Study On Effects Of Lipopolysaccharide On The Transcriptional Activity Of GRP78 And CHOP In Rat Renal Tubular Epithelial Cells

Background and ObjectivesInfection is closely related to the occurrence and progress of chronic kidney disease. Lipopolysaccharide is produced by gram-negative bacteria,which can lead to kidney injury directly or indirectly. Endoplasmic reticulum stress is a kind of critical response mechanism in a variety of harmful stimulation of cells. Excessive endoplasmic reticulum stress can cause dysfunction of endoplasmic reticulum and even cell apoptosis. Glucose regulated protein 78 (GRP78) is a sign protein of endoplasmic reticulum stress, and apoptosis signaling molecule CHOP involve in the endoplasmic reticulum stress and apoptosis in cell injury. Currently it is rarely reported about the presence of endoplasmic reticulum stress in the course of renal tubular epithelial cells' injury by lipopolysaccharide. In this study, different concentrations of lipopolysaccharide continued to stimulate incubated renal tubular epithelial cells (NRK-52E cells) for different time, transcriptional activity of endoplasmic reticulum stress proteins GRP78 and apoptosis signaling molecule CHOP was detected by reverse transcription polymerase chain reaction,in oreder to investigate the role and mechanism of endoplasmic reticulum stress in LPS induced the injury of rat renal tubular epithelial cell.MethodsThe recovered renal tubular epithelial cells (NRK-52E cells), were cultured with 10% fetal calf serum,100 U/ml penicillin and 100 U/ml streptomycin in DMEM/F12 1:1 medium, placed in CO2 incubator 37℃,5% CO2,95% humidity, after 24 to 48 hours, growing to 80%-90% confluence,the cells were passaged.When re-growing to 80% confluence, the cells were cultured in the medium without fetal calf serum for 16 hours to synchronize for the experiment.The cultured cells were incubated in a medium contained LPS in various concentration (Ong/ml,10ng/ml, 100ng/ml,1000ng/ml) and stimulus duration (12h,24h or 36h), total RNA was extracted and was reversely transcribed into cDNA,the expression of GRP78 and CHOPmRNA change were detected by electrophoresis after PCR amplification.Results1. GRP78 and CHOPmRNA had a low basic expression in NRK-52E cells without lipopolysaccharide stimulation in various duration,and there were no significant changes of basic expression with the changes of cultured time.2. RT-PCR results showed that:the lower concentration of lipopolysaccharide (10ng/ml) stimulation in a short time (12h) could induce high expression of GRP78 and CHOP mRNA in NRK-52E cells, compared with the concentration of Ong/ml, the difference was statistically significant (P<0.01).3. RT-PCR results showed that:GRP78 and CHOP transcription activity of NRK-52E cells was increased by LPS at a relatively low concentration (10ng/ml) and a short duration(12h), and increased depended on LPS concentration and incubating duration; The expressions of GRP78 and CHOP were significantly higher in concentration of 100ng/ml, 1000ng/ml and stimulus duration 12h,24h than that of 0mg/ml,10ng/ml (P< 0.01); The expressions of GRP78 and CHOP were significantly higher in concentration of 10ng/ml,100ng/ml, 1000ng/ml and stimulus duration 12h,24h,36h than that of Omg/ml, (P< 0.01).While the comparation of expressions of GRP78 and CHOP in concentration of 100ng/ml,1000ng/ml and stimulus duration 12h,24h were not significant (P>0.05)Conclusions1. LPS induced endoplasmic reticulum stress in NRK-52E cells.2. LPS increased the transcriptional activity of GRP78 and CHOP of NRK-52E cells depended on its concentration and incubating duration.