Renal 20-HETE Research Articles

20-HETE As an Emerging Target to Improve Renal Health in Sickle Cell Disease

The kidney is one of the most severely affected organs in sickle cell disease (SCD), with glomerular hyperfiltration and microalbuminuria beginning in childhood, and progressing to chronic kidney disease (CKD) in majority of adults. Sickle individuals also suffer from acute kidney injury (AKI) often during characteristic vaso-occlusive crisis. Renal pathology in sickle cell nephropathy includes extensive microvascular congestions and thrombotic microangiopathy. The hypercoagulable and vasoconstrictive environment of SCD, in conjunction with medullary hypoperfusion and cortical hyperperfusion contributes to vaso-occlusive injury and deregulates intrarenal hemostasis leading to CKD development. Ischemic injury increases arachidonic acid hydroxylation producing 20-hydroxyeicosatetraenoic acid (20-HETE), which in turn triggers thrombosis and promotes vasoconstriction in the kidney. However, the role of 20-HETE in regulating renal hemostasis in SCD nephropathy has never been reported. We found that plasma 20-HETE is significantly elevated in SCD patients (n=9-11; p<0.01) compared to normal individuals. Similarly, transgenic SCD mice (SS), homozygous for human hemoglobin S, have increased plasma 20-HETE compared to control mice (AA), homozygous for human hemoglobin A (n=6; p<0.01). We have previously shown that elevated circulating heme mimicking hemolytic crisis triggers clinically relevant acute kidney injury (AKI) in SS mice (Blood (2020) 135(13): 1044-1048). We postulated that hemolytic stress may exacerbate 20-HETE production which in turn aggravates renal microvascular occlusion and vasoconstriction, leading to renal damage in SCD. Immunohistochemistry and biochemical experiments showed that the expression of CYP4A12a, a cytochrome 450-dependent enzyme responsible 20-HETE production in mice, is substantially elevated in the kidneys of SS mice compared to AA mice, associated with increased vascular congestion and renal thrombogenesis. Renal CYP4A12a and plasma 20-HETE were increased substantially following heme-induced AKI in SS mice, associated with a heightened expression of renal angiotensin II receptor 1 (ATIIR1), thus indicating induced vasoconstrictive activity. In vitro, we found that 20-HETE induced ATIIR1 expression in human renal glomerular endothelial cells (hRGEC), which was further exacerbated by low-concentration of heme challenge (5 mM for 24 hour). Nitro-oleic acid (NO 2-OA) is a nitrated fatty acid that binds ATIIR1 and inhibits angiotensin II induced vasoconstriction. Pretreatment with NO 2-OA reduced 20-HETE-dependent ATIIR1 expression in hRGEC, while NO 2-OA prophylaxis during hemin-induced AKI inhibited albuminuria, normalized plasma creatinine levels, and lowered the expression of ATIIR1 in SS mice kidneys. Overall, our data show that elevated 20-HETE levels during hemolytic stress may aggravate renal injury in SCD which can be prevented by NO 2-OA prophylaxis. Ongoing experiments will determine the direct effect of elevated 20-HETE in renal hemostasis and CKD development in SCD.

Introduction of a carboxylic acid group into pyrazolylpyridine derivatives increased selectivity for inhibition of the 20-HETE synthase CYP4A11/4F2

20-Hydroxyeicosatetraenoic acid (20-HETE) is a lipid mediator and one of the major arachidonic acid metabolites whose formation is mainly catalyzed by the enzymes cytochrome P450 (CYP) 4F2 and CYP4A11. Several studies have suggested that 20-HETE is involved in the pathogenesis of renal diseases, including diabetic nephropathy and autosomal dominant polycystic kidney disease, and we previously reported compound 1 as a dual inhibitor of CYP4A11/4F2 with therapeutic potential against renal fibrosis. Subsequent studies revealed that compound 1, the dual CYP4A11/4F2 inhibitor, however, exhibited low selectivity over another CYP4F subtype, CYP4F22, which catalyzes ω-hydroxylation of ultra-long-chain fatty acids (ULCFAs); ULCFAs are important for the formation of acylceramides, which play a role in skin barrier formation. Therefore, we sought to develop a CYP4A11/4F2 inhibitor that would show greater CYP4A11/4F2 selectivity against CYP4F22, to avoid potential dermatological side effects. We re-evaluated a series of compounds from our 20-HETE program and identified pyrazolylpyridine derivatives containing a carboxylic acid group showing only weak CYP4F22 inhibition. Subsequent optimization studies from these derivatives led to identification of compound 15, which showed CYP4A11/4F2 inhibition with improved selectivity against CYP4F22. Compound 15 inhibited 20-HETE production in both human and rat renal microsomes and did not inhibit ω-hydroxylation of ULCFAs in human keratinocytes. Compound 15 also significantly inhibited renal 20-HETE production after oral administration.

Discovery of Novel Pyrazolylpyridine Derivatives for 20-Hydroxyeicosatetraenoic Acid Synthase Inhibitors with Selective CYP4A11/4F2 Inhibition.

