Study Of γδT Cell In Renal Tubular Epithelial Cells By Crystallization Of Calcium Oxalate

Background:γδT cell plays an important role in regulating renal inflammatory immune microenvironment.However,the effect of γδT cell on calcium oxalate-inducedrenal injuryis still uncertain.Objective:This study aims to explore thedistribution of γδT cell in calcium oxalate-induced renal injury and its effect on apoptosis of renal tubular epithelial cells.Methods:First,the mice model(calcium oxalate renal injury)was established.Flow cytometry was used to analyzethe apoptosis of renal tubular epithelial cells,γδT cell infiltration and activation.Cell viability was detected by CCK-8 after co-culture of primary renal tubular epithelial cell and γδT cell.The expression of IL-17 and caspase-3 were detected by ELISA and Western blot,respectively.Moreover,γδT cells,isolated from the kidney,were sequenced by immune repertoire sequencing(IR-seq)to reveal thedistribution and abundance of TCR clones in γδT cell.Finally,immunohistochemical staining was used to measure the impact of γδT cell on the deposition of renal calcium oxalate crystals,the apoptosis of renal tubular epithelial cell and the secretion of IL-17 in TCRδknockout mouse model.Transcriptome sequencing(RNA-seq)and Western blot explored the mechansim of γδT cells in apoptosis.Results:Calcium oxalate-induced renal injury resulted in γδT cell infiltration,elevated level of IL-17 and enhanced renal tubular epithelial cells apoptosis,suggesting that γδT cell activation is involved in the apoptosis of renal tubular epithelial cells induced by calcium oxalate crystallization.In addition,IR-seq showed thehighly tissue-specificTCR V usage.The proportion of T cells expressing trdv5 increased after the deposition of calcium oxalate crystals.Although the use of most V,J,VJ and VDJ combinations is similar,the deposition of calcium oxalate crystal leads to the diversification of clone types of VJ and VDJ combinations.Moreover,significantly elevated CDR3 clones and variants were observed in the glyoxylate group,indicating that calcium oxalate crystal deposition resulted inenhanced TCR diversitycaused by VDJ recombination.The rearrangement of TCR immune repertoire of γδT cell is helpful to recognize renal autoantigens and ectopic antigens,which induce inflammatory milieu and renal injury.γδT cell deficient mice can alleviate calcium oxalate crystal deposition,inhibited IL-17 signaling pathway and decreased the phosphorylationof NFκb,which led to reducerenal tubular epithelial cell apoptosis and kidney injury.