Kidney Injury by Unilateral Ureteral Obstruction in Mice Lacks Sex Differences.

INTRODUCTION: Renal fibrosis is a critical event in the development and progression of chronic kidney disease (CKD), and it is considered the final common pathway for all types of CKD. The prevalence of CKD is higher in females; however, males have a greater prevalence of end-stage renal disease. In addition, low birth weight and low nephron number are associated with increased risk for CKD. This study examined the development and severity of unilateral ureter obstruction (UUO)-induced renal fibrosis in male and female wild-type (ROP +/+) and mutant (ROP Os/+) mice, a mouse model of low nephron number. METHODS: Male and female ROP +/+ and ROP Os/+ mice were subjected to UUO, and kidney tissue was collected at the end of the 10-day experimental period. Kidney histological analysis and mRNA expression determined renal fibrosis, tubular injury, collagen deposition, extracellular matrix proteins, and immune cell infiltration. RESULTS: Male and female UUO mice demonstrated marked renal injury, kidney fibrosis, and renal extracellular matrix production. Renal fibrosis and α-smooth muscle actin were increased to a similar degree in ROP +/+ and ROP Os/+ mice with UUO of either sex. There were also no sex differences in renal tubular cast formation or renal infiltration of macrophage in ROP +/+ and ROP Os/+ UUO mice. Interestingly, renal fibrosis and α-smooth muscle actin were 1.5-3-fold greater in UUO-ROP +/+ compared to UUO-ROP Os/+ mice. Renal inflammation phenotypes following UUO were also 30-45% greater in ROP +/+ compared to ROP Os/+ mice. Likewise, expression of extracellular matrix and renal fibrotic genes was greater in UUO-ROP +/+ mice compared to UUO-ROP Os/+ mice. In contrast to these findings, ROP Os/+ mice with UUO demonstrated glomerular hypertrophy with 50% greater glomerular tuft area compared to ROP +/+ with UUO. Glomerular hypertrophy was not sex-dependent in any of the genotypes of ROP mice. These findings provide evidence that low nephron number contributes to UUO-induced glomerular hypertrophy in ROP Os/+ mice but does not enhance renal fibrosis, inflammation, and renal tubular injury. CONCLUSION: Taken together, we demonstrate that low nephron number contributes to enhanced glomerular hypertrophy but not kidney fibrosis and tubular injury. We also demonstrate that none of the changes caused by UUO was affected by sex in any of the ROP mice genotypes.

Kidney in the net of acute and long-haul coronavirus disease 2019: a potential role for lipid mediators in causing renal injury and fibrosis.

PURPOSE OF REVIEW: Severe COVID-19 disease is often complicated by acute kidney injury (AKI), which may transition to chronic kidney disease (CKD). Better understanding of underlying mechanisms is important in advancing therapeutic approaches. RECENT FINDINGS: SARS-CoV-2-induced endothelial injury initiates platelet activation, platelet-neutrophil partnership and release of neutrophil extracellular traps. The resulting thromboinflammation causes ischemia-reperfusion (I/R) injury to end organs. Severe COVID-19 induces a lipid-mediator storm with massive increases in thromboxane A2 (TxA2) and PGD2, which promote thromboinflammation and apoptosis of renal tubular cells, respectively, and thereby enhance renal fibrosis. COVID-19-associated AKI improves rapidly in the majority. However, 15-30% have protracted renal injury, raising the specter of transition from AKI to CKD. SUMMARY: In COVID-19, the lipid-mediator storm promotes thromboinflammation, ischemia-reperfusion injury and cytotoxicity. The thromboxane A2 and PGD2 signaling presents a therapeutic target with potential to mitigate AKI and transition to CKD. Ramatroban, the only dual antagonist of the thromboxane A2/TPr and PGD2/DPr2 signaling could potentially mitigate renal injury in acute and long-haul COVID. Urgent studies targeting the lipid-mediator storm are needed to potentially reduce the heavy burden of kidney disease emerging in the wake of the current pandemic.

SARS-CoV-2 spike protein causes cardiovascular disease independent of viral infection.

