Skeletal and mineral metabolic effects of risedronate in a rat model of high-turnover renal osteodystrophy.

INTRODUCTION: High-turnover bone disease is a major consequence of SHPT and may explain the high risk for fracture in patients with advanced chronic kidney disease (CKD). Bisphosphonates suppress bone turnover and improve bone strength, but their effects have not been fully characterized in advanced CKD with severe SHPT. Bisphosphonates also increase 1,25-dihydroxyvitamin D levels in normal and uremic rats, but the underlying mechanism remains to be determined. MATERIALS AND METHODS: We investigated the skeletal and mineral metabolic effects of RIS, a pyridinyl bisphosphonate, in rats with severe SHPT induced by 5/6 nephrectomy plus a high phosphate diet. RESULTS: Nephrectomized rats developed severe SHPT, along with hyperphosphatemia, low 1,25-dihydroxyvitamin D, and markedly increased FGF23. Moreover, these rats exhibited characteristic features of high-turnover renal osteodystrophy, including increased indices of trabecular bone turnover, decreased cortical bone thickness, inferior cortical biomechanical properties, and a prominent increase in peritrabecular fibrosis. RIS treatment increased bone volume and partially attenuated trabecular bone remodeling, cortical bone loss, and mechanical properties, whereas it produced a marked improvement in peritrabecular fibrosis along with a corresponding decrease in osteogenic gene markers. RIS treatment also suppressed the elevation of FGF23, which was associated with increased 1,25-dihydroxyvitamin D. CONCLUSIONS: In a rat model of severe SHPT, treatment with RIS partially attenuated histological manifestations of high-turnover bone disease. RIS treatment also suppressed the elevation of FGF23, which may explain the increased 1,25-dihydroxyvitamin D production during the treatment.

Feasibility of photodynamic therapy for secondary hyperparathyroidism in chronic renal failure rats.

BACKGROUND: Feasibility of photodynamic therapy (PDT) for secondary hyperparathyroidism (SHPT) was examined in a rat model of SHPT. METHODS: A photosensitizer, 5-aminolevulinic acid (5-ALA), was injected intraperitoneally, and the parathyroid glands were irradiated either after surgical exposure with 385-nm light or transdermally with 630-nm light from a light-emitting diode (LED) lamp. RESULTS: PDT with high 5-ALA and irradiation doses caused severe hypoparathyroidism in SHPT rats within two days. Low-dose invasive PDT reduced intact parathyroid hormone (iPTH) levels in all rats from 748.9 ± 462.6 pg/mL at baseline to 138.7 ± 117.5 pg/mL at week 6, followed by a further decrease to 80.5 ± 54.0 pg/mL at week 9 in 60 % of rats or an increase to 970.0 ± 215.6 pg/mL at week 9 in 40 % of rats. Low-dose noninvasive PDT reduced iPTH levels from 1612.5 ± 607.8 pg/mL at baseline to 591.9 ± 480.1 pg/mL at week 4 in all rats. Thereafter, iPTH levels remained low in 43 % of rats and were 233.7 ± 51.6 pg/mL at week 9, whereas 57 % showed an increase, reaching 3305.9 ± 107.3 pg/mL at week 9. Control SHPT rats had iPTH levels of 2487.8 ± 350.9 and 2974.6 ± 372.1 pg/mL at week 4 and 9, respectively. The parathyroid glands of the rats with low iPTH levels were atrophied and had few parathyroid cells surrounded by fibrotic materials and no recognizable blood vessels. Those of the rats with high iPTH levels showed well-preserved gland structure, clusters of parathyroid cells, and blood vessels. CONCLUSION: These results demonstrate that 5-ALA-mediated PDT for SHPT is feasible.

[New Developments in CKD-MBD. Cell Biology of parathyroid in CKD].

Parathyroid monitors the calcium concentration in blood by signals from calcium-sensing receptors, adjusts secretion of parathyroid hormone to keep constant calcium concentration in the body. Although parathyroid parenchymal cells consist of chief cells which secrete PTH, and oxyphil cells which are rich in mitochondria, all hardly perform mitotic proliferation in normal status. However, in CKD, PTH hypersecretion and hyperplasia are started by hyperphosphatemia, hypocalcemia, and activated-vitamin-D deficiency, and the secondary hyperparathyroidism develops. While treatment with cinacalcet hydrochloride salt induced apoptosis into the parathyroid cell, a possibility of promoting the transdifferentiation to oxyphil cells from chief cells was suggested. The specific accumulation to the parathyroid of an oncotropic photosensitizer suggests the possibility of photodynamic diagnosis and treatment of hyperparathyroidism.