20-Hydroxyeicosatetraenoic acid (20-HETE) is one of the major oxidized arachidonic acid (AA) metabolites produced by cytochrome P450 (CYP) 4A11 and CYP4F2 isozymes in the human liver and kidney. Numerous studies have suggested the involvement of 20-HETE in the pathogenesis of renal diseases, and suppression of 20-HETE production by inhibition of CYP4A11 and CYP4F2 may be an attractive therapeutic strategy for renal diseases. At first, we identified methylthiazole derivative 2 as a potent dual inhibitor of CYP4A11 and CYP4F2. An optimization study of a series of derivatives with a molecular weight of around 300 to improve aqueous solubility and selectivity against drug-metabolizing CYPs while maintaining the CYP4A11- and CYP4F2-inhibitory activities led to the identification of acetylpiperidine compound 11c. Compound 11c inhibited 20-HETE production in both human and rat renal microsomes and exhibited a favorable pharmacokinetic profile. Furthermore, 11c also significantly inhibited renal 20-HETE production in Sprague-Dawley rats after oral dosing at 0.1 mg/kg.

A synthetic epoxyeicosatrienoic acid analogue prevents the initiation of ischemic acute kidney injury.

Imbalances in cytochrome P450 (CYP)-dependent eicosanoid formation may play a central role in ischemic acute kidney injury (AKI). We reported previously that inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) action ameliorated ischemia/reperfusion (I/R)-induced AKI in rats. Now we tested the hypothesis that enhancement of epoxyeicosatrienoic acid (EET) actions may counteract the detrimental effects of 20-HETE and prevent the initiation of AKI. Male Lewis rats underwent right nephrectomy and ischemia was induced by 45min clamping of the left renal pedicle followed by up to 48h of reperfusion. Circulating CYP-eicosanoid profiles were compared in patients who underwent cardiac surgery with (n=21) and without (n=38) developing postoperative AKI. Ischemia induced an about eightfold increase of renal 20-HETE levels, whereas free EETs were not accumulated. To compensate for this imbalance, a synthetic 14,15-EET analogue was administered by intrarenal infusion before ischemia. The EET analogue improved renal reoxygenation as monitored by in vivo parametric MRI during the initial 2h reperfusion phase. The EET analogue improved PI3K- as well as mTORC2-dependent rephosphorylation of Akt, induced inactivation of GSK-3β, reduced the development of tubular apoptosis and attenuated inflammatory cell infiltration. The EET analogue also significantly alleviated the I/R-induced drop in creatinine clearance. Patients developing postoperative AKI featured increased preoperative 20-HETE and 8,9-EET levels. Pharmacological interventions targeting the CYP-eicosanoid pathway could offer promising new options for AKI prevention. Individual differences in CYP-eicosanoid formation may contribute to the risk of developing AKI in clinical settings.

Proximal tubular‐specific over‐expression of the Cyp4a12‐20‐HETE synthase in male mice promotes salt‐sensitive hypertension

20‐Hydroxyeicosatetraenoic acid (20‐HETE), the ω‐hydroxylation metabolite of arachidonic acid produced by cytochrome P450 (CYP) enzymes of the 4A and 4F gene families, has been linked to the regulation of blood pressure (BP) via actions on the renal microvasculature and tubules. In renal microvessels, 20‐HETE promotes vasoconstriction, endothelial dysfunction and vascular remodeling; all of which contribute to hypertension, whereas in renal tubules 20‐HETE inhibits sodium transport and contributes to lowering of BP. However, some studies suggested that 20‐HETE promotes sodium reabsorption by actions on proximal and distal tubule transport mechanisms. In order to assess the contribution of renal tubular 20‐HETE to hypertension, we generated transgenic mice with proximal tubule‐specific overexpression of the Cyp4a12‐20‐HETE synthase (PEPCK‐Cyp4a12 mice). 20‐HETE levels in kidney cortex of PEPCK‐4a12 males showed a 2.5‐fold increase compared to littermate WT (966.63 ± 209.63 vs. 248.87 ± 69.46 pg/mg protein; p<0.05). In females, renal cortical 20‐HETE levels were not different between PEPCK‐4a12 and corresponding littermate WT mice (120.88 ± 14.50 vs. 91.65 ± 11.08 pg/mg protein; p=0.99). The absence of a significant increase in 20‐HETE levels in female PEPCK‐4a12 mice was associated with a lack of increases in CYP4a12 mRNA and protein levels in the kidney cortex compared to littermate WT (mRNA: 1.57 ± 0.39 vs. 1.07 ± 0.18; p=0.644; protein: 0.416 ± 0.046 vs. 0.330 ± 0.013; p=0.631). In contrast, renal cortical Cyp4a12 mRNA and protein in male PEPCK‐4a12 mice significantly increased compared to WT (mRNA: 2.61 ± 0.52 vs. 1.01 ± 0.09; p=0.05; protein: 0.824 ± 0.10 vs. 0.295 ± 0.023; p<0001). Males PEPCK‐Cyp4a12 mice displayed elevated BP (142 ± 1.4 vs 111 ± 4 mmHg in WT, p<0.0001), whereas BP in females PEPCK‐Cyp4a12 mice was not significantly different from corresponding WT (118 ± 2 vs 117 ± 2 mmHg for WT; p=0.98). In male PEPCK‐4a12 mice, BP decreased when transitioned from a control salt (0.4%) to a low salt diet (0.075%) from 135 ± 4 to 120 ± 6 mmHg (p=0.10) and increased to 153 ± 5 mmHg (p<0.05) when placed on a high salt diet (4%). Female PEPCK‐4a12 mice did not show changes in BP on either low or high salt diet (120 ± 2, 123 ± 4, and 120 ± 3 mmHg on control, low and high salt diet, respectively). Urine volume (2.15 ± 0.18 vs. 3.17 ± 0.15 mL/day; p=0.02) and sodium excretion (5.75 ± 0.45 vs 8.65 ± 1.11 μmol/g BW/day; t‐test P=0.05; 2‐way ANOVA p=0.21) tend to be lower in PEPCK‐4a12 males compared to WT. In female PEPCK‐4a12 urine volume (1.53 ± 0.23 vs. 1.13 ± 0.20 ml/day; p=0.45) and sodium excretion (9.38 ± 1.15 vs. 7.73 ± 0.77 μmol/g BW/day; t‐test P=0.27; 2‐way ANOVA p=0.57) were not different compared to WT. In conclusion, the expression of Cyp4a12 driven by the PEPCK promoter is gender‐specific probably due to the X‐linked PEPCK. The salt‐sensitive hypertension seen in PEPCK‐4a12 male mice may be due to a potential anti‐natriuretic activity of 20‐HETE.Support or Funding InformationSupported by NIH grants PO1‐034300 and HL‐139793This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of cytochrome P450 4A14 contributes to angiotensin II-induced renal fibrosis in mice