The SARS-CoV-2 virus that results in COVID-19 has been found to damage multiple organs beyond the lung. Interestingly, the SARS-CoV-2 spike (S) protein can be found circulating in the blood of COVID-19 patients. Experimental findings are demonstrating that the circulating S protein can bind to receptors resulting in inflammation and cell, tissue, and organ damage. Avolio et al. previously determined that the S protein acting through the cluster of differentiation 147 (CD147) receptor, and another unknown mechanism had detrimental effects on human cardiac pericytes (Clin Sci (Lond) (2021) 135 (24): 2667-2689. DOI: 10.1042/CS20210735). These findings support the notion that circulating SARS-CoV-2 S protein could contribute to cardiovascular disease independent of viral infection. Future studies are needed to determine the effect of the S protein on pericytes in other organs and evaluate the effectiveness of CD147 receptor-blocking therapies to decrease organ damage caused by the S protein.

Kidney-Targeted Epoxyeicosatrienoic Acid Analog, EET-F01, Reduces Inflammation, Oxidative Stress, and Cisplatin-Induced Nephrotoxicity.

Although epoxyeicosatrienoic acid (EET) analogs have performed well in several acute and chronic kidney disease models, targeted delivery of EET analogs to the kidney can be reasonably expected to reduce the level of drug needed to achieve a therapeutic effect and obviate possible side effects. For EET analog kidney-targeted delivery, we conjugated a stable EET analog to folic acid via a PEG-diamine linker. Next, we compared the kidney targeted EET analog, EET-F01, to a well-studied EET analog, EET-A. EET-A or EET-F01 was infused i.v. and plasma and kidney tissue collected. EET-A was detected in the plasma but was undetectable in the kidney. On the other hand, EET-F01 was detected in the plasma and kidney. Experiments were conducted to compare the efficacy of EET-F01 and EET-A for decreasing cisplatin nephrotoxicity. Cisplatin was administered to WKY rats treated with vehicle, EET-A (10 mg/kg i.p.) or EET-F01 (20 mg/kg or 2 mg/kg i.p.). Cisplatin increased kidney injury markers, viz., blood urea nitrogen (BUN), N-acetyl-ß-(D)-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), and thiobarbituric acid reactive substances (TBARS). EET-F01 was as effective as EET-A in decreasing BUN, NAG, KIM-1, TBARS, and renal histological injury caused by cisplatin. Despite its almost 2×-greater molecular weight compared with EET-A, EET-F01 was comparably effective in decreasing renal injury at a 10-fold w/w lower dose. EET-F01 decreased cisplatin nephrotoxicity by reducing oxidative stress and inflammation. These data demonstrate that EET-F01 targets the kidney, allows for a lower effective dose, and combats cisplatin nephrotoxicity. In conclusion, we have developed a kidney targeted EET analog, EET-F01, that demonstrates excellent potential as a therapeutic for kidney diseases.

Eicosanoid blood vessel regulation in physiological and pathological states.

Arachidonic acid can be metabolized in blood vessels by three primary enzymatic pathways; cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP). These eicosanoid metabolites can influence endothelial and vascular smooth muscle cell function. COX metabolites can cause endothelium-dependent dilation or constriction. Prostaglandin I2 (PGI2) and thromboxane (TXA2) act on their respective receptors exerting opposing actions with regard to vascular tone and platelet aggregation. LO metabolites also influence vascular tone. The 12-LO metabolite 12S-hydroxyeicosatrienoic acid (12S-HETE) is a vasoconstrictor whereas the 15-LO metabolite 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) is an endothelial-dependent hyperpolarizing factor (EDHF). CYP enzymes produce two types of eicosanoid products: EDHF vasodilator epoxyeicosatrienoic acids (EETs) and the vasoconstrictor 20-HETE. The less-studied cross-metabolites generated from arachidonic acid metabolism by multiple pathways can also impact vascular function. Likewise, COX, LO, and CYP vascular eicosanoids interact with paracrine and hormonal factors such as the renin-angiotensin system and endothelin-1 (ET-1) to maintain vascular homeostasis. Imbalances in endothelial and vascular smooth muscle cell COX, LO, and CYP metabolites in metabolic and cardiovascular diseases result in vascular dysfunction. Restoring the vascular balance of eicosanoids by genetic or pharmacological means can improve vascular function in metabolic and cardiovascular diseases. Nevertheless, future research is necessary to achieve a more complete understanding of how COX, LO, CYP, and cross-metabolites regulate vascular function in physiological and pathological states.

Dual soluble epoxide hydrolase inhibitor/PPAR-γ agonist attenuates renal fibrosis.

