Fischereder Michael, Schröppel Bernd, Wiese Patrick, Fink Monika, Banas Bernhard, Schmidbauer Stefan, Schlöndorff Detlef
Medical Policlinic, Ludwig-Maximilians-University, Munich, Germany.
J Nephrol. 2003 Jan-Feb;16(1):103-9.
Risk factors for peritoneal fibrosis and mesothelial cell (MsC) injury in CAPD are infections and bioincompatibility of the dialysate, including high glucose concentrations. To study a potential link between dialysate and glucose toxicity in MsC, we investigated the expression of facilitative glucose transporters (GLUT), which could contribute to glucose toxicity.
After induction of cell differentiation, MsC were incubated in regular medium or medium with 60 mM D-glucose, 30 mM glucose plus 30 mM mannitol, 60 mM mannitol, PD effluent, or with a cytokine mix. Expression of GLUT1, GLUT3, SGLT and GAPDH/L32 was studied by RNase protection assay. MsC were incubated under identical conditions with 14C-fluoro-deoxy-glucose for 30 minutes and glucose uptake was measured. To estimate Vmax and Km, 14C-fluoro-deoxy-glucose uptake rates were determined over a range of 0.6 to 10 mM unlabeled glucose.
The cytokine mix significantly stimulated GLUT1 expression (3-fold) and GLUT3 (1.7-fold). There was a 1.4-fold increase in GLUT1 (p<0.05) and a 1.7-fold increase in GLUT3 (p<0.05) after incubation in high glucose but not in mannitol or PD-effluent controls. Glucose uptake studies confirmed this increase after incubation in 30 mM (p<0.05) and 60 mM glucose solutions. Kinetic studies showed the Km was approximately 3.7 mM for this transport.
GLUT mRNA expression and glucose uptake are induced by high ambient glucose concentrations and cytokines. Unlike many other cells, MsC are not able to protect themselves from increased glucose concentrations by downregulation of GLUTs. The intracellular glucose concentration may therefore increase during CAPD, affecting growth factor expression and glycosylation, and contributing to glucose toxicity.
持续性非卧床腹膜透析（CAPD）中腹膜纤维化和间皮细胞（MsC）损伤的危险因素包括感染和透析液的生物不相容性，其中透析液的高葡萄糖浓度也有影响。为研究透析液与MsC中葡萄糖毒性之间的潜在联系，我们调查了促进性葡萄糖转运蛋白（GLUT）的表达，其可能与葡萄糖毒性有关。
诱导细胞分化后，将MsC置于常规培养基或含60 mM D-葡萄糖、30 mM葡萄糖加30 mM甘露醇、60 mM甘露醇、腹膜透析流出液的培养基或含细胞因子混合物的培养基中孵育。通过核糖核酸酶保护试验研究GLUT1、GLUT3、钠-葡萄糖协同转运蛋白（SGLT）和甘油醛-3-磷酸脱氢酶/β-肌动蛋白（GAPDH/L32）的表达。将MsC在相同条件下与14C-氟脱氧葡萄糖孵育30分钟并测量葡萄糖摄取。为估算最大反应速度（Vmax）和米氏常数（Km），在0.6至10 mM未标记葡萄糖范围内测定14C-氟脱氧葡萄糖摄取率。
细胞因子混合物显著刺激GLUT1表达（增加3倍）和GLUT3表达（增加1.7倍）。在高糖环境中孵育后，GLUT1增加1.4倍（p<0.05），GLUT3增加1.7倍（p<0.05），而在甘露醇或腹膜透析流出液对照中未出现这种情况。葡萄糖摄取研究证实，在30 mM（p<0.05）和60 mM葡萄糖溶液中孵育后葡萄糖摄取增加。动力学研究表明，这种转运的Km约为3.7 mM。
高环境葡萄糖浓度和细胞因子可诱导GLUT mRNA表达及葡萄糖摄取。与许多其他细胞不同，MsC无法通过下调GLUT来保护自身免受葡萄糖浓度升高的影响。因此，在CAPD过程中细胞内葡萄糖浓度可能会升高，影响生长因子表达和糖基化，并导致葡萄糖毒性。
