High Glucose Regulates The Expression Of Hexokinase Via P38MAPK And ERK1/2 Signal Pathway In Human Peritoneal Mesothelial Cells

Introduction and objectiveUltrafiltration failture is a serious complication in continuous ambulatory peritoneal dialysis ( CAPD) and has been a main cause for patients in end-stage renal failure withdrawn from the effective renal replacement therapy. Alterations in peritoneal transport associated with the time going on PD, this is followed by faster glucose reabsorption, and a dissipation of the transperitoneal osmotic gradient , as a consequence, a reduced ultrafilation. The increased glucose absorption is an important characteristic for decreased ultrafiltration, so it is necessary to study the mechanism of peritoneal glucose absorption.Glucose uptake and utilization are the functional coupling and combining effect of both glucose transporter ( GLUT) and hexokinase ( HK). HK plays a central role in this process via phosphorylating glucose to yield glucose -6-phosphate. By this mechanism, HK keeps the favorable downhill concentration gradient that permits facilitated glucose enters the cells. In addition, they initiate all subsequent pathways of glucose utilization. As a biological barrier, the human peritoneal mesothelial cells ( HPMCs) are continuously exposed to peritoneal dialysate during dialysis, the activity and regulation of HK in HPMCs may be relevant to the uptake of glucose by peritoneum. However, the expression and regulation of HK in HPMCs have not been fully addressed.Duing peritoneal dialysis, the peritoneum is directly and continuously exposed to unphysiologic peritoneal dialysis fluid because of its low pH, hyperos-molality and high glucose content. Continuous exposure of the peritoneal membrane to high glucose may result in changes in HPMC biology and finally lead toperitoneal functional injury. Numerous studies have revealed that protein kinase C (PKC) is activated in diabetic and high glucose conditions. The mechanism responsible for the activation of PKC by high glucose is related to an elevation of de novo diacylglycerol (DAG) levels from the glycolytic intermediates. In addition, DAG-PKC was also activated in HPMCs. PKC propagates the physiologic responses via an array of down-stream signals, such as mitogen-activated protein kinase ( M APK). Ultimately, transmitted signals regulate the transcripton of gene responsible for the key cellular responses f such as proliferation, differentiation, and apoptosis.The MAPK family includes the extracellular signal-regulated protein kinase ( ERK1/2) and p38 mitogen-activated protein kinase ( p38MAPK) , ERK1/2 as well as p38 MAPK are activated in diabetic conditions and high glucose media. PKC plays an important role in the process of ERK1/2 activation. The mechanism of high glucose-induced p38 MAPK activation in mesangial cells was shown to be mediated possibly via reactive oxygen species rather than PKC activation. In contrast, in vascular smooth muscle cells, PKC-dependent activation of p38 MAPK has been proposed, and glucose at extremely elevated levels can also activate p38 MAPK by hyperosmolarity via the PKC-independent pathway. In HPMCs, it has been reported that the activation of p38MAPK pathway by high glucose was not due to changes in osmolality. But the mechanism of p38MAPK and ERK1/2 activation remains undefined in HPMCs when the concentration of glucose equals to that of peritoneal dialysis fluid.Because activation of PKC-MAPK pathway could play a crucial role in mediating the development and progression of high glucose-induced pathophysioligic injury, the use of inhibitors of PKC and MAPK would be valuable therapeutic strategies. Vitamin E ( d-a-tocopherol) can prevent glomerular hyperfiltration as well as albuminuria in streptozotocin (STZ) -induced diabetic rats by inhibiting PKC activation through decreasing DAG levels without changing plasma glucose level. During PD, the HPMCs are exposed to dialysates containing high concentration of glucose. Does this kind of exposure stimulate the expression of HK in HPMCs? The expression and regulation of HK in HPMCs have not been reported. The present study investigated the effect of high glucose on the expressionof HK and the regulatory effect of p38MAPK and ERKl/2 pathway, which might provide theoretical basis on how to decrease the absorption of glucose and increase ultrafiltration.Methods1. HPMCs were isolated from omentum and subcultured after enzymatic digestion. Morphological and immunocytochemical method were used for identification.2. HPMCs were exposed to glucose and mannitol at the concentration of 0. 