Angiotensin II (AngII) plays an important role in the pathogenesis of hypertension and associated renal injuries. To elucidate the molecular mechanism by which AngII induces renal damage, we found that AngII infusion significantly induced CYP4A14 expression in renal proximal tubule cells (RPTCs) with marked increases in blood pressure and proteinuria. Renal production of the major CYP4A metabolite, 20-HETE, was also significantly increased in the AngII-treated mice. Compared to wild-type (WT) mice, CYP4A14 knockout (CYP4A14−/−) mice exhibited significantly lower levels of blood pressure, renal 20-HETE production, proteinuria and renal fibrosis following AngII infusion. Furthermore, AngII-induced renal expression of profibrotic genes and proinflammatory genes was significantly attenuated in CYP4A14−/− mice. In vitro studies using cultured RPTCs demonstrated that AngII significantly induced CYP4A14 expression and 20-HETE production via the MAPK signaling pathway. AngII treatment increased TGF-β and collagen expression, which was attenuated by the CYP4A inhibitor, TS-011. Moreover, 20-HETE treatment potently induced CYP4A14 expression and TGF-β and collagen levels. Collectively, these findings suggest that attenuated renal fibrosis in AngII-treated CYP4A14−/− mice may result from both reduced systemic blood pressure and renal 20-HETE production. Therefore, CYP4A14 may represent a useful target for the treatment of AngII-associated renal damage.

Fenofibrate Attenuates Hypertension in Goldblatt Hypertensive Rats: Role of 20-Hydroxyeicosatetraenoic Acid in the Nonclipped Kidney

BackgroundThere is vast evidence that the renin-angiotensin system is not the sole determinant of blood pressure (BP) elevation in human renovascular hypertension or the relevant experimental models. This study tested the hypothesis that kidney deficiency of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of cytochrome P450 (CYP)-dependent ω-hydroxylase pathway of arachidonic acid metabolism, is important in the pathophysiology of the maintenance phase of 2-kidney, 1-clip (2K1C) Goldblatt hypertension. Materials and MethodsIn 2K1C Goldblatt rats with established hypertension, angiotensin II, angiotensin 1-7, 20-HETE concentrations and gene expression of CYP4A1 enzyme (responsible for 20-HETE formation) of the nonclipped kidney were determined. We examined if 14 days׳ administration of fenofibrate, a lipid-lowering drug, would increase CYP4A1 gene expression and renal 20-HETE formation, and if increased 20-HETE concentrations in the nonclipped kidney would decrease BP (telemetric measurements). ResultsCYP4A1 gene expression, 20-HETE and angiotensin 1-7 concentrations were lower and angiotensin II levels were higher in the nonclipped kidney of 2K1C rats than in sham-operated rats. Fenofibrate increased CYP4A1 gene expression and 20-HETE concentration in the nonclipped kidney and significantly decreased BP in 2K1C rats but did not restore it to normotensive range. The treatment did not change BP in sham-operated rats. ConclusionsOur results suggest that alterations in the RAS and CYP-dependent ω-hydroxylase metabolites of arachidonic acid in the nonclipped kidneys are both important in the pathophysiology of the maintenance phase of 2K1C Goldblatt hypertension. Therefore, fenofibrate treatment effectively attenuated hypertension, probably via stimulation of 20-HETE formation in the nonclipped kidney.

