Abstract. Aldosterone (Aldo) accelerates hypertension, proteinuria, and glomerulosclerosis in animal models of malignant hypertension or chronic renal failure. Aldo may exert these deleterious renal effects by elevating renal vascular resistance and glomerular capillary pressure. To test this possibility, directly examined were the action of Aldo on the afferent (Af) and efferent (Ef) arterioles (Arts). Examined were the effect of Aldo added to both the bath and lumen on the intraluminal diameter (measured at the most responsive point) of rabbits. Aldo caused dose-dependent constriction in both arterioles with a higher sensitivity in Ef-Arts. Vasoconstrictor action of Aldo was not affected by a mineralocorticoid receptor antagonist spironolactone and was reproduced by membrane-impermeable albumin-conjugated Aldo, suggesting that the vasoconstrictor actions are nongenomic. Pretreatment with neomycin (a specific inhibitor of phospholipase C) abolished the vasoconstrictor action of Aldo in both arterioles. In addition, the vasoconstrictor action of Aldo on Af-Arts was inhibited by both nifedipine and efonidipine, whereas that on Ef-Arts was inhibited by efonidipine but not nifedipine. The results demonstrate that Aldo causes nongenomic vasoconstriction by activating phospholipase C with a subsequent calcium mobilization thorough L-or T-type voltage-dependent calcium channels in Af-or Ef-Arts, respectively. These vasoconstrictor actions on the glomerular microcirculation may play an important role in the pathophysiology and progression of renal diseases by elevating renal vascular resistance and glomerular capillary pressure.There is increasing evidence that renin-angiotensin-aldosterone system (RAAS) plays an important role in the pathogenesis and progression of renal diseases (1-5). Although angiotensin II (AngII) has been identified as the primary mediator of the system, recent studies have raised the possibility that aldosterone (Aldo), independent of renin-angiotensin, also participates in mediating renal injury (5-10). Greene et al. (7) have evaluated the ability of Aldo to reverse the renal protective effects of RAAS blockade in the 5/6 nephrectomy model of hypertension and glomerulosclerosis. They found that pharmacologic blockade of RAAS with angiotensin-converting enzyme inhibitor and AngII receptor blocker reduces plasma Aldo levels, systolic BP, proteinuria and renal lesions, all of which were reversed when Aldo was infused concurrently. Rocha et al. (8) also demonstrated that renal-protective effects of captopril, an angiotensin-converting enzyme inhibitor, were reversed by an Aldo infusion in stroke-prone spontaneously hypertensive rat (SHRSP), an animal model of malignant hypertension. They found that captopril treatment prevented the development of proteinuria and glomerular-renal vascular lesions with reducing endogenous Aldo levels, whereas subsequent Aldo infusion reversed these protective effects of captopril. Although the underlying mechanisms for these actions are not well defined, Aldo may e...

Although angiotensin II type 2 (AT 2 ) receptor has recently been cloned, its functional role is not well understood. We tested the hypothesis that selective activation of AT 2 receptor causes vasodilation in the preglomerular afferent arteriole (Af-Art), a vascular segment that accounts for most of the preglomerular resistance. We microperfused rabbit AfArts at 60 mmHg in vitro, and examined the effect of angiotensin II (Ang II; 10 Ϫ 11 -10 Ϫ 8 M) on the luminal diameter in the presence or absence of the Ang II type 1 receptor antagonist CV11974 (CV; 10 Ϫ 8 M). Ang II was added to both the bath and lumen of preconstricted Af-Arts. Ang II further constricted Af-Arts without CV (by 74 Ϯ 7% over the preconstricted level at 10 Ϫ 8 M; P Ͻ 0.01, n ϭ 7). In contrast, in the presence of CV, Ang II caused dose-dependent dilation; Ang II at 10 Ϫ 8 M increased the diameter by 29 Ϯ 2% ( n ϭ 7, P Ͻ 0.01). This dilation was completely abolished by pretreatment with an AT 2 receptor antagonist PD123319 (10 Ϫ 7 M, n ϭ 6), suggesting that activation of AT 2 receptor causes vasodilation in Af-Arts. The dilation was unaffected by inhibiting either nitric oxide synthase ( n ϭ 7) or cyclooxygenase ( n ϭ 7), however, it was abolished by either disrupting the endothelium ( n ϭ 10) or inhibiting the cytochrome P-450 pathway, particularly the synthesis of epoxyeicosatrienoic acids (EETs, n ϭ 7). These results suggest that in the Af-Art activation of the AT 2 receptor may cause endothelium-dependent vasodilation via a cytochrome P-450 pathway, possibly by EETs. ( J. Clin. Invest. 1997. 100:2816-2823.)

