John D. Imig

Soluble epoxide hydrolase as a therapeutic target for cardiovascular diseases

The cardiovascular effects of epoxyeicosatrienoic acids (EETs) include vasodilation, antimigratory actions on vascular smooth muscle cells and anti-inflammatory actions. These endogenous lipid mediators are broken down into diols by soluble epoxide hydrolase (sEH), and so inhibiting this enzyme would be expected to enhance the beneficial cardiovascular properties of EETs. sEH inhibitors (sEHIs) that are based on 1,3-disubstituted urea have been rapidly developed, and have been shown to be antihypertensive and anti-inflammatory, and to 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 the first clinical trials of sEHIs has highlighted the therapeutic potential of these agents.

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

An Orally Active Epoxide Hydrolase Inhibitor Lowers Blood Pressure and Provides Renal Protection in Salt-Sensitive Hypertension

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 mm Hg 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.

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.

Receptor-Mediated Intrarenal Angiotensin II Augmentation in Angiotensin II–Infused Rats

Chronic low-dose angiotensin II (Ang II) infusion for 13 days mimics two-kidney, one clip Goldblatt hypertension and increase intrarenal Ang II levels. We performed studies to determine the time course for the enhancement of intrarenal Ang II levels and whether the increased intrarenal Ang II is a tissue-specific event and requires a receptor-mediated step. Male Sprague-Dawley rats were uninephrectomized, and either vehicle or Ang II (40 ng/min) was infused via a subcutaneous osmotic minipump. Plasma and renal Ang II levels were measured 3, 7, 10, and 13 days after minipump implantation. Compared with controls (126 +/- 2 mm Hg), systolic pressure in Ang II-infused rats exhibited a detectable increase by day 6 (146 +/- 2 mm Hg) and continued to increase to 189 +/- 5 mm Hg by day 12. Plasma Ang II levels were elevated by day 3, whereas intrarenal Ang II levels were not significantly elevated until 10 days of Ang II infusion. Renal injury characterized by focal and segmental glomerulosclerosis was evident after 13 days of Ang II infusion. Losartan (30 mg/kg per day) prevented the development of hypertension in the Ang II-infused rats for the duration of the infusion period (125 +/- 1 mm Hg) and reduced the degree of glomerular injury. Plasma renin activity was suppressed in the Ang II-infused group but was elevated markedly in both losartan-treated groups. Plasma Ang II levels were elevated in the Ang II-infused rats and were even higher during losartan treatment. Intrarenal Ang II levels were enhanced significantly (354 +/- 60 versus 164 +/- 23 fmol/g) in the Ang II-infused rats. However, losartan treatment prevented the augmentation of intrarenal Ang II caused by Ang II infusion. Heart and adrenal Ang II levels were not significantly increased in the Ang II-infused rats but were significantly elevated during losartan treatment. These results suggest that the tissue-specific elevations of intrarenal Ang II levels caused by chronic Ang II infusion are mediated by angiotensin type 1 receptor activation, which leads to either receptor-mediated internalization of Ang II, enhancement of intrarenal Ang II formation, or both.

Identification of a Putative Microvascular Oxygen Sensor

The vascular response to changes in oxygen levels in the blood and tissue is a highly adaptive physiological response that functions to match tissue oxygen supply to metabolic demand. Defining the cellular mechanisms that can sense physiologically relevant changes in PO2 and adjust vascular diameter are vital to our understanding of this process. A cytochrome P450 (P450) enzyme of the 4A family of omega-hydroxylases was localized in renal microvessels, renal cortex, and a striated muscle microvascular bed (cremaster) of the rat. In the presence of molecular oxygen, this P450 enzyme catalyzes formation of 20-HETE from arachidonic acid (AA). Prior studies have shown that 20-HETE potently contracts renal and cerebral arteries and arterioles. The present study demonstrates that 20-HETE constricts striated muscle arterioles as well. In both intact renal microvessels and enriched renal cortical microsomal enzyme preparations, the formation of 20-HETE was linearly dependent on PO2 between 20 and 140 mm Hg. Homogenates of cremaster tissue produced 20-oxygen HETE when incubated with AA. They also expressed message for P450 4A enzyme, as determined by Southern and Western blots. Administration of 17-octadecynoic acid (17-ODYA), which is a P450 4A inhibitor, attenuated the constriction of third-order cremasteric arterioles in response to elevation of superfusion solution PO2 from approximately equal to 3 to 5 mm Hg to approximately equal to 35 mm Hg. 17-ODYA had no effect on basal vascular tone or response of cremaster arterioles to vasoactive compounds. These results demonstrate the existence of P450 omega-hydroxylase activity and 20-HETE formation in the vasculature and parenchyma of at least two microvascular beds. Our data suggest that a P450 enzyme of the 4A family has the potential to function as an oxygen sensor in mammalian microcirculatory beds and to regulate arteriolar caliber by generating 20-HETE in an oxygen-dependent manner.

