Evocalcet is an allosteric modulator of the calcium-sensing receptor (CaSR) that effectively suppresses parathyroid hormone (PTH) secretion in both primary and secondary hyperparathyroidism. However, its effects on the PTH-calcium setpoint and parathyroid cell proliferation remain unclear. We investigated these effects using the PC mouse model of primary hyperparathyroidism, which is characterized by parathyroid-targeted cyclin D1 overexpression . Evocalcet was administered orally at a dose of 0.025 mg/g diet. The PTH-calcium setpoint was evaluated, and the antiproliferative effect of evocalcet on parathyroid cells was assessed using 5-bromo-2′-deoxyuridine (BrdU) incorporation assays. The effects of evocalcet were compared with those of cinacalcet. Expression levels of the vitamin D receptor (VDR) and CaSR in parathyroid glands were also examined. Evocalcet significantly reduced the PTH-calcium setpoint in PC mice, restoring it to levels comparable to those observed in wild-type controls. Evocalcet treatment markedly decreased the proportion of BrdU-positive parathyroid cells, indicating suppression of parathyroid cell proliferation. This antiproliferative effect was comparable to that observed with cinacalcet. Neither evocalcet nor cinacalcet altered VDR or CaSR expression in the parathyroid glands. Evocalcet, similar to cinacalcet, lowers the PTH-calcium setpoint and inhibits parathyroid cell proliferation in a mouse model of primary hyperparathyroidism. These findings suggest that evocalcet may not only reduce PTH secretion but also attenuate disease progression in hyperparathyroidism.

The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system plays an important role in defense against oxidative stress, and its involvement has been implicated in body weight reduction under obese conditions. While the effect on white adipose tissue (WAT) has been intensely studied, focusing on the anti-inflammatory and anti-oxidative stress effects exerted by Nrf2 activation, the involvement of skeletal muscle has not been investigated. We assessed the body weight changes induced by Nrf2 activation by comparing its effect on WAT with those on skeletal muscle. We evaluated TBE-31, a potent Nrf2 activator, in a high-fat diet (HFD)-induced obesity model. Notably, TBE-31 significantly suppressed HFD-induced body weight gain compared with vehicle treatment. While treatment with TBE-31 induced a significant WAT weight decrease compared with vehicle, skeletal muscle weight was not affected. In addition, body weight changes were significantly correlated with WAT but not with skeletal muscle. Lipid deposition was remarkably improved in the adipose tissue, but muscle histology was not affected. A gene expression analysis revealed that Ucp-1 was upregulated and Il-6 downregulated by TBE-31 treatment in an Nrf2-dependent manner. Taken together, these findings suggest that pharmacological activation of Nrf2 suppressed HFD-induced body weight gain by affecting WAT.

BACKGROUND:Nuclear factor erythroid 2-related factor 2 (Nrf2) is crucial for defense against oxidative stress. In the Phase 2 TSUBAKI study, bardoxolone methyl, an Nrf2 activator, was shown to increase the glomerular filtration rate (GFR) in patients with diabetic kidney disease (DKD). Though non-clinical reports suggest that bardoxolone methyl acts mainly through Nrf2-mediated antioxidant response activation, this has not been proven in clinical settings. The present study assessed its effects using plasma and urine from the TSUBAKI study. METHODS:Patients received either bardoxolone methyl or placebo daily for 16 weeks. Urine and plasma samples were collected at baseline, after 16 weeks of dosing, and 4 weeks post-dosing and were cryopreserved for an analysis. A total of 45 patients in the bardoxolone methyl group and 52 in the placebo group were subjected to proteomic and metabolic analyses. Proteomic profiling was conducted using the SOMAscan assay, while metabolomics analyses were performed by Human Metabolome Technologies, Inc. RESULTS:Plasma proteomics revealed that two oxidative stress related pathways have been significantly changed by bardoxolone methyl treatment. Among these two pathways, antioxidative proteins and proteins that enhance the antioxidative process were significantly increased. Some of these increased proteins were known to be Nrf2 target proteins. Similarly, urine proteomics revealed that four pathways were changed, and antioxidative proteins that are the target proteins of Nrf2 were increased. Furthermore, seven metabolites that potentiate the antioxidative effect were significantly increased in urine. CONCLUSIONS:This study demonstrated the first evidence in humans that bardoxolone methyl activates an antioxidant response.