Abstract-We tested the hypothesis that blockade of angiotensin II type 1 receptors reduces oxidative stress markers in parallel with urinary albumin and type IV collagen excretions. Sixty-six diabetic patients with nephropathy were randomly assigned to either the angiotensin II receptor blocker (ARB; nϭ33) or trichlormethiazide (nϭ33) group. The majority of patients had been treated with angiotensin-converting enzyme inhibitors or calcium channel blockers for Ն1 year before the present study. Reduction of blood pressure was not different between the 2 groups, and HbA1c levels did not change over the study period (8 weeks). Treatment with ARB (candesartan 8 mg/day, nϭ11 or valsartan 80 mg/day, nϭ22) for 8 weeks reduced the levels of plasma monocyte chemoattractant protein 1, interleukin 6, urinary 8-epi-prostaglandin F2␣, 8-hydroxydeoxyguanosine, albumin, and type IV collagen, whereas the levels of these markers were not altered with trichlormethiazide (2 mg/day). Significant correlation was observed between the reduction of the urinary 8-epi-prostaglandin F2␣ and 8-hydroxydeoxyguanosine and those of the urinary albumin and type IV collagen. Subjects with large oxidative stress had large reduction rates because of ARB administration and showed large urinary albumin suppression. These results suggest that ARBs reduce oxidative stress and inflammation in diabetic patients independent of their effects on blood pressure. In addition, increases in oxidative stress caused by angiotensin II may play an important role in the progression of diabetic nephropathy. Our results may help to explain the clinical observation that ARB reduces urinary albumin excretion very efficiently in some patients but not in others.

