Adebayo Oyekan

Endothelin-1 and CYP450 arachidonate metabolites interact to promote tissue injury in DOCA-salt hypertension

Inhibition of cytochrome P-450 (CYP450) enzymes with cobalt chloride (CoCl2) prevented hypertension, organ hypertrophy, and renal injury induced by DOCA and salt (1% NaCl) in uninephrectomized (UNx) rats. Systolic blood pressure (SBP) rose to 193 +/- 6 mmHg by day 21 from control levels of 150 +/- 7 mmHg in response to DOCA-salt treatment, a rise that was prevented by CoCl2 (24 mg. kg-1. 24 h-1). The effects of DOCA-salt treatment, which increased protein excretion to 88.3 +/- 6.9 mg/24 h on day 21 from 9.0 +/- 1.1 mg/24 h on day 3, were prevented by CoCl2. CoCl2 also attenuated the renal and left ventricular hypertrophy and the increase in media-to-lumen ratio in hypertensive rats. DOCA-salt treatment increased excretion of endothelin (ET)-1 from 81 +/- 17 to 277 +/- 104 pg. 100 g body wt-1. 24 h-1 associated with a fourfold increase in 20-hydroxyeicosatetraenoic acid (20-HETE) excretion from 3.0 +/- 1.1 to 12.2 +/- 1.9 ng. 100 g body wt-1. 24 h-1 (days 3 vs. 21). CoCl2 blunted these increases by 58 and 72%, respectively. In aortic rings pulsed with [3H]thymidine, ET-1 increased its incorporation. Dibromododec-11-enoic acid, an inhibitor of 20-HETE synthesis, attenuated ET-1-induced increases in [3H]thymidine incorporation. We distinguished effects of CoCl2 acting via CO generation vs. suppression of CYP450-arachidonic acid metabolism by treating UNx-salt-DOCA rats with 1-aminobenzotriazole (ABT), which suppresses CYP450 enzyme activity, and compared these results to those produced by CoCl2. ABT reduced hypertension, as did CoCl2. Unlike CoCl2, ABT did not prevent organ hypertrophy and proteinuria, suggesting that these effects were partially related to CO formation. Blockade of the ETA receptor with BMS-182874 reduced SBP, organ hypertrophy, and proteinuria, indicating the importance of ET-initiated abnormalities to the progression of lesions in UNx-salt-DOCA.

Relationship between PPARα activation and NO on proximal tubular Na+ transport in the rat

BackgroundNitric oxide (NO) regulates renal proximal tubular (PT) Na+ handling through modulation of Na+-K+ ATPase. Peroxisome Proliferator Activated Receptorα (PPARα), a nuclear transcription factor, is expressed in PTs and has been reported to influence NO generation/activity in renal tissues. This study tested the hypothesis that PPARα interacts with NO and thereby affects renal tubular Na+ transport. Urinary excretion of nitrite (UNOXV) and Na+ (UNaV) and PT Na+ transport (Na+-K+ ATPase activity) were determined in rats treated with clofibrate (250 mg/kg i.p) or WY14643 (45 mg/kg; i.p.), a PPARα ligand, 2% NaCl (orally), clofibrate/NaCl, L-NAME, an inhibitor of NO production (100 mg/kg; orally), L-NAME/Clofibrate.ResultsClofibrate or WY14643 increased PPARα expression by 106 ± 7% (p < 0.05) and 113 ± 8% (p < 0.05), respectively. Similarly, clofibrate and WY14643 increased expression of MCAD, a downstream target protein of PPARα by 123 ± 8% (p < 0.05) and 143 ± 8% (p < 0.05), respectively. L-NAME attenuated clofibrate-induced increase in PPARα expression by 27 ± 2% (p < 0.05) but did not affect MCAD expression. UNOXV excretion increased 3–4 fold in rats treated with clofibrate, WY14643 or NaCl from 44 ± 7 to 170 ± 15, 144 ± 18 or 132 ± 11 nmol/24 hr, respectively (p < 0.05). Similarly, clofibrate, WY14643 or NaCl elicited a 2–5 fold increase in UNaV. L-NAME significantly reduced basal UNOXV and UNaV and abolished the clofibrate-induced increase. Clofibrate, WY14643, NaCl or clofibrate + NaCl treatment reduced Na+-K+-ATPase activity in the PT by 89 ± 23, 62 ± 10, 43 ± 9 and 82 ± 15% (p < 0.05), respectively. On the contrary, L-NAME or ODQ, inhibitor of sGC, abolished the inhibition of Na+-K+-ATPase activity by clofibrate (p < 0.05). Clofibrate either alone or with NaCl elicited ~2-fold increase in the expression of the α1 subunit of Na+-K+ ATPase in the PT while L-NAME abolished clofibrate-induced increase in Na+-K+ ATPase expression.ConclusionThese data suggest that PPARα activation, through increased NO generation promotes renal excretion of Na+ through reduced Na+-K+ ATPase activity in the PT probably via post translational modification of Na+-K+-ATPase.

