The Effects Of Augmenter Of Liver Regeneration On The Hydrogen Peroxide-Induced Endoplasmic Reticulum Stress In Renal Tubule Epithelial Cells

PARTⅠTHE ESTABLISHMENT OF ENDOPLASMICRETICULUM STRESS MODEL OF RENAL TUBULAREPITHELIAL CELLS INDUCED BY HYDROGENPEROXIDEObjective:To establish the in vitro model of endoplasmic reticulum stress by stimulating renal tubular epithelial cells with hydrogen peroxide which simulate renal ischemia-reperfusion.To investigate the expression of augmenter of liver regeneration in endoplasmic reticulum stress.Methods:In order to simulate the process of renal ischemia-reperfusion injury,in the experiment we stimulated human renal tubular epithelial cells（HK-2 cell line）in vitro with hydrogen peroxide to establish endoplasmic reticulum stress cell model.Hydrogen peroxide was added to the medium to induce cellular ER stress.Different concentrations of hydrogen peroxide（100μM,200μM or 400μM）were incubated for different time periods（6 h,12 h and 24 h）in order to decide upon the optimal conditions by CCK-8assay.Cellular reactive oxygen species（ROS）levels were measured by flow cytometry using DCFH-DA reagent.The total protein was extracted after treatment of hydrogen peroxide of 3h,6h,12h,24h and 48h.The protein expression level of ALR was detected by Western blot.And the protein expression level of GRP78 was detected by Western blot.Results:H₂O₂ inhibited the survival rate of HK-2 cells in a concentration-dependent manner.The CCK-8 results showed that ROS level was increased in renal tubular epithelial cells with prolonged hydrogen peroxide treatment.Western blot results showed that ALR protein expression gradually increased at 12 h,compared with cells without H₂O₂treatment（p<0.05）,and decreased after 48 h compared with the expression at 12 h.The Western blot of GRP78 showed that the expression of GRP78 in the cells with hydrogen peroxide treatment was significantly higher than that without hydrogen peroxide treatment.Conclusion:1.The in vitro cell model of endoplasmic reticulum stress induced by hydrogen peroxide in renal tubular epithelial cells was constructed.2.ALR may be involved in the endoplasmic reticulum stress process induced by hydrogen peroxide stimulation,and its protein expression level increased gradually with hydrogen peroxide prolonged,reached the highest level at 12h and then gradually decreased.PARTⅡTHE INVESTIGATION OF THE EFFECT OF OVEREXPRESSION OF ALR IN ENDOPLASMIC RETICULUM STRESS IN RENAL TUBULAR EPITHELIAL CELLSObjective: To establish a renal tubular epithelial cell line which stably overexpressing augmenter of liver regeneration.To investigate the role of overexpressing of augmenter of liver regeneration in the endoplasmic reticulum stress induced by hydrogen peroxide.Methods: The sequences of ALR gene were cloned into a lentiviral vector GV287 and then transfected into renal tubular epithelial cells.HK-2 cells were infected with the Lenti-ALR-EGFP（hereafter referred to as ALR overexpression,ALR-OE）or Lenti-EGFP（hereafter referred to as vector）.The cells were observed by fluorescence microscope,the ALR mRNA level was detected by PCR,and the protein level of ALR was detected by Western blot.The subcellular distribution of ALR in renal tubule cells was detected by confocal microscopy.The ER fraction and mitochondria fraction was isolated by ER protein extraction kit or mitochondrial protein extraction kit.The protein level of ALR was detected by Western blot.The cell viability was detected by CCK-8 assay.The ROS level of HK-2 cells was determined by DHE using flow cytometry.The apoptosis level of HK-2 cells was determined by Annexin V using flow cytometry.The morphology of the ERwas observed by transmission electron microscopy.Results: PCR results showed that the ALR mR NA levels of the ALR-OE group were significantly increased compared with that of the vector group or wild-type group（WT）（p < 0.05）.Western blot results showed that the expression level of the ALR protein in the ALR-OE group was significantly higher than that of the vector group or WT group（p < 0.05）.Western blot results showed that the expression level of ALR protein extracted from ER in the ALR-OE group was significantly higher than that of the vector group or WT group（p<0.05）.Similarly,the expression level of ALR protein extracted from mitochondria in the ALR-OE group was significantly higher than that of the vector group or WT group（p < 0.05）.Confocal microscopy results showed that ALR extensively overlapped with the ER compartment in HK-2 cells,particularly in the ALR-OE group.Meanwhile,confocal analysis of mitochondria and ALR showed that ALR was colocalized in mitochondria.CCK-8 results showed that 200 μM of H2O2,the ALR-OE group showed a significantly higher cell survival rate from 6 to 24 h,compared with that of the vector group or WT group（p < 0.05）.The determination of ROS showed that the fluorescence intensity of the ALR-OE group was significantly decreased compared with that of the vector group.The apoptosis results showed that the proportion（%）of apoptotic cells in the vector group was higher than that of the ALR-OE group.The TEM results showed that the lumen of ER in the ALR-OE group weredilated to a lesser extent than that of the vector group.Conclusion: 1.A renal tubular epithelial cell line which stably overexpressing augmenter of liver regeneration is established.2.ALR is expressed in the endoplasmic reticulum of HK-2 cells and can protects the morphology of ER induced by hydrogen peroxide.3.Overexpression of ALR inhibits hydrogen peroxide-induced apoptosis in HK-2 cells.PARTⅢ PRELIMINARY STUDY ON THE MECHANISM OF AUGMENTER OF LIVER REGENERATION IN ENDOPLASMIC RETICULUM STRESSObjective: To investigate the mechanism of ALR in endoplasmic reticulum stress and the subsequent cell apoptosis.To investigate the role of ALR in the Calcium homeostasis in endoplasmic reticulum.Methods: Endoplasmic reticulum stress-related protein GRP78,p-PERK,PERK,p-e IF2α,e IF2α and CHOP was detected by Western blot.The apoptosis related protein Bcl-2,Bax and the cleaved Caspase-3 was detected by Western blot.The calcium was added by Rhod 2-AM probe and measured by fluorescence microscopy or the Varioskan LUX multimode microplate reader.Results: the protein expression of GRP78 was significantly decreased in the ALR-OE group compared with that of the vector group（p < 0.05）.Furthermore,the phosphorylation of PERK or eIF2α was significantly upregulated in the vector group（p < 0.05）,compared with that of the ALR-OE group.In addition,the expression of CHOP,a critical mediator of ER stress-induced apoptosis,was significantly up-regulated in the vector group,compared with that of the ALR-OE group（p < 0.05）.The Bax expression increased in both groups,but particularly in the vector group compared with that of the ALR-OE group.The expression of bcl-2decreased in both groups under H2O2 insult（p < 0.05）,and the expression of Bcl-2 protein decreased to a lesser extent in the ALR-OE group compared with that of the vector group（p < 0.05）.The expression of cleaved Caspase-3 was upregulated significantly in the vector group compared to the ALR-OE group（p < 0.05）.The calcium detection showed that the increment of fluorescence intensity of the ALR-OE group was significantly less than that of the vector group（p < 0.05）.Consistent with the microscopy results,kinetic measurement by a multimode microplate reader showed that overexpression of ALR led to a slower increase of Ca2+ concentration compared with that of the vector group（p < 0.05）.Conclusion: 1.Overexpression of ALR may attenuate apoptosis of HK-2 cells by inhibiting PERK/CHOP pathway.2.Overexpression of ALR reduces the movement of calcium from endoplasmic reticulum to mitochondria thereby maintaining the endoplasmic reticulum calcium homeostasis.