemodynamics and electrolyte excretion of the non-clipped kidney to left renal artery infusions of inhibitors of EETs or 20-HETE formation (MS-PPOH and DDMS, respectively) were measured.

Results

In 2K1C rats the urinary EETs excretion was lower and 20-HETE excretion was higher than in sham-operated animals. Intrarenal inhibition of EETs significantly decreased renal hemodynamics and sodium excretion in sham-operated but not in 2K1C rats. Intrarenal inhibition of 20-HETE decreased sodium excretion in sham-operated rats but elicited increases in renal hemodynamics and sodium excretion in 2K1C rats.

Conclusions

The results indicate that the nonclipped kidney of Goldblatt 2K1C rats in the phase of sustained hypertension exhibits decreased intrarenal EETs and elevated 20-HETE levels as compared with the kidney of sham-operated animals. This suggests that altered production and action of CYP-derived metabolites in this phase contributes to the mechanism of Goldblatt 2K1C hypertension.

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 normal (7% fat) or high fat (36% fat) diet for 6 weeks and osmotic pumps were implanted to deliver angiotensin II (ANG) or vehicle for four additional weeks. High fat diet treatment did not alter ANG-induced hypertension compared to normal diet (174±6 vs. 170±5 mmHg, respectively). High fat diet treatment increased body weight gain and plasma leptin levels and induced insulin resistance in normotensive and ANG hypertensive rats. Plasma TBARs, a measure of oxidative stress, was elevated in high fat diet fed rats compared to control (11.2±1 vs. 8.4±1 nmol/ml, respectively) and was further increased in ANG hypertensive rats fed high fat diet (18.8±2.2 nmol/ml). Urinary nitrite excretion was also decreased in rats fed high fat diet with or without ANG infusion compared to control. Afferent arteriolar relaxation to acetylcholine was impaired in high fat fed rats with or without ANG infusion. Renal cortical TNF-α, COX-2, and phospho-IKK expression increased in high fat diet compared to normal diet fed rats. The increases in phospho-IKK and COX-2 expression were further elevated in ANG hypertensive rats fed high fat diet. These data suggest that ANG-induced hypertension exacerbates oxidative stress and renal inflammation without further impairment in vascular dysfunction in high fat diet-induced obesity.

Preface

Cardiovascular effects of epoxyeicosatrienoic acids (EETs) include vasodilation, vascular smooth muscle cell anti-migratory actions, and anti-inflammatory actions. These endogenous lipid mediators are broken down to diols by soluble epoxide hydrolase (sEH), and so inhibiting this enzyme would be expected enhance the beneficial cardiovascular properties of EETs. The rapid development of 1,3-disubstituted urea based sEH inhibitors (sEHIs) has resulted in a number of studies demonstrating cardiovascular protection, and it has been shown that sEHIs are anti-hypertensive, anti-inflammatory, and protect the brain, heart and kidney from damage. Although challenges for the future exist — including improving the drug like properties of sEHIs and finding better ways to target sEHIs to specific tissues — the recent initiation of first in human clinical trials has highlighted the promise of sEHIs as a therapeutic target.

Epoxyeicosatrienoic acids (EETs) contribute importantly to the regulation of vascular tone and blood pressure control. The purpose of this study was to develop stable EET analogs and test their in vivo blood pressure lowering effects in hypertensive rats. Using the pharmacophoric moiety of EETs, ether EET analogs were designed with improved solubility and resistance to auto-oxidation and metabolism by soluble epoxide hydrolase. Ether EET analogs were chosen based on their ability to dilate afferent arterioles and subsequently tested for blood pressure lowering effects in rodent models of hypertension. Initially, 11,12-ether-EET-8-ZE failed to lower blood pressure in angiotensin hypertension or spontaneously hypertensive rats (SHR). Esterification of the carboxylic group of 11,12-ether-EET-8-ZE prevented blood pressure increase in SHR when injected at 2 mg/day for 12 days (MAP Δ change at day 8 of injection was −0.3 ± 2 for treated and 12 ± 1 mmHg for control SHR). Amidation of the carboxylic group with aspartic acid produced another EET analog (NUDSA) with a blood pressure lowering effect when injected at 3 mg/day in SHR for 5 days. Amidation of the carboxylic group with lysine amino acid produced another analog with minimal blood pressure lowering effect. These data suggest that esterification of the carboxylic group of 11,12-ether-EET-8-ZE produced the most effective ether-EET analog in lowering blood pressure in SHR and provide the first evidence to support the use of EET analogs in treatment of cardiovascular diseases.