Study of the Effects of Deuterium-Depleted Water on the Expression of GLUT4 and Insulin Resistance in the Muscle Cell Line C2C12.

Deuterium-depleted water (DDW) is used in the treatment of many diseases, including cancer and diabetes. To detect the effect of DDW on gene expression and activation of the insulin-responsive transporter GLUT4 as a mechanism for improving the pathology of diabetes, we investigated the GLUT4 expression and glucose uptake at various concentrations of DDW using the myoblast cell line C2C12 differentiated into myotubes. GLUT4 gene expression significantly increased under deuterium depletion, reaching a maximum value at a deuterium concentration of approximately 50 ppm, which was approximately nine times that of natural water with a deuterium concentration of 150 ppm. GLUT4 protein also showed an increase at similar DDW concentrations. The membrane translocation of GLUT4 by insulin stimulation reached a maximum value at a deuterium concentration of approximately 50-75 ppm, which was approximately 2.2 times that in natural water. Accordingly, glucose uptake also increased by up to 2.2 times at a deuterium concentration of approximately 50 ppm. Drug-induced insulin resistance was attenuated, and the glucose uptake was four times higher in the presence of 10 ng/mL TNF-α and three times higher in the presence of 1 µg/mL resistin at a deuterium concentration of approximately 50 ppm relative to natural water. These results suggest that DDW promotes GLUT4 expression and insulin-stimulated activation in muscle cells and reduces insulin resistance, making it an effective treatment for diabetes.

Cinacalcet induces apoptosis in parathyroid cells in patients with secondary hyperparathyroidism: histological and cytological analyses.

BACKGROUND/AIMS: Previous studies reported a reduction in parathyroid gland volume during treatment with cinacalcet in patients with secondary hyperparathyroidism (SHPT). However, it remains to be determined whether cinacalcet accelerates apoptosis of hyperplastic parathyroid cells in these patients. METHODS: The study subjects were 16 hemodialysis patients who had undergone parathyroidectomy for severe SHPT. We compared the expression of the apoptotic marker TUNEL and the proliferative marker Ki67 by immunohistochemistry and the expression of CYP27B1 by quantitative real-time PCR in hyperplastic parathyroid glands from patients treated with cinacalcet (cinacalcet group; n = 8) and those not treated with cinacalcet (non-cinacalcet group; n = 8). We also examined the effect of cinacalcet on parathyroid cell death in in vitro cell culture with TUNEL staining, using parathyroid cells from SHPT patients. RESULTS: Compared with the non-cinacalcet group, the expression of TUNEL was significantly increased but was accompanied with significantly increased Ki67 expression in the parathyroid glands from the cinacalcet group. In vitro examination showed dose- and time-dependent increases of apoptotic cells by adding cinacalcet into culture medium. We also found that the expression of CYP27B1 showed a three-fold increase in glands from the cinacalcet group compared to that of the non-cinacalcet group. CONCLUSION: Our data suggest that cinacalcet induces apoptosis of human parathyroid cells, but this effect may be overcome by more aggressive proliferation of parathyroid cells in patients with severe, cinacalcet-resistant SHPT.

Evaluation of bioartificial renal tubule device prepared with human renal proximal tubular epithelial cells cultured in serum-free medium.

Bioartificial renal tubule devices (BTD) use cell therapy to improve conditions commonly observed in recipients of artificial kidneys for treatment of kidney diseases. We previously reported significant improvement of the condition of acute kidney injury (AKI) animals after treatment with BTD prepared with lifespan-extended human renal proximal tubular cells (hRPTEC). However, a major obstacle to use of BTD for patients is their biological safety, because hRPTEC are cultured in medium containing fetal calf serum. To establish the biological safety of BTD, we prepared BTD with lifespan-extended hRPTEC cultured in a newly developed serum-free medium and compared these with BTD prepared with hRPTEC cultured in serum-containing conventional medium. Lifespan-extended hRPTEC cultured in serum-free medium (hRPTEC-SFM) can proliferate similar to hRPTEC cultured in serum-containing conventional medium (hRPTEC-CM). Comparison of leakage and of reabsorption of small molecules for BTD prepared with hRPTEC-SFM (BTD-SFM) with those for our previous BTD prepared with hRPTEC-CM (BTD-CM) showed transportation in these two types of BTD was almost identical. When AKI goats were treated with BTD-SFM for 26 h, increase of survival time and reduction of cytokine expression in blood cells were almost same as for AKI goats treated with BTD-CM. Quantification of the expression of some genes of hRPTEC in BTD revealed significant changes during BTD treatment for AKI goats. In conclusion, lifespan-extended hRPTEC-SFM work as well as hRPTEC-CM, and the biological safety of BTD for patients could be elevated without loss of function by preparation from hRPTEC-SFM.