Little is known about the effect of the recently developed calcimimetic evocalcet (Evo) on parathyroid calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) expression. We examined the effects of Evo and cinacalcet (Cina) on CaSR and VDR expression in 5/6 nephrectomized Sprague-Dawley rats fed a high-phosphorus diet for 4 weeks to develop secondary hyperparathyroidism (SHPT). These uremic rats were divided into 4 groups-baseline control (Nx4W) and groups with additional treatment with either the Vehicle, Evo, or Cina for 2 weeks; normal rats were used as normal controls (NC). Blood parameters and parathyroid tissue were analyzed. CaSR and VDR expression levels were determined using immunohistochemistry. The degree of kidney injury and hyperphosphatemia was similar in the uremic groups (Nx4W, Vehicle, Cina, and Evo). Serum parathyroid hormone levels were significantly higher in the Nx4W and Vehicle groups than in the NC group. This increase was significantly suppressed in the Cina and Evo groups compared with that in the Vehicle group. Serum calcium levels were significantly and equally lower in the Cina and Evo groups relative to those in the Vehicle group. CaSR expression was significantly lower in the Nx4W and Vehicle groups than in the NC group. This downregulation was of an equally lesser magnitude in the Cina and Evo groups. A similar trend was observed for VDR expression. These results indicate that Evo and Cina treatment can increase parathyroid CaSR and VDR expression in uremic rats with SHPT, which could provide better control of mineral and bone disorder markers.

Mitochondrial dysfunction plays a critical role in the pathogenesis of kidney diseases via ATP depletion and reactive oxygen species overproduction. Nonetheless, few studies have reported the renal mitochondrial status clinical settings, partly due to a paucity of methodologies. Recently, a positron emission tomography probe, 18F-BCPP-BF, was developed to non-invasively visualize and quantitate the renal mitochondrial status in vivo. Here, 18F-BCPP-BF positron emission tomography was applied to three mechanistic kidney disease models in rats: kidney ischemia-reperfusion, 5/6 nephrectomy and anti-glomerular basement membrane glomerulonephritis. In rats with ischemia-reperfusion, a slight decrease in the kidney uptake of 18F-BCPP-BF was accompanied by morphological abnormality of the mitochondria in the proximal tubular cells after three hours of reperfusion, when the kidney function was slightly declined. In 5/6 nephrectomy and rats with anti-glomerular basement membrane glomerulonephritis, the kidney uptake of 18F-BCPP-BF cumulatively decreased with impairment of the kidney function, which was accompanied by a reduction of mitochondrial protein and a pathological tubulointerstitial exacerbation rather than glomerular injury. The 18F-BCPP-BF uptake in the injured kidney was suggested to represent the volume of healthy tubular epithelial cells with normally functioning mitochondria. Thus, this positron emission tomography probe can be a powerful tool for studying the pathophysiological meanings of the mitochondrial status in kidney disease.

BACKGROUND:Elevated parathyroid hormone (PTH) levels in secondary hyperparathyroidism (SHPT) lead to vascular calcification, which is associated with cardiovascular events and mortality. Increased PTH production is caused by the excessive proliferation of parathyroid gland cells, which is accelerated by abnormal mineral homeostasis. Evocalcet, an oral calcimimetic agent, inhibits the secretion of PTH from parathyroid gland cells and has been used for the management of SHPT in dialysis patients. We observed the effects of evocalcet on ectopic calcification and parathyroid hyperplasia using chronic kidney disease (CKD) rats with SHPT.METHODS:CKD rats with SHPT induced by adenine received evocalcet orally for 5 weeks. The calcium and inorganic phosphorus content in the aorta, heart and kidney was measured. Ectopic calcified tissues were also assessed histologically. To observe the effects on the proliferation of parathyroid gland cells, parathyroid glands were histologically assessed in CKD rats with SHPT induced by 5/6 nephrectomy (Nx) after receiving evocalcet orally for 4 weeks.RESULTS:Evocalcet prevented the increase in calcium and inorganic phosphorus content in the ectopic tissues and suppressed calcification of the aorta, heart and kidney in CKD rats with SHPT by reducing the serum PTH and calcium levels. Evocalcet suppressed the parathyroid gland cell proliferation and reduced the sizes of parathyroid cells in CKD rats with SHPT.CONCLUSIONS:These findings suggest that evocalcet would prevent ectopic calcification and suppress parathyroid hyperplasia in patients with SHPT.