Fenofibrate Attenuates Malignant Hypertension by Suppression of the Renin-angiotensin System: A Study in Cyp1a1-Ren-2 Transgenic Rats

BackgroundMalignant hypertension is a life-threatening condition, and its pathophysiology is still poorly understood. The present study was designed to evaluate the role of interaction of the renin-angiotensin system with 20-hydroxyeicosatetraenoic acid (20-HETE), a product of cytochrome P450 (CYP)–dependent ω-hydroxylase pathway, in the pathophysiology of angiotensin II (ANG II)–dependent malignant hypertension in Cyp1a1-Ren-2 transgenic rats. MethodsMalignant hypertension was induced by 12 days׳ dietary administration of 0.3 % indole-3-carbinol (I3C), a natural xenobiotic that activates a mouse renin gene. We hypothesized that chronic administration of fenofibrate, 190mg/kg body weight, a lipid-lowering drug, should increase renal production of 20-HETE, a tubular transport inhibitor; an expected increase in sodium excretion would oppose the development of ANG II–dependent malignant hypertension. Blood pressure was monitored by radiotelemetry, and at the end of the experiment rats were prepared for renal functional studies to evaluate in vivo the pressure-natriuresis relationship in response to stepwise reductions in renal arterial pressure (RAP). ResultsIn I3C-induced rats, the treatment with fenofibrate significantly attenuated hypertension and improved the slope of the pressure-natriuresis relationship. Although fenofibrate treatment increased kidney gene and protein expression of CYP4A1, a major isoform responsible for 20-HETE formation, it did not increase renal 20-HETE concentration. On the contrary, fenofibrate treatment significantly suppressed renin gene expression, plasma renin activity and plasma and kidney ANG II levels. ConclusionsFenofibrate treatment significantly attenuated the course of malignant hypertension in I3C-induced CYP1a1-Ren-2 transgenic rats, and the mechanism responsible for antihypertensive action was fenofibrate-induced suppression of renin-angiotensin system activity.

Abstract P267: Sex Differences in Cyp450 Activity and the Development of Hypertension and Renal Injury in the Dahl S Rat

It is well documented that a sexual dimorphism exists in the regulation of blood pressure in both the human population as well as experimental animal models, however evidence of a sex difference is lacking in the Dahl S rat. Thus, we hypothesize that alterations in CYP450 expression and 20-HETE production contribute to the progression of renal injury in Dahl S rats. Consistent with what we have previously reported, no difference was noted in the blood pressure of male or female Dahl SSJr rat (213.8±12 vs 196.8±13 mmHg, ns) following 4 weeks of a high salt diet (8%NaCl). However, proteinuria (148±25 vs 355±22 mg/day, p<0.05) and renal injury (1.9±0.01 vs 2.5±0.2) were lower in female relative to male rats. In addition, GFR was significantly reduced in male vs female rats (392.4±89 vs 829.5±98 μl/min/g, p<0.05) following at high salt challenge. Renal cortical (11.3.8±16 vs 20.99±2.8 pmol/min/mg, p<0.05) and outer medullary (19.4±3 vs 6.9±1.8 pmol/min/mg, p<0.05) 20-HETE production was elevated in female versus male rats. Furthermore, renal vascular 20-HETE production was elevated in the renal vessels compared to males (0.53±0.23 vs 3.2±1.2 pmol/min/mg, p<0.05). Thus, alterations in the production of renal eicosanoids may contribute to the delay in renal injury in females relative to male Dahl SSJr rats. AHA 14SDG20160020

Abstract P134: Hypertension in Postmenopausal Women: Role of Renin-angiotensin System and Eicosanoids

Hyperandrogenemic postmenopausal women have elevated blood pressure (BP) and the mechanisms responsible for it have not been elucidated. We tested the hypothesis that activated renin-angiotensin system (RAS) in combination with cytochrome P450 (CYP 450) arachidonic acid metabolites in renal microvasculature contribute to the elevated BP in post-estrous cycling hyperandrogenemic female (PMHAF) rats. Female SD rats were implanted with DHT (7.5mg/90d) or placebo pellets (n=12/grp) beginning at 6 wks of age; pellets were changed every 85 d. At 14 months of age, baseline mean arterial pressure (MAP, telemetry) was measured in control and PMHAF rats. Each group was then divided into two groups, one group received non-specific CYP 450 inhibitor, 1-aminobenzotriazole (1-ABT; 100mg/kg/day, i.p.), and the other received Losartan (40mg/kg/day, P.O.) for 10 days. For the following 10 days all rats received both drugs. Baseline BP was significantly higher in PMHAF than controls (112±3 vs 126±3 mmHg, p=0.008). 1-ABT alone had no effect on BP in either control or PMHAF rats. Losartan alone reduced MAP in both groups (112±3 vs 97±3 mmHg, p<0.05 in control and 126±3 vs 100±3 mmHg, p<0.05 in PMHAF). In control rats first given 1-ABT, losartan had no effect on BP (112±3 vs 113±2 mmHg, p=NS). In control rats first given losartan, BP continued to decrease over the next 10 days (97±3 vs 87±2 mmHg, p=0.046). Just as in control rats, in PMHAF rats given 1-ABT first, losartan had no further effect on BP (130±2 vs 126±4 mmHg, P=NS). Unlike in control rats, PMHAF rats given 1-ABT after losartan, the BP increased to back to baseline levels (126±3 vs 116±5 mmHg, p=NS). This data suggests that BP in aging female control rats is mediated by the RAS and likely greater vasoconstrictor to vasodilator ratios of eicosanoids. BP control in PMHAF rats is more complex with the RAS playing a major role. Data suggests that vasodilators (EETs) in the renal microvasculature and 20-HETE in the tubules contribute to BP control which would explain the increase in BP back to baseline with 1-ABT + losartan. Future studies will be necessary to determine exact mechanisms responsible for the hypertension in PMHAF rats, and may explain why in hyperandrogenemic women BP is difficult to control to normotensive levels. PO1HL51971.

