Mohammad Newaz

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.

Modulation of Nitric Oxide Synthase Activity in Brain, Liver, and Blood Vessels of Spontaneously Hypertensive Rats by Ascorbic Acid: Protection from Free Radical Injury

End organ damage in essential hypertension has been linked to increased oxygen free radical generation, reduced antioxidant defense, and/or attenuation of nitric oxide synthase (NOS) activity. Ascorbic acid (AA), a water-soluble antioxidant, has been reported as a strong defense against free radicals in both aqueous and nonaqueous environment. In this study we examined the hypothesis that antioxidant ascorbic acid may confer protection from increased free radical activity in brain, liver, and blood vessels of spontaneously hypertensive rats (SHR). Male SHRs were divided into groups: SHR + AA (treated with AA, 1 mg/rat/day; for 12 weeks) or SHR (untreated). Wister-Kyoto rats (WKY) served as the control. Mean systolic blood pressure (SBP) in treated and untreated SHR was 145 ± 7 mmHg and 142 ± 8 mmHg, respectively. AA treatment prevented the increase in systolic blood pressure in SHR by 37 ± 1% (p < 0.05). NOS activity in the brain, liver, and blood vessels of WKY rat was 1.82 ± 0.02, 0.14 ± 0.003, and 1.54 ± 0.06 pmol citruline/mg protein, respectively. In SHR, total NOS activity was significantly reduced by 52 ± 1%, 21 ± 3%, and 44 ± 4%, respectively. AA increased NOS activity in brain, liver, and blood vessels of SHR from 0.87 ±.03, 0.11 ±.01, and 0.87 ±.08 pmol citruline/mg protein to 0.93 ± 0.01, 0.13 ± 0.001, and 1.11 ± 0.03 pmol citruline/mg protein (p < 0.05), respectively. Lipid peroxides in the brain, liver, and blood vessels from WKY rats were 0.87 ± 0.06, 0.11 ± 0.005, and 0.47 ± 0.04 nmol MDA equiv/mg protein, respectively. In SHR, lipid peroxides in brain, liver, and blood vessels were significantly increased by 40 ± 3%, 64 ± 3%, and 104 ± 13%, respectively. AA reduced lipid peroxidation in liver and blood vessels by 17 ± 1% and 34 ± 3% but not in brain. Plasma lipid peroxides were almost doubled in SHR (p < 0.01) together with a reduction in total antioxidant status (6 ± 0.1%; p < 0.05), nitrite (53 ± 2%; p < 0.05) and superoxide dismutase (SOD) activity (36 ± 2%; p < 0.05). AA treatment reduced plasma lipid peroxide (p < 0.001), and increased TAS (p < 0.001), nitrite (p < 0.001), and SOD activity (p < 0.001). From this study, we conclude that brain, liver, and blood vessels in SHR are susceptible to free radical injury, which reduces the availability of NO either by scavenging it or by reducing its production via inhibiting NOS. In addition, brain, liver, and blood vessels in SHR; may be protected by antioxidant, which improves total antioxidant status, and SOD thus may prevent high blood pressure and its complications.

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.

Ciglitazone, a Peroxisome Proliferator-Activated Receptor γ Inducer, Ameliorates Renal Preglomerular Production and Activity of Angiotensin II and Thromboxane A2 in Glycerol-Induced Acute Renal Failure

Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear transcription factor, modulates vascular responses to angiotensin II (AII) or thromboxane A2 (TxA2) via regulation of their gene/receptor. Increased vasoconstriction and deteriorating renal function in glycerol-induced acute renal failure (ARF) may be attributed to down-regulation of PPARγ. In this study, we investigated the effect of ciglitazone (CG), a PPARγ inducer, on AII and TxA2 production and activity in glycerol-induced ARF. Vascular responses to AII or 9,11-dideoxy-11α,9α-epoxymethano prostaglandin F2α (U46619), a TxA2 mimetic, were determined in preglomerular vessels following induction of ARF with glycerol. Renal damage and function were assessed in CG-treated (9 nmol/kg for 21 days) rats. PPARγ protein expression and activity, which were significantly lower in ARF rats, were enhanced by CG (26 and 30%). CG also increased PPARγ mRNA by 67 ± 6%, which was reduced in ARF. In ARF, there was significant tubular necrosis and apoptosis, a 5-fold increase in proteinuria and a 2-fold enhancement in vasoconstriction to AII and U46619. CG reduced proteinuria (49 ± 3%), enhanced Na+ (124 ± 35%) and creatinine excretion (92 ± 25%), markedly diminished tubular necrosis, and reduced ARF-induced increase in AII (40 ± 3%) and TxA2 (39 ± 2%) production, the attending increase in vasoconstriction to AII (36 ± 2%) and U46619 (50 ± 11%), and the increase in angiotensin receptor-1 (AT1) (23 ± 3%) or thromboxane prostaglandin (TP) receptor (13 ± 1%). CG reduced free radical generation by 55 ± 14% while elevating nitrite excretion (65 ± 13%). Our results suggest that enhanced activity of AII and TxA2, increased AT1 or TP receptor expression, and renal injury in glycerol-induced ARF are consequent to down-regulation of PPARγ gene. CG ameliorated glycerol-induced effects through maintaining PPARγ gene.

