Ichiro Takamatsu

A case–control study of calciphylaxis in Japanese end-stage renal disease patients

BACKGROUND Calciphylaxis, also called calcific uremic arteriolopathy, is a rare and often fatal complication of end-stage renal disease and is characterized by painful skin ulceration, necrosis, medial calcification and intimal proliferation of small arteries. Studies in western countries have reported incidences ranging from 1 to 4% in chronic hemodialysis patients. Since no systematic studies of calciphylaxis have ever been performed in Japan, we conducted a nationwide survey and a case-control study to identify the characteristics of calciphylaxis in the Japanese dialysis population. METHODS Firstly, we sent a questionnaire to 3760 hemodialysis centers in Japan, asking whether calciphylaxis cases had been encountered in the past, and detailed clinical data regarding each case were then collected from the centers. In addition, two control dialysis patients matched for age and duration of hemodialysis to each calciphylaxis case were identified at the participating centers, and their data were analyzed to identify risk factors for calciphylaxis. RESULTS Responses to the questionnaire were obtained from 1838 centers (48.3%), and 151 centers reported that a total of 249 cases had been encountered. Sixty-four centers agreed to participate in the case-control study, and detailed clinical data in regard to 67 cases were obtained. In 28 of the 67 cases, a definite diagnosis of calciphylaxis was made by our study group based on the clinical characteristics and skin biopsy findings. A univariate logistic regression model comparing them with 56-matched controls identified warfarin therapy [odds ratio (OR) 11.4, 95% confidence interval (CI)] 2.7-48.1, P=0.0009], each 1 g/dL decline in serum albumin level (OR 19.8, 95% CI 4.4-89.5, P=0.0001), each 100 mg/dL increment in plasma glucose level (OR 3.74, 95% CI 1.08-12.9, P=0.037) and each 1 mg/dL increment in adjusted serum calcium level (OR 3.2, 95% CI 1.63-6.30, P=0.0008) at the time of diagnosis as significantly associated with calciphylaxis, but no significant associations were found with female gender, vitamin D analog therapy, serum phosphate level, adjusted calcium-phosphate products or serum alkaline-phosphatase level. Warfarin therapy and lower serum albumin levels were still significant risk factors after a multivariate logistic regression model analysis. CONCLUSION The results of this study showed that warfarin therapy and lower serum albumin levels are significant and strong risk factors for the development of calciphylaxis in chronic hemodialysis patients in Japan.

Pioglitazone Lowers Systemic Asymmetric Dimethylarginine by Inducing Dimethylarginine Dimethylaminohydrolase in Rats

Peroxisome proliferator activated receptor-γ (PPARγ) ligands increase nitric oxide (NO) production and reduce systemic blood pressure. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which has two isoforms, DDAH-I and -II. In order to elucidate the mechanism whereby PPARγ ligands affect NO metabolism, their effects on the DDAH-ADMA pathway were investigated. Six-week-old male Wister-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were maintained with or without pioglitazone (PIO), a PPARγ ligand. After 4 weeks, serum ADMA levels and urinary daily NO excretion were analyzed. Tissue DDAH expression was examined by real-time polymerase chain reaction (PCR), immunoblotting, and immunohistochemistry. The results showed that PIO decreased serum ADMA and increased urinary NO excretion in both WKY and SHR. Also in both strains, the expression level of DDAH-II in the kidney was increased at transcriptional levels, although the DDAH-I level was unaffected. PIO lowered blood pressure in SHR, but not in WKY. We also demonstrated that PIO induced DDAH-II protein expression in Marbin-Dubin Canine Kidney (MDCK) cells, a renal tubular cell line. In conclusion, a PPARγ ligand was here found to increase NO production partly by upregulating tissue DDAH-II expression and decreasing systemic ADMA levels. This mechanism constitutes a direct action on renal tubular cells, but is less likely to be responsible for the blood pressure-lowering effects of PPARγ ligands. Since ADMA is one of the risk factors for cardiovascular events, this study provides compelling evidence that PPARγ ligands have the potential for reducing cardiovascular risks.