Abstract P284: Angiotensin II Upregulates Cytochrome-450 4A Expression in Rat Kidney Through Type 1 Receptor

20-hydroxyeicosatetraenoic acids (20-HETE) is a cytochrome P-450 (CYP) 4A-dependent metabolite of arachidonic acid and regulates vascular tone and renal tubular function. Previous studies showed that angiotensin II (Ang II) stimulated the renal CYP activity and 20-HETE production through the Ang II type 1 (AT1) receptor and that the Ang II-increased the 20-HETE was linked to the Ang II type 2(AT2) receptor. Thus, the study was designed to clarify the role of Ang II in CYP4A isoforms expression in the rat kidney. Male Sprague-Dawley rats were infused Ang II at low dose (AL, 0.17mg/kg/min, sc) and high dose (AH, 0.70mg/kg/day, sc) by using osmotic mini pump, with or without AT1 receptor blocker candesartan (1 and 3mg/kg/day, po) for 1 week. The protein expression of CYP4A isoforms, AT1 receptor and AT2 receptor in the renal cortex, outer medulla, and inner medulla was examined by immunoblot analysis. The mRNA expression of CYP4A isoforms was examined by reverse transcription and polymerase chain reaction (RT-PCR). Ang II at high dose increased systolic blood pressure (control, 109±2; AH, 164±8 mmHg, p<0.01), creatinine (control, 0.24±0.00; AH, 0.29±0.01 mg/dl, p<0.01) and urinary albumin excretion (control, 20.3±5.9; AH, 2398.6±303.6 μg/mg creatinine, p<0.01). In the control group, the CYP4A1, 4A2, and 4A8 proteins were highly expressed in the renal cortex, lowly expressed in the outer medulla, barely detected in the inner medulla. The AT1 receptor was expressed in kidney sections; highly in the outer and inner medulla, the AT2 receptor was only detected in the outer medulla. Ang II dose-dependently increased all CYP4A isoform proteins in the renal cortex and outer medulla (CYP4A1, 24% and 222%; CYP4A2, by 51% and 258%; CYP4A8, by 52% and 550%, p<0.05). Ang II also increased all CYP4A isoform mRNAs in the renal cortex and outer medulla. The candesartan treatment dose-dependently inhibited the Ang II-increased blood pressure, creatinine, urinary albumin excretion and CYP4A isoform expressions. These results indicated that Ang II increases CYP4A isoform expressions in the kidney through AT1 receptor. The Ang II-upregulated CYP4A expressions may play an important role in hypertension and renal function.

20-HETE and CYP4A2 ω-hydroxylase contribute to the elevated blood pressure in hyperandrogenemic female rats.

In male rats, androgen supplements increase 20-hydroxyeicosatetraenoic acid (20-HETE) via cytochrome P-450 (CYP)4A ω-hydroxylase and cause an increase in blood pressure (BP). In the present study, we determined the roles of 20-HETE and CYP4A2 on the elevated BP in hyperandrogenemic female rats. Chronic dihydrotestosterone (DHT) increased mean arterial pressure (MAP) in female Sprague-Dawley rats (96 ± 2 vs. 108 ± 2 mmHg, P < 0.05) and was associated with increased renal microvascular CYP4A2 mRNA expression (15-fold), endogenous renal 20-HETE (5-fold), and ω-hydroxylase activity (3-fold). Chronic DHT also increased MAP in low salt-fed Dahl salt-resistant female rats (81 ± 4 vs. 95 ± 1 mmHg, P < 0.05) but had no effect on MAP in Dahl salt-sensitive female rats (154 ± 3 vs. 153 ± 3 mmHg), which are known to be 20-HETE deficient. To test the role of CYP4A2, female CYP4A2(-/-) and SS.5(Bn) (wild type) rats were treated with DHT. DHT increased MAP in SS.5(Bn) female rats (104 ± 1 vs. 128 ± 1 mmHg, P < 0.05) but had no effect in CYP4A2(-/-) female rats (118 ± 1 vs. 120 ± 1 mmHg). Renal microvascular 20-HETE was reduced in control CYP4A2(-/-) female rats and was increased with DHT in SS.5(Bn) female rats (6-fold) but not CYP4A2(-/-) female rats. ω-Hydroxylase activity was 40% lower in control CYP4A2(-/-) female rats than in SS.5(Bn) female rats, and DHT decreased ω-hydroxylase activity in SS.5(Bn) female rats (by 50%) but significantly increased ω-hydroxylase activity in CYP4A2(-/-) female rats (3-fold). These data suggest that 20-HETE via CYP4A2 contributes to the elevation in BP in hyperandrogenemic female rats. The data also suggest that 20-HETE synthesis inhibition may be effective in treating the elevated BP in women with hyperandrogenemia, such as women with polycystic ovary syndrome.