SUMMARY

In the present study, we determined the role of hypertension, oxidative stress and inflammation on kidney damage in a rodent model of obesity and diabetes. Hypertension was induced in male obese (db/db) mice and lean (db/m) mice by implantation of deoxycorticosterone acetate (DOCA) pellets and mice were allowed to drink water containing 1% salt. Mice were divided into six groups as follows: obese and lean control, obese and lean 1% salt (salt) and obese and lean DOCA plus 1% salt (DOCA-salt).Blood pressure was significantly increased in lean and obese DOCA-salt groups relative to their respective control; however, there was no difference in blood pressure between the lean and obese control and salt groups. Urinary 8-isoprostane was increased in obese control compared with lean control mice (1464 ± 267 vs 493 ± 53 pg/μmol creatinine, respectively) and this elevation was further increased in the obese DOCA-salt mice (2430 ± 312 pg/μmol creatinine). Urinary monocyte chemoattractant protein-1 excretion and CD68-positive cells were also increased in both obese and lean DOCA-salt groups compared with their respective controls. Furthermore, DOCA-salt administration increased collagen IV excretion in both obese and lean mice compared with controls, but there was no difference between obese and lean DOCA-salt groups. Urinary albumin excretion was significantly increased in the obese compared with the lean DOCA-salt mice (507 ± 160 vs 202 ± 48 μg/day, respectively).These data suggest that obese DOCA-salt hypertensive mice exhibit greater renal injury than lean DOCA-salt hypertensive mice in a manner independent of blood pressure and that this renal injury is associated with obesity related pre-existing renal oxidative stress.

Peroxisome proliferator-activated receptors (PPARs) are members of a steroid hormone receptor superfamily that responds to changes in lipid and glucose homeostasis. Peroxisomal proliferator-activated receptor subtype γ (PPARγ) has received much attention as the target for antidiabetic drugs, as well as its role in responding to endogenous compounds such as prostaglandin J2. However, thiazolidinediones (TZDs), the synthetic agonists of the PPARγ are tightly associated with fluid retention and edema, as potentially serious side effects. The epithelial sodium channel (ENaC) represents the rate limiting step for sodium absorption in the renal collecting duct. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. The role of PPARγ agonists on ENaC activity remains controversial. While PPARγ agonists were shown to stimulate ENaC-mediated renal salt absorption, probably via Serum- and Glucocorticoid-Regulated Kinase 1 (SGK1), other studies reported that PPARγ agonist-induced fluid retention is independent of ENaC activity. The current paper provides new insights into the control and function of ENaC and ENaC-mediated sodium transport as well as several other epithelial channels/transporters by PPARs and particularly PPARγ. The potential contribution of arachidonic acid (AA) metabolites in PPAR-dependent mechanisms is also discussed.

Studies suggest that the inflammatory cytokine, TNF-α plays a role in the prognosis of end-stage renal diseases. We have previously shown that TNF-α inhibition slowed the progression of hypertension and renal damage in angiotensin II salt-sensitive hypertension. Thus, we hypothesize that TNF-α contributes to renal inflammation in a model of mineralocorticoid-induced hypertension. Four groups of rats (n=5-6) were studied for 3 weeks with the following treatments 1) placebo, 2) placebo + TNF-α inhibitor, etanercept (1.25 mg/kg/day, sc), 3) deoxycorticosterone acetate plus 0.9 % NaCl to drink (DOCA-salt), or 4) DOCA-salt + etanercept. Mean arterial blood pressure (MAP) measured by telemetry increased in DOCA-salt rats compared to baseline (177±4 vs. 107±3 mmHg, P<0.05) and TNF-α inhibition had no effect in the elevation of MAP in these rats (177±8 mmHg). Urinary protein excretion significantly increased in DOCA-salt rats compared to placebo (703±76 vs. 198±5 mg/day, respectively); etanercept lowered the proteinuria (514±64 mg/day, P < 0.05 vs. DOCA-salt alone). Urinary albumin excretion followed a similar pattern in each group. Urinary MCP-1 and ET-1 excretion were also increased in DOCA-salt rats compared to placebo (MCP-1: 939±104 vs. 43±7 ng/day, and ET-1: 3.30±0.29 vs. 1.07±0.03 fmol/day, respectively, both P<0.05); TNF-α inhibition significantly decreased both MCP-1 and ET-1 excretion (409±138 ng/day and 2.42±0.22 fmol/day, respectively, both P < 0.05 vs. DOCA-salt alone). Renal cortical NFκB activity also increased in DOCA-salt hypertensive rats and etanercept treatment significantly reduced this effect. These data support the hypothesis that TNF-α contributes to the increase in renal inflammation in DOCA-salt rats.