Saxagliptin, a potent and selective DPP-4 inhibitor, exhibits a slow dissociation from DPP-4. We investigated the sustained effects of saxagliptin on renal DPP-4 activity in a washout study using renal tubular (HK-2) cells, and in a pharmacodynamic study using normal rats. In HK-2 cells, the inhibitory potency of saxagliptin on DPP-4 activity persisted after washout, while that of sitagliptin was clearly reduced. In normal rats, a single treatment of saxagliptin or sitagliptin inhibited the plasma DPP-4 activity to similar levels. The inhibitory action of saxagliptin on the renal DPP-4 activity was retained, even when its inhibitory effect on the plasma DPP-4 activity disappeared. However, the inhibitory action of sitagliptin on the renal DPP-4 activity was abolished in correlation with the inhibition of the plasma DPP-4 activity. In situ staining showed that saxagliptin suppressed the DPP-4 activity in both glomerular and tubular cells and its inhibitory effects were significantly higher than those of sitagliptin. Saxagliptin exerted a sustained inhibitory effect on the renal DPP-4 activity in vitro and in vivo. The long binding action of saxagliptin in renal tubular cells might involve the sustained inhibition of renal DPP-4.

Saxagliptin, a potent and selective DPP-4 inhibitor, is characterized by its slow dissociation from DPP-4 and its long half-life and is expected to have a potent tissue membrane-bound DPP-4-inhibitory effect in various tissues. In the present study, we examined the effects of saxagliptin on in situ cardiac DPP-4 activity. We also examined the effects of saxagliptin on isoproterenol-induced the changes in the early stage such as, myocardial remodeling and cardiac diastolic dysfunction. Male SD rats treated with isoproterenol (1 mg/kg/day via osmotic pump) received vehicle or saxagliptin (17.5 mg/kg via drinking water) for 2 weeks. In situ cardiac DPP-4 activity was measured by a colorimetric assay. Cardiac gene expressions were examined and an echocardiographic analysis was performed. Saxagliptin treatment significantly inhibited in situ cardiac DPP-4 activity and suppressed isoproterenol-induced myocardial remodeling and the expression of related genes without altering the blood glucose levels. Saxagliptin also significantly ameliorated cardiac diastolic dysfunction in isoproterenol-treated rats. In conclusion, the inhibition of DPP-4 activity in cardiac tissue by saxagliptin was associated with suppression of myocardial remodeling and cardiac diastolic dysfunction independently of its glucose-lowering action in isoproterenol-treated rats. Cardiac DPP-4 activity may contribute to myocardial remodeling in the development of heart failure.

Although previous studies have shown an important role of renal dipeptidyl peptidase-4 (DPP-4) inhibition in ameliorating kidney injury in hypertensive rats, the renal distribution of DPP-4 and mechanisms of renoprotective action of DPP-4 inhibition remain unclear. In this study, we examined the effects of the DPP-4 inhibitor saxagliptin on DPP-4 activity in renal cells (using in situ DPP-4 staining) and on renal gene expression related to inflammation and fibrosis in the renal injury in hypertensive Dahl salt-sensitive (Dahl-S) rats. Male rats fed a high-salt (8% NaCl) diet received vehicle (water) or saxagliptin (12.7mg/kg/day) for 4 weeks. Blood pressure (BP), serum glucose and 24-h urinary albumin and sodium excretions were measured, and renal histopathology was performed. High salt-diet increased BP and urinary albumin excretion, consequently resulting in glomerular sclerosis and tubulointerstitial fibrosis. Although saxagliptin did not affect BP and blood glucose levels, it significantly ameliorated urinary albumin excretion. In situ staining showed DPP-4 activity in glomerular and tubular cells. Saxagliptin significantly suppressed DPP-4 activity in renal tissue extracts and in glomerular and tubular cells. Saxagliptin also significantly attenuated the increase in inflammation and fibrosis-related gene expressions in the kidney. Our results demonstrate that saxagliptin inhibited the development of renal injury independent of its glucose-lowering effect. Glomerular and tubular DPP-4 inhibition by saxagliptin was associated with improvements in albuminuria and the suppression of inflammation and fibrosis-related genes. Thus, local glomerular and tubular DPP-4 inhibition by saxagliptin may play an important role in its renoprotective effects in Dahl-S rats.

