Direct exposure of renal tubular brush-border membranes (BBM) to free cadmium (Cd) causes a reduction in phosphate (Pi) transport capacity. Biochemical mechanism of this reduction was investigated in the present study. Renal proximal tubular brush-border membrane vesicles (BBMV) were isolated from rabbit kidney outer cortex by Mg precipitation method. Vesicles were exposed to 50~200 muM CdCl2 for 30 min, then the phosphate transporter activity was determined. The range of Cd concentration employed in this study was comparable to that of the unbound Cd documented in renal cortical tissues of Cd-exposed animals at the time of onset of renal dysfunction. The rate of sodium-dependent phosphate transport (Na+-Pi cotransport) by BBMV was determined by 32P-labeled inorganic phosphate uptake, and the number of Na+/-Pi cotransporters in the BBM was assessed by Pi-protectable 14C-labeled phosphonoformic acid ((14C)PFA) binding. The exposure of BBMV to Cd decreased the Na+-Pi cotransport activity in proportion to the Cd concentration in the preincubation medium, but it showed no apparent effect on the Pi-protectable PFA binding. These results indicate that an interaction of renal BBM with free Cd induces a reduction in Na+-Pi cotransport activity without altering the carrier density in the membrane. This, in turn, suggest that the suppression of phosphate transport capacity (Vmax) observed in Cd-treated renal BBM is due to a reduction in Na+-Pi translocation by existing carriers, possibly by Cd-induced fall in membrane fluidity.
Animals ; Cadmium ; Cadmium Chloride ; Foscarnet* ; Kidney ; Membrane Fluidity ; Membranes* ; Phosphate Transport Proteins*

Spirodela polyrrhiza is a floating plant widely used in biomass utilization and eutrophication phytoremediation. It becomes a common aquatic plant everywhere with the increasingly serious eutrophication. It has been reported that S. polyrrhiza has a good effect on the remediation of eutrophication water. In order to study the absorption and transportation of phosphorus in S. polyrrhiza, we extracted RNA from S. polyrrhiza and then reverse transcribed it into cDNA, which was used as a template to amplify a specific fragment. The full-length sequence of the open reading frame (ORF) was 1 620 bp, encoding 539 amino acids, named SpPHT1;1, and the accession number in GenBank was MN720003. Bioinformatical analysis showed that SpPHT1;1 had no intron. The protein it encoded was a stable, hydrophobic protein with 11 transmembrane domains. SpPHT1;1 structure was similar to that of major facilitator superfamily (MFS) superfamily members. The cluster analysis showed that SpPHT1;1 was closely related to ZMPHT2 in maize and SBPHT1-8 in sorghum. So, it might belong to plant PHT1 family. The expression of SpPHT1;1 in leaf was significantly more than that of root under normal phosphorus condition. Low phosphorus condition could promote gene expression, and the relative expression level of SpPHT1;1 arrived at the peak at 48 h both in root and leaf. High phosphorus condition could inhibit gene expression. These results indicated that SpPHT1;1 expression would be affected by external phosphorus concentration. The results of this study are helpful for further research on the function of phosphate transporter. It also can provide theoretical basis for further development and utilization of S. polyrrhiza.
Araceae/genetics* ; Biodegradation, Environmental ; Cloning, Molecular ; DNA, Complementary ; Phosphate Transport Proteins/genetics*

Cisplatin treatment increases the excretion of inorganic phosphate in vivo. However, the mechanism by which cisplatin reduces phosphate uptake through renal proximal tubular cells has not yet been elucidated. We examined the effect of cisplatin on Na+-dependent phosphate uptake in opossum kidney (OK) cells, an established proximal tubular cell line. Cells were exposed to cisplatin for an appropriate time period and phosphate uptake was measured using [32P]-phosphate. Changes in the number of phosphate transporter in membranes were evaluated by kinetic analysis, [14C]phosphonoformic acid binding, and Western blot analysis. Cisplatin inhibited phosphate uptake in a time- and dose-dependent manner, and also the Na+-dependent uptake without altering Na+-independent uptake. The cisplatin inhibition was not affected by the hydrogen peroxide scavenger catalase, but completely prevented by the hydroxyl radical scavenger dimethylthiourea. Antioxidants were ineffective in preventing the cisplatin-induced inhibition of phosphate uptake. Kinetic analysis indicated that cisplatin decreased Vmax of Na+-dependent phosphate uptake without any change in the Km value. Na+-dependent phosphonoformic acid binding was decreased by cisplatin treatment. Western blot analysis showed that cisplatin caused degradation of Na+-dependent phosphate transporter protein. Taken together, these data suggest that cisplatin inhibits phosphate transport in renal proximal tubular cells through the reduction in the number of functional phosphate transport units. Such effects of cisplatin are mediated by production of hydroxyl radicals.
Antioxidants ; Blotting, Western ; Catalase ; Cell Line ; Cisplatin ; Epithelial Cells* ; Foscarnet ; Hydrogen Peroxide ; Hydroxyl Radical ; Kidney ; Kinetics ; Membranes ; Opossums ; Phosphate Transport Proteins

