Kazuhisa Takeuchi

Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells1

Angiotensin (A) II plays a critical role in vascular remodeling, and its action is mediated by type 1 AII receptor (AT1R). Recently, 15-deoxy-Δ12,14-prostaglandin J2 and thiazolidinediones have been shown to be ligands for peroxisome proliferator-activated receptor (PPAR)-γ and activate PPAR-γ. In the present work, we have studied the effect of PPAR-γ on AT1R expression in rat vascular smooth muscle cells (VSMCs). We observed that: 1) endogenous AT1R expression was significantly decreased by PPAR-γ ligands both at messenger RNA and protein levels, whereas AT1R messenger RNA stability was not affected; 2) AII-induced increase of 3H-thymidine incorporation into VSMCs was inhibited by PPAR-γ ligands; 3) rat AT1R gene promoter activity was significantly suppressed by PPAR-γ ligands, and PPAR-γ overexpression further suppressed the promoter activity; 4) transcriptional analyses using AT1R gene promoter mutants revealed that a GC-box-related sequence within the −58/−34 region of the AT1R gene promoter was respo...

Possible Role of P-450 Metabolite of Arachidonic Acid in Vasodilator Mechanism of Angiotensin II Type 2 Receptor in the Isolated Microperfused Rabbit Afferent Arteriole

Although angiotensin II type 2 (AT2) receptor has recently been cloned, its functional role is not well understood. We tested the hypothesis that selective activation of AT2 receptor causes vasodilation in the preglomerular afferent arteriole (Af-Art), a vascular segment that accounts for most of the preglomerular resistance. We microperfused rabbit Af-Arts at 60 mmHg in vitro, and examined the effect of angiotensin II (Ang II; 10(-11)-10(-8) M) on the luminal diameter in the presence or absence of the Ang II type 1 receptor antagonist CV11974 (CV; 10(-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+/-7% over the preconstricted level at 10(-8) M; P < 0.01, n = 7). In contrast, in the presence of CV, Ang II caused dose-dependent dilation; Ang II at 10(-8) M increased the diameter by 29+/-2% (n = 7, P < 0.01). This dilation was completely abolished by pretreatment with an AT2 receptor antagonist PD123319 (10(-7) M, n = 6), suggesting that activation of AT2 receptor causes vasodilation in Af-Arts. The dilation was unaffected by inhibiting either nitric oxide synthase (n = 7) or cyclooxygenase (n = 7), however, it was abolished by either disrupting the endothelium (n = 10) or inhibiting the cytochrome P-450 pathway, particularly the synthesis of epoxyeicosatrienoic acids (EETs, n = 7). These results suggest that in the Af-Art activation of the AT2 receptor may cause endothelium-dependent vasodilation via a cytochrome P-450 pathway, possibly by EETs.

Nongenomic Vascular Action of Aldosterone in the Glomerular Microcirculation

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.

Angiotensin II Type 1 Receptor Blockers Reduce Urinary Oxidative Stress Markers in Hypertensive Diabetic Nephropathy

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; n=33) or trichlormethiazide (n=33) group. The majority of patients had been treated with angiotensin-converting enzyme inhibitors or calcium channel blockers for ≥1 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, n=11 or valsartan 80 mg/day, n=22) for 8 weeks reduced the levels of plasma monocyte chemoattractant protein 1, interleukin 6, urinary 8-epi-prostaglandin F2&agr;, 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&agr; 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.

Rat kidney thromboxane receptor: molecular cloning, signal transduction, and intrarenal expression localization.

Thromboxane (TX) plays important roles in control of renal hemodynamics and water and electrolyte metabolism, and is involved in the pathophysiology of many renal diseases. The aim of the present study is to isolate a rat kidney cDNA encoding functional TX receptor, and to reveal its intrarenal expression localization. A clone (rTXR2) was isolated from a rat kidney cDNA library by a homology screening approach. rTXR2 was shown to encode the amino acid sequence containing seven transmembrane spanning domains representing rat (r) TX receptor. The membrane from COS-7 cells transiently transfected with rTXR2 cDNA was shown to be specifically bound by a thromboxane receptor antagonist, SQ29548. Either in Xenopus oocyte expression or in transfected COS-7 cells, rTX receptor was shown to be linked with Ca2+ messenger system. TX receptor-mediated increase in cytosolic Ca2+ was also observed in cultured glomerular mesangial cells. In situ hybridization showed that rTX receptor mRNA was detected in renal glomeruli, smooth muscle cells in renal arterioles, and transitional cell epithelium of renal pelvis. Reverse transcription linked to PCR applied to microdissected nephron segments indicated the presence of rTX receptor mRNA exclusively in the glomerulus. In conclusion, we have cloned a functional rat kidney TX receptor, which is expressed specifically in renal glomerulus, arterial smooth muscle cells, and transitional cell epithelium of renal pelvis. The present study will provide important insights into the etiology and pathophysiology of renal diseases with relation to TX metabolism.

