The Effect Of NLRP3on High Glucose Induced Tubular Epithelial To Mesenchymal Transition And The Relationship Between Its Expression And Galectin-1

Background:Diabetic Kidney disease(DKD) is the most common microvascular complication of diabetes. The renal tubulointerstitial inflammation and fibrosis are closely related to the decline of renal function. It was reported that tubular epithelial to mesenchymal transition (TEMT) was a key link in diabetic renal tubulointerstitial fibrosis. NLRP3and the inflammasome it participates in assembling was found to play an important role in the development of diabetic nephropathy, and they were main involved in renal tubular interstitial inflammation and fibrosis. However, the specific mechanism was uncertain and whether NLRP3cause tubular interstitial fibrosis through participating in the process of TEMT induced by high glucose has not been reported. Recent papers delivered that galectin-1had the function to suppress the tubular interstitial fibrosis, which was opposite to NLRP3. Whether there exists an interaction between them has also not been reported.Objective:①.Detect the expression of NLRP3、IL-18、α-SMA、 E-cadherin and galectin-1in renal tubular epithelial cells(HK-2) which cultrued in different conditions (different concentration of glucose for48h or undergo different time with30mM glucose) and discuss the relationship between them.②. Use NLRP3siRNA to silence its expression in HK-2cells and then futher discuss the effect of NLRP3on high glucose induced TEMT and the relationship between its expression and galectin-1.Methods:HK-2cells were cultrued routinely. According to different purposes of experimental research, which were divided into three groups. One of them was distributed into five small groups according to different concentration of glucose (time was fixed to48h), including normal control group (5.6mM glusose,NG),15mM high glucose(HG) group,30mM HG group,45mM HG group and high penetration control group (5.6mM glusose plus39.6mM mannitol). The another group was distributed into three small groups according to different time of intervene(concentration of glucose was fixed to30mM), including12h、24h and48h group. The last group including normal control group、30mM HG group、NLRP3siRNA plus30mM HG group and irrelevant siRNA plus30mM HG group. Optical microscope was used to observe the change of cell morphology, and real-time PCR and Western blot were used to detect the gene and protein expression of NLRP3、IL-18、α-SMA、 E-cadherin and galectin-1.Result:①. Under the condition of HG, cobblestone appearance HK-2cells changed to spindle cells in a concentration dependent manner.②. Under the condition of HG, the mRNA and protein expression of NLRP3in HK-2cells were up-regulated in a concentration and time dependent manner, moreover, the protein of its downstream proinflammatory cytokine IL-18was also up-regulated (*P<0.05、**P<0.01VS NG group); At the same time, the mRNA and protein expression of a-SMA in HK-2cells were up-regulated, whereas the mRNA and protein expression of E-cadherin were down-regulated (*P<0.05、**P<0.01VS NG group); Moreover the protein expression of galectin-1in HK-2cells was up-regulated in a concentration and time dependent manner(*P<0.05、**P<0.01VS NG group); the above indexs expressed in high penetration control group had no different with NG group.③. Under the condition of30mM HQ the mRNA and protein expression of NLRP3in HK-2cells were up-regulated, however this expression was significantly suppressed by NLRP3siRNA transfection (*P<0.05VS30mM HG group); Under the condition of30mM HQ the mRNA and protein expression of a-SMA were up-regulated, whereas E-cadherin were down-regulated, NLRP3siRNA transfection in HK-2cells could suppress the expression of a-SMA and upregulate the expression of a-SMA both in mRNA and protein level (*P<0.05VS30mM HG group); Under the condition of30mM HQ the protein expression of galectin-1was up-regulated, NLRP3siRNA transfection in HK-2can enhance its expression (*P<0.05VS30mM HG group) Unrelated tranfection of siRNA in HK-2had no effect on the expression of the indexs metioned above.Conclusion:①.The expression of NLRP3in renal tubular epithelial cells could be enhanced by high glucose in a concentration and time dependent manner, and the expression of its downstream cytokine IL-18could also be enhanced; NLRP3and the inflammasome assembled run NLRP3for may participated in TEMT induced by high glucose.②. The expression of galectin-1in renal tubular epithelial cells could also enhanced by high glucose in a concentration and time dependent manner. After the transfection of NLRP3siRNA, renal tubular epithelial cells expressed higher galectin-1compared to30mM HG group, which indicated that NLRP3might regulate the expression of galectin-1negatively. We were firstly to prove the relationship between NLRP3and high glucose induced TEMT and galectin-1expression, which would provide experimental basis for searching therapy for diabetic renal interstitial fibrosis target at NLRP3.