Thromboxane (TX)A Thromboxane (TX)1 A 2 is a labile metabolite of arachidonic acid synthesized by TX synthase (1). TX is known to exert many biological effects such as platelet aggregation, contraction and growth of vascular smooth muscle cells (VSMCs) (2, 3), and renal electrolyte metabolism (4). TX is also known to play a pathophysiological role in the inflammatory diseases such as atherosclerosis (5) and glomerulonephritis (6). The biological action of TX is mediated via its specific membrane TX receptor (TXR). We previously isolated a cDNA for rat TXR (7), localized it in either the kidney (8) or testis (9), and identified its chromosomal localization (10). Moreover, we have isolated 5Ј-flanking region (FL) of the rat TXR gene and studied its transcription regulation in VSMCs (11).Peroxisome proliferator-activated receptor (PPAR)-␥ is a nuclear hormone receptor that was shown to transactivate adipocyte-specific genes and induce adipocyte differentiation (12). Either insulin-sensitizing thiazolidinediones including troglitazone (TRO) or 15-deoxy-⌬ 12,14 -prostaglandin J 2 (PGJ 2 ) (13, 14) has been identified as a ligand of PPAR-␥. Recently, PPAR-␥ has been shown to be present not only in adipocytes but also in vascular tissues including VSMCs (15), and an inhibitory effect of PPAR-␥ on gene expression in atherosclerosis has been studied. Activation of PPAR-␥ with its ligands suppressed expression of plasminogen activator inhibitor type 1 (16) in vascular endothelial cells and that of matrix metalloproteinase-9 in VSMCs (15). Moreover, we have observed that PPAR-␥ can suppress TX synthase gene transcription in macrophages (17).In the present study, we examined the role of PPAR-␥ in TXR gene expression in VSMCs. We observed that PPAR-␥ inhibited the TX-mediated cell growth of VSMCs and TXR mRNA expression. Suppression of TXR gene transcription was confirmed, and the suppression was shown to be dependent on a GC box-related sequence present at the Ϫ22/Ϫ7 region of TXR gene promoter (upstream of transcription start site), which was bound by Sp1 but not by PPAR-␥. PPAR-␥ was shown to interact physically with Sp1 by glutathione S-transferase (GST) pull-down assays. Taken together, PPAR-␥ was suggested to suppress TXR gene transcription via a protein-protein interaction with Sp1. An antiatherosclerotic action of PPAR-␥ by inhibiting TXR gene expression in VSMCs may be suggested. EXPERIMENTAL PROCEDURESPlasmids-Previously reported (11) and newly subcloned chimeric constructs containing rat TXR gene promoter and luciferase cDNA were used for transient transfection studies: Ϫ989/ϩ184-luc (989-bp 5Ј-flanking region (FL) and 184-bp 5Ј-untranslated region (UTR) of rat TXR gene); Ϫ809/ϩ184-luc (809-bp 5Ј-FL and 184-bp 5Ј-UTR); Ϫ489/ϩ184-luc (489-bp 5Ј-FL and 184-bp 5Ј-UTR); Ϫ213/ϩ184-luc (213-bp 5Ј-FL and 184-bp 5Ј-UTR); Ϫ78/ϩ184-luc (78-bp 5Ј-FL and 184-bp 5Ј-UTR); Ϫ78/ϩ120-luc (78-bp 5Ј-FL and 120-bp 5Ј-UTR); Ϫ47/ϩ120-luc (47-bp 5Ј-FL and 120-bp 5Ј-UTR); Ϫ39/ϩ120-luc (39-bp 5Ј-FL and 120-bp 5Ј-UTR); Ϫ22/ϩ120-luc (22-bp ...

Abstract-We have recently demonstrated that aldosterone causes nongenomic vasoconstriction by activating phospholipase C (PLC) in the preglomerular afferent arteriole (Af-Art). In the present study, we tested the hypothesis that endothelium modulates this vasoconstrictor action by releasing nitric oxide (NO). In addition, to study the post-PLC mechanism, we examined possible contributions of phosphoinositol hydrolysis products. Rabbit Af-Arts were microperfused at 60 mm Hg in vitro, and increasing doses of aldosterone (10 Ϫ10 to 10 Ϫ8 mol/L) were added to the bath and lumen. Aldosterone caused dose-dependent vasoconstriction (within 10 minutes); significant (PϽ0.01) constriction was observed from 5ϫ10 Ϫ9 mol/L, and at 10 Ϫ8 mol/L, intraluminal diameter decreased by 29%Ϯ3% (nϭ9). Disrupting the endothelium augmented vasoconstriction; significant constriction was observed from 10 Ϫ10 mol/L, and at 10 Ϫ8 mol/L, the diameter decreased by 38%Ϯ2% (nϭ6). NO synthesis inhibition reproduced this augmentation (nϭ7). Pretreatment with chelerythrine (10 Ϫ6 mol/L), a protein kinase C (PKC) inhibitor, slightly attenuated the constriction; aldosterone at 10 Ϫ8 mol/L now decreased the diameter by 18%Ϯ3% (nϭ7). However, in Af-Arts treated with thapsigargin (10 Ϫ6 mol/L) or dantrolene (3ϫ10 Ϫ5 mol/L), which blocks inositol 1,4,5-triphosphate (IP 3 )-induced intracellular calcium release, aldosterone at 10 Ϫ8 mol/L decreased the diameter by only 9%Ϯ1% (nϭ6) or 9%Ϯ2% (nϭ5), respectively. These results demonstrate that in the Af-Art endothelium-derived NO modulates vasoconstrictor actions of aldosterone that are mediated by the activation of both IP 3 and PKC pathways. Such vasoconstrictor actions of aldosterone may contribute to the development or aggravation of hypertension by elevating renal vascular resistance in cardiovascular diseases associated with endothelium dysfunction.