Regulation of Renin Expression by Β1-Integrin in As4.1 Juxtaglomerular Line Cells.

(1) Background: Renal dysfunction and hypertension are mutually aggravating factors; however, the details of their interaction remain unclear. In a study using renal tissue from diabetic rats, we found that ß1-integrin, a cell-substrate adhesion molecule, is specifically phosphorylated in juxtaglomerular cells that secrete renin, a blood pressure regulator. (2) Methods: A mouse juxtaglomerular cell line (As4.1 cells) was used for the following experiments: drug-induced promotion of ß1-integrin phosphorylation/dephosphorylation; knockdown of ß1-integrin and the cell adhesion molecule connexin-40 (a candidate for the main body of baroreceptor); and pressurization to atmospheric pressure + 100 mmHg. culture in hypotonic liquid medium. The expression of renin under these conditions was measured by qRT-PCR. (3) Results: Phosphorylation of ß1-integrin suppressed the expression of renin, while dephosphorylation conversely promoted it. ß1-integrin and connexin-40 knockdown both promoted the expression of renin. Pneumatic pressurization and hypotonic medium culture both decreased the expression of renin, which was restored by the knockdown of ß1-integrin. (4) Conclusions: ß1-integrin plays an inhibitory role in the regulation of the expression of renin, which may be controlled by phosphorylation and dephosphorylation. It is hypothesized that ß1-integrin and other adhesion factors regulate the expression of renin by altering the sensitivity of baroreceptors on the plasma membrane.

Diselenide-bond replacement of the external disulfide bond of insulin increases its oligomerization leading to sustained activity.

Seleno-insulin, a class of artificial insulin analogs, in which one of the three disulfide-bonds (S-S's) of wild-type insulin (Ins) is replaced by a diselenide-bond (Se-Se), is attracting attention for its unique chemical and physiological properties that differ from those of Ins. Previously, we pioneered the development of a [C7UA,C7UB] analog of bovine pancreatic insulin (SeIns) as the first example, and demonstrated its high resistance against insulin-degrading enzyme (IDE). In this study, the conditions for the synthesis of SeIns via native chain assembly (NCA) were optimized to attain a maximum yield of 72%, which is comparable to the in vitro folding efficiency for single-chain proinsulin. When the resistance of BPIns to IDE was evaluated in the presence of SeIns, the degradation rate of BPIns became significantly slower than that of BPIns alone. Furthermore, the investigation on the intermolecular association properties of SeIns and BPIns using analytical ultracentrifugation suggested that SeIns readily forms oligomers not only with its own but also with BPIns. The hypoglycemic effect of SeIns on diabetic rats was observed at a dose of 150 µg/300 g rat. The strategy of replacing the solvent-exposed S-S with Se-Se provides new guidance for the design of long-acting insulin formulations.

Effects of cinacalcet treatment on serum soluble Klotho levels in haemodialysis patients with secondary hyperparathyroidism.

BACKGROUND: Klotho is a transmembrane protein that acts as a cofactor for fibroblast growth factor 23 (FGF23). Klotho also exists as a soluble circulating protein, but its role in secondary hyperparathyroidism (SHPT) is largely unknown. METHODS: We measured serum soluble Klotho levels in 51 haemodialysis patients, who participated and completed a 52-week, multicentre, open-label single-arm trial that examined the effectiveness of cinacalcet for treating SHPT. RESULTS: After 12 weeks of cinacalcet treatment, serum soluble Klotho decreased significantly (P = 0.03) but only marginally from 398 pg/mL [interquartile range (IQR), 268-588 pg/mL] to 378 pg/mL (IQR, 266-568 pg/mL) and returned to baseline levels. There were no significant associations between the changes in soluble Klotho levels and changes in any other parameters of mineral metabolism, including serum calcium, phosphorus, intact parathyroid hormone and FGF23. CONCLUSION: Despite significant alterations in mineral and bone metabolism during treatment with cinacalcet, this resulted in only small and transient reductions in serum levels of soluble Klotho.

