The Protective Effect And Mechanism Of ATRA On Hypoxia-induced Injury In Renal Tubular Epithelial Cells

Background and Aim: Acute renal injury(AKI)is characterized by inflammatory infiltration,renal tubular epithelial cells(RTECs)damage and death.Maladaptive repair of injured tubules after AKI is considered as a risk for chronic kidney disease(CKD)and end-stage renal disease.All-trans retinoic acid(ATRA)has been reported protective in renal fibrosis,but the mechanism is not fully understood.This experiment was designed to investigate whether ATRA alleviated RTECs injury induced by OGD/R through RARα/RXRα-LMX1 B pathway.Methods: An RTECs(NRK-52 E cells)model of oxygen and glucose deprivation/reperfusion(OGD/R)was established.Cells were divided into four groups:(i)normal control group: NRK-52 E cells subjected normoxic condition;(ii)OGD/R group: NRK-52 E cells subjected to deprivation of oxygen and glucose for 6 hours and reperfusion for 12 hours;(iii)DMSO+OGD/R group: NRK-52 E cells pretreated with DMSO for 6 hours before being subjected to OGD/R;(iv)ATRA+OGD/R group: NRK-52 E cells pretreated with ATRA dissolved in DMSO for 6 hours before being subjected to OGD/R.The morphological changes of the cells were observed under inverted phase contrast microscope;the cell viability was evaluated by CCK-8 assay;the cell apoptosis rate was detected by flow cytometry;and the protein expressions of transforming growth factor-beta 1(TGF-β1),α-smooth muscle actin(α-SMA),retinoic acid receptor alpha(RARα),retinoid X receptor alpha(RXRα)and LIM homeobox transcription factor 1-beta(LMX1B)were measured by Western Blot.Results: 1.It was observed that NRK-52 E cells turned wrinkled and rounded,the edges of cells became blurred,intercellular space got widened and the number of suspended cells increased under an inverted phase contrast microscope after OGD/R treatment.Lower cell viability(p<0.05)and higher cell apoptosis rate(13.20% ± 0.91% vs 5.09% ± 0.39%,p<0.05)were observed in the OGD/R group than those in the NC group.2.The result of Western Blot showed that the protein expressions of TGF-β1 and α-SMA in the OGD/R group were significantly higher than those in the NC group(p<0.05),but decreased after being pretreated with ATRA(p<0.05).3.Further detection of RARα,RXRα and LMX1 B expressions was found that the levels of protein expressions of the three indicators above were downregulated after being treated OGD/R(p<0.05).In the ATRA+OGD/R group,the expressions of RARα and LMX1 B increased(p<0.05),while the expression of RXRα got even less(p<0.05)when compared to the DMSO+OGD/R group.4.Correlation analysis showed that the protein expressions of RARα,RXRα and LMX1 B were not only negatively correlated with TGF-β1(r=-0.643,-0.879,-0.839,p<0.05),but also with α-SMA(r=-0.755,-0.894,-0.816,p<0.05).LMX1 B was positively correlated with RARα and RXRα(r=0.766,0.776,p<0.05),while RARα was also positively correlated with RXRα(r=0.755,p<0.05).Conclusion: The protein expressions of RARα,RXRα and LMX1 B in injured NRK-52 E cells induced by OGD/R significantly decreased,while TGF-β1 and α-SMA significantly increased.ATRA could increase the expressions of RARα and LMX1 B,and decrease the expressions of TGF-β1,α-SMA and RXRα.The protein expressions of RARα,RXRα and LMX1 B were positively correlated with each other and negatively correlated with TGF-β1 and α-SMA,suggesting that all of them were involved in the OGD/R-induced RTECs injury.The anti-fibrosis and anti-epithelial-mesenchymal transition(EMT)effects of ATRA were observed in kidney,and its mechanism might be the regulation of LMX1 B expression through upregulating RARα and downregulating RXRα.