Effect Of High Concentration Glucose On Activity Of Aldose Reductase In Peritoneal Mesothelial Cells

Effect of high concentration of glucose on activity of aldose reductase in peritoneal mesothelial cellsPrefaceAlthough it is wildly assumed that exposure of peritoneal mesothelial cells (PMC) to high glucose - based dialysis solution contributes to the damage of peritoneal membrane which effect the long - term peritoneal dialysis, the precise role of glucose (GS) in the pathogenesis of damage to peritoneal membrane is unclearly. The activation of aldose reductase ( AR) ,the rate - limiting enzyme of polyol pathway (PP) ,by increasing glucose concentrations, has been widely implicated in the pathogenesis of diabetic complications such as retinopathy, neuropathy, and nephropathy. Part of this evidence relates to morphological studies of the peritoneal membrane that have identified vascular alterations similar to those described for diabetic microangiopathy, the peritoneum of peritoneal dialysis patients is constantly exposed to glucose concentrations far in excess of those found in diabetic plasma, therefore it is presumed that polyol pathway may also be implicated in the pathogenesis of the impact of glucose on PMC . Peritoneal inflammation and peritoneal dialysis can stimulate peritoneal macrophage to produce a vast amount of nitric oxide (NO) and NO can modulate the polyol pathway. We observed the effect of high glucose, hyperosmolality and NO on AR activity and AR gene expression of cultered human peritoneal mesothelial cells (HPMC) and study the inhibitive effect of Aminoguanidine ( AG) on AR activity of PMC. To explore the mechanism that high glucose impair PMC in the aspect of PP and provide a rationale for the use of aldose reductase inhibitors (ARI) to alleviate glucose -induced damage in PMC.Materials and MethodsMAIN REAGENTSMl99 medium and DL - glyceraldehyde were bought from Shenyang Boer-mei Reagent corporation. TRIZOL, Aldose reductase primers and Reverse Transcription Polymerase Chain Reaction ( RT - PCR) kit were purchased from Shenyang Lianxing Reagent corporation. The PCR amplification apparatus was Bi-ometra TP .Methods1. HPMC culture and treatmentMesothelial cells were isolated from the omental tissue of consenting patients undergoing elective abdominal surgery by typsin - EDTA disaggregation. HPMCs were grown to confluence and then changed to 2% fetal calf serum ( FCS) medium for 24 hours for growth arrest prior to stimulation.To examine the effect of glucose or mannitol on AR activity and AR gene expression,growth - arrest HPMC were incubated with D - glucose(1. 5% ,2. 5% ,4. 25% ) or mannitol ( 1. 5% ,2. 5% ,4. 25% ) for time periods up to 48 hours.To examine the effect of NO on AR activity and AR gene expression, HPMCs were treated with D - glucose (1.5%) in presence of increasing NO concentration(0. 5mmol/L, 1. 0mmol/L,2. Ommol/L) .To examine the effect of AG on AR activity, Confluent HPMCs were maintained in M199 medium supplemented with 10% FCS, 1. 5% GS andlOmmol/ LAG for 6 days.2. MethodsAldose reductase activity was measured on spectrophotometer by monitoring the decrease in absorbance of NADPH at 340nm. AR mRNA expression was measured by semi - quantive reverse transcript PCR.3. Statistical analysisStatistical analysis between treatments was performed using analysis of variance and students t test. P value less then 0.05 was regarded as significant.Result1. Effect of glucose or mannitol on AR activity of in PMC 1.1 Dose - dependent relation between hyperglycemin or hyperosmolality and AR activity in PMCAR activity increased as glucose or mannitol concentration increased, but the effect of mannitol was lower than that of glucose , at the concentration of 2. 5% and 4.25% AR activity of glucose groups has remarkably increased as compared with mannitol groups.1. 2 Time - dependent relation between hyperglycemin and AR activity in PMCGlucose induced a time -dependent increase in AR activity . This increase became significant at 24 and 48 hours.2. Effect of glucose or mannitol on AR mRNA expression by PMCThe increase in AR mRNA expression was caused by increasing concentration of glucose or mannitol, however, the effect of mannitol was significantly lower than that of glucose.3. Effect of NO on AR activity in PMC under high glucose3.1 Dose dependence of the effect of NO on AR activity in PMCWhen incubated for 6 hours a dose - dependent increase in AR activity wasobserved with increasing NO concentration. This increase became significant at1. Ommol/L and 2. Ommol/L3. 2 Time dependence of the effect of NO on AR activity in PMC Incubation of PMC with glucose in the presence of NO( 1. Ommol/L) resulted in a time — dependent increase in AR activity. A significant increase was observed at 6 and 12 hours. In contrast in the presence of NO glucose -induced increase of AR activity was greater than glucose alone. There was a significant increase at 6 and 12 hours.4. Effect of NO on AR mRNA expression by PMC under high glucose The amount of AR mRNA increased significantly as NO concentration increased.5. Effect of AG on AR activity in PMCWhen PMC was incubated with glucose in the presence of AG (lOmmol/L) for 6 days,there was a decrease in AR activity,but the difference compared to control became not significant.Conclusions1. High glucose can up - regulate AR activity and its gene expression of peritoneal mesothelial cells.2. Hyperosmolality can up -regulate AR activity and its gene expression of peritoneal mesothelial cells.3. Nitric oxide can up - regulate AR activity and its gene expression of peritoneal mesothelial cells under high glucose.4. Aminoguanidine can not significantly inhibit AR activity of peritoneal mesothelial cells.