Renal fibrosis is a contributor to chronic kidney disease and an important predictor of long-term prognosis. We developed a dual soluble epoxide hydrolase inhibitor-PPAR-γ agonist (sEHi/PPAR-γ), RB394, and investigated its ability to attenuate renal fibrosis in a mouse unilateral ureteral obstruction (UUO) model. RB394 efficacy was compared to an sEH inhibitor (sEHi), a PPAR-γ agonist rosiglitazone (Rosi), or their combination (sEHi + Rosi). All interventional treatments were administrated in drinking water 3 days after UUO induction surgery and continued for 7 days. UUO mice developed renal fibrosis with higher collagen formation and RB394 significantly attenuated fibrosis (P < 0.05). Renal expression of α-smooth muscle actin (α-SMA) was elevated in UUO mice and all treatments except sEHi significantly attenuated renal α-SMA expression. Renal mRNA expression fibrotic and fibrosis regulators were higher in UUO mice and RB394 and sEHi + Rosi treatments attenuated their expression. Renal inflammation was evident in UUO mice with increased infiltration of CD45 and F4/80 positive cells. RB394 and sEHi + Rosi treatments attenuated renal inflammation in UUO mice. UUO mice had renal tubular and vascular injury. Renal tubular and vascular injuries were attenuated to a greater extent by RB394 and sEHi + Rosi than sEHi or Rosi treatment alone. Renal mRNA expression of oxidative stress markers were significantly higher in UUO mice (P < 0.05). RB394 and sEHi + Rosi attenuated expression of oxidative stress markers to a greater extent than other interventional treatments (P < 0.05). These findings demonstrate that RB394 can attenuate renal fibrosis by reducing renal inflammation, oxidative stress, tubular injury, and vascular injury. In conclusion, RB394 demonstrates exciting potential as a therapeutic for renal fibrosis and chronic kidney disease.

Epoxides and soluble epoxide hydrolase in cardiovascular physiology.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites that importantly contribute to vascular and cardiac physiology. The contribution of EETs to vascular and cardiac function is further influenced by soluble epoxide hydrolase (sEH) that degrades EETs to diols. Vascular actions of EETs include dilation and angiogenesis. EETs also decrease inflammation and platelet aggregation and in general act to maintain vascular homeostasis. Myocyte contraction and increased coronary blood flow are the two primary EET actions in the heart. EET cell signaling mechanisms are tissue and organ specific and provide significant evidence for the existence of EET receptors. Additionally, pharmacological and genetic manipulations of EETs and sEH have demonstrated a contribution for this metabolic pathway to cardiovascular diseases. Given the impact of EETs to cardiovascular physiology, there is emerging evidence that development of EET-based therapeutics will be beneficial for cardiovascular diseases.

Epoxyeicosatrienoic acid analog EET-B attenuates post-myocardial infarction remodeling in spontaneously hypertensive rats.

Epoxyeicosatrienoic acids (EETs) and their synthetic analogs have cardiovascular protective effects. Here, we investigated the action of a novel EET analog EET-B on the progression of post-myocardial infarction (MI) heart failure in spontaneously hypertensive rats (SHR). Adult male SHR were divided into vehicle- and EET-B (10 mg/kg/day; p.o., 9 weeks)-treated groups. After 2 weeks of treatment, rats were subjected to 30-min left coronary artery occlusion or sham operation. Systolic blood pressure (SBP) and echocardiography (ECHO) measurements were performed at the beginning of study, 4 days before, and 7 weeks after MI. At the end of the study, tissue samples were collected for histological and biochemical analyses. We demonstrated that EET-B treatment did not affect blood pressure and cardiac parameters in SHR prior to MI. Fractional shortening (FS) was decreased to 18.4 ± 1.0% in vehicle-treated MI rats compared with corresponding sham (30.6 ± 1.0%) 7 weeks following MI induction. In infarcted SHR hearts, EET-B treatment improved FS (23.7 ± 0.7%), markedly increased heme oxygenase-1 (HO-1) immunopositivity in cardiomyocytes and reduced cardiac inflammation and fibrosis (by 13 and 19%, respectively). In conclusion, these findings suggest that EET analog EET-B has beneficial therapeutic actions to reduce cardiac remodeling in SHR subjected to MI.