Peroxisome Proliferator-Activated Receptor γ Ligands Inhibit Rho/Rho Kinase Pathway by Inducing Protein Tyrosine Phosphatase SHP-2

Although peroxisome proliferator-activated receptor γ (PPARγ) ligands have an antihypertensive effect in vivo, the precise mechanism has not been fully elucidated. We examined their effects on Rho/Rho kinase pathway, a key regulator of vascular tone. In cultured rat aortic smooth muscle cells (RASMC), Rho kinase stimulated by angiotensin II was suppressed by the pretreatment with pioglitazone and troglitazone, and these effects were explained by the inhibition of the Rho translocation to the cell membrane. We evaluated the role of Vav, a GTP/GDP exchange factor upregulating Rho kinase activity, and Src homology region 2–containing protein tyrosine phosphatase-2 (SHP-2), a protein tyrosine phosphatase that dephosphorylated Vav and subsequently inactivated Rho kinase. Both pioglitazone and troglitazone upregulated SHP-2, particularly in the cytosolic fraction, and the SHP-2-bound Vav, and reduced the phosphorylation of Vav. Furthermore, 4-week treatment with pioglitazone lowered systolic blood pressure in spontaneously hypertensive rats (SHR) and suppressed the Rho/Rho kinase activity in aortic tissues isolated from SHR. Consistently, the expression of SHP-2 was upregulated in vascular tissues from pioglitazone-treated SHR. The phosphorylated Vav was increased in SHR, compared with that in normotensive Wistar–Kyoto rats (WKY), which was mitigated by pioglitazone. Finally, both basal and angiotensin II–stimulated levels of Rho kinase activity were greater in RASMC from SHR than those from WKY, and the enhanced Rho kinase activity was blocked by pioglitazone or troglitazone in both strains. Collectively, PPARγ ligands inhibit the Rho/Rho kinase pathway through upregulation of cytosolic SHP-2 expression and inactivation of Vav, and may contribute to the hemodynamic, in addition to metabolic, action in hypertensive metabolic syndrome. The full text of this article is available online at http://circres.ahajournals.org.

Rho-kinase as a molecular target for insulin resistance and hypertension

Rho‐kinase plays an important role in hypertension and is reported to interfere with insulin signaling through serine phosphorylation of insulin receptor substrate‐1 (IRS‐1) in cultured vascular smooth muscle cells. We therefore examined the role of Rho‐kinase in the development of insulin resistance in Zucker obese rats. In skeletal muscles and aortic tissues of Zucker obese rats, activation of RhoA/Rho‐kinase was observed. Long‐term Rho‐kinase inhibition by 4 wk treatment with fasudil (a Rho‐kinase inhibitor) not only reduced blood pressure but corrected glucose and lipid metabolism, with improvement in serine phosphorylation of IRS‐1 and insulin signaling in skeletal muscles. Direct visualization of skeletal muscle arterioles with an intravital CCD videomicroscope demonstrated that both acetylcholine‐ and sodium nitroprusside‐induced vasodilations were blunted, which were restored by the fasudil treatment. Furthermore, both fasudil and Y‐27632 prevented the serine phosphorylation of IRS‐1 induced by insulin and/or tumor necrosis factor‐α in skeletal muscle cells. Collectively, Rho‐kinase is responsible for the impairment of insulin signaling and may constitute a critical mediator linking between metabolic and hemodynamic abnormalities in insulin resistance.

Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.

Objective To assess the in-vivo action on the renal microvasculature of the calcium antagonists nifedipine (L-type blocker), efonidipine (L/T-type blocker), and mibefradil (predominant T-type blocker). Design An intravital needle-type charge-coupled device (CCD) camera videomicroscope was introduced to visualize the renal microcirculation directly in vivo. Methods In anesthetized mongrel dogs, nifedipine (0.01–1 mg/kg per min), efonidipine (0.033–0.33 mg/kg per min), or mibefradil (0.01–1 mg/kg per min) was infused intravenously after the insertion of a CCD probe into the kidney. Renal microvascular responses to calcium antagonists were directly evaluated, with concomitant observation of renal clearance. Results Each calcium antagonist caused modest vasodepressor action without affecting heart rate. Nifedipine (1 mg/kg per min, n = 9) increased renal plasma flow (RPF) (14 ± 4%, P < 0.05) and glomerular filtration rate (GFR) (19 ± 5%, P < 0.05), and tended to increase the filtration fraction (5 ± 2% increment, P = 0.07). Efonidipine (0.33 mg/kg per min, n = 9), however, had no effect on filtration fraction, with 14 ± 6% increments in RPF (P < 0.05) and 14 ± 7% increments in GFR (P = 0.08). Rather, mibefradil (1 mg/kg per min, n = 9) elicited 6 ± 2% decreases in filtration fraction (P < 0.05), with slight increments in RPF (6 ± 3%) and no changes in GFR. In direct in-vivo microvasculature observations, nifedipine caused predominant (22 ± 2%) dilatation of afferent arterioles (from 15.5 ± 0.4 to 18.9 ± 0.4μm, n = 5), compared with that of efferent arterioles (10 ± 2%; from 11.0 ± 0.4 to 12.1 ± 0.3μm). In contrast, efonidipine caused a similar magnitude of vasodilatation (16 ± 4%) compared with 18 ± 2%;n = 6), and mibefradil caused greater dilatation of efferent arterioles (20 ± 4%, n = 7) than that of afferent arterioles (13 ± 4%). Conclusions There exists marked heterogeneity in action of nifedipine, efonidipine and mibefradil on the renal microvascular in canine kidneys in vivo. Furthermore, our current observations suggest an important contribution of T-type calcium channel activity to efferent arteriolar tone in vivo.

