Ikuji Hatamura

Stretch-Induced Collagen Synthesis in Cultured Smooth Muscle Cells from Rabbit Aortic Media and a Possible Involvement of Angiotensin II and Transforming Growth Factor-β

Mechanical strain reportedly stimulates the synthesis of collagen in vascular smooth muscle cells (SMCs). The present study was designed to investigate a possible involvement of angiotensin II (Ang II) and transforming growth factor (TGF)-β in stretch-induced collagen synthesis of cultured SMCs derived from the rabbit aortic media. SMCs were cyclically stretched at a rate of 10% elongation and 30 cycles/min for 24 h using the Flexercell® strain unit (Flexcell International Corp., McKeesport, Pa.). A two-fold increase in collagen synthesis and a concurrent increase in total protein synthesis were noted in stretched SMCs. Concentration of immunoreactive Ang II in the conditioned medium was elevated under the mechanical strain. Stretch-induced collagen and total protein synthesis were inhibited by either a selective antagonist to Ang II (saralasin), an angiotensin I-converting enzyme inhibitor (captopril) or an antisense oligonucleotide for angiotensinogen mRNA. An elevated secretion of TGF-β, both active and latent forms, was found in the medium of stretched SMCs. Saralasin inhibited the stretch-induced secretion of TGF-β from SMCs. Stretch-induced collagen and total protein synthesis was further inhibited by either an anti-TGF-β1 neutralizing antibody or an adenovirus-mediated transfer of a truncated TGF-β type II receptor. Elevated expression of collagen α1(III) chain and TGF-β1 mRNAs, and its reversal by saralasin were also demonstrated in stretched SMCs. Results indicate that the stretch-induced collagen and total protein synthesis appears to be mediated via an autocrine-paracrine mechanism of Ang II and TGF-β released from SMCs.

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.

Regulation of Fibroblast Growth Factor 23 Production in Bone in Uremic Rats

Background: Fibroblast growth factor 23 (FGF23) regulates renal phosphate reabsorption and 1α,25-dihydroxyvitamin D [1,25(OH)2D3] metabolism. Patients with chronic kidney disease (CKD) have increased levels of circulating FGF23, but the direct regulation of this elevation of FGF23 is incompletely understood. Method:We measured plasma parameters in uremic rats fed a high-phosphorus diet and then performed parathyroidectomy (PTX) to determine its effect. We also investigated FGF23 mRNA expression in various tissues to identify the major source of circulating FGF23. Result: The uremic rats displayed dramatic changes in plasma FGF23 levels, consistent with increased expression of FGF23 in bone. Elevated FGF23 was associated with phosphate and parathyroid hormone (PTH). After PTX, the elevated FGF23 had decreased, consistent with decreased expression of FGF23 in bone. Significant decreases in plasma FGF23 were associated with PTH and 1,25(OH)2D3, but not phosphate. Conclusion: Elevated plasma FGF23 levels in uremic rats reflect the increased expression of FGF23 in bone. The expression of FGF23 in bone may be regulated by a PTH-1,25(OH)2D3 axis-dependent pathway and another PTH-dependent and 1,25(OH)2D3-independent pathway in uremic rats. The pathway may be decided by the degree of renal dysfunction.

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.

