Masahide Mizobuchi

Combination Therapy with an Angiotensin-Converting Enzyme Inhibitor and a Vitamin D Analog Suppresses the Progression of Renal Insufficiency in Uremic Rats

Monotherapy with angiotensin-converting enzyme inhibitors has been shown to be beneficial in suppressing the progression of experimentally induced kidney diseases. Whether such therapy provides additional benefits when combined with vitamin D or an analog of vitamin D has not been established. Rats were made uremic by 5/6 nephrectomy and treated as follows: Uremic + vehicle (UC), uremic + enalapril (30 mg/L in drinking water; E), uremic + paricalcitol (19-nor; 0.8 microg/kg, three times a week), and uremic + enalapril + paricalcitol (E + 19-nor). A group of normal rats served as control (NC). BP was significantly elevated in the UC and 19-nor groups compared with the NC group but was indistinguishable from normal in the E and E + 19-nor groups. The decrease in creatinine clearance and the increase in the excretion of urinary protein that were observed in the UC group were ameliorated by the use of E alone or by E + 19-nor (P < 0.05 versus UC). The glomerulosclerotic index was significantly decreased in both the 19-nor (P < 0.01) and E + 19-nor groups (P < 0.01) compared with the UC group. Tubulointerstitial volume was significantly decreased in both the E (P < 0.05) and E + 19-nor groups (P < 0.01) compared with the UC group. Both macrophage infiltration (ED-1-positive cells) and production of the chemokine monocyte chemoattractant protein-1 were significantly blunted in E + 19-nor compared with E group. TGF-beta1 mRNA and protein expression were increased in the UC group (mRNA: 23.7-fold; protein: 29.1-fold versus NC). These increases were significantly blunted in the 19-nor group (mRNA: 7.1-fold; protein: 8.0-fold versus NC) and virtually normalized in the E + 19-nor group (protein: 0.8-fold versus NC). Phosphorylation of Smad2 was also elevated in the UC group (7.6-fold versus NC) but less so in the 19-nor-treated rats (5.5-fold versus NC). When rats were treated with E + 19-nor, the phosphorylation of Smad2 was normal (1.1-fold versus NC). Thus, 19-nor can suppress the progression of renal insufficiency via mediation of the TGF-beta signaling pathway, and this effect is amplified when BP is controlled via renin-angiotensin system blockade.

Calcimimetic Compound Upregulates Decreased Calcium-Sensing Receptor Expression Level in Parathyroid Glands of Rats with Chronic Renal Insufficiency

The reduced expression level of the calcium-sensing receptor (CaR) is attributed to the hyposensitivity of parathyroid cells to extracellular calcium concentration [Ca2+]o, which plays a crucial role in the pathogenesis of secondary hyperparathyroidism (SHPT) in patients and rats with chronic renal insufficiency (CRI). Calcimimetic compounds have been demonstrated to improve the decreased sensitivity of CaR to extracellular calcium concentration and to suppress both parathyroid hormone (PTH) oversecretion and parathyroid cell proliferation. However, the effect of calcimimetics on the reduced CaR expression level in parathyroid cells in CRI remains unclarified. The aim of this investigation was to examine the effect of the calcimimetic compound NSP R-568 (R-568) on the CaR expression in the parathyroid cells of rats with experimental CRI. Subtotally nephrectomized rats were fed a high-phosphorus diet for 8 (n = 12; Nx-8 group) or 9 wk (n = 11; Nx-9 group) to induce severe SHPT. Another group of uremic rats were fed a high-phosphorus diet for 8 wk and then orally administered R-568 (100 micromol/kg body wt) once a day for 7 d (n = 11; Nx+R-568 group). Sham-operated rats that were fed a standard diet for 9 wk were used as controls (n = 8). R-568 treatment induced a significant reduction in plasma PTH level with significant decrease in serum calcium and without change in serum phosphorus concentration. Serum 1,25(OH)2D3 level was not affected by R-568 administration. CaR mRNA and protein levels in the Nx-8 and Nx-9 groups significantly decreased compared with those in the controls; however, no significant difference in these parameters was observed between the Nx-8 and Nx-9 groups. In the Nx+R-568 group, CaR mRNA and protein levels significantly increased compared with those in either the Nx-8 or Nx-9 group. R-568 was effective in reducing the number of proliferating cell nuclear antigen-positive cells along with parathyroid gland growth suppression in the Nx+R-568 group compared with that in the Nx-9 group. The results suggest that the calcimimetic compound R-568 upregulates decreased CaR expression, and the upregulation possibly has an enhancement effect on PTH secretion and parathyroid cell hyperplasia through the improved sensitivity of CaR to [Ca2+]o.

Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells

Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10(-4)M), or 10% cyclodextrin, for 6h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro.

Biochemical and Cellular Effects of Direct Maxacalcitol Injection into Parathyroid Gland in Uremic Rats

The most important etiological factors of resistance to medical treatments for secondary hyperparathyroidism are the decreased contents of the vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in parathyroid cells and a severely swollen parathyroid gland (PTG) as a result of hyperplasia. The effects of direct maxacalcitol (OCT) injection into PTG in terms of these factors were investigated in this study. The PTG of Sprague-Dawley rats that were 5/6 nephrectomized and fed a high-phosphate diet were treated by a direct injection of OCT (DI-OCT) or vehicle (DI-vehicle). The changes in serum intact parathyroid hormone (PTH), Ca(2+), and phosphorus levels, in VDR and CaSR expression levels in parathyroid cells, and in Ca(2+)-PTH curves were examined. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA electrophoresis for PTG. DI-OCT markedly decreased serum intact PTH level, and a significant difference in this level between DI-OCT and DI-vehicle was observed. However, serum Ca(2+) and phosphorus levels did not changed markedly in both groups. The upregulations of both VDR and CaSR, the clear shift to the left downward in the Ca(2+)-PTH curve, and the induction of apoptosis after DI-OCT were observed. These findings were not observed in the DI-vehicle-treated rats. Moreover, these effects of DI-OCT were confirmed by the DI-OCT into one PTG and DI-vehicle alone into another PTG in the same rat. DI-OCT may introduce simultaneous VDR and CaSR upregulations and the regression of hyperplastic PTG, and these effects may provide a strategy for strongly suppressing PTH levels in very severe secondary hyperparathyroidism.

Involvement of alpha-klotho and fibroblast growth factor receptor in the development of secondary hyperparathyroidism.

Background/Aims: Fibroblast growth factor 23 (FGF23) has been shown to suppress parathyroid hormone (PTH) secretion. α-Klotho has been demonstrated to function as a fibroblast growth factor receptor (FGFR) cofactor for FGF23. Thus, both α-Klotho and FGFR may play roles in PTH synthesis and/or secretion. Functions of α-Klotho and FGFR in secondary hyperparathyroidism (SHPT) remain to be studied. The present studies explore the role of α-Klotho and FGFR in SHPT. Methods: Hyperplastic parathyroid glands (n = 44) were obtained from patients with SHPT. Results: Immunohistochemical study showed that both α-Klotho and FGFR1c expression in hyperplastic glands was significantly decreased compared with that in normal glands (Klotho p < 0.01, and FGFR1c p < 0.05). A significant positive correlation was observed between α-Klotho and FGFR1c (r2 = 0.375, p < 0.01) indicating a cooperative system. Both α-Klotho (r2 = 0.235, p < 0.05) and FGFR1c (r2 = 0.181, p < 0.05) correlated positively with the calcium-sensing receptor (CaR), which plays an important role in the development of SHPT. In addition, expression of α-Klotho correlated negatively with parathyroid cell proliferation, as confirmed by Ki67 staining (r2 = 0.148, p < 0.05). Conclusion: Decreased expression of α-Klotho and FGFR1c in parallel with CaR expression and parathyroid cell growth may be involved in the pathogenesis of SHPT.