PPARα ligand clofibrate ameliorates blood pressure and vascular reactivity in spontaneously hypertensive rats

Aim:Peroxisome proliferator activated receptors (PPARs) are nuclear transcription factors that regulate numerous genes influencing blood pressure. The aim of this study was to examine the effects of clofibrate, a PPARα ligand, on blood pressure in spontaneously hypertensive rats (SHR).Methods:Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR), 8–9 weeks old, were randomly allocated into groups treated with vehicle or clofibrate (250 mg·kg−1·d−1, ip for 21 d). Systolic blood pressure (SBP) was measured before and after the study period using tail-cuff plethysmography. Rats were sacrificed under anesthesia and blood, urine and tissue samples were processed for subsequent analysis.Results:SHR rats showed significantly higher SBP compared with WKY rats (198±6 mmHg vs 93±7 mmHg), and a 3-fold increase in urinary protein excretion. Clofibrate treatment reduced SBP by 26%±2% and proteinuria by 43%±9% in SHR but not in WKY rats. The urinary nitrite/nitrate excretion in SHR rats was nearly 2-fold greater than that in WKY, and was further increased by 30%±4% and 48%±3%, respectively, following clofibrate treatment. In addition, PPARα protein expression and PPARα activity were significantly lower in SHR than that in WKY rats. Clofibrate treatment significantly increased PPARα protein expression and PPARα activity in SHR rats, but not in WKY rats. Moreover, the vasoconstrictor response of aortic ring was markedly increased in SHRs, which was blunted after clofibrate treatment.Conclusion:PPARα contributes to regulation of blood pressure and vascular reactivity in SHR, and clofibrate-mediated reduction in blood pressure and proteinuria is probably through increased NO production.

Acrolein-induced inflammatory signaling in vascular smooth muscle cells requires activation of serum response factor (SRF) and NFκB

Abstract Background: Modulation of inflammatory signaling has been elucidated in several disease models. Acrolein, an environmental pollutant, has been linked to diseases such as atherosclerosis and to the inflammatory process involving nuclear factor κB (NFκB). Serum response factor (SRF), a transcription factor, regulates cell development, differentiation and proliferation through signaling molecules such as extracellular signal-regulated kinase 1/2 (ERK1/2) and CD36. We hypothesized that acrolein toxicity involves SRF in the process of activating NFκB and may involve CD36/ERK1/2. Methods: Vascular smooth muscle cells (VSMCs) were exposed to acrolein (0.5 μg/mL) in the presence or absence of 10 nM QNZ (NFκB inhibitor), 300 nM CCG1423 (SRF inhibitor) and 50 μM PD98059 (ERK1/2 inhibitor). Protein and RNA were isolated. Changes in expression were determined by Western blot and polymerase chain reaction (PCR) array. Results: Subtoxic doses of acrolein increased ERK1/2, SRF and NFκB protein expression, whereas CD36 expression was unchanged. Increase in NFκB expression was accompanied by an increase in activity. ERK1/2 inhibition only blunted SRF expression. SRF inhibition blunted NFκB expression but not that of ERK1/2. CD36 expression was unchanged in the presence of either inhibitor. PCR array analysis indicated up-regulation of nine genes (>4- to 50-fold) and down-regulation of six genes (>4- to 12-fold) involved in inflammatory signaling. Conclusions: We propose that SRF is required in acrolein activation of NFκB and is ERK1/2 dependent.

The Role of Hypoxia-Inducible Factor/Prolyl Hydroxylation Pathway in Deoxycorticosterone Acetate/Salt Hypertension in the Rat