5, 1.0, 1.5,2.5,4.25%, respectively. Standard G6PDH-coupled assay and temperature sensitive assay were used to detect the activity of total HK and its isozyme. RT-PCR and western blot were performed to determine mRNA and protein expression respectively. Immunocytochemical staining was used for observation of intracellular location.3. HPMCs were exposed to glucose and mannitol at the concentration of 1. 5, 2.5, 4.25% respectively. The expression of phosphorylated p38MAPK and ERKl/2 were detected by western blot with phospho-specific antibodies, P38MAPK and ERKl/2.4. HPMCs were exposed to high glucose at the concentration of 2.5% with or without the p38MAPK inhibitor SB203580, or the upstream kinase of ERK1/ 2 (MEK1/2) inhibitor PD98059. RT-PCR and western blot were performed to determine HK mRNA and protein expression respectively.5. HPMCs were exposed to high glucose at the concentration of 2.5% with or without d-a-tocopherol. The expression of phosphorylated p38MAPK and ERKl/2 were detected by western blot with phospho-specific antibodies, p38MAPK and ERKl/2. RT-PCR and western blot were performed to determine HK mRNA and protein expression, respectively. Standard G6PDH-coupled assay and temperature sensitive assay were used to detect the activity of total HK and its isozyme. The net glucose utilization was assayed by glucose disappearance from medium using hexokinase method.Results1. High glucose increased total HK activity which was due to the selective increase of HK II in HPMCs. The HK II mRNA and protein expression and its activity were increased when exposed to 1.5% glucose. However, the activity of HK I did not change.2. Hyperosmosis increased the HK II mRNA and protein expression and its activity in HPMCs. The HKIImRNA was increased, but the protein and its activity did not change when exposed to 1. 5% mannitol. However, the protein and activity of HK II was increased when the concentrations of mannitol was higher than 2. 5%.3. After exposed to high glucose and hyperosmosis, the HK H protein translocated from cytoplasm to the perinuclear. HK II staining was light brown in the cytoplasm of HPMCs in normal group, but it became stronger and accumulated to perinuclear when exposed to 1.5% glucose or 2. 5% mannitol.4. High glucose and hyperosmosis increased the protein expression of phos-phorylated p38MAPK in HPMCs. Protein of phosphorylated p38MAPK increased when exposed to 1.5% glucose or 2. 5% mannitol. But the protein expression was stronger in glucose.5. High glucose instead of hyperosmosis increased the protein expression of phosphorylated ERK1/2. Protein of phosphorylated ERK1/2 increased in HPMCs when exposed to 1.5% glucose, but it did not change when exposed to mannitol.6. The p38MAPK inhibitor SB203580 decreased the high glucose-induced HK D expression at both the protein and mRNA levels.7. The upstream kinase of ERK1/2 ( MEK1/2) inhibitor PD98059 decreased the high glucose-induced HK H expression at both the protein and mRNA levels.8. d-a-tocopherol decreased the high glucose-induced protein expression of phosphorylated p38MAPK and ERK1/2.9. d-a-tocopherol decreased the high glucose-induced mRNA and proteinexpression of HK II , as well as its activity which lead to the decrease of total HK activity. In addition, the net glucose utilization was decreased.Conclusions1. High glucose and hyperosmosis increased the HK H mRNA and protein expression and its activity in HPMCs. The HK II protein translocated from cytoplasm to the perinuclear.2. High glucose might regulate the expression of HK II through p38MAPK and ERK1/2 pathway in HPMCs.3. Hyperosmosis might regulate the expression of HK II through p38MAPK pathway in HPMCs.4. D-a-tocopherol might prevent high glucose-induced HK II expression by inhibiting p38MAPK and ERK1/2 pathway.