Abstract P610: Alterations in 20-HETE Production Contribute to End Organ Damage in Dahl S Rats

It is well documented that a sexual dimorphism exists in the regulation of blood pressure in both the human population as well as experimental animal models, such that males have higher blood pressures than females of the same age. While there is a clear disparity in the development of hypertension and progression of renal injury in many rodent models, evidence of a sex difference is lacking in the Dahl S rat. While the current reports present conflicting data, we hypothesize that alterations in CYP450 expression and 20-HETE production contributes to the relative resistance of female Dahl S rats to target organ damage compared to males. Consistent to what we have previously reported, the time course for the development of proteinuria and renal injury were significantly reduced in female Dahl SSJr rats challenged with a high salt diet relative to male rats. In addition, renal cortical 20-HETE production was elevated in female (120.8±4.2 pmol/min/mg) versus male rats (45.1±11.78 pmol/min/mg), while no difference was noted in the outer medulla (19.6±1.8 vs 17.15± 3.9 pmol/min/mg). Introgression of the CYP4A1 gene into the Dahl S genetic background, resulted in significant elevations in 20-HETE production both on low salt and high salt diets. Furthermore the rise in mean arterial pressure was attenuated in CYP4A1 overexpressing rats in both sexes (Δ19mmHg in CYP females vs 61mmg in Dahl S females; 20mmHg in CYP males vs 50mmHg in Dahl S males). Moreover, the degree of glomerular injury was reduced in CYP rats, both male and female, compared to Dahl S rats in response to a high salt diet. Therefore, increases in the CYP gene expression and 20-HETE production prevent the rise in mean arterial pressure and kidney injury in male Dahl S rats. AHA 14SDG20160020

Deficiency in the Formation of 20-Hydroxyeicosatetraenoic Acid Enhances Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion (IR) injury is the most common cause of AKI. The susceptibility to develop AKI varies widely among patients. However, little is known about the genes involved. 20-Hydroxyeicosatetraenoic acid (20-HETE) has an important role in the regulation of renal tubular and vascular function and has been implicated in IR injury. In this study, we examined whether a deficiency in the renal formation of 20-HETE enhances the susceptibility of Dahl salt-sensitive (SS) rats to ischemic AKI. Transfer of chromosome 5 containing the CYP4A genes responsible for the formation of 20-HETE from the Brown Norway (BN) rat onto the SS genetic background increased renal 20-HETE levels after ischemia and reduced plasma creatinine levels (±SEM) 24 hours after IR from 3.7±0.1 to 2.0±0.2 mg/dl in an SS.5(BN)-consomic strain. Transfer of this chromosome also prevented the secondary decline in medullary blood flow and ischemia that develops 2 hours after IR in the susceptible SS strain. Blockade of the synthesis of 20-HETE with HET0016 reversed the renoprotective effects in SS.5(BN) rats. Similar results were observed in an SS.5(Lew)-congenic strain, in which a smaller region of chromosome 5 containing the CYP4A genes from a Lewis rat was introgressed onto the SS genetic background. These results indicate that 20-HETE has a protective role in renal IR injury by maintaining medullary blood flow and that a genetic deficiency in the formation of 20-HETE increases the susceptibility of SS rats to ischemic AKI.

Abstract 298: Protective Role of Endogenous 20-HETE in Renal Ischemia-Reperfusion Injury

The present study compared renal ischemia-reperfusion (IR) injury in Dahl salt-sensitive (SS) rats that have a deficiency in the renal formation of 20-HETE versus CYP4A1 transgenic SS (SS.4A1) rats in which the renal production of 20-HETE is restored. The concentrations of free 20-HETE in the renal cortex and outer medulla were significantly greater in SS.4A1 than in SS rats. Renal 20-HETE levels rose to a greater extent in SS.4A1 than in SS rats following renal IR. Plasma creatinine level rose to 3.7 ± 0.1 in SS versus 1.8 ± 0.3 mg/dl in SS.4A1 rats (respectively, n=6) following 30 min of ischemia and 24 h reperfusion. The % of necrotic tubules and apoptotic cells were 4-fold higher in SS than in SS.4A1 rats. Administration of the 20-HETE synthesis inhibitor (HET0016, 10 mg/kg) abolished the resistance of SS.4A1 rats to renal IR injury and plasma creatinine level rose to 3.8 ± 0.1 mg/dl (n=6). Cortical blood flow in SS, SS.4A1 and HET0016 treated SS.4A1 rats immediately returned to control following IR. However, medullary blood flow in SS and HET0016 treated SS.4A1 rats fell to 30 % of control 3 h after IR (n=5), and it remained depressed for 24 h. In contrast, medullary blood flow did not decline following IR in SS.4A1 rats. Proximal intratubular pressure rose from 13 to approximately 40 mmHg, 2 h after IR in both SS and SS.4A1 rats. Proximal intratubular pressure remained much higher in SS than in SS.4A1 rats 24 h after IR (32 vs 19 mmHg). These data indicate that normalization of renal CYP4A activity and 20-HETE production opposes renal IR injury by preventing secondary fall in medullary blood flow and the prolonged renal medullary ischemia.

Mechanism of impaired afferent arteriole myogenic response in Dahl salt-sensitive rats: role of 20-HETE.