Trps1 Functions Downstream of Bmp7 in Kidney Development

During embryonic development, the mesenchyme of the lungs, gut, kidneys, and other tissues expresses Trps1, an atypical member of the GATA-type family of transcription factors. Our previous work suggested the possibility that Trps1 acts downstream of bone morphogenic protein 7 (Bmp7), which is essential for normal renal development. To examine the role of Trps1 during early renal development, we generated Trps1-deficient mice and examined their renal histology. Compared with wild-type mice, Trps1-deficient newborn mice had fewer tubules and glomeruli, an expanded renal interstitium, and numerous uninduced metanephric mesenchymal cells, which resulted in fewer nephrons. In wild-type kidneys, Trps1 expression was present in ureteric buds, cap mesenchyme, and renal vesicles, whereas Trps1 was virtually absent in Bmp7-deficient kidneys. Furthermore, Trps1-deficient kidneys had low levels of Pax2 and Wt1, which are markers of condensed mesenchymal cells, suggesting that a lack of Trps1 affects the differentiation of cap mesenchyme to renal vesicles. In cultured metanephric mesenchymal cells, Bmp7 induced Trps1 and E-cadherin and downregulated vimentin. Knockdown of Trps1 with small interference RNA inhibited this Bmp7-induced mesenchymal-to-epithelial transition. Last, whole-mount in situ hybridization of Wnt9b and Wnt4 demonstrated prolonged branching of ureteric buds and sparse cap mesenchyme in the kidneys of Trps1-deficient mice. Taken together, these findings suggest that normal formation of nephrons requires Trps1, which mediates mesenchymal-to-epithelial transition and ureteric bud branching during early renal development.

Trps1 plays a pivotal role downstream of Gdf5 signaling in promoting chondrogenesis and apoptosis of ATDC5 cells

Tricho‐rhino‐phalangeal syndrome (TRPS) is an autosomal dominant skeletal disorder caused by mutations of TRPS1. Based on the similar expression patterns of Trps1 and Gdf5, we hypothesized a possible functional interaction between these two molecules. Using a chondrogenic cell line (ATDC5), we investigated the association of Gdf5‐mediated signaling pathways with Trps1 and the phenotypic changes of ATDC5 cells due to over‐expression or suppression of Trps1. Treatment of cells with Gdf5 enhanced Trps1 protein levels and phosphorylation of p38 mitogen‐activated protein kinase (MAPK) in a dose‐dependent manner. Nuclear translocation of Trps1 was also induced by Gdf5. These effects were blocked by a dominant negative form of activin‐linked kinase 6 (dn‐Alk6) and by SB203580, an inhibitor of the p38 MAPK pathway. Conversely, Gdf5 expression was suppressed by the over‐expression of Trps1. Trps1‐overexpressing ATDC5 (O/E) cells differentiated into chondrocytes more quickly than mock‐infected control cells, whereas cells transfected with dn‐Alk6 showed slower differentiation. On the other hand, O/E cells showed an increase of apoptosis along with the up‐regulation of cleaved caspase 3 and down‐regulation of Bcl‐2, whereas dn‐Alk6 cells showed suppression of apoptosis. In conclusion, Trps1 acts downstream of the Gdf5 signaling pathway and promotes the differentiation and apoptosis of ATDC5 cells.

TNF-α deficiency accelerates renal tubular interstitial fibrosis in the late stage of ureteral obstruction

TNF-alpha and TGF-beta1 have a complementary relationship in fibrogenesis. This study was performed to investigate the role of TNF-alpha in renal tubular interstitial fibrosis. We compared the extent of renal tubular interstitial fibrosis after unilateral ureteral obstruction (UUO) between wild-type and TNF-alpha-deficient mice by using immunohistochemistry, enzyme-linked immunoassay, and the real-time polymerase chain reaction (PCR). In comparison with wild-type mice, there was no significant difference in the extent of renal fibrosis in the TNF-alpha-deficient mice at 2 weeks after UUO. By 4 weeks after UUO, however, fibrosis marked an increase in the TNF-alpha-deficient mice to exceed that in the wild-type mice. Immunohistochemistry, enzyme-linked immunoassay, and real-time PCR demonstrated an increase of extracellular matrix in the kidneys of TNF-alpha-deficient mice that was caused by upregulation of the expression of TGF-beta1 and Snail, which in turn resulted from an increase of infiltrating macrophages. Real-time PCR revealed an increase in expression of the TNF-alpha type 2 receptor at 4 weeks after UUO, which explained the difference in the extent of renal fibrosis between TNF-alpha-deficient and wild-type mice. In the chronic stage of renal fibrosis, TNF-alpha suppresses the infiltration of macrophages by inducing TNF-alpha type 2 receptor expression, resulting in the amelioration of fibrosis.