Hypertension and type II diabetes are co-morbid diseases that lead to the development of nephropathy. Soluble epoxide hydrolase (sEH) inhibitors are reported to provide protection from renal injury. We hypothesized that the sEH inhibitor 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) protects the kidney from the development of nephropathy associated with hypertension and type II diabetes. Hypertension was induced in spontaneously diabetic Goto-Kakizaki rats using angiotensin II and a high salt diet. Hypertensive Goto-Kakizaki rats were treated for two weeks with either AUDA or its vehicle added to drinking water. Mean arterial pressure increased from 118 ± 2 mmHg to 182 ± 20 and 187 ± 6 mmHg for vehicle and AUDA treated hypertensive Goto-Kakizaki rats, respectively. AUDA treatment did not alter blood glucose. Hypertension in Goto-Kakizaki rats resulted in a 17-fold increase in urinary albumin excretion that was decreased with AUDA treatment. Renal histological evaluation determined that AUDA treatment decreased glomerular and tubular damage. In addition, AUDA treatment attenuated macrophage infiltration and inhibited urinary excretion of MCP-1 and kidney cortex MCP-1 gene expression. Taken together, these data provide evidence that sEH inhibition with AUDA attenuates the progression of renal damage associated with hypertension and type II diabetes.

Experiments were designed to test the hypothesis that antioxidant treatment would increase the antihypertensive actions of endogenous kinins during angiotensin converting enzyme (ACE) inhibition. Four groups of rats, all given angiotensin II (Ang II) for 2 weeks, were studied: 1) control, 2) enalapril, 3) tempol or 4) both tempol and enalapril. Ang II significantly increased systolic blood pressure (BP) when compared with the baseline (170± 8 vs. 128± 4 mm Hg, P<0.05). Neither enalapril nor tempol alone was able to attenuate the elevation in BP (165± 7 and 164± 6 mm Hg, respectively). In contrast, combined administration of tempol and enalapril prevented the increase in BP (137± 5 mmHg). Plasma 8-isoprostane increased in Ang II infused rats when compared with control untreated rats (69± 14 vs. 23± 0.5 pg/ml, P<0.05). Tempol alone or tempol plus enalapril significantly attenuated the increase in plasma 8-isoprostane (29± 6 and 34± 7 pg/ml, respectively). In additional experiments, we used the bradykinin B2 antagonist, icatibant to determine if increased B2 receptor contributes to the antihypertensive effect of combined tempol and enalapril in Ang II infused rats. Icatibant decreased the ability of this combination to lower arterial pressure. Additionally, a significant increase in B1 receptor protein expression in renal cortex of Ang II infused rats was observed compared to control suggesting that the bradykinin receptors activation could account for the effect of enalapril to enhance the actions of tempol. These data support the hypothesis that combined reduction of superoxide along with enhanced endogenous kinins may facilitate blood pressure lowering in Ang II hypertension.

Background/Aims

Disrupting the enzyme Cyp4a14 in mice leads to hypertension, which is more severe in the male mice and appears to be due to androgen excess. Because the Cyp4a14 enzyme is located in the proximal tubule of the kidney, we hypothesized that there could be dysregulation of transport in this segment that could contribute to the hypertension.

Methods

Wild-type (SV/129) mice and mice that had targeted disruption of the Cyp4a14 gene were studied. Proximal convoluted tubules (PCT) from knockout and wild-type mice were dissected and perfused in vitrofor measurement of volume absorption (JV). Expression of the sodium-hydrogen exchanger 3 (NHE3), the predominant transporter responsible for sodium transport in this segment, was measured by immunoblot. Renal vascular (afferent arteriole) responses to angiotensin and endothelin were also measured.

Results

PCT volume absorption was elevated in tubules from the Cyp4a14 knockout mice as compared to the wild-type mice. Brush border membrane NHE3 expression was almost 2-fold higher in Cyp4a14 knockout mice than in wild-type mice. No difference was found in the afferent arteriolar response.

Conclusion

Thus, hypertension in the Cyp4a14 knockout mice appears to be driven by excessive fluid reabsorption in the proximal tubule, which is secondary to overexpression of NHE3.