Oxidative stress-associated vascular aging is xanthine oxidase-dependent but not NAD(P)H oxidase-dependent.

Abstract: Vascular aging is characterized by endothelial dysfunction that is primarily attributed to increased superoxide production, the exact source of which remains ambiguous. This study compared the NAD(P)H and xanthine oxidase (XO) systems as sources of superoxide and impaired vascular function in aging. Male Sprague Dawley rats, 4-months-old (young) and 18-months-old (Aging), were used. Systolic blood pressure was higher (36 ± 3%) in the aging group compared with young rats, and this was accompanied by reduced acetylcholine-induced renal vasodilatation. Urinary excretion of nitrite was lower in the aging rats (P < 0.05), and this was associated with reduced nitric oxide synthase (NOS) activity and reduced eNOS and iNOS protein expression in the aorta. Aged rats showed a n approximately twofold increase in free radical generation, as evident by increased plasma 8-isoprostane level, and an approximately fourfold increase in proteinuria compared with the young rats. Vascular NADP(H) oxidase was unchanged between both groups, as was the expression of p67phox or p47phox components of NAD(P)H oxidase. However, XO activity was increased (19 ± 1%; P < 0.05) as well as XO expression in the aorta of aging rats. These results suggest that increased free radical generation-associated increase in SBP in aging rats is XO but not NAD(P)H oxidase-dependent.

PPARs and their Effects on the Cardiovascular System

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily that undergo transactivation or transrepression by distinct mechanisms leading to induction or repression of expression of target genes. The PPAR family consists of three isoforms, α, γ, and ß/δ, which share similar structural organization, possess distinct functions, and vary in their ligand affinity, expression, and activity in different metabolic pathways in different tissues. PPARs are involved in many functions especially those involved in the regulation of vascular tone, inflammation and energy homeostasis and therefore represent important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. PPARs may influence the inflammatory response either by direct transcriptional downregulation of proinflammatory genes via mechanisms involving transrepression, or indirectly via their transcriptional effects on lipid metabolism. On account of their pleiotropic effects, they are now known to be active participants in many disease conditions and they represent potent targets for the development of therapy of a wide array of diseases.

Contribution of cytochrome P450 4A isoforms to renal functional response to inhibition of nitric oxide production in the rat