Differential Regulation of Elevated Renal Angiotensin II in Chronic Renal Ischemia

The present study was undertaken to clarify the role of intrarenal angiotensin (Ang) II and its generating pathways in clipped and nonclipped kidneys of 4-week unilateral renal artery stenosis in anesthetized dogs. After 4 weeks, renal plasma flow (RPF) decreased in clipped and nonclipped kidneys (baseline, 59±3; clipped, 16±1; nonclipped, 44±2 mL/min;P <0.01, n=22). Renal Ang I levels increased only in clipped, whereas intrarenal Ang II contents were elevated in both clipped (from 0.7±0.1 to 2.0±0.2 pg/mg tissue) and nonclipped kidneys (from 0.6±0.1 to 2.5±0.3 pg/mg tissue). Intrarenal ACE activity was increased in nonclipped kidneys but was unaltered in clipped kidneys. An angiotensin receptor antagonist (olmesartan medoxomil) given into the renal artery markedly restored RPF, and dilated both afferent and efferent arterioles (using intravital videomicroscopy). Furthermore, in clipped kidneys, the elevated Ang II was suppressed by a chymase inhibitor, chymostatin (from 2.1±0.6 to 0.8±0.1 pg/mg tissue;P <0.05), but not by cilazaprilat. In nonclipped kidneys, in contrast, cilazaprilat, but not chymostatin, potently inhibited the intrarenal Ang II generation (from 2.4±0.3 to 1.5±0.2 pg/mg tissue;P <0.05). Finally, [Pro11-d-Ala12]Ang I (an inactive precursor that yields Ang II by chymase but not by ACE; 1 to 50 nmol/kg) markedly elevated intrarenal Ang II in clipped, but not in nonclipped, kidneys. In conclusion, renal Ang II contents were elevated in both clipped and nonclipped kidneys, which contributed to the altered renal hemodynamics and microvascular tone. Furthermore, the mechanisms for intrarenal Ang II generation differ, and chymase activity is enhanced in clipped kidneys, whereas ACE-mediated Ang II generation is possibly responsible for elevated Ang II contents in nonclipped kidneys.

Role of Rho-Kinase and p27 in Angiotensin II-Induced Vascular Injury

Angiotensin II enhances the development of atherosclerotic lesion in which cellular proliferation and/or migration are critical steps. Although cyclin-dependent kinase inhibitor, p27, and Rho/Rho-kinase pathway have recently been implicated as factors regulating these events cooperatively, their role in vivo has not been fully elucidated. We evaluated the contribution of p27 and Rho-kinase to angiotensin II-induced vascular injury using p27-deficient mice. Two-week angiotensin II (1500 ng/kg per minute SC) infusion elicited similar degrees of elevation in systolic blood pressure in wild-type mice (159±5 mm Hg) and p27-deficient mice (157±5 mm Hg; P>0.05). Angiotensin II infusion to wild-type mice resulted in increases in the medial thickness of aorta, proliferating cell number, and monocyte/macrophage infiltration within the vasculature. In p27-deficient mice, however, these changes were more prominent than those in wild-type mice. Treatment of wild-type mice with fasudil, a selective Rho-kinase inhibitor, did not alter blood pressure but significantly upregulated p27 expression, decreased medial thickness of aorta, reduced proliferating cell number, and prevented monocyte/macrophage infiltration. These protective effects of fasudil were attenuated in p27-deficient mice. In conclusion, p27 constitutes an important modulator of angiotensin II–induced monocyte/macrophage infiltration and vascular remodeling, which is mediated in part by Rho-kinase stimulation. Inhibition of Rho-kinase activity improves angiotensin II–induced vascular injury through p27-dependent and p27-independent mechanisms.