Evaluation of bioartificial renal tubule device prepared with lifespan-extended human renal proximal tubular epithelial cells.

BACKGROUND: Acute kidney injury (AKI), accompanied by the development of systemic inflammatory response syndrome and multiorgan dysfunction syndrome, is associated with a high risk of death. Bioartificial renal tubule device (BTD) is a cell therapy that improves the conditions common to artificial kidney recipients treated for kidney diseases. In this paper, we describe the establishment of BTD with lifespan-extended human renal proximal tubular epithelial cells. METHODS: AKI goats were established by performing bilateral nephrectomy followed by lipopolysaccharide administration. The AKI goats were treated with BTD or sham-BTD, and the two groups of animals were compared by measuring the respective life spans and the levels of blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and serum electrolytes. The expression levels of inflammatory cytokines were detected by reverse transcription-polymerase chain reaction, and plasma interleukin (IL)-6 levels were measured by enzyme-linked immunosorbent assay. RESULTS: The life span of AKI goats was extended: the lifetime with the BTD treatment compared with sham-BTD. BTD and sham-BTD showed a similar degree of small solute clearance. The expression levels of inflammatory cytokines and plasma IL-6 levels were decreased by the BTD treatment. CONCLUSIONS: BTD treatment results in less damage from endotoxin shock and increased life span in AKI goats. These results suggest that BTD may be a useful component of bioartificial kidneys and should be considered in the next generation of renal replacement therapies.

Development of bioartificial renal tubule devices with lifespan-extended human renal proximal tubular epithelial cells.

BACKGROUND: The bioartificial renal tubule device is a cell therapy system for renal failure. The major obstacle in the development of the bioartificial renal tubule device is the obtainment of a large number of viable renal tubule cells to seed on the inner surface of hollow fibers. Although our previous studies had used a transformed cell line, they may be dangerous for clinical uses. Therefore, different approaches to amplify renal proximal tubular epithelial cells (RPTEC) in culture without oncogenes, vectors and carcinogens have been required. METHODS: The limitation of the replicative lifespan of human RPTEC, which is ∼12 population doublings (PDs), was extended by invalidating messenger RNA of cell cycle-related genes with antisense oligonucleotide or small interfering RNA (siRNA). RESULTS: Periodic transfection of siRNA to a tumor suppressor p53 or a cyclin-dependent kinase inhibitor p16(INK4a) extended the lifespan by 33 and 63 PDs, respectively, in 3 months of culture. The siRNA-mediated lifespan extension was controllable because cell division ceased within 2 weeks after the transfection was discontinued. Expressions of γ-glutamyltransferase 1 and glucose transporter 1 were recovered in siRNA-transfected RPTEC cultured on porous membranes. Bioartificial renal tubule devices (0.8 m(2)) constructed with these cells showed reabsorption of water (122.3 ± 4.2 mL/30 min), sodium (18.1 ± 0.7 mEq/30 min) and glucose (121.7 ± 4.4 mg/30 min) after 1 week of circulation. Furthermore, ß2-microglobulin and pentosidine were metabolized by RPTEC in mini-devices (65 cm(2)) within 48 h of circulation. CONCLUSIONS: These approaches enabled us to yield a high enough number of RPTEC for construction of bioartificial renal tubule devices repeatedly. Lifespan-extended RPTEC could recover their specific characteristics by culturing on porous membranes, and bioartificial renal tubule devices constructed with these cells showed good performances of reabsorption and metabolism. SUMMARY: A large number of human renal tubular cells required for construction of the bioartificial renal tubule device were prepared by extending the lifespan of the primary cells by invalidating mRNA of cell cycle-related genes. Constructed bioartificial renal tubule devices with lifespan-extended cells showed good performances of in vitro examination of reabsorption and metabolism. Requiring no oncogenes, vectors or cell cloning, the RNAi-mediated lifespan extension can help advance tissue-replacement therapy as well as basic research.

Regulation of Blood Pressure and Phosphorylation of ß1-integrin in Renal Tissue in a Rat Model of Diabetic Nephropathy.