20‐Hydroxyeicosatetraenoic acid (20‐HETE), a major renal eicosanoid, regulates renal function and contributes to renal responses following withdrawal of nitric oxide (NO). However, the role of 20‐HETE‐synthesizing isoforms in renal function resulting from NO inhibition is unknown. The present study evaluated the role of cytochrome (CYP)4A1, ‐4A2 and ‐4A3 isoforms on renal function in the presence and absence of NO. Antisense oligonucleotides (ASODN) to CYP4A1, ‐4A2 and ‐4A3 reduced 20‐HETE synthesis and downregulated the expression of CYP4A isoforms in renal microsomes. Nω‐L‐nitromethyl arginine ester (L‐NAME, 25 mg kg−1), an inhibitor of NO production, increased mean arterial blood pressure (MABP, Δ=+18 to 26 mmHg), reduced renal blood flow (RBF, Δ= ‐1.8 to 2.9 ml min−1), increased renal vascular resistance (RVR, Δ=+47 to 54 mmHg ml−1 min−1), reduced glomerular filtration rate (GFR), but increased sodium excretion (UNaV). ASODN to CYP4A1 and ‐4A2 but not ‐4A3 reduced basal MABP and RVR and increased basal GFR, while ASODN to CYP4A2 significantly reduced basal UNaV suggesting a differential role for CYP4A isoforms in the regulation of renal function. ASODN to CYP4A2 but not ‐4A1 or ‐4A3 blunted the increase in MABP by L‐NAME (38 ± 9 %, P < 0.05). ASODN to CYP4A1, ‐4A2 and ‐4A3 attenuated the reduction in RBF and the consequent increase in RVR by L‐NAME with a potency order of CYP4A2 = CYP4A1 > CYP4A3. ASODN to CYP4A1 and ‐4A2 but not ‐4A3 attenuated L‐NAME‐induced reduction in GFR, but ASODN to all three CYP4A isoforms blunted the L‐NAME‐induced increase in UNaV (CYP4A3 > CYP4A1 >> CYP4A2). We conclude from these data that CYP4A isoforms contribute to different extents to basal renal function. Moreover, CYP4A2 contributes greatest to haemodynamic responses while CYP4A3 contributes greatest to tubular responses following NO inhibition. We therefore propose that NO differentially regulates the function of CYP4A1, ‐4A2, and ‐4A3 isoforms in the renal vasculature and the nephron.

The role of the RhoA/Rho-kinase signaling pathway in renal vascular reactivity in endothelial nitric oxide synthase null mice.

Background Smooth muscle contraction is regulated by the small GTPase RhoA and its target, Rho-kinase and recent evidence indicates that nitric oxide (NO) causes vasodilation through inhibition of the RhoA/Rho-kinase (ROCK) signaling pathway. Aim This study tested the hypothesis that the enhanced renal vascular tone and systemic hypertension in endothelial nitric oxide synthase (eNOS) null mice is due to disinhibition of the ROCK signaling pathway. Methods Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and the isolated Krebs-perfused kidney preparation was used to evaluate renal vascular responses in C57BL/6 (wild type, WT) and eNOS knockout (KO) mice treated with Y-27632, a ROCK inhibitor. Results Compared with the WT mice, Rho kinase activity was higher in eNOS KO mice (37 ± 8%, P < 0.05) as was SBP (33 ± 4%, P < 0.05), basal renal perfusion pressure (31 ± 4%, P < 0.05) and renal vascular resistance (35 ± 4%, P < 0.05). Y-27632 abolished these differences. Vasoconstriction elicited by angiotensin II (Ang II) or phenylephrine (PE), G-protein-coupled receptor (GPCR) agonists, but not that elicited by arachidonic acid or KCl, was greater in eNOS KO mice. Y-27632 eliminated the amplified vasoconstriction elicited by Ang II or phenylephrine but to a greater extent in eNOS KO mice. Similarly, responses elicited by guanosine 5′-γ-thiotriphosphate (GTPγS), a non-hydrolyzable GTP analog, or sodium tetrafluoride (NaF4), an activator of G-proteins, was greater in eNOS KO mice, 53 ± 14 and 50 ± 3%, respectively. Y-27632 normalized the difference. Y-27632 also elicited a dose-dependent renal vasodilation that was greater in eNOS KO mice. Conclusions These results show that the ROCK signaling pathway is amplified in the eNOS KO mouse kidney and that the enhanced renal vascular tone and selective increase in reactivity to GPCR agonists supports a role for ROCK in the hypertension and vascular dysfunction in the eNOS KO mice.