The afferent arteriole (Af-Art) controls glomerular capillary pressure, an important determinant of glomerular injury. Af-Art myogenic response is mediated by ATP, and ATP signaling is in turn mediated by 20-HETE. Dahl salt-sensitive rats (Dahl SS) have decreased renal 20-HETE production. We hypothesized that Dahl SS have an impaired myogenic response and constrictor response to ATP, due to decreased 20-HETE. Af-Arts from Dahl SS or Dahl salt-resistant rats (Dahl SR) were microdissected and perfused. When myogenic response was induced by increasing Af-Art perfusion pressure from 60 to 140 mmHg, luminal Af-Art diameter decreased in Dahl SR but not in Dahl SS (-3.1 ± 0.8 vs. 0.5 ± 0.8 μm, P < 0.01). The 20-HETE antagonist 20-HEDE (10(-6) M) blocked the myogenic response in Dahl SR but had no effect in Dahl SS. Addition of a subconstrictor concentration of 20-HETE (but not a subconstrictor concentration of norepinephrine) restored the myogenic response in Dahl SS. We then perfused Af-Arts at 60 mmHg and tested the effects of the ATP analog α,β-methylene-ATP (10(-6) M). Maximum ATP-induced constriction was attenuated in Dahl SS compared with Dahl SR (1.5 ± 0.5 vs. 7.4 ± 0.8 μm, P < 0.001). 20-HEDE attenuated ATP-induced Af-Art constriction in Dahl SR but not in Dahl SS, and consequently, ATP-induced constriction was no longer different between strains. In conclusion, Dahl SS have an impaired myogenic response and ATP-induced Af-Art constriction due to a decrease in Af-Art 20-HETE. The impaired myogenic responses may contribute to the nephrosclerosis that develops in Dahl SS.

Abstract 159: Inhibition of Ace Does Not Prevent 20-hete-mediated Vascular Injury in Cyp4a12tg Mice

20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite in the microcirculation, increases smooth muscle contraction and proliferation, stimulates endothelial dysfunction and activation and promotes hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells, 20-HETE increases angiotensin converting enzyme (ACE) mRNA by 3.1-fold (± 0.16; p&lt;0.05), ACE protein by 4.2-fold (±0.99; p&lt;0.05) and cellular and extracellular ACE activity by 2.2-fold (±0.09; p&lt;0.05) and 1.96-fold (±0.16; p&lt;0.05), respectively. The contribution of ACE to vascular 20-HETE-mediated microvascular remodeling in hypertension was assessed using the Cyp4a12 transgenic mouse in which the expression of the Cyp4a12-20-HETE synthase is driven by a tetracyclin (doxycycline, DOX)-sensitive promoter. Treatment of Cyp4a12tg mice with DOX increased blood pressure (133±6 vs. 107±3 mmHg; p&lt;0.05) and remodeling of renal resistance arteries (M/L, 0.15±0.01 vs. 0.07±0.01; p&lt;0.05); these increases were abrogated by co-treatment with the 20-HETE antagonist, 20-HEDGE. Renal ACE protein expression was significantly higher in DOX-treated (1.6 ± 0.17 fold; p&lt;0.05) as compared to untreated Cyp4a12tg mice; co-treatment with 20-HEDGE prevented the increase in ACE expression. Lisinopril treatment of Cyp4a12tg mice concurrently receiving DOX prevented the blood pressure increase (133±6 vs. 104±2 mmHg; p&lt;0.05) but did not interfere with renal microvascular 20-HETE production (1.05±0.18 vs 1.09±0.30 ng/mg). Co-treatment with lisinopril and DOX attenuated but did not prevent remodeling of renal microvessels (M/L, 0.07±0.0 vs. 0.15±0.01 vs. 0.11±0.01; p&lt;0.05 in water, DOX and DOX+Lisinopril, respectively). In contrast, co-treatment with 20-HEDGE prevented DOX-induced remodeling of resistance arteries of Cyp4a12tg mice. This study demonstrates that 20-HETE is a key determinant of microvascular remodeling in DOX-induced Cyp4a12tg hypertensive mice. 20-HETE’s mechanism only partially relies on activation of the RAS. Thus, the 20-HETE-driven microvascular remodeling does not fully depend on the upregulation of ACE and elevation of blood pressure.

Abstract 283: Protective Role of Endogenous 20-HETE in Renal Ischemia-Reperfusion Injury