Altered Renal Vascular Responsiveness to Vasoactive Agents in Rats with Angiotensin II-Dependent Hypertension and Congestive Heart Failure.

OBJECTIVE: We evaluated the hypothesis that the development of renal dysfunction and congestive heart failure (CHF) caused by volume overload in rats with angiotensin II (ANG II)-dependent hypertension is associated with altered renal vascular responsiveness to ANG II and to epoxyeicosatrienoic acids (EETs). METHODS: Ren-2 transgenic rats (TGRs) were used as a model of ANG II-dependent hypertension. CHF was induced by volume overload achieved by the creation of the aorto-caval fistula (ACF). Renal blood flow (RBF) responses were determined to renal arterial administration of ANG II, native 11,12-EET, an analog of 14,15-EETs (EET-A), norepinephrine (NE), acetylcholine (Ach) and bradykinin (Bk) in healthy (i.e., sham-operated) TGR and ACF TGR (5 weeks after ACF creation). RESULTS: Selective intrarenal administration of neither vasoactive drug altered mean arterial pressure in any group. Administration of ANG II caused greater decreases in RBF in ACF TGR than in sham-operated TGR, whereas after administration of NE the respective decreases were comparable in the 2 groups. Administration of Ach and Bk elicited significantly higher RBF increases in ACF TGR as compared with sham-operated TGR. In contrast, administration of 11,12-EET and EET-A caused significantly smaller RBF increases in ACF TGR than in sham-operated TGR. CONCLUSION: The findings show that 5 weeks after creation of ACF, the TGR exhibit exaggerated renal vasoconstrictor responses to ANG II and reduced renal vasodilatory responses to EETs, suggesting that both these alterations might play an important role in the development of renal dysfunction in this model of CHF.

A novel dual PPAR-γ agonist/sEH inhibitor treats diabetic complications in a rat model of type 2 diabetes.

AIMS/HYPOTHESIS: The metabolic syndrome is a cluster of risk correlates that can progress to type 2 diabetes. The present study aims to evaluate a novel molecule with a dual action against the metabolic syndrome and type 2 diabetes. METHODS: We developed and tested a novel dual modulator, RB394, which acts as a soluble epoxide hydrolase (sEH) inhibitor and a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist in rat models of the metabolic syndrome-the obese spontaneously hypertensive (SHROB) rat and the obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat. In SHROB rats we studied the ability of RB394 to prevent metabolic syndrome phenotypes, while in ZSF1 obese diabetic rats we compared RB394 with the ACE inhibitor enalapril in the treatment of type 2 diabetes and associated comorbid conditions. RB394 (10 mg/kg daily) and enalapril (10 mg/kg daily) were administered orally for 8 weeks. RESULTS: RB394 blunted the development of hypertension, insulin resistance, hyperlipidaemia and kidney injury in SHROB rats and reduced fasting blood glucose and HbA1c, improved glucose tolerance, reduced blood pressure and improved lipid profiles in obese ZSF1 rats. A reduction in liver fibrosis and hepatosteatosis was evident in RB394-treated obese ZSF1 rats. Unlike RB394, enalapril did not demonstrate any positive effects in relation to diabetes, hyperlipidaemia or liver dysfunction in obese ZSF1 rats. RB394 ameliorated diabetic nephropathy by reducing renal interstitial fibrosis and renal tubular and glomerular injury in obese diabetic ZSF1 rats. Intriguingly, enalapril demonstrated a weaker action against diabetic nephropathy in obese ZSF1 rats. CONCLUSIONS/INTERPRETATION: These findings demonstrate that a novel sHE inhibitor/PPAR-γ agonist molecule targets multiple risk factors of the metabolic syndrome and is a glucose-lowering agent with a strong ability to treat diabetic complications.

Infarct size-limiting effect of epoxyeicosatrienoic acid analog EET-B is mediated by hypoxia-inducible factor-1α via downregulation of prolyl hydroxylase 3.