Renal function and vasomotor activity in mice lacking the Cyp4a14 gene.

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney is thought to be involved in the control of renal vascular tone and tubular sodium and chloride reabsorption. Cytochrome (Cyp) P-450 enzymes of the Cyp4a family in the mouse, namely 4a10, -12 and 14, are involved in 20-HETE synthesis. Recent advances in the molecular genetics of the mouse have produced mice in which Cyp4a isoforms have been disrupted and the consequence of such an approach is examined. This study evaluated the effect of deletion of the Cyp4a14 gene on blood pressure, renal vascular responses and tubular function. When compared with the wild-type (WT) litter mates, systolic blood pressure was greater in Cyp4a14 null (KO) mice as were renal vascular responses to angiotensin II or phenyephrine, G protein-coupled receptor (GPCR) agonists, but not KCl, a non-GPCR agonist. Renal vascular responses to guanosine 5′-O-(gamma-thio)triphosphate, a non-hydrolyzable GTP analog, or NaF4, an activator of G-proteins, were also enhanced. However, vasodilation to bradykinin or apocynin but not sodium nitroprusside was blunted in Cyp4a14 null (KO) kidneys. These changes in KO mice were accompanied by increased 20-HETE synthesis, reduced renal production of nitric oxide (NO), increased lipid hydroperoxides and increased apocynin-inhibitable vascular NADPH oxidase activity that was prevented by administration of NO synthase (NOS) inhibitor, suggesting endothelial nitric oxide synthase (eNOS) uncoupling. Cyp4a14 KO mice also exhibited a diminished capacity to excrete an acute sodium load (0.9% NaCl, 2.5 mL/kg). These data suggest that deletion of the Cyp4a gene conferred a prohypertensive status via mechanisms involving increased 20-HETE synthesis and eNOS uncoupling leading to increased oxidative stress, enhanced vasoconstriction but diminished vasodilation as well as a defect in the renal excretory capacity in Cyp4a14 KO mice. These mechanisms suggest that the Cyp4a14-deficient mouse may be a useful model for evaluation of NO/20-HETE interactions.

Nitric oxide/cytochrome P450 interactions in cyclosporin A-induced effects in the rat.

Introduction The present study evaluated the contribution of 20-hydroxyeicosatetraenoic acid (20-HETE) and its interaction with nitric oxide (NO) in cyclosporin A-induced nephrotoxicity and hypertension. Methods and results The treatment of rats with cyclosporin A (25 mg/kg) for 7 days increased the renal microsomal conversion of arachidonic acid (AA) to 20-HETE (93 ± 6%, P < 0.05), increased systolic blood pressure (SBP), reduced the urinary excretion of nitrite (53 ± 8%, P < 0.05), induced renal damage as indicated by a marked increase in protein excretion (163 ± 14%, P < 0.05), increased renal vasoconstrictor responses to AA (82 ± 5%, P < 0.05) but not endothelin-1 or phenylephrine, and decreased vasodilator responses to bradykinin (42 ± 10%, P < 0.05) and sodium nitroprusside (SNP; 56 ± 13%, P < 0.05) in the renal preglomerular vessel treated with indomethacin and NO synthase inhibitor. The pretreatment of rats with HET0016 (10 mg/kg) or 1-aminobenzotriazole (50 mg/kg), inhibitors of cytochrome P450 (CYP450) activity, attenuated or prevented cyclosporin A-induced increases in 20-HETE production, SBP, and protein excretion, as did L-arginine (4 g/l), a substrate for NO synthase. L-Arginine but not HET0016 or 1-aminobenzotriazole blunted the cyclosporin A-induced decrease in nitrite excretion. Similarly, L-arginine blunted the enhanced vasoconstriction by AA as did HET0016 or 1-aminobenzotriazole. However, cyclosporin A-blunted dilator responses to bradykinin and SNP were not affected by L-arginine, HET0016, or 1-aminobenzotriazole. Conclusions These data suggest that cyclosporin A-induced nephrotoxicity can be accounted for by reduced NO production and a consequent increase in 20-HETE. The cyclosporin A-induced nephrotoxicity is thus an ideal model for evaluating NO/CYP450 interactions.