Previous studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) protect against ischemia reperfusion injury (IRI) in the brain and heart. However, the role of endogenous 20-HETE in renal IRI has not been well characterized. The present study compared the susceptibility to IRI in Dahl salt-sensitive (S) rats in which the renal formation of 20-HETE is impaired versus that seen in normal Sprague-Dawley (SD) rats and in SS.5LEW 4A+ (4A+) congenic and SS.BN5 (BN5) consomic strains in which the CYP4A region of chromosome 5 was transferred from the Lewis or Brown Norway rats into the S genetic background. S, 4A+ and BN5 rats were fed a low salt diet and SD rats were fed a standard diet. IRI was induced by bilateral renal ischemia for 30 minutes followed by reperfusion. The 20-HETE production was measured using liquid chromatography-mass spectrometry (LC/MS). The renal production level of 20-HETE was significantly greater in SD, 4A+ and BN5 rats as compared to S rats and renal 20-HETE levels rose to a greater extent in these strains than in S rats following renal ischemia reperfusion. Plasma creatinine concentration rose to 3.7 ± 0.1 (n=6), 2.0 ± 0.4 (n=6), 1.9 ± 0.3 (n=6) and 2.2 ± 0.3 (n=6) mg/dl 24 hours following renal ischemia in S, 4A+, BN5 and SD rats, respectively. The kidney of S rats following IRI exhibited renal tubular necrosis and protein casts. The % of necrotic tubules in the corticomedullary regions of S rats (42.5 ± 3.5 %) was significantly higher than that seen in 4A+ (10.8 ± 3.8 %) or BN5 (10.2 ± 1.5 %) rats. The number of apoptotic tubular cells in the corticomedullary regions of S rats (125.3 ± 8.2 cells/field) was significantly higher than that seen in BN5 rats (46.8 ± 5.1 cells/field). Administration of the 20-HETE antagonist (6, 15-20-hydroxyeicosadecanoic acid) or the 20-HETE synthesis inhibitor (HET0016) abolished the resistance of 4A+ and BN5 rats to renal IRI and plasma creatinine levels rose to 4.0 ± 0.4 (n=4) and 3.8 ± 0.1 (n=4) mg/dl, respectively. The % of necrotic tubules respectively increased to 33.6 ± 6.9 % and 38.5 ± 5.5 % in 4A+ and BN5 rats and the number of apoptotic tubular cells increased to 119.2 ± 10.7 cells/field in BN5 rats. These data indicate that endogenously released 20-HETE plays a protective role in renal IRI.

Upregulation of renal medullary 20‐HETE production opposes the development of hypertension in Sleeping Beauty Transposon CYP4A1 transgenic Dahl S rats

A genetic deficiency in the renal formation of 20‐HETE, reported to reduce sodium transport in the TALH, contributes to the development of hypertension in Dahl S (S) rats. Therefore, we hypothesized that increased expression of CYP4504A protein and renal formation of 20‐HETE attenuates the rise in MAP in S rats. We created a transgenic rat in which the CYP4A1 gene responsible for the production of 20‐HETE was introduced into the S genetic background. We found the production of 20‐HETE in the renal outer medulla was similar in S and CYP4A transgenic rats on standard (0.3%NaCl) chow (8 ±4 vs 10±3 pmol/mg/min). After 28 days on 8%NaCl diet, renal medullary 20‐HETE production increased markedly and was 3‐fold higher in CYP4A1 transgenic animals compared to S rats (127±22 vs 40±16 pmol/mg/min). MAP was significantly lower in the CYP4A1 (n=10) transgenic rats as compared to S rats (139±5 vs 177±10 mmHg, n=8, p&lt;0.05). Protein excretion was significantly lower in CYP4A1 transgenic rats relative to S rats (175±22 vs 295±22 mg/day, p&lt;0.05) and the degree of renal injury was greatly reduced. These results indicate that the renal medullary production of 20‐HETE is elevated on a high salt diet and upregulation of the expression of CYP4A1 gene in the S genetic background increases the renal production of 20‐HETE, improves proteinuria and opposes the development of hypertension. 1T32HL105324‐01, AHA11POST7520052, HL36279, HL29587.

Piroxicam Reverses Endotoxin‐Induced Hypotension in Rats: Contribution of Vasoactive Eicosanoids and Nitric Oxide

Nitric oxide (NO) produced by inducible NO synthase (iNOS) is responsible for endotoxin-induced vascular hyporeactivity and hypotension resulting in multiple organ failure. Endotoxic shock is also characterized by decreased expression of constitutive cyclooxygenase (COX-1), cytochrome P450 (CYP) 4A and endothelial NOS (eNOS). Our previous studies demonstrated that dual inhibition of iNOS and COX with a selective COX-2 inhibitor, NS-398, or a non-selective COX inhibitor, indomethacin, restores blood pressure presumably because of increased production of 20-hydroxyeicosatetraenoic acid (20-HETE) derived from arachidonic acid (AA) by CYP4A in endotoxaemic rats. The aim of this study was to investigate the effects of piroxicam, a preferential COX-1 inhibitor, on the endotoxin-induced changes in blood pressure, expression of COX-1, inducible COX (COX-2), CYP4A1, eNOS, iNOS and heat shock protein 90 (hsp90), and production of PGI(2), PGE(2), 20-HETE and NO. Injection of endotoxin (10 mg/kg, i.p.) to male Wistar rats caused a fall in blood pressure and an increase in heart rate associated with elevated renal 6-keto-PGF(1α) and PGE(2) levels as well as an increase in COX-2 protein expression. Endotoxin also caused an elevation in systemic and renal nitrite levels associated with increased renal iNOS protein expression. In contrast, systemic and renal 20-HETE levels and renal expression of eNOS, COX-1 and CYP4A1 were decreased in endotoxaemic rats. The effects of endotoxin, except for renal COX-1 and eNOS protein expression, were prevented by piroxicam (10 mg/kg, i.p.), given 1 hr after injection of endotoxin. Endotoxin did not change renal hsp90 protein expression. These data suggest that a decrease in the expression and activity of COX-2 and iNOS associated with an increase in CYP4A1 expression and 20-HETE synthesis contributes to the effect of piroxicam to prevent the hypotension during rat endotoxaemia.