Endothelium-Derived Nitric Oxide Modulates Vascular Action of Aldosterone in Renal Arteriole

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 (10−10 to 10−8 mol/L) were added to the bath and lumen. Aldosterone caused dose-dependent vasoconstriction (within 10 minutes); significant (P <0.01) constriction was observed from 5×10−9 mol/L, and at 10−8 mol/L, intraluminal diameter decreased by 29%±3% (n=9). Disrupting the endothelium augmented vasoconstriction; significant constriction was observed from 10−10 mol/L, and at 10−8 mol/L, the diameter decreased by 38%±2% (n=6). NO synthesis inhibition reproduced this augmentation (n=7). Pretreatment with chelerythrine (10−6 mol/L), a protein kinase C (PKC) inhibitor, slightly attenuated the constriction; aldosterone at 10−8 mol/L now decreased the diameter by 18%±3% (n=7). However, in Af-Arts treated with thapsigargin (10−6 mol/L) or dantrolene (3×10−5 mol/L), which blocks inositol 1,4,5-triphosphate (IP3)-induced intracellular calcium release, aldosterone at 10−8 mol/L decreased the diameter by only 9%±1% (n=6) or 9%±2% (n=5), 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 IP3 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.

Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-gamma via an interaction with Sp1 in vascular smooth muscle cells

Thromboxane (TX) A2exerts contraction and proliferation of vascular smooth muscle cells (VSMCs) via its specific membrane TX receptor (TXR), possibly leading to the progression of atherosclerosis. A nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)-γ, has recently been reported to be expressed in VSMCs. Here we examined a role of PPAR-γ in TXR gene expression in VSMCs. PPAR-γ ligands 15-deoxy-Δ12,14-prostaglandin J2 and troglitazone reduced TXR mRNA expression levels as well as cell growth as assessed by [3H]thymidine incorporation. Transcriptional activity of the TXR gene promoter was suppressed with PPAR-γ ligands, and the suppression was augmented further by PPAR-γ overexpression. By deletion and mutation analyses, the transcription suppression was shown to be the result of a −22/−7 GC box-related sequence (upstream of transcription start site). Electrophoretic mobility shift assays also showed that the sequence was bound by Sp1 but not by PPAR-γ, and the formation of a Sp1·DNA complex was inhibited either by coincubation with PPAR-γ or PPAR-γ ligand treatment of VSMCs. Moreover, glutathione S-transferase pull-down assays demonstrated a direct interaction between PPAR-γ and Sp1. In conclusion, PPAR-γ suppresses TXR gene transcription via an interaction with Sp1. PPAR-γ may possibly have an antiatherosclerotic action by inhibiting TXR gene expression in VSMCs.

Bezafibrate reduces blood glucose in type 2 diabetes mellitus

The clinical efficacy of bezafibrate was examined with special reference to glucose metabolism in patients with type 2 diabetes mellitus (DM2). In protocol 1, 342 patients with DM2 and hyperlipidemias were randomly divided into 2 groups, 16-week bezafibrate treatment (n = 174) and no bezafibrate treatment (n = 168). In protocol 2, 20 DM2 patients were randomly divided into 2 groups, 8-week bezafibrate treatment (n = 10) and no bezafibrate treatment (n = 10), and a meal tolerance test (MTT) was performed. In protocol 1, bezafibrate treatment significantly reduced the fasting levels of triglyceride (TG) by 50% +/- 1.6%, total cholesterol (TC) by 12% +/- 1.1%, plasma glucose (PG) from 151.3 +/- 3.5 to 128.6 +/- 3.4 mg/dL, and hemoglobin A1c (HbA1c) from 7.2% +/- 0.1% to 6.9% +/- 0.1%, and significantly increased high-density lipoprotein cholesterol (HDL-C) by 20% +/- 0.8%. In protocol 2, fasting TG, PG, and insulin levels were significantly reduced by bezafibrate treatment. Moreover, in the MTT, postprandial increments of TG were significantly blunted after bezafibrate treatment, whereas postprandial PG and insulin levels were not significantly changed. Leptin levels were significantly decreased, while tumor necrosis factor alpha (TNF-alpha) levels were not changed. In conclusion, both hyperglycemia and hyperlipidemia can be improved by bezafibrate treatment in DM2.

Effects of Monotherapy of Temocapril or Candesartan with Dose Increments or Combination Therapy with Both Drugs on the Suppression of Diabetic Nephropathy

We examined the effects of increasing the recommended initial doses of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs), or of switching to combination therapy with both drugs, on diabetic nephropathy. Hypertensive type 2 diabetic patients with urinary albumin excretion (ACR) between 100 and 300 mg/g creatinine (Cre) were assigned to the following five groups in which an antihypertensive drug was administered at a recommended initial dose for 48 weeks, and then either the dose was doubled or an additional drugs was added to regimen for the following 48 weeks: N, nifedipine-CR (N) 20 mg/day (initial dose); T, ACEI temocapril (T) 2 mg/day; C, ARB candesartan (C) 4 mg/day; T+C, T first and then addition of C; C+T, C first and then addition of C. ACR decreased in the T (n=34), C (n=40), T+C (n=37) and C+T (n=35) groups, but not in the N group (n=18). However, the anti-proteinuric effect was less in the T than in the C, T+C or C+T groups, while no differences existed among the latter three. In each group, there were significant linear relationships between attained BP and ACR; however, the regression lines were shifted toward lower ACR level in the renin-angiotensin system–inhibition groups compared with the N group. These results indicate that an ACEI and/or ARB is superior to a CCB in retarding diabetic nephropathy, while the combination of low doses of ACEI and ARB has effects similar to those of high-dose ARB. Even among patients treated with an ACEI and/or ARB, lowering BP is important.

Acarbose lowers serum triglyceride and postprandial chylomicron levels in type 2 diabetes

Aim:  This study was designed to examine the therapeutic effect of acarbose on serum triglyceride (TG), free fatty acid (FFA), very low‐density lipoprotein (VLDL) and chylomicron (CM) in the meal tolerance test (MTT) before and after acarbose treatment in type 2 diabetes mellitus (DM2).