Effects Of Lipopolysaccharide On Glucose Transporter-1 Expression In Cultured Human Mesothelial Cells

ObjectivePeritoneal dialysis (PD) , is the important therapy way of renal failure, whose principle is that water and solute in blood and dialysate are exchanged through peritoneum,a semipermeable membrane, so the unnecessary water and metabolic waste can be cleared. But patients who use dialysate of hypertonic glucose may cause peritoneum infection easily, which is the primary reason of uLtrafiltration failure.Ultrafitration failure (UF) may be devided into four types, in which type Ⅰ is characterized by rapid absorption of glucose and earlier disappearance of osmotic pressure. However,the mechanism about how type Ⅰ UF happen is not very clear now, and it relates to the long - term application with dialysate of high glucose and peritonitis. It is suggested that peritonitis may induce inflammatory cells release many kind of mediators of inflammation and cytokines, so that the regulation of matrix protein and proliferation of blood capillary may happen, and solute of micromolecule including glucose is transported more rapidly and the clearance of peritoneum to water descend. This manifest UF. On ultramicro-structure, our study try to illuminate how peritonitis lead to UF.It is reportered that there are three glucose transporers in Human peritonealmesothelial cells ( HPMCs) , in which glucose transporter - 1 is primary glucose transporter in HPMCs. It has not been reportered that how the expression of GLUTl change during peritonitis. By studiing lipopolysaccharide (LPS) stimulating HPMCs and observing the expressiong of HPMCs GLUTl, this study try to reveal the mechanism of UF.Methods1. Enzymatic disaggregation was used for primary culture and passage of HPMCs.2. Indirect immunofluorescence was used for identification of HPMCs.3. GLUTl expression was detected by immunocytochemistry, and the result was analyzed by image analytical system.4. GLUTl mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) technology.5. Hexokinase assay was used for measurement of concentration of glucose in cell culture fluid, and then net glucose utilization was calculated.6. Statistical analysis : All data were processed with SPSS 10.0. T test and ANOVA was used to test the differences between goups, and all data were presented as x ± s . P <0. 05 was considered to be statistically significant.Results1. Identification of HPMCs: the result of indirect immunofluorescence showed that the antibody of cytokeratin dyeing presented green fluorescence in endochylema and that is the character of HPMCs.2 . HPMCs GLUTl expression in normal glucose when different -concentration LPS stimulated. (1) The result of Immunocytochemical staining in light microscope showed that HPMCs express GLUTl generally which is yellow and both in plasma membrane and endochylema, while not in cell nucleus;(2) Expression of GLUTl rised while the concentration of LPS increased compared to the control ( P <0. 01 ).3. HPMCs GLUT1 expression in normal glucose when LPS stimulated for different periods by immunocytochemical staining. When stimulated for one hour, GLUT1 expressed mainly in the cell membrane which presented 11 a ring" , while few in the endochylema;after 6 hour GLUT1 expression rised gradually and to the most at 24 hour( P < 0. 01).4. The result of GLUT1 mRNA detected by RT - PCR showed that (1) GLUT1 mRNA expression rised when the concentration of LPS increased. (2) GLUT1 mRNA expression went up when LPS stimulate gradually.5. The effect of LPS on net glucose utilization: LPS accelerated net glucose utilization in a dose - dependent manner and time - dependent manner.6. Compared to the control, LPS may promote HPMCs GLUT1 expression significantly in high glucose.Conclusion1. HPMCs express GLUT1 generally.2. LPS may promote GLUT1 expression on protein and mRNA of HPMCs in a dose - dependent and time - depenent manner.3. LPS may accelerate net glucose utilization of HPMCs significantly.4. LPS may promote GLUT1 expression of HPMCs in high glucose significantly.5. LPS may participate the process of UF by stimulating HPMCs to up -regulate GLUT1 expression.