OBJECTIVE: Induction of hypertension by diabetic nephropathy (DN) may be dependent on increased renin secretion from juxtaglomerular cells (JGC). To reveal that the mechanisms of cell adhesion and cell motility associated with ß1-integrin phosphorylation contribute to pressure sensing in JGC, we tested the ß1-integrin phosphorylation levels in renal tissue and the relationship between ß1-integrin phosphorylation and the expression of renin. METHODS: The DN rat model was generated by intravenous injection of streptozotocin (STZ, 60 mg/kg body weight). Immunohistochemistry and an imaging analysis were performed to detect and evaluate the ß1-integrin phosphorylation levels in renal tissue. Quantitative real-time polymerase chain reaction was also performed to evaluate renin mRNA levels. RESULTS: We found that the serine-785 and threonine-788/789 sites of ß1-integrin are specifically phosphorylated in macula densa and JGC, respectively, and that changes in their expression during the progression of DN are associated with the production of renin. Phosphorylation of these ß1-integrins increased or decreased with changes of the renin expression during the progression of DN. In particular, phosphorylation of threonine-788/789 was negatively correlated with the expression of renin. CONCLUSION: These findings suggest that the phosphorylation of ß1-integrin may contribute to the regulatory mechanism of renin production in JGC.

Parathyroid hormone-producing cells exist in adipose tissues surrounding the parathyroid glands in hemodialysis patients with secondary hyperparathyroidism.

Possible ectopic parathyroid hormone (PTH) production in adipose tissues surrounding hyperplastic parathyroid glands was examined in patients with secondary hyperparathyroidism (SHPT). In vitro culture of adipose tissues from 31 patients excised during parathyroidectomy showed PTH secretion in 23 (74.2%) patients. In vitro PTH secretion was detected in adipose tissues adhered to the parathyroid glands from 22 (71.0%) patients, in not-adhered adipose from 11 (35.5%) and in the thymus from four (28.6%) patients. Immunohistochemistry revealed colonies of PTH- and GCM2-positive cells intricately intertwined with adipocytes in excised adipose tissues prior to culture. When pieces of parathyroid parenchyma from SHPT patients were transplanted into the thyroid of immunodeficient nude rats with induced SHPT, the transplants secreted human PTH for one to three-and-half months after transplantation and expressed adipocyte markers, PPARγ2 and perilipin A, that the transplants did not express prior to transplantation. These findings indicate the importance of thoroughly removing adipose tissues surrounding the parathyroid glands when performing parathyroidectomy. We speculate that these ectopic PTH-producing cells are parathyroid parenchymal cells pushed out from the glands along with adipocyte progenitors during nodular growth of hyperplastic parenchymal cells and that these cells proliferate in SHPT, forming colonies PTH-producing cells intricately intertwined with adipocytes.

Suppression of parathyroid hormone production in vitro and in vivo by RNA interference.

We used RNA interference, which causes sequence-specific degradation of target mRNAs to suppress the production of parathyroid hormone by cells of patients with secondary hyperparathyroidism in vitro and in vivo. Transfection of small interfering RNA (siRNA) against human parathyroid hormone into monolayers of parathyroid cells cultured from these patients caused a dose-dependent decrease of secretion and mRNA levels with 80% or more suppression using 40 nM siRNA. Parathyroid cells cultured on non-adherent plastic produced spheroid cell aggregates which secreted parathyroid hormone for more than 150 days. Transfection of these spheroids with 50 nM targeted siRNA decreased parathyroid hormone production to 20% of the control level, with half of them being suppressed for 50 days. When parathyroid cells were transplanted into the livers of athymic nude mice, plasma human parathyroid hormone rose to 100-300 pg/ml within one month and remained at about this level for at least 39 days. Systemic delivery of hormone-targeted siRNA into these mice caused a dose-dependent suppression of circulating human parathyroid hormone for at least one month, with a maximum 80% suppression achieved by 80 microg of siRNA. Our study shows that hormone secretion by parathyroid cells of patients with secondary hyperparathyriodism can be suppressed both in vitro and in vivo by targeted siRNAs.

Enhancement of permeability in endothelial cells for the development of an antithrombogenic bioartificial hemofilter.