We have studied the transcription regulation of the rat thromboxane synthase (TXS) gene by peroxisome proliferator-activated receptor ␥ (PPAR␥) in macrophages. The transcription activity of a cloned 5-flanking region (1.6 kilobases) of the rat TXS gene (5FL-TXS) was examined by luciferase reporter gene assay. TXS mRNA expression and the transcription activity of 5FL-TXS were inhibited by PPAR␥ ligands, 15-deoxy-⌬ 12,14 -prostaglandin J 2 (PGJ 2 ), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPAR␥ also significantly suppressed transcription, and further addition of PGJ 2 or TRO augmented the suppression. Deletion analysis showed that the element responsible for the PPAR␥ effect is located in a region containing the nuclear factor E2 (NF-E2)/ AP-1 site (؊98/؊88), which was indicated to be the major promoter of the TXS gene. By electrophoretic mobility shift assay using the NF-E2/AP-1 site and nuclear extracts from macrophages, we observed a specific protein-DNA complex formation, which was inhibited by a specific antibody against the transcription factor NRF2 (NF-E2-related factor 2). Moreover, the complex was decreased with PGJ 2 , TRO, or in vitro translated PPAR␥. The transcription suppression by PPAR␥ was confirmed using this truncated NRF2-binding element (؊98/؊88) by the reporter gene assay. Finally, a direct interaction between PPAR␥ and NRF2 was confirmed by glutathione S-transferase pull-down assay. In conclusion, the NRF2-binding site (؊98/؊88) is the major promoter of 5FL-TXS which can be suppressed by activated PPAR␥ via a protein-protein interaction with NRF2 in macrophages.

Background-A natural retinoid all-trans retinoic acid (ATRA) contains various beneficial effects on vasculature, including suppression of neointima formation after balloon injury. However, little is known about the effects of ATRA on vascular endothelial function. We therefore studied its role in nitric oxide (NO) production of vascular endothelial cells (ECs). Methods and Results-Human dermal microvascular ECs, human umbilical vein ECs, and SV40-transformed rat lung vascular ECs were incubated with or without ATRA (1 mol/L) for 48 hours. Their NO production was determined with the use of a fluorescent NO indicator, diaminofluorescein-2 diacetate. ATRA significantly increased their basal as well as acetylcholine-induced NO production. Treatment with N -nitro-L-arginine methyl ester or carboxy-PTIO suppressed their fluorescence. Increase of NO production was also observed by incubation with retinoic acid receptor (RAR) agonist Am580. ATRA-induced NO increase was abolished by coincubation with RAR antagonist LE540. Moreover, the NO increase was completely inhibited by the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and LY294002. ATRA as well as Am580 enhanced endothelial NO synthase (eNOS) phosphorylation at Ser-1177 as well as Akt phosphorylation at Ser-473 without changing their protein expression. Overexpression of dominant-negative Akt inhibited the eNOS phosphorylation. Moreover, ATRA increased PI3K activity as well as PI3K catalytic subunit p110␤ protein expression, which was completely inhibited by LE540 treatment. Real-time polymerase chain reaction analyses demonstrated that ATRA increased PI3K catalytic subunit p110␤ mRNA expression without affecting its stability. Finally, ATRA-induced NO increase was observed in COS-1 cells transfected with wild-type eNOS and RAR␣, but not with mutated eNOS whose Ser-1177 was substituted. Conclusions

Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (ϳ2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility "supershift" analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.