Epoxyeicosatrienoic acids (EETs) decrease cardiac ischemia-reperfusion injury; however, the mechanism of their protective effect remains elusive. Here, we investigated the cardioprotective action of a novel EET analog, EET-B, in reperfusion and the role of hypoxia-inducible factor (HIF)-1α in such action of EET-B. Adult male rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion. Administration of 14,15-EET (2.5 mg/kg) or EET-B (2.5 mg/kg) 5 min before reperfusion reduced infarct size expressed as a percentage of the area at risk from 64.3 ± 1.3% in control to 42.6 ± 1.9% and 46.0 ± 1.6%, respectively, and their coadministration did not provide any stronger effect. The 14,15-EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (2.5 mg/kg) inhibited the infarct size-limiting effect of EET-B (62.5 ± 1.1%). Similarly, the HIF-1α inhibitors 2-methoxyestradiol (2.5 mg/kg) and acriflavine (2 mg/kg) completely abolished the cardioprotective effect of EET-B. In a separate set of experiments, the immunoreactivity of HIF-1α and its degrading enzyme prolyl hydroxylase domain protein 3 (PHD3) were analyzed in the ischemic areas and nonischemic septa. At the end of ischemia, the HIF-1α immunogenic signal markedly increased in the ischemic area compared with the septum (10.31 ± 0.78% vs. 0.34 ± 0.08%). After 20 min and 2 h of reperfusion, HIF-1α immunoreactivity decreased to 2.40 ± 0.48% and 1.85 ± 0.43%, respectively, in the controls. EET-B blunted the decrease of HIF-1α immunoreactivity (7.80 ± 0.69% and 6.44 ± 1.37%, respectively) and significantly reduced PHD3 immunogenic signal in ischemic tissue after reperfusion. In conclusion, EET-B provides an infarct size-limiting effect at reperfusion that is mediated by HIF-1α and downregulation of its degrading enzyme PHD3. NEW & NOTEWORTHY The present study shows that EET-B is an effective agonistic 14,15-epoxyeicosatrienoic acid analog, and its administration before reperfusion markedly reduced myocardial infarction in rats. Most importantly, we demonstrate that increased hypoxia-inducible factor-1α levels play a role in cardioprotection mediated by EET-B in reperfusion likely by mechanisms including downregulation of the hypoxia-inducible factor -1α-degrading enzyme prolyl hydroxylase domain protein 3.

Role of the cytochrome P-450/ epoxyeicosatrienoic acids pathway in the pathogenesis of renal dysfunction in cirrhosis.

Background: Hepatorenal syndrome (HRS) is a life-threatening complication of advanced liver cirrhosis that is characterized by hemodynamic alterations in the kidney and other vascular beds. Cytochrome P(CYP)-450 enzymes metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acids. These eicosanoids regulate blood pressure, vascular tone and renal tubular sodium transport under both physiological and pathophysiological states. Methods: Experiments were performed to investigate the role of the CYP system in the pathogenesis of renal dysfunction during cirrhosis. Rats underwent bile duct ligation (BDL) or sham surgery and were studied at 2, 4 and 5 weeks post-surgery. In additional experiments, post-BDL rats were treated with three daily intraperitoneal doses of either the selective epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MSPPOH) or a vehicle, starting on Day 22 after surgery. Results: BDL led to progressive renal dysfunction that was associated with reduced renal cortical perfusion but without any overt histologic changes, consistent with HRS. CYP isoform enzyme expression was significantly altered in BDL rats. In the kidney, CYP2C23 expression was upregulated at both the mRNA and protein levels in BDL rats, while CYP2C11 was downregulated. Histologically, the changes in CYP2C23 and CYP2C11 expression were localized to the renal tubules. EET production was increased in the kidneys of BDL rats as assessed by urinary eicosanoid levels. Finally, treatment with the selective epoxygenase inhibitor MSPPOH significantly reduced renal function and renal cortical perfusion in BDL rats, suggesting a homeostatic role for epoxygenase-derived eicosanoids. Conclusions: The CYP/EET pathway might represent a novel therapeutic target for modulating renal dysfunction in advanced cirrhosis.

Combined Inhibition of Soluble Epoxide Hydrolase and Renin-Angiotensin System Exhibits Superior Renoprotection to Renin-Angiotensin System Blockade in 5/6 Nephrectomized Ren-2 Transgenic Hypertensive Rats with Established Chronic Kidney Disease.