For the development of an antithrombogenic bioartificial hemofilter, in which the inner surface of hollow fibers is lined by endothelial cells, it is essential to increase the permeability of the cells in order to achieve a sufficient ultrafiltrate. We tried to increase it by using an actin microfilament polymerization inhibitor, cytochalasin B (CyB). Fifty microg/mL CyB was added for 2 h to the culture medium of confluent rat glomerular endothelial cells (RGEC) and human umbilical vein endothelial cells (HUVEC). Under the 130 mmHg hydrostatic pressure, the CyB-treated group produced significantly more ultrafiltration than the non-treated control group and this increase was maintained for at least 7 days. Horseradish peroxidase (HRP) permeability acutely and reversibly increased in the CyB-treated group compared with the non-treated control group. Scanning electron microscopy revealed a larger average diameter and increased number of fenestrae on the CyB-treated endothelial cells, compared with the non-treated cells. This phenomenon also lasted for at least 7 days. The platelet adherence test showed that CyB did not deteriorate the antithrombogenic property of endothelial cells. These results indicate that CyB is potentially applicable for the enhancement of endothelial cell permeability in an antithrombogenic bioartificial hemofilter.

Prevention of LLC-PK(1) cell overgrowth in a bioartificial renal tubule device using a MEK inhibitor, U0126.

A common approach to construct a bioartificial renal tubule system is to utilize renal tubular cells seeded in porous polymer membrane hollow fibers. We have reported that overgrowth of renal tubular cells was not beneficial for the transport and reabsorption functions of bioartificial tubules. Therefore, long-term maintenance of a confluent monolayer of cells in hollow fibers is essential and technically challenging. In this study, we examined whether MEK inhibitor, U0126, could maintain the monolayer of Lewis-lung cancer porcine kidney 1 (LLC-PK(1)) cells on polystyrene plates and in a dialysis module housing hollow fibers made of ethylene vinyl alcohol (EVAL). We also evaluated the leakage of urea nitrogen (UN) and creatinine (Cr) through the cell-lined hollow fibers, and reabsorption of glucose and sodium by the cells, comparing the U0126-treated cells with nontreated cells in the module. Treatment with 50micromol l(-1) U0126 prevented the overgrowth of cells cultured on polystyrene plates. Moreover, U0126-treatment reduced the leakage of UN, and increased the reabsorption of electrolytes in 65cm(2) modules. Scanning electron microscopy revealed that it also prevented the overconfluence of cells in modules. Therefore, application of U0126 is a potentially effective method to improve the performance of the device.

Vitamin D receptor agonist VS-105 directly modulates parathyroid hormone expression in human parathyroid cells and in 5/6 nephrectomized rats.

Vitamin D receptor (VDR) agonists (VDRAs) are commonly used to treat secondary hyperparathyroidism (SHPT) associated with chronic kidney disease (CKD). Current VDRA therapy often causes hypercalcemia, which is a critical risk for vascular calcification. Previously we have shown that a novel VDRA, VS-105, effectively suppresses serum parathyroid hormone (PTH) without affecting serum calcium levels in 5/6 nephrectomized (NX) uremic rats. However, it is not known whether VS-105 directly regulates PTH gene expression. To study the direct effect of VS-105 on modulating PTH, we tested VS-105 and paricalcitol in the spheroid culture of parathyroid cells from human SHPT patients, and examined the time-dependent effect of the compounds on regulating serum PTH in 5/6 NX uremic rats (i.p. 3x/week for 14days). In human parathyroid cells, VS-105 (100nM) down-regulated PTH mRNA expression (to 3.6% of control) and reduced secreted PTH (to 43.9% of control); paricalcitol was less effective. VS-105 effectively up-regulated the expression of VDR (1.9-fold of control) and CaSR (1.8-fold of control) in spheroids; paricalcitol was also less effective. In 5/6 NX rats, one single dose of 0.05-0.2µg/kg of VS-105 or 0.02-0.04µg/kg of paricalcitol effectively reduced serum PTH by >40% on Day 2. Serum PTH remained suppressed during the dosing period, but tended to rebound in the paricalcitol groups. These data indicate that VS-105 exerts a rapid effect on suppressing serum PTH, directly down-regulates the PTH gene, and modulates PTH, VDR and CaSR gene expression more effectively than paricalcitol.

Upregulation of α3ß1-Integrin in Podocytes in Early-Stage Diabetic Nephropathy.