Saxagliptin, a potent dipeptidyl peptidase-4 (DPP-4) inhibitor, is currently used to treat type 2 diabetes mellitus, and it has been reported to exhibit a slower rate of dissociation from DPP-4 compared with another DPP-4 inhibitor, sitagliptin. In this study, we compared the effects of saxagliptin and sitagliptin on hypertension-related renal injury and the plasma and renal DPP-4 activity levels in Dahl salt-sensitive hypertensive (Dahl-S) rats. The high-salt diet (8% NaCl) significantly increased the blood pressure and quantity of urinary albumin excretion and induced renal glomerular injury in the Dahl-S rats. Treatment with saxagliptin (14mg/kg/day via drinking water) for 4 weeks significantly suppressed the increase in urinary albumin excretion and tended to ameliorate glomerular injury without altering the blood glucose levels and systolic blood pressure. On the other hand, the administration of sitagliptin (140mg/kg/day via drinking water) did not affect urinary albumin excretion and glomerular injury in the Dahl-S rats. Meanwhile, the high-salt diet increased the renal DPP-4 activity but did not affect the plasma DPP-4 activity in the Dahl-S rats. Both saxagliptin and sitagliptin suppressed the plasma DPP-4 activity by 95% or more. Although the renal DPP-4 activity was also inhibited by both drugs, the inhibitory effect of saxagliptin was more potent than that of sitagliptin. These results indicate that saxagliptin has a potent renoprotective effect in the Dahl-S rats, independent of its glucose-lowering actions. The inhibition of the renal DPP-4 activity induced by saxagliptin may contribute to ameliorating renal injury in hypertension-related renal injury.

Aldosterone-induced activation of mineralocorticoid receptor, a member of the nuclear receptor family, results in increased tissue damage such as vascular inflammation and cardiac and perivascular fibrosis. Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for hypertension and angina. Benidipine exhibits pleiotropic pharmacological features such as renoprotective and cardioprotective effects through triple blockade of L-, N-, and T-type calcium channels. However, the mechanism of additional beneficial effects on end-organ damage is poorly understood. Here, we examined the effects of benidipine and other calcium channel blockers on aldosterone-induced mineralocorticoid receptor activation using luciferase reporter assay system. Benidipine showed more potent activity than efonidipine, amlodipine, or azelnidipine. Benidipine depressed the response to higher concentrations of aldosterone, whereas pretreatment of eplerenone, a steroidal mineralocorticoid receptor antagonist, did not. Binding studies using [(3)H] aldosterone indicated that benidipine and other calcium channel blockers competed for binding to mineralocorticoid receptor. Benidipine and other calcium channel blockers showed antagonistic activity on Ser810 to Leu mutant mineralocorticoid receptor, which is identified in patients with early-onset hypertension. On the other hand, eplerenone partially activated the mutant. Results of analysis using optical isomers of benidipine indicated that inhibitory effect of aldosterone-induced mineralocorticoid receptor activation was independent of its primary blockade of calcium channels. These results suggested that benidipine directly inhibits aldosterone-induced mineralocorticoid receptor activation, and the antagonistic activity might contribute to the drug's pleiotropic pharmacological features.

Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for treatment of hypertension and angina. Benidipine exerts pleiotropic pharmacological features, such as renoprotective and cardioprotective effects. In pathophysiological conditions, the antidiuretic hormone aldosterone causes development of renal and cardiovascular diseases. In adrenal glomerulosa cells, aldosterone is produced in response to extracellular potassium, which is mainly mediated by T-type voltage-dependent Ca2+ channels. More recently, it has been demonstrated that benidipine inhibits T-type Ca2+ channels in addition to L-type Ca2+ channels. Therefore, effect of calcium channel blockers, including benidipine, on aldosterone production and T-type Ca2+ channels using human adrenocortical cell line NCI-H295R was investigated. Benidipine efficiently inhibited KCI-induced aldosterone production at low concentration (3 and 10 nM), with inhibitory activity more potent than other calcium channel blockers. Patch clamp analysis indicated that benidipine concentration-dependently inhibited T-type Ca2+ currents at 10, 100 and 1000 nM. As for examined calcium channel blockers, inhibitory activity for T-type Ca2+ currents was well correlated with aldosterone production. L-type specific calcium channel blockers calciseptine and nifedipine showed no effect in both assays. These results indicate that inhibition of T-type Ca2+ channels is responsible for inhibition of aldosterone production in NCI-H295R cells. Benidipine efficiently inhibited KCI-induced upregulation of 11-beta-hydroxylase mRNA and aldosterone synthase mRNA as well as KCI-induced Ca2+ influx, indicating it as the most likely inhibition mechanism. Benidipine partially inhibited angiotensin II-induced aldosterone production, plus showed additive effects when used in combination with the angiotensin II type I receptor blocker valsartan. Benidipine also partially inhibited angiotensin II-induced upregulation of the above mRNAs and Ca2+ influx inhibitory activities of benidipine for aldosterone production. T-type Ca2+ channels may contribute to additional benefits of this drug for treating renal and cardiovascular diseases, beyond its primary anti-hypertensive effects from blocking L-type Ca2+ channels. (c) 2008 Elsevier B.V. All rights reserved.

In safety pharmacology studies, the effects on the QT interval of electrocardiograms are routinely assessed using a telemetry system in cynomolgus monkeys. However, there is a lack of integrated databases concerning in vivo QT assays in conscious monkeys. As part of QT Interval Prolongation: Project for Database Construction (QT PRODACT), the present study examined 10 positive compounds with the potential to prolong the QT interval and 6 negative compounds considered to have no such effect on humans. The experiments were conducted at 7 facilities in accordance with a standard protocol established by QT PRODACT. The vehicle or 3 doses of each test compound were administered orally to male cynomolgus monkeys (n=3-4), and telemetry signals were recorded for 24 h. None of the negative compounds prolonged the corrected QT using Bazett's formula (QTcB) interval. On the other hand, almost all of the positive compounds prolonged the QTcB interval, but haloperidol, terfenadine, and thioridazine did not. The failure to detect the QTcB interval prolongation appeared to be attributable for the differences in metabolism between species and/or disagreement with Bazett's formula for tachycardia. In the cynomolgus monkeys, astemizole induced Torsade de Pointes and cisapride caused tachyarrhythmia at lower plasma concentrations than those observed in humans and dogs. These results suggest that in vivo QT assays in conscious monkeys represent a useful model for assessing the risks of drug-induced QT interval prolongation.

Postmortem coronary angiography was performed in 20 beagles and 15 mongrels, and the origin and distribution of the sinus node arteries (SNAs) were subsequently investigated using soft X-ray radiography, the tissue clearing method, and histological examination. In 19 of 20 beagles, the SNAs consisted of a single atrial branch originating from the right coronary artery, and the distribution pattern of the atrial branch seemed to be uniform. In contrast, the following three different patterns were recognized in the atrial branches forming the SNAs of mongrels: (1) distribution by two atrial branches, i.e., one originating from the right coronary artery and the other from the left circumflex artery, (2) dual distribution by two atrial branches originating from the right coronary artery, and (3) distribution by a single atrial branch originating from the right coronary artery or from the left circumflex artery. In 26 of the 30 dogs which were histologically examined, the SNAs ran outside of the sinus node and were not centrally located. In the beagles, the proximal atrial branch from the right coronary artery reached the atrioventricuolar node area and supplied blood to the atrioventricular node together with the arterial branches derived from the anterior septal artery and posterior septal artery. In addition, the arterial branch of the SNAs reached the atrioventricular junction area. These findings should contribute to clinical, pharmacological, and pathological studies of the cardiovascular system, including studies on cardiac arrhythmias in beagles.