Role of Endothelium-Derived Hyperpolarizing Factor in ACE Inhibitor-Induced Renal Vasodilation in Vivo

Abstract—Although the angiotensin-converting enzyme (ACE) inhibitor-induced bradykinin enhances nitric oxide (NO) release, bradykinin may also stimulate the production of an additional vasodilator, endothelium-derived hyperpolarizing factor (EDHF). This study examined the role of EDHF in mediating the NO-independent action of ACE inhibitors in canine renal microcirculation in vivo. We used intravital CCD camera videomicroscopy that allowed direct visualization of renal microcirculation in superficial and juxtamedullary nephrons in an in vivo, in situ, and relatively intact setting. In the presence of E4177 (an angiotensin receptor blocker), cilazaprilat (30 &mgr;g/kg) had no effect on diameter of superficial afferent arterioles (Aff), but it increased renal contents of bradykinin and nitrate plus nitrite, and it elicited dilation of juxtamedullary Aff (from 24.0±0.2 to 28.2±0.8 &mgr;m), juxtamedullary efferent arterioles (Eff) (from 24.2±0.2 to 28.0±0.8 &mgr;m), and superficial Eff (from 18.2±0.2 to 19.7±0.2 &mgr;m). These changes in diameters were prevented by N&agr;-adamantaneacetyl-d-Arg-[Hyp3,Thi5,8,D-Phe7]bradykinin, a bradykinin receptor antagonist. The pretreatment with nitro-l-arginine methylester (l-NAME) plus E4177 eliminated the dilator response of juxtamedullary/superficial Eff and the increase in renal nitrate plus nitrite levels induced by cilazaprilat. In contrast, in the presence of E4177+l-NAME, cilazaprilat still caused 8%±3% dilation of juxtamedullary Aff, which was completely eliminated by proadifen, a cytochrome-P450 and KCa channel blocker. Collectively, the ACE inhibitor exerts multiple vasodilator mechanisms, including the inhibition of angiotensin II formation; blockade of angiotensin II activity appears to be a dominant mechanism in superficial Aff, whereas the bradykinin-induced NO acts on superficial Eff and juxtamedullary Aff/Eff. Furthermore, a putative EDHF is an additional mechanism for the ACE inhibitor-induced vasodilation of juxtamedullary Aff in vivo.

Free Radical Activity Depends on Underlying Vasoconstrictors in Renal Microcirculation

We examined the role of free radicals in renal microvascular tone induced by various vasoactive stimuli. Isolated perfused rat hydronephrotic kidneys were used for direct visualization of renal microcirculation. The effect of tempol on angiotensin II‐, norepinephrine‐, KCl‐, and pressure‐induced afferent arteriolar constriction was evaluated. Under angiotensin II‐induced constriction, tempol (3 mmol/L) caused 57 ± 8% dilation of afferent arterioles. In contrast, tempol elicited only 38 ± 8% and 26 ± 9% dilation of norepinephrine‐ and KCl‐induced constriction. Similarly, myogenic response induced by elevating renal arterial pressure from 80 to180 mmHg was resistant to the vasodilator action of tempol (22 ± 7% inhibition). Furthermore, tempol failed to reverse nitro‐L‐arginine methylester‐induced afferent constriction, nor had vasodilator effect on the angiotensin II‐induced constriction in the presence of nitro‐L‐arginine methylester. In contrast, nitroprusside elicited marked vasodilation of angiotensin II‐ (97 ± 5% reversal) and norepinephrine‐induced afferent constriction (89 ± 6% reversal), but had less effect on KCl‐ (46 ± 8% reversal) and pressure‐induced constriction (26 ± 9% reversal). These different actions were also observed when polyethylene‐glycolated superoxide dismutase was used as an antioxidant. In conclusion, the role of free radicals in afferent arteriolar tone varies, depending on the underlying vasoconstrictor stimuli, with greater contribution of free radicals to angiotensin II‐induced constriction. The heterogeneity in the responsiveness to free radical scavengers is attributed to both magnitude of free radicals produced and sensitivity of the underlying vasoconstrictors to nitric oxide.

Impaired nitric oxide- and endothelium-derived hyperpolarizing factor-dependent dilation of renal afferent arteriole in Dahl salt-sensitive rats.

Background and Aims:  We previously demonstrated that acetylcholine elicited nitric oxide‐dependent sustained and endothelium‐derived hyperpolarizing factor (EDHF)‐dependent transient dilation of afferent arterioles. The present study examined whether free radicals interacted with nitric oxide‐dependent and EDHF‐dependent vasodilator mechanisms in renal microvessels of salt‐sensitive hypertension, using the isolated perfused hydronephrotic kidney.