| IntroductionPeritoneal Dialysis ( PD) is an adecpuate and effective renal replacement therapy for end - stage renal failure. The longevity of PD depends on the long - term preservation of the peritoneum as a dialytic membrane. During dialysis, the Peritoneal Mesothelial Cells (PMCs) acting as a biological barrier is continuously exposed to unphysiologic peritoneal dialysate because of its low pH, hyperosmolality and high glucose content. The unphysiologic dialysate resulted in significant morphological changes; this include increase of the PMCs volume, loss of microvilli, denudation of PMCs, increased accumulation of extracellular matrix (ECM) under the PMCs. Thickening and diabeti-form reduplication of basement membrane of mesothelium and stroma blood vessels of peritoneum were encounted in some patients. The extremely high glucose concentration (83 to 234 mmol/L) in the dialysate was supposed to contribute to these changes. The changes in the structure of peritoneum may influence its dialysis efficiency, peritoneum function changed markedly: increased permeability, increased glucose absorption and decreased ultrafiltration ( UF) . Therefore highconcentration glucose (Glc) may severely damage the structure and functions of PMCs, which is considered to be the primary cause of the decline in UF and dialysis efficacy. It is necessary to study the mechanism of peritoneal glucose absorption and to investigate the mechanism by which high glucose damage PMCs during PD. The primary culture of PMCs will provide the cell model for the research.Glucose uptake and utilization are the functional coupling and combining effect of both glucose transporters ( GLUTs) and Hexoki-nases ( HKs). HKs take the central play in this process via phospho-rylating Glc to yield Glc -6 - phorsphate. By this mechanism, HKs keep the favorable downhill concentration gradient that permits facilitated Glc entry cells. In addition, they initiate all subsequent pathways of Glc utilization. So the change of HKs activity may determine the Glc utilization.The accomplished animal experiments showed that protein kinase C ( PKC ) induced the activity of HKs in primary cultured rat mesang-ial cells and increased net Glc utilization. This plays an important role in the progression of diabetic nephropathy. During PD, the PMCs are exposed to dialysates containing high concentration of glucose. Does this kind of exposure stimulate the expression of HKs in PMCs?The expression and regulation of HKs in PMCs have not been explored. The present study investigated the effect of high glucose and phorbal esters on HKs activity in primary cultured PMCs and the association between HKs and glucose uptake, which might provide the theories basis for how to reduce the absorption of glucose and increase UF.Materials1. Experimental animals: Albino male rats of the Sprague Dawley (SD) strain were used.2. Experimental cells; Rat peritoneal mesothelial cells were isolated by primary culture.3. Reagents relating to cell culture and identification.4. Reagents relating to measurement of hexokinases activity and glucose concentration.Methods1. Enzymatic disaggregation used for primary culture2. Immunofluorescent staining used for identification3. Standard G6PDH - coupled assay used for measurement of HKs4. Observing the regulating effect of each treatment factor5. Colorimetric assay used for measurement of net Glc utilizationResults1. Confluent mesothelial cells were obtained by limited treatment with trypsin, it took 5 to 8 days to reach confluency. Immunofluorescent staining of confluent cells demonstrated that all cells reacted positively to cytokeratin.2. The basic activity of HKs in rat PMCs of primary culture was (4. 80 +0.39) U/g protein.3. Glucose induced the expression of HKs in a dose - dependent manner. At concentration of 2. 5 % and 4. 25 % Glc, the increase in HKs activity were 65.9 % and 80 % ( P <0. 05) , respectively above the level in the control, the activity of HKs induced by 4.25 %... |
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