KKidney disease could result from hypertension and ischemia/hypoxia. Key mediators of cellular adaptation to hypoxia are oxygen-sensitive hypoxia inducible factor (HIF)s which are regulated by prolyl-4-hydroxylase domain (PHD)-containing dioxygenases. However, HIF activation can be protective as in ischemic death or promote renal fibrosis in chronic conditions. This study tested the hypothesis that increased HIF-1α consequent to reduced PHD expression contributes to the attendant hypertension and target organ damage in deoxycorticosterone acetate (DOCA)/salt hypertension and that PHD inhibition ameliorates this effect. In rats made hypertensive by DOCA/salt treatment (DOCA 50 mg/kg s/c; 1% NaCl orally), PHD inhibition with dimethyl oxallyl glycine (DMOG) markedly attenuated hypertension (P<0.05), proteinuria (P<0.05) and attendant tubular interstitial changes and glomerular damage (P<0.05). Accompanying these changes, DMOG blunted the increased expression of kidney injury molecule (KIM)-1 (P<0.05), a marker of tubular injury and reversed the decreased expression of nephrin (P<0.05), a marker of glomerular injury. DMOG also decreased collagen I staining (P<0.05), increased serum nitrite (P<0.05) and decreased serum 8-isopostane (P<0.05). However, the increased HIF-1α expression (P<0.01) and decreased PHD2 expression (P<0.05) in DOCA/salt hypertensive rats was not affected by DMOG. These data suggest that reduced PHD2 expression with consequent increase in HIF-1α expression probably results from hypoxia induced by DOCA/salt treatment with the continued hypoxia and reduced PHD2 expression evoking hypertensive renal injury and collagen deposition at later stages. Moreover, a PHD inhibitor exerted a protective effect in DOCA/salt hypertension by mechanisms involving increased nitric oxide production and reduced production of reactive oxygen species.

Antioxidant U74389G Improves Glycerol-Induced Acute Renal Failure without Affecting PPARγ Gene

Oxygen metabolites play an important role in the pathogenesis of myoglobinuric acute renal failure (ARF). Previously, we have reported a down regulation of peroxisome proliferator activated receptor γ (PPARγ) in glycerol-induced ARF, and the induction of PPARγ has been shown to provide renal protection. In this study, we determined the protective influence of U74389G, a hydroxyl radical scavenger in myoglobinuric ARF, and its association with PPARγ-mediated renal protection in the rat. Vascular responses to AII were determined in renal pre-glomerular vessels following the induction of ARF with glycerol (50%, v/v, i.m.). The extent of renal damage and function were assessed with or without pre-treatment with U74389G (10 mg/kg × 21 days). In ARF, AII vasoconstriction was enhanced (97%; p < 0.05), and AII production was doubled. U74389G reduced AII vasoconstriction and production by 42% (p < 0.05) and 40% (p < 0.05), respectively. U74389G reduced proteinuria (85%; p < 0.05), which was four times higher in ARF. Similarly, U74389G enhanced Na+ excretion twofold while reducing plasma creatinine (24%; p < 0.05) and BUN (31%; p < 0.05). U74389G attenuated free radical generation in ARF while nitrite excretion was unchanged. In renal pre-glomerular vessel, PPARγ expression, activity, and mRNA were significantly lower in ARF rats; this was unchanged with U74389G treatment. On the other hand, U74389G significantly reduced NFκB protein expression, which was elevated in ARF by 25% (p < 0.05). We suggest that antioxidant U74389G blunted renal injury and improved renal function in glycerol-induced ARF through the reduction of free radical production and/or inhibition of NFκB without affecting PPARγ.

Chronic Endopeptidase Inhibition in DOCA‐Salt Hypertension: Mechanism of Cardiovascular Protection

These studies examined the interactions of neutral endopeptidase (NEP), endothelin‐1 (ET‐1), and nitric oxide (NO) in deoxycorticosterone acetate (DOCA)‐induced hypertension. Male Sprague–Dawley rats (n = 35) were uninephrectomized (UNx) or uninephrectomized and treated with DOCA (25 mg pellet implanted subcutaneously). Candoxatril (30 mg/kg day−1), a NEP inhibitor, was given orally for 3 weeks in UNx or DOCA rats. Sham nephrectomized rats (SHAM) served as controls. Except SHAM, all other groups received 1% NaCl in drinking water ad libitum. Measurements were taken of systolic blood pressure (SBP), left ventricle (LV), and aortic weight (AW), plasma ET‐1, and urinary excretion of nitrite and Na+. Whole body vascular hypertrophy and morphometric analysis of histological sections of the heart were also determined. In DOCA rats, SBP increased from 113 ± 5 to 170 ± 5 mmHg without significant changes in body weight (BW). Candoxatril reduced the increase in SBP to 135 ± 9 mmHg (P < 0.05), abolished the increased LV wall thickness (P < 0.05), and increased the reduced LV lumen diameter (P < 0.05) in DOCA‐salt rats. Candoxatril also reduced plasma ET‐1 by 88 ± 9% and 89 ± 17% (P < 0.05) in UNx and DOCA rats, respectively, and elicited increases in urinary excretion of nitrite. These effects were accompanied by a marked increase in urinary excretion of Na+ (UNaV) (P < 0.05) and a blunting of the proteinuria (32 ± 5%; P < 0.05) in DOCA rats. We conclude that endopeptidase inhibition in DOCA‐salt hypertension reduced the increase in blood pressure and the attendant tissue hypertrophy and renal injury. These effects suggest a correlation between endopeptidase‐related reduction in ET‐1 production and protection in DOCA‐salt hypertension.