MafB interacts with Gcm2 and regulates parathyroid hormone expression and parathyroid development.

Serum calcium and phosphate homeostasis is critically regulated by parathyroid hormone (PTH) secreted by the parathyroid glands. Parathyroid glands develop from the bilateral parathyroid‐thymus common primordia. In mice, the expression of transcription factor Glial cell missing 2 (Gcm2) begins in the dorsal/anterior part of the primordium on embryonic day 9.5 (E9.5), specifying the parathyroid domain. The parathyroid primordium then separates from the thymus primordium and migrates to its adult location beside the thyroid gland by E15.5. Genetic ablation of gcm2 results in parathyroid agenesis in mice, indicating that Gcm2 is essential for early parathyroid organogenesis. However, the regulation of parathyroid development at later stages is not well understood. Here we show that transcriptional activator v‐maf musculoaponeurotic fibrosarcoma oncogene homologue B (MafB) is developmentally expressed in parathyroid cells after E11.5. MafB expression was lost in the parathyroid primordium of gcm2 null mice. The parathyroid glands of mafB+/− mice were mislocalized between the thymus and thyroid. In mafB−/− mice, the parathyroid did not separate from the thymus. Furthermore, in mafB−/− mice, PTH expression and secretion were impaired; expression levels of renal cyp27b1, one of the target genes of PTH, was decreased; and bone mineralization was reduced. We also demonstrate that although Gcm2 alone does not stimulate the PTH gene promoter, it associates with MafB to synergistically activate PTH expression. Taken together, our results suggest that MafB regulates later steps of parathyroid development, that is, separation from the thymus and migration toward the thyroid. MafB also regulates the expression of PTH in cooperation with Gcm2.

Trps1 deficiency enlarges the proliferative zone of growth plate cartilage by upregulation of Pthrp

We have reported that elongation of the columnar proliferative zone of long bone growth plates in Trps1-/- mice during the late fetal stage in the previous study [1]. Since expression of Trps1 protein was found to overlap with that of mRNAs for Indian hedgehog (Ihh), PTH/PTHrP receptor (PPR), and PTHrP, we hypothesized that Trps1 may inhibit the hypertrophic differentiation of chondrocytes by interacting with the Ihh/PTHrP feedback loop. To investigate whether Trps1 has a role in this Ihh/PTHrP feedback loop, we compared the growth plates of Trps1-/- mice and wild-type (Trps1+/+) mice. Immunohistochemistry showed that Trps1 protein was strongly expressed in the periarticular and prehypertrophic zones of the fetal growth plate in wild-type mice on embryonic day 18.5 (E18.5). On the other hand, Ihh, PPR, and PTHrP mRNAs were predominantly expressed in the prehypertrophic zone at this stage of development. While expression of Ihh and PPR by prehypertrophic chondrocytes was unaffected in the growth plates of Trps1-/- mice, the range of PTHrP expression was expanded toward the proliferating zone in these mice. Quantitative real-time PCR analysis demonstrated upregulation of PTHrP in the epiphyseal growth plates of Trps1-/- mice. Furthermore, promoter analysis combined with the chromatin immunoprecipitation (ChIP) assay demonstrated that direct binding of Trps1 to the PTHrP promoter suppressed the transcription of PTHrP. Finally, organ culture of E14.5 tibiae in the absence or the presence of Pthrp revealed that the proliferative zone of the tibial growth plate was elongated by culture with Pthrp compared to that of control tibiae. Taken together, these data provide the first genetic evidence that lack of Trps1 leads to overexpression of PTHrP, and that Trps1 is required to maintain the normal organization of chondrocytes in the growth plate.