Influence On GRP75 Expression Of NRK-52E Cells In High Glucose Ambience By Regulating QDPR Gene

Objectives To explore the effect of quinoid dihydropteridine reductase(QDPR)gene expression level on glucose-regulated protein 75(GRP75)in renal tubular epithelial cell NRK-52 E in high glucose ambience and the probable underlying mechanism of QDPR gene in diabetic nephropathy(DN).Methods Western blot was used to detect the content of GRP75 proetin in renal cortex of DN rats(OLETF rats)and control rats(LETO rats);transfected lentivirus to NRK-52 E cells,and established the models of QDPR gene overexpression and knockdown and respective control.After cultured all groups for 72 hours in 5.4mmol/L and 30mmol/L glucose medium respectively,the cells were divided into several groups: 1 NRK-52 E control group(NC)2 NRK-52 E exposed to high glucose group(NHG)3 Lentivirus overexpression control group(LV-OCON)4 Lentivirus overexpression control exposed to high glucose group(LV-OCON-HG)5 Lentivirus overexpressed QDPR group(LV-QDPR)6 Lentivirus overexpressed QDPR exposed to high glucose group(LV-QDPR-HG)7 Lentivirus knockdown SHDNA control group(LV-SHCON)8 Lentivirus knowdown SHDNA control exposed to high glucose group(LV-SHCON-HG)9 Lentivirus knockdown QDPR SHDNA group(LV-SHQDPR)10 Lentivirus knockdown QDPR SHDNA exposed to high glucose group(LV-SHQDPR-HG).The expression level of GRP75 protein in cell groups were measured by Western blot and cell cycle profiles were determined by flow cytometric propidium iodide method.Results 1 The Western blot analysis showed that the amount of GRP75 protein significantly decreased in OLETF rats renal cortex than that in contorl rats[(1.53±0.27)vs(0.79±0.26),P<0.01];2 Compared with NC group,NHG group downregulated the expression of GRP75 protein[(0.62±0.03)vs(0.46±0.06),P<0.05];3 Compared with NC group,NHG group increased the G0/G1 phase cells population[(39.80±1.31)% vs(50.35±0.33)%,P<0.05] and decreased the S phase cells population[(48.55±1.85)% vs(37.17±0.10)%,P<0.05];4 The NRK-52 E cell of overexpression QDPR gene were established successfully;5 The NRK-52 E cell of knockdown QDPR gene were established successfully;6 GRP75 expression level remained similar both in LV-QDPR and LVOCON group(P > 0.05)when cells were cultured in normal glucose environment;compared with LV-QDPR and LV-OCON-HG group,the expression of GRP75 protein was significantly decreased in LV-QDPR-HG group[(0.95±0.07)?(0.85±0.10)vs(0.45±0.16),P< 0.05];7 Compared with LV-OCON-HG group,LV-QDPR-HG group increased the G0/G1 phase cells population[(43.73±0.48)% vs(61.87±0.17)%,P<0.01] and decreased the S phase cells population[(42.42±0.66)% vs(25.29±0.12)%,P<0.01)];8 The content of GRP75 in LV-SHQDPR-HG and LV-SHCON-HG showed no difference;9 Compared with LV-SHCON-HG group,LV-SHQDPR-HG group decreased the S phase cells population[(42.78±1.46)% vs(38.59±0.16)%,P<0.01],increased the G2/M phase cells population [(13.86±0.87)% vs(16.69±0.50)%,P<0.01],but had no influence on G0/G1 phase cells population.Conclusions 1 GRP75 protein decreased in DN,which suggested that GRP75 was involed in the pathogenesis of DN;2 Overexpression QDPR gene in NRK-52 E downregulated GRP75 protein and induced cell cycle arrest,which suggested QDPR gene may influence the occurrence and process of DN by regulating the expression of GRP75 protein and cell cycle in high glucose ambience.