BACKGROUNDC-Jun N-terminal kinase (JNK) signaling pathway and ankylosis gene (ANK) play a critical role in endplate chondrocytes degeneration. The purpose of this study was to investigate whether the expression levels of ANK was associated with the activation of JNK.

METHODSCartilage endplates of 49 patients were divided into the control group (n = 19) and the experimental group (n = 30). The patients in the control group were graded 0 and those in the experimental group were graded I-III according to Miller's classification. Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro. The inverted phase contrast microscope, teluidine blue staining, HE staining, real time RT-PCR, and MTT were used to observe morphological appearances, biological characteristics, and growth curve of endplate chondrocytes from the cartilage endplate of the two groups. Real time RT-PCR and Western blotting were used to analyze the mRNA and protein expression levels of associated factors in the degeneration process in the cultured endplate chondrocytes with or without subjected SP600125.

RESULTSThe expression levels of type II collagen, aggrecan, and ANK in endplate chondrocytes of experimental group were lower than that of control group and phosphorylation level of JNK in the experimental group which was higher than that in the control group. Application of JNK phosphorylation inhibitor to degeneration chondrocytes resulted in a marked decrease in the phosphorylation level of JNK and a significant increase in the expression levels of type II collagen, aggrecan, and ANK.

CONCLUSIONThe degeneration of the human cervical endplate chondrocytes might be promoted by JNK phosphorylation by down-regulating the expression of ANK.

Adult ; Aged ; Anthracenes ; pharmacology ; Cells, Cultured ; Cervical Vertebrae ; metabolism ; pathology ; Chondrocytes ; metabolism ; pathology ; Down-Regulation ; Female ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Middle Aged ; Phosphate Transport Proteins ; genetics ; physiology ; Phosphorylation

Three-dimensional (3D) cultures are known to promote cell differentiation. Previously, we investigated the differentiation of rat dermal fibroblasts to α-smooth muscle actin (α-SMA)-positive myofibroblasts through transforming growth factor (TGF)-β production using a 3D culture model. Here, we investigated the phenotypic change from dermal mesenchymal cells (mostly fibroblasts) to osteoblast-like cells, being inspired by the roles of smooth muscle cells or fibroblasts during vascular calcification. Spindle-shaped cells that grew in heterologous populations out of dermal explants from 2-day-old Wistar rats were cultured within a collagen matrix. α-SMA and alkaline phosphatase (ALP) messenger RNA (mRNA) levels initially increased, followed by a rise in Runx2 and osteocalcin (OCN) mRNA levels without calcification. Calcium deposits were produced in the presence of a high concentration of inorganic phosphate (2.1 mM) or β-glycerophosphate (βGP, 10 mM) after 2 weeks of culture, and both were sensitive to an inhibitor of type III phosphate transporters. An ALP inhibitor decreased only βGP-induced calcification. Inhibition of TGF-β type-I receptors attenuated ALP mRNA levels and βGP-induced calcification, suggesting that endogenous TGF-β stimulates ALP activity and then βGP breakdown. An increase in the number of cells embedded in the collagen gel enhanced the mRNA levels of Runx2 and OCN, but not of ALP. Collectively, several factors are likely to promote the differentiation of dermal mesenchymal cells into osteoblast-like cells and ectopic calcification in a 3D collagen matrix, implying the utility of these cells as a potential autologous cell source for tissue engineering.
Actins ; Alkaline Phosphatase ; Animals ; Calcium ; Cell Differentiation ; Collagen ; Dermis ; Fibroblasts ; Myocytes, Smooth Muscle ; Myofibroblasts ; Osteocalcin ; Phosphate Transport Proteins ; Rats* ; Rats, Wistar ; RNA, Messenger ; Tissue Engineering ; Transforming Growth Factors ; Vascular Calcification