The epoxygenase metabolite, 11,12-epoxyeicosatrienoic acid (11,12-EET), has renal vascular actions. 11,12-EET analogs have been developed to determine the structure activity relationship for 11,12-EET and as a tool to investigate signaling mechanisms responsible for afferent arteriolar dilation. We hypothesized that 11,12-EET mediated afferent arteriolar dilation involves increased phosphoprotein phosphatase 2A (PP2A) and large-conductance calcium activated K+ (KCa) channels. We evaluated the chemically and/or metabolically stable 11,12-EET analogs: 11,12-EET-N-methylsulfonimide (11,12-EET-SI), 11-nonyloxy-undec-8(Z)-enoic acid (11,12-ether-EET-8-ZE), and 11,12-trans-oxidoeicosa-8(Z)-eonoic acid (11,12-tetra-EET-8-ZE). Afferent arteriolar responses were assessed. Activation of KCa channels by 11,12-EET analogs were established by single cell channel recordings in renal myocytes. Assessment of renal vascular responses revealed that 11,12-EET analogs increased afferent arteriolar diameter. Vasodilator responses to 11,12-EET analogs were abolished by K+ channel or PP2A inhibition. 11,12-EET analogs activated renal myocyte large-conductance KCa channels. 11,12-EET analogs increased cAMP by 2-fold and PP2A activity increased 3-8 fold in renal myocytes. PP2A inhibition did not significantly affect the 11,12-EET analog mediated increase in cAMP and PP2A increased renal myocyte KCa channel activity to a much greater extent than PKA. These data support the concept that 11,12-EET utilizes PP2A dependent pathways to activate large-conductance KCa channels and dilate the afferent arteriole.

Obesity and hypertension have been identified as cardiovascular risk factors that contribute to the progression of end-stage renal disease. To examine the mechanisms by which high fat diet and hypertension contribute to endothelial dysfunction and renal injury, 8 week old male spontaneously hypertensive rats and Wistars were fed high fat (36% fat) or normal fat (7% fat) diet for 10 weeks. High fat diet increased body weight in Wistar and hypertensive rats by 25g and 31g respectively. Systolic blood pressure was higher in the hypertensive rats compared with Wistars, however blood pressure was unaltered by high fat diet. Afferent arteriole response to acetylcholine was impaired in high fat groups after just 3 weeks. Renal macrophage infiltration was increased in the hypertensive high fat group compared with others and monocyte chemoattractant protein-1 excretion was increased in both high fat fed groups. Renal PCR arrays displayed significant increases in 2 inflammatory genes in hypertensives fed a normal diet, 1 gene was increased in high fat fed Wistars, whereas 12 genes were increased in high fat fed hypertensives. Urinary albumin excretion was increased in the hypertensives compared with Wistars, which was further exacerbated by the high fat diet. Glomerular nephrin expression was reduced and desmin was increased by high fat diet in the hypertensives. Our results indicate that endothelial dysfunction precedes renal injury in normotensive and spontaneously hypertensive rats fed a high fat diet, and hypertension with obesity induces a powerful inflammatory response, and disruption of the renal filtration barrier.

Soluble epoxide hydrolase (sEH) inhibitors have been demonstrated to have cardiovascular protective actions. This hydrolase enzyme converts fatty acid epoxides to their corresponding diols, and this conversion can alter the biologic activity of these metabolites. We hypothesized that 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), a sEH inhibitor, would protect stroke-prone spontaneously hypertensive rats from cerebral ischemia. AUDA was administered to 6-week-old male rats for 6 weeks, during which blood pressure was measured by telemetry. Cerebral ischemia was induced by middle cerebral artery occlusion, the size of the cerebral infarct was assessed after 6 hours of ischemia, and the results were expressed as a percentage of the hemisphere infarcted (%HI). Vascular structure and function were assessed using a pressurized arteriograph. Plasma levels of AUDA at the end of the treatment period averaged 5.0 ± 0.4 ng/mL, and the urinary excretion rate was 99 ± 21 ng/d. AUDA-treated rats had significantly smaller cerebral infarcts than control rats (36 ± 4% vs 53 ± 4% HI, treated versus control, P < 0.05, n = 6). This difference occurred independently of changes in blood pressure. AUDA treatment increased the passive compliance of the cerebral vessels but had no effect on vascular structure. The results of this study provide novel evidence suggesting that the sEH inhibitor AUDA is a possible therapeutic agent for ischemic stroke.