Vascular responses to endothelin-1, angiotensin-II, and U46619 in glycerol-induced acute renal failure.

Angiotensin II and endothelin-1, major endogenous vasoconstrictors in acute renal failure (ARF), can modulate the effects of each other. This study aimed to evaluate the interaction between these vasoconstrictors in glycerol-induced ARF by evaluating their effects in the isolated perfused kidney in the presence of their respective antagonists. In ARF, angiotensin II (2.5–25 ng) caused an increase in perfusion pressure. Saralasin, 1 &mgr;M, a nonselective angiotensin receptor antagonist, reduced these responses by 61± 6% (p < 0.05). Surprisingly, SQ29548, 1 &mgr;M, a selective PGH 2 /thromboxane A 2 receptor blocker, also reduced angiotensin II responses (62 ± 4%; p < 0.05). BQ610 1 &mgr;M, an ET A -selective receptor antagonist, was without effect, but BQ788 1 &mgr;M, an ET B -selective antagonist, attenuated the response by 70 ± 4% (p < 0.05). In ARF, in contrast to angiotensin II, vasoconstriction by endothelin-1 (5–25 ng) was diminished. Saralasin further attenuated endothelin-1 response by 65 ± 2% (p < 0.05), whereas SQ29548 was without effect. BQ788 reduced the responses by 67 ± 7% (p < 0.05), whereas BQ610 was without effect (42 ± 30%; p > 0.05). BQ610 and BQ788 combination further reduced vasoconstriction by 89 ± 3% (p < 0.05). Responses to U46619 were not changed in ARF. However, saralasin and BQ788, but not BQ610, attenuated its vasoconstrictor action. We conclude that vascular responses in ARF may be attributed to enhanced responses to angiotensin II through activation of ET B and/or PGH 2 /thromboxane A 2 receptors. We also suggest that the vasoconstrictor response to endothelin-1 in ARF is predominantly ET B receptor-mediated.

Interactions of PPAR-alpha and adenosine receptors in hypoxia-induced angiogenesis.

Hypoxia and adenosine are known to upregulate angiogenesis; however, the role of peroxisome proliferator-activated receptor alpha (PPARα) in angiogenesis is controversial. Using transgenic Tg(fli-1:EGFP) zebrafish embryos, interactions of PPARα and adenosine receptors in angiogenesis were evaluated under hypoxic conditions. Epifluorescent microscopy was used to assess angiogenesis by counting the number of intersegmental (ISV) and dorsal longitudinal anastomotic vessel (DLAV) at 28 h post-fertilization (hpf). Hypoxia (6h) stimulated angiogenesis as the number of ISV and DLAV increased by 18-fold (p<0.01) and 100 ± 8% (p<0.001), respectively, at 28 hpf. Under normoxic and hypoxic conditions, WY-14643 (10 μM), a PPARα activator, stimulated angiogenesis at 28 hpf, while MK-886 (0.5 μM), an antagonist of PPARα, attenuated these effects. Compared to normoxic condition, adenosine receptor activation with NECA (10 μM) promoted angiogenesis more effectively under hypoxic conditions. Involvement of A2B receptor was implied in hypoxia-induced angiogenesis as MRS-1706 (10nM), a selective A2B antagonist attenuated NECA (10 μM)-induced angiogenesis. NECA- or WY-14643-induced angiogenesis was also inhibited by miconazole (0.1 μM), an inhibitor of epoxygenase dependent production of eicosatrienoic acid (EET) epoxide. Thus, we conclude that: activation of PPARα promoted angiogenesis just as activation of A2B receptors through an epoxide dependent mechanism.