Influence Of Mutual Negative Feedback Between Slit2 And Gremlin1 On Epithelial-mesenchymal Transition In Renal Tubular Epithelial Cells Induced By High Glucose

Background and purpose:Tubulointerstitial fibrosis(TIF)is an important cause of the progression of diabetic nephropathy(DN)to end stage renal disease(ESRD).Previous studies have shown that renal tubular epithelial cells can stimulate oxidative stress,secrete cytokines,cause interstitial inflammation and fibrosis,and participate in the occurrence of DN under the stimulation and development of DN inflammatory state,metabolic disorders and high urine protein.However,the specific pathogenesis of DN has not been fully clarified,so it is necessary to further explore its related mechanisms.Recent studies have shown that there is a negative regulatory relationship between the Slit2/ROBO signaling pathway and the Gremlin/BMP2 signaling pathway.As it is known that BMP7 deficiency and activity reduction in diabetic nephropathy are one of the important factors in the progression of diabetic nephropathy,We speculate that there may be a relationship between Slit2\Gremlin\BMP7,which ultimately leads to an increase in the fibrosis of Gremlin cells,while the anti-fibrosis of Slit2 and BMP7 is weakened,but these need to be confirmed.This study aims to explore the transdifferentiation of Slit2/ROBO1,Gremlin/BMP7 signaling pathway and its negative feedback in renal tubular epithelial mesenchymal transition in human kidney 2 cell(HK2)in high glucose environment.The role and mechanism of the two anti-inflammatory cell signaling pathways in the renal tubules are expected to provide a new therapeutic strategy for DN TIF.Research Method:This experiment is divided into three parts:Part I: The effect of Slit2 on high glucose-induced renal tubular epithelial mesenchymal transition and its mechanismThe culture of