The present study tested the hypothesis that increasing epoxyeicosatrienoic acids by inhibition of soluble epoxide hydrolase (sEH) would lower blood pressure and ameliorate renal damage in salt-sensitive hypertension. Rats were infused with angiotensin and fed a normal-salt diet or an 8% NaCl diet for 14 days. The sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), was given orally to angiotensin-infused animals during the 14-day period. Plasma AUDA metabolite levels were measured, and they averaged 10±2 ng/mL in normal-salt angiotensin hypertension and 19±3 ng/mL in high-salt angiotensin hypertension on day 14 in the animals administered the sEH inhibitor. Mean arterial blood pressure averaged 161±4 mm Hg in normal-salt and 172±5 mmHg in the high-salt angiotensin hypertension groups on day 14. EH inhibitor treatment significantly lowered blood pressure to 140±5 mm Hg in the normal-salt angiotensin hypertension group and to 151±6 mm Hg in the high-salt angiotensin hypertension group on day 14. The lower arterial blood pressures in the AUDA-treated groups were associated with increased urinary epoxide-to-diol ratios. Urinary microalbumin levels were measured, and ED-1 staining was used to determine renal damage and macrophage infiltration in the groups. Two weeks of AUDA treatment decreased urinary microalbumin excretion in the normal-salt and high-salt angiotensin hypertension groups and macrophage number in the high-salt angiotensin hypertension group. These data demonstrate that sEH inhibition lowers blood pressure and ameliorates renal damage in angiotensin-dependent, salt-sensitive hypertension.

Clinical use of prostaglandin synthase–inhibiting NSAIDs is associated with the development of hypertension; however, the cardiovascular effects of antagonists for individual prostaglandin receptors remain uncharacterized. The present studies were aimed at elucidating the role of prostaglandin E2 (PGE2) E-prostanoid receptor subtype 1 (EP1) in regulating blood pressure. Oral administration of the EP1 receptor antagonist SC51322 reduced blood pressure in spontaneously hypertensive rats. To define whether this antihypertensive effect was caused by EP1 receptor inhibition, an EP1-null mouse was generated using a “hit-and-run” strategy that disrupted the gene encoding EP1 but spared expression of protein kinase N (PKN) encoded at the EP1 locus on the antiparallel DNA strand. Selective genetic disruption of the EP1 receptor blunted the acute pressor response to Ang II and reduced chronic Ang II–driven hypertension. SC51322 blunted the constricting effect of Ang II on in vitro–perfused preglomerular renal arterioles and mesenteric arteriolar rings. Similarly, the pressor response to EP1-selective agonists sulprostone and 17-phenyltrinor PGE2 were blunted by SC51322 and in EP1-null mice. These data support the possibility of targeting the EP1 receptor for antihypertensive therapy.

The epoxyeicosatrienoic acids (EETs) have been identified as endothelium-derived hyperpolarizing factors. Metabolism of the EETs to the dihydroxyeicosatrienoic acids is catalyzed by soluble epoxide hydrolase (sEH). Administration of urea-based sEH inhibitors provides protection from hypertension-induced renal injury at least in part by lowering blood pressure. Here, we investigated the hypothesis that a mechanism by which sEH inhibitors elicit their cardiovascular protective effects is via their action on the vasculature. Mesenteric resistance arteries were isolated from Sprague-Dawley rats, pressurized, and constricted with the thromboxane A2 agonist U46619 (9,11-dideoxy-11,9-epoxymethano-prostaglandin F2α). Mesenteric arteries were then incubated with increasing concentrations of the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA). AUDA resulted in a concentration-dependent relaxation of mesenteric arteries, with 10 μM resulting in a 48 ± 7% relaxation. Chain-shortened analogs of AUDA had an attenuated vasodilatory response. Interestingly, at 10 μM, the sEH inhibitors 1-cyclohexyl-3-dodecylurea, 12-(3-cyclohexyl-ureido)dodecanoic acid, and 950 [adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea] were significantly less active, resulting in a 25 ± 8%, 10 ± 9%, and −8 ± 3% relaxation, respectively. Treatment of mesenteric arteries with tetraethyl-ammonium, iberiotoxin, ouabain, or glibenclamide did not alter AUDA-induced relaxation. The AUDA-induced relaxation was completely inhibited when constricted with KCl. In separate experiments, denuding mesenteric resistance vessels did not alter AUDA-induced relaxation. Taken together, these data demonstrate that adamantyl-urea inhibitors have unique dilator actions on vascular smooth muscle compared with other sEH inhibitors and that these dilator actions depend on the adamantyl group and carbon chain length.