Vitamin D receptor activators inhibit vascular smooth muscle cell mineralization induced by phosphate and TNF-α

BACKGROUND Vascular calcification is a highly regulated process. Tumor necrosis factor-α (TNF-α) has been shown to accelerate the highly regulated osteogenic process in vascular smooth muscle cells (VSMCs). Vitamin D receptor activators (VDRAs) have been associated with beneficial cardiovascular outcomes in patients with chronic kidney disease. We examined whether maxacalcitol, a vitamin D(3) analog, exhibits a suppressive effect on VSMC mineralization induced by phosphate and TNF-α. METHODS Human VSMCs were treated with either vehicle, maxacalcitol (10(-9) to 10(-7) M), or calcitriol (10(-9) to 10(-7) M) in 2.5 mM of phosphate media with TNF-α (1 ng/mL) for 9 days. VSMC mineralization was determined and expression of genes associated with the osteogenic process was examined by real-time reverse transcription-polymerase chain reaction. Expression of matrix metalloproteinase-2 (MMP-2) messenger RNA (mRNA) in VSMCs and MMP-2 protein in media was also analyzed. RESULTS Vehicle-treated VSMCs exhibited massive mineralization, which was inhibited by maxacalcitol in a concentration-dependent manner. Calcitriol also inhibited the mineralization. While vehicle-treated VSMCs exhibited increased mRNA expression of genes associated with the osteogenic process (Cbfa1/Runx2 and osteocalcin) compared with VSMCs grown in normal media without TNF-α (control), maxacalcitol and calcitriol suppressed the increase in mRNA species. Furthermore, vehicle-treated VSMCs exhibited increased MMP-2 mRNA and protein in the media that were suppressed notably by maxacalcitol. CONCLUSIONS Both the VDRAs abrogated the acceleration of the osteogenic process induced by phosphate and TNF-α in VSMCs, which was linked to inhibition of mineralization in VSMCs. MMP-2 blockade by VDRAs may contribute to an inhibitory effect on vascular calcification.

Vitamin D and vascular calcification in chronic kidney disease

Vascular calcification is frequently observed and is closely associated with cardiovascular mortality in patients with chronic kidney disease (CKD). Vascular calcification is largely divided into two types. One is atherosclerotic intimal layer calcification and the other is medial layer calcification (Monckebergs calcification). The latter is more common in patients with CKD than in general population. Evidence is growing that vascular calcification is a regulated active process as well as a passive process resulting from elevated serum phosphate (P) and an increase in the calcium phosphate (Ca x P) product leading to oversaturated plasma. Proving the active process, in vitro studies have demonstrated that the transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells is a crucial mechanism in the progression of vascular calcification. Reduction of the activity of systemic and local inhibitors has also been recognized to be important. The link between vitamin D and vascular calcification is complex. Experimental and clinical researches have revealed that both vitamin D excess and vitamin D deficiency have been shown to be associated with vascular calcification in uremic milieu. On the other hand, although there are some biases, recent large observational studies have demonstrated that vitamin D has beneficial effects on the mortality of patients with CKD independent of serum Ca, P, and parathyroid hormone levels, likely due to its activation of the vitamin D receptor in vasculature and cardiac myocytes. Further prospective studies are necessary to evaluate the direct effect of vitamin D on vascular calcification in order to improve the cardiovascular health of patients with CKD.