Physiological role for P2X1 receptors in renal microvascular autoregulatory behavior

This study tests the hypothesis that P2X1 receptors mediate pressure-induced afferent arteriolar autoregulatory responses. Afferent arterioles from rats and P2X1 KO mice were examined using the juxtamedullary nephron technique. Arteriolar diameter was measured in response to step increases in renal perfusion pressure (RPP). Autoregulatory adjustments in diameter were measured before and during P2X receptor blockade with NF279 or A1 receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Acute papillectomy or furosemide perfusion was performed to interrupt distal tubular fluid flow past the macula densa, thus minimizing tubuloglomerular feedback‐dependent influences on afferent arteriolar function. Under control conditions, arteriolar diameter decreased by 17% and 29% at RPP of 130 and 160 mmHg, respectively. Blockade of P2X1 receptors with NF279 blocked pressuremediated vasoconstriction, reflecting an attenuated autoregulatory response. The A1 receptor blocker DPCPX did not alter autoregulatory behavior or the response to ATP. Deletion of P2X1 receptors in KO mice significantly blunted autoregulatory responses induced by an increase in RPP, and this response was not further impaired by papillectomy or furosemide. WT control mice exhibited typical RPPdependent vasoconstriction that was significantly attenuated by papillectomy. These data provide compelling new evidence indicating that tubuloglomerular feedback signals are coupled to autoregulatory preglomerular vasoconstriction through ATP-mediated activation of P2X1 receptors.

Neuronal nitric oxide synthase modulates rat renal microvascular function

This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 +/- 0.2 microns) and efferent (18.5 +/- 0.2 microns) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-L-thiocitrulline (L-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses to L-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, N omega-nitro-L-arginine (L-NNA) were only attenuated by papillectomy. L-SMTC (10 microM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, L-NNA (100 microM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary micro-vasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 ± 0.2 μm) and efferent (18.5 ± 0.2 μm) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-l-thiocitrulline (l-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses tol-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, N ω-nitro-l-arginine (l-NNA) were only attenuated by papillectomy.l-SMTC (10 μM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, l-NNA (100 μM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary microvasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.

Epoxygenase Metabolites Contribute to Nitric Oxide-Independent Afferent Arteriolar Vasodilation in Response to Bradykinin

In the kidney, epoxyeicosatrienoic acids (EETs) have been suggested to be endothelium-derived hyperpolarizing factors (EDHFs). The aim of the present study was to determine the contribution of EETs to the preglomerular vasodilation elicited by bradykinin. Sprague-Dawley rats were studied utilizing an in vitro perfused juxtamedullary nephron preparation. The afferent arteriolar diameter was determined and the diameter averaged 19 ± 1 µm (n = 26) at a renal perfusion pressure of 100 mm Hg. Addition of 1, 10 and 100 nM bradykinin to the perfusate dose-dependently increased afferent arteriolar diameter by 5 ± 1, 12 ± 2 and 17 ± 2%, respectively. The nitric oxide inhibitor Nω-nitro-L-arginine reduced bradykinin-induced afferent arteriolar vasodilation by 50%, and the diameter increased by 9 ± 2% in response to 100 nM bradykinin. Epoxygenase inhibitors N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide or miconazole greatly attenuated the nitric oxide-independent component of the vasodilation elicited by bradykinin. Cyclooxygenase (COX) inhibition attenuated the nitric oxide-independent vasodilation elicited by 1 nM bradykinin but did not significantly affect the vascular response to 100 nM bradykinin. Combined inhibition of nitric oxide, COX and epoxygenase pathways completely abolished bradykinin-mediated afferent arteriolar vasodilation. In additional studies, renal microvessels were isolated and incubated with bradykinin and samples were analyzed by NICI/GC/MS. Under control conditions, renal microvascular EET levels averaged 49 ± 9 pg/mg/20 min (n = 7). In the presence of bradykinin, EET levels were significantly higher and averaged 81 ± 11 pg/mg/20 min (n = 7). These data support the concept that EETs are EDHFs and contribute to the nitric oxide-independent afferent arteriolar vasodilation elicited by bradykinin.

Renal Accumulation of Circulating Angiotensin II in Angiotensin II–Infused Rats

Previous studies have demonstrated that low-dose angiotensin II (Ang II) infusion for 14 days mimics two-kidney, one clip Goldblatt hypertension and increases intrarenal Ang II levels. The objective of the present study was to determine whether the augmented intrarenal Ang II is due to intrarenal accumulation of the infused Ang II and/or to an increase in intrarenal formation of endogenous Ang II. Male Sprague-Dawley rats were uninephrectomized and divided into three groups: control (N=6), those infused with [Ile5]Ang II (endogenous form) (N=6), and those infused with [Val5]Ang II (n=8). [Ile5]Ang II or [Val5]Ang II was infused at 40 ng/min via an osmotic minipump implanted subcutaneously. By day 12, systolic blood pressure increased significantly in both [Val5]Ang II-infused rats (197 +/- 7 mm Hg) and [Ile5]Ang II-infused rats (173 +/- 3 mm Hg). Blood and kidney samples were harvested, subjected to high-performance liquid chromatography to separate [Val5]Ang II from [Ile5]Ang II, and then measured by radioimmunoassay. Plasma renin activity was markedly suppressed in both [Ile5]Ang II- and [Val5]Ang II-infused rats. Plasma Ang II levels were elevated in rats infused with both [Ile5]Ang II (121 +/- 24 fmol/mL) and [Val5]Ang II (119 +/- 14 fmol/mL) compared with controls (69 +/- 15 fmol/mL). Both [Ile5]Ang II- and [Val5]Ang II-infused rats exhibited an enhancement of total intrarenal Ang II. Only [Ile5]Ang II (358 +/- 53 fmol/g) was detected in the kidneys of rats infused with -Ile5-Ang II. In [Val5]Ang II-infused rats, a significant portion of total renal Ang II (371 +/- 57 fmol/g) was in the form of [Val5]Ang II (256 +/- 44 fmol/g). Renal [Ile5]Ang II levels were maintained in the [Val5]Ang II-infused rats (116 +/- 15 fmol/g) compared with control rats (116 +/- 11 fmol/g) despite marked suppression of renin release. These results support the hypothesis that infused circulating ANG II is bound to receptor or taken up intrarenally in a manner that protects against degradation.