BACKGROUND/AIMS: We found recently that increasing renal epoxyeicosatrienoic acids (EETs) levels by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, shows renoprotective actions and retards the progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX). This prompted us to examine if additional protection is provided when sEH inhibitor is added to the standard renin-angiotensin system (RAS) blockade, specifically in rats with established CKD. METHODS: For RAS blockade, an angiotensin-converting enzyme inhibitor along with an angiotensin II type receptor blocker was used. RAS blockade was compared to sEH inhibition added to the RAS blockade. Treatments were initiated 6 weeks after 5/6 NX in TGR and the follow-up period was 60 weeks. RESULTS: Combined RAS and sEH blockade exhibited additional positive impact on the rat survival rate, further reduced albuminuria, further reduced glomerular and tubulointerstitial injury, and attenuated the decline in creatinine clearance when compared to 5/6 NX TGR subjected to RAS blockade alone. These additional beneficial actions were associated with normalization of the intrarenal EETs deficient and a further reduction of urinary angiotensinogen excretion. CONCLUSION: This study provides evidence that addition of pharmacological inhibition of sEH to RAS blockade in 5/6 NX TGR enhances renoprotection and retards progression of CKD, notably, when applied at an advanced stage.

Cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids contribute to insulin sensitivity in mice and in humans.

AIMS/HYPOTHESIS: Insulin resistance is frequently associated with hypertension and type 2 diabetes. The cytochrome P450 (CYP) arachidonic acid epoxygenases (CYP2C, CYP2J) and their epoxyeicosatrienoic acid (EET) products lower blood pressure and may also improve glucose homeostasis. However, the direct contribution of endogenous EET production on insulin sensitivity has not been previously investigated. In this study, we tested the hypothesis that endogenous CYP2C-derived EETs alter insulin sensitivity by analysing mice lacking CYP2C44, a major EET producing enzyme, and by testing the association of plasma EETs with insulin sensitivity in humans. METHODS: We assessed insulin sensitivity in wild-type (WT) and Cyp2c44 -/- mice using hyperinsulinaemic-euglycaemic clamps and isolated skeletal muscle. Insulin secretory function was assessed using hyperglycaemic clamps and isolated islets. Vascular function was tested in isolated perfused mesenteric vessels. Insulin sensitivity and secretion were assessed in humans using frequently sampled intravenous glucose tolerance tests and plasma EETs were measured by mass spectrometry. RESULTS: Cyp2c44 -/- mice showed decreased glucose tolerance (639 ± 39.5 vs 808 ± 37.7 mmol/l × min for glucose tolerance tests, p = 0.004) and insulin sensitivity compared with WT controls (hyperinsulinaemic clamp glucose infusion rate average during terminal 30 min 0.22 ± 0.02 vs 0.33 ± 0.01 mmol kg-1 min-1 in WT and Cyp2c44 -/- mice respectively, p = 0.003). Although glucose uptake was diminished in Cyp2c44 -/- mice in vivo (gastrocnemius Rg 16.4 ± 2.0 vs 6.2 ± 1.7 µmol 100 g-1 min-1, p < 0.01) insulin-stimulated glucose uptake was unchanged ex vivo in isolated skeletal muscle. Capillary density was similar but vascular KATP-induced relaxation was impaired in isolated Cyp2c44 -/- vessels (maximal response 39.3 ± 6.5% of control, p < 0.001), suggesting that impaired vascular reactivity produces impaired insulin sensitivity in vivo. Similarly, plasma EETs positively correlated with insulin sensitivity in human participants. CONCLUSIONS/INTERPRETATION: CYP2C-derived EETs contribute to insulin sensitivity in mice and in humans. Interventions to increase circulating EETs in humans could provide a novel approach to improve insulin sensitivity and treat hypertension.

Soluble epoxide hydrolase in podocytes is a significant contributor to renal function under hyperglycemia.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of renal failure, and podocyte dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH, encoded by Ephx2) is a conserved cytosolic enzyme whose inhibition has beneficial effects on renal function. The aim of this study is to investigate the contribution of sEH in podocytes to hyperglycemia-induced renal injury. MATERIALS AND METHODS: Mice with podocyte-specific sEH disruption (pod-sEHKO) were generated, and alterations in kidney function were determined under normoglycemia, and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia. RESULTS: sEH protein expression increased in murine kidneys under HFD- and STZ-induced hyperglycemia. sEH deficiency in podocytes preserved renal function and glucose control and mitigated hyperglycemia-induced renal injury. Also, podocyte sEH deficiency was associated with attenuated hyperglycemia-induced renal endoplasmic reticulum (ER) stress, inflammation and fibrosis, and enhanced autophagy. Moreover, these effects were recapitulated in immortalized murine podocytes treated with a selective sEH pharmacological inhibitor. Furthermore, pharmacological-induced elevation of ER stress or attenuation of autophagy in immortalized podocytes mitigated the protective effects of sEH inhibition. CONCLUSIONS: These findings establish sEH in podocytes as a significant contributor to renal function under hyperglycemia. GENERAL SIGNIFICANCE: These data suggest that sEH is a potential therapeutic target for podocytopathies.