Detection of Peripheral Artery Disease by Duplex Ultrasonography among Hemodialysis Patients

BACKGROUND AND OBJECTIVES Peripheral arterial disease (PAD) is a known predictor of cardiovascular morbidity and mortality among hemodialysis patients. Although ankle-brachial BP index (ABI) is a simple and reliable test for PAD screening, its sensitivity has been suggested to decrease among dialysis patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We performed a cross-sectional outpatient cohort study to examine prevalence of PAD among hemodialysis patients using duplex ultrasonography of the lower extremity artery. We also evaluate the influence of increased arterial stiffness on impaired accuracy of ABI for PAD screening. RESULTS Of 315 total patients, 23.8% had PAD. PAD was associated with younger age, diabetes, current smoking, atherosclerotic comorbidities, increased total cholesterol levels, increased triglyceride levels, and lower Kt/V. The receiver operating characteristic analysis (area under the receiver operating characteristic curve = 0.846) showed that sensitivity and specificity of ABI values for PAD were 49.0 and 94.8%, respectively. An ABI cut-off value of 1.05 resulted in the best sensitivity (74.5%) and specificity (84.4%). There was a significant difference in sensitivity of ABI levels <0.9 for detecting PAD among patients in different brachial-ankle pulse wave velocity quartiles. In patients with the highest brachial-ankle pulse wave velocity quartile, PAD was most prevalent (46.5%), and ABI had the highest accuracy in detecting PAD (area under the curve, 0.933). CONCLUSIONS These results suggest that duplex ultrasonography was a useful tool for screening asymptomatic PAD among hemodialysis patients and that the diagnostic value of ABI for PAD was affected by various factors.

Involvement of matrix metalloproteinase-2 in the development of medial layer vascular calcification in uremic rats.

Vascular calcification is the most important cause of cardiovascular disease in patients with chronic kidney disease (CKD). Medial layer vascular calcification, which is recognized to be an active process (i.e. the transformation of vascular smooth muscle cells into osteoblast‐like cells), is common in CKD patients. We have recently reported the possibility of an interaction between elastin degradation and medial layer vascular calcification. Matrix metalloproteinase‐2 (MMP‐2), which induces the degradation of elastin, has been implicated in the elastic calcification in arteries of dialysis patients; however, the precise mechanisms by which elastin degradation interacts with the development of vascular calcification remain to be studied. To clarify the mechanisms by which elastin degradation is involved in the development of medial layer vascular calcification in the uremic milieu, we induced aortic medial layer calcification in 5/6 nephrectomized uremic rats (male Sprague–Dawley rats) fed a diet containing high phosphate (1.2%) and lactate (20%). After 10 weeks, the rats were euthanized for the measurement of serum chemistry profiles and histological analyses. The uremic rats showed significant increases in blood pressure, serum creatinine, phosphate, and parathyroid hormone levels compared with normal rats. Von Kossa staining showed medial layer aortic calcification in the uremic rats. In calcified lesions, thin elastic lamellae were observed by elastin staining, indicating that elastin degradation could occur in the area. Furthermore, MMP‐2 expression determined by immunohistochemistry was also observed in the same area. Elastin degradation accompanied by MMP‐2 expression might be involved in the development of medial layer vascular calcification in uremic rats.

PTH-dependence of the effectiveness of cinacalcet in hemodialysis patients with secondary hyperparathyroidism

Cinacalcet lowers parathyroid hormone levels. Whether it can prolong survival of people with chronic kidney disease (CKD) complicated by secondary hyperparathyroidism (SHPT) remains controversial, in part because a recent randomized trial excluded patients with iPTH <300 pg/ml. We examined cinacalcet’s effects at different iPTH levels. This was a prospective case-cohort and cohort study involving 8229 patients with CKD stage 5D requiring maintenance hemodialysis who had SHPT. We studied relationships between cinacalcet initiation and important clinical outcomes. To avoid confounding by treatment selection, we used marginal structural models, adjusting for time-dependent confounders. Over a mean of 33 months, cinacalcet was more effective in patients with more severe SHPT. In patients with iPTH ≥500 pg/ml, the reduction in the risk of death from any cause was about 50% (Incidence Rate Ratio [IRR] = 0.49; 95% Confidence Interval [95% CI]: 0.29–0.82). For a composite of cardiovascular hospitalization and mortality, the association was not statistically significant, but the IRR was 0.67 (95% CI: 0.43–1.06). These findings indicate that decisions about using cinacalcet should take into account the severity of SHPT.