Background. Podocyte injury plays an important role in the onset and progression of diabetic nephropathy (DN). Downregulation of α3ß1-integrin expression in podocytes is thought to be associated with podocyte detachment from the glomerular basement membrane, although the mechanisms remain obscure. To determine the mechanism of podocyte detachment, we analyzed the expression levels of α3ß1-integrin in podocytes in early and advanced stages of DN. Methods. Surgical specimens from DN patients were examined by in situ hybridization, and the expression levels of α3- and ß1-integrin subunits in glomeruli of early (n = 6) and advanced (n = 8) stages were compared with those of normal glomeruli (n = 5). Heat-sensitive mouse podocytes (HSMP) were cultured with TGF-ß1 to reproduce the microenvironment of glomeruli of DN, and the expression levels of integrin subunits and the properties of migration and attachment were examined. Results. Podocytes of early-stage DN showed upregulation of α3- and ß1-integrin expression while those of advanced stage showed downregulation. Real-time PCR indicated a tendency for upregulation of α3- and ß1-integrin in HSMP cultured with TGF-ß1. TGF-ß1-stimulated HSMP also showed enhanced in vitro migration and attachment on collagen substrate. Conclusions. The results suggested that podocyte detachment during early stage of DN is mediated through upregulation of α3ß1-integrin.

Macho-1 functions as transcriptional activator for muscle formation in embryos of the ascidian Halocynthia roretzi.

Various kinds of maternal factor that play crucial roles in embryogenesis are present and localized in the ascidian egg cytoplasm. Localized maternal mRNA of the macho-1 gene is a muscle determinant in the embryo of the ascidian Halocynthia roretzi. The macho-1 protein has a zinc-finger domain and accumulates in nuclei, being expected to function as a DNA-binding transcription factor. In the present study, we show that macho-1 is, indeed, a DNA-binding transcriptional activator, and directly or indirectly regulates the expression of six downstream genes. Macho-1 was required and sufficient for expression of the muscle actin, myosin, calcium transporter, myogenic factor, Tbx6, and snail genes, whose expression is initiated in muscle blastomeres at the cleavage stages in normal embryos. Furthermore, when macho-1 conjugated with a transcription-repression domain of Drosophila engrailed (En(R)) was expressed in embryos, it repressed expression of these downstream genes. In contrast, expression of macho-1 fused with a transcription-activation domain of VP16 caused ectopic expression of these muscle genes in non-muscle blastomeres. PCR-assisted binding-site selection and gel-retardation assay showed that macho-1 protein binds to the consensus target sequence (TGGGTGGTC) for GLI/Zic-family proteins, and that three guanine residues with underlines are crucial for the specificity. The 5' promoter region of the muscle actin gene supported expression of the reporter gene only in muscle cells at late stage. By contrast, when the target sequence was added to the promoter, it well reproduced early expression of muscle actin at the cleavage stage, indicating that macho-1 can recognize the target sequence in vivo. In conclusion, the maternal muscle determinant macho-1 functions as a transcription factor that positively regulates gene expression for muscle formation in ascidian embryos.

Impact of parathyroidectomy on serum FGF23 and soluble Klotho in hemodialysis patients with severe secondary hyperparathyroidism.

CONTEXT: Klotho is a transmembrane protein that functions as a coreceptor for fibroblast growth factor 23 (FGF23). Klotho is cleaved and released into the circulation; however, the main site of production, physiological role, and regulation of soluble Klotho in humans are largely unknown. OBJECTIVE: The aim of this study was to determine the impact of parathyroidectomy (PTx) on serum FGF23 and soluble Klotho levels in patients with severe secondary hyperparathyroidism. DESIGN AND SETTING: This was a prospective, single-arm trial conducted at Tokai University School of Medicine. PATIENTS: Thirteen hemodialysis patients with severe secondary hyperparathyroidism who were candidates for PTx participated in the study. INTERVENTIONS: All patients underwent total PTx with forearm autotransplantation. MAIN OUTCOME MEASURES: We evaluated changes in serum FGF23 and soluble Klotho levels for 90 days after PTx. Other biochemical parameters related to mineral and bone metabolism were also assessed. RESULTS: At baseline, serum FGF23 levels were markedly elevated, whereas serum soluble Klotho levels were modestly decreased. PTx resulted in a marked, progressive decline in serum FGF23 levels together with significant reductions in serum calcium, phosphorus, and intact PTH levels. The serum soluble Klotho levels were reduced 13% from baseline on the day after PTx; however, these levels then increased progressively, reaching 34% above the postoperative values. CONCLUSIONS: Our results suggest that the parathyroid gland is not the major site of soluble Klotho production in patients with end-stage renal disease, and the production of Klotho by other organ(s) is affected by alterations in mineral metabolism or medications taken after PTx.