Orally Active Epoxyeicosatrienoic Acid Analogs.

Biologically active epoxyeicosatrienoic acid (EET) regioisomers are synthesized from arachidonic acid by cytochrome P450 epoxygenases of endothelial, myocardial, and renal tubular cells. EETs relax vascular smooth muscle and decrease inflammatory cell adhesion and cytokine release. Renal EETs promote sodium excretion and vasodilation to decrease hypertension. Cardiac EETs reduce infarct size after ischemia-reperfusion injury and decrease fibrosis and inflammation in heart failure. In diabetes, EETs improve insulin sensitivity, increase glucose tolerance, and reduce the renal injury. These actions of EETs emphasize their therapeutic potential. To minimize metabolic inactivation, 14,15-EET agonist analogs with stable epoxide bioisosteres and carboxyl surrogates were developed. In preclinical rat models, a subset of agonist analogs, termed EET-A, EET-B, and EET-C22, are orally active with good pharmacokinetic properties. These orally active EET agonists lower blood pressure and reduce cardiac and renal injury in spontaneous and angiotensin hypertension. Other beneficial cardiovascular actions include improved endothelial function and cardiac antiremodeling actions. In rats, EET analogs effectively combat acute and chronic kidney disease including drug- and radiation-induced kidney damage, hypertension and cardiorenal syndrome kidney damage, and metabolic syndrome and diabetes nephropathy. The compelling preclinical efficacy supports the prospect of advancing EET analogs to human clinical trials for kidney and cardiovascular diseases.

The epoxyeicosatrienoic acid analog PVPA ameliorates cyclosporine-induced hypertension and renal injury in rats.

The introduction of calcineurin inhibitors (CNI) into clinical practice in the late 1970s transformed organ transplantation and led to significant improvement in acute rejection episodes. However, despite their significant clinical utility, the use of these agents is hampered by the development of hypertension and nephrotoxicity, which ultimately lead to end-stage kidney disease and overt cardiovascular outcomes. There are currently no effective agents to treat or prevent these complications. Importantly, CNI-free immunosuppressive regimens lack the overall efficacy of CNI-based treatments and put patients at risk of allograft rejection. Cytochrome P-450 epoxygenase metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), have potent vasodilator and antihypertensive properties in addition to many cytoprotective effects, but their effects on CNI-induced nephrotoxicity have not been explored. Here, we show that PVPA, a novel, orally active analog of 14,15-EET, effectively prevents the development of hypertension and ameliorates kidney injury in cyclosporine-treated rats. PVPA treatment reduced proteinuria and renal dysfunction induced by cyclosporine. PVPA inhibited inflammatory cell infiltration into the kidney and decreased renal fibrosis. PVPA also reduced tubular epithelial cell apoptosis, attenuated the generation of reactive oxygen species, and modulated the unfolded protein response that is associated with endoplasmic reticulum stress. Consistent with the in vivo data, PVPA attenuated cyclosporine-induced apoptosis of NRK-52E cells in vitro. These data indicate that the cytochrome P-450/EET system offers a novel therapeutic strategy to treat or prevent CNI-induced nephrotoxicity.

Radiation-induced afferent arteriolar endothelial-dependent dysfunction involves decreased epoxygenase metabolites.

Chronic kidney disease is a known complication of hematopoietic stem cell transplant (HSCT) and can be caused by irradiation at the time of the HSCT. In our rat model there is a 6- to 8-wk latent period after irradiation that leads to the development of proteinuria, azotemia, and hypertension. The current study tested the hypothesis that decreased endothelial-derived factors contribute to impaired afferent arteriolar function in rats exposed to total body irradiation (TBI). WAG/RijCmcr rats underwent 11 Gy TBI, and afferent arteriolar responses to acetylcholine were determined at 1, 3, and 6 wk. Blood pressure and blood urea nitrogen were not different between control and irradiated rats. Afferent arteriolar diameters were not altered in irradiated rats. Impaired endothelial-dependent responses to acetylcholine were evident at 3 and 6 wk following TBI. Nitric oxide synthase (NOS), cyclooxygenase (COX), and epoxygenase (EPOX) contribution to acetylcholine dilator responses were evaluated. NOS inhibition with N(G)-nitro-l-arginine methyl ester (l-NAME) reduced acetylcholine responses by 50% in controls and 90% in 3-wk TBI rats. COX inhibition with indomethacin did not significantly alter the acetylcholine response in the presence or absence of l-NAME. EPOX inhibition with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide significantly decreased acetylcholine responses (35%) in controls but did not significantly alter acetylcholine responses (4%) in TBI rats. Biochemical analysis revealed decreased urinary EPOX metabolites but no change in COX, NOS, or reactive oxygen species at 3 wk TBI. Taken together, these results indicate that afferent arteriolar endothelial dysfunction involves a decrease in EPOX metabolites that precedes the development of proteinuria, azotemia, and hypertension in irradiated rats.

Epoxyeicosatrienoic acid analogue mitigates kidney injury in a rat model of radiation nephropathy.

Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by CYP epoxygenases, and EETs are kidney protective in multiple pathologies. We determined the ability of an EET analogue, EET-A, to mitigate experimental radiation nephropathy. The kidney expression of the EET producing enzyme CYP2C11 was lower in rats that received total body irradiation (TBI rat) compared with non-irradiated control. At 12 weeks after TBI, the rats had higher systolic blood pressure and impaired renal afferent arteriolar function compared with control, and EET-A or captopril mitigated these abnormalities. The TBI rats had 3-fold higher blood urea nitrogen (BUN) compared with control, and EET-A or captopril decreased BUN by 40-60%. The urine albumin/creatinine ratio was increased 94-fold in TBI rats, and EET-A or captopril attenuated that increase by 60-90%. In TBI rats, nephrinuria was elevated 30-fold and EET-A or captopril decreased it by 50-90%. Renal interstitial fibrosis, tubular and glomerular injury were present in the TBI rats, and each was decreased by EET-A or captopril. We further demonstrated elevated renal parenchymal apoptosis in TBI rats, which was mitigated by EET-A or captopril. Additional studies revealed that captopril or EET-A mitigated renal apoptosis by acting on the p53/Fas/FasL (Fas ligand) apoptotic pathway. The present study demonstrates a novel EET analogue-based strategy for mitigation of experimental radiation nephropathy by improving renal afferent arteriolar function and by decreasing renal apoptosis.

A dual COX-2/sEH inhibitor improves the metabolic profile and reduces kidney injury in Zucker diabetic fatty rat.

Cyclooxygenase (COX) and soluble epoxide hydrolase (sEH) inhibitors have therapeutic potential. The present study investigated efficacy of a novel dual acting COX-2/sEH inhibitor, PTUPB in type 2 diabetic Zucker Diabetic Fatty (ZDF) rats. Male ZDF rats were treated with vehicle or PTUPB (10mg/kg/d, i.p.) for 8 weeks. At the end of the 8-week experimental period, ZDF rats were diabetic (fasting blood glucose, 287±45mg/dL) compared to Zucker Diabetic Lean rats (ZDL, 99±6mg/dL), and PTUPB treatment improved glycemic status in ZDF rats (146±6mg/dL). Kidney injury was evident in ZDF compared to ZDL rats with elevated albuminurea (44±4 vs 4±2mg/d) and nephrinurea (496±127 vs 16±4µg/d). Marked renal fibrosis, tubular cast formation and glomerular injury were also present in ZDF compared to ZDL rats. In ZDF rats, PTUPB treatment reduced kidney injury parameters by 30-80% compared to vehicle. The ZDF rats also demonstrated increased inflammation and oxidative stress with elevated levels of urinary monocyte chemoattractant protein-1 excretion (862±300 vs 319±75ng/d), renal macrophage infiltration (53±2 vs 37±4/mm(2)) and kidney malondialdehyde/protein ratio (10±1 vs 5±1µmol/mg). PTUPB treatment decreased these inflammatory and oxidative stress markers in the kidney of ZDF rats by 25-57%. These data demonstrate protective actions of a novel dual acting COX-2/sEH inhibitor on the metabolic abnormalities and kidney function in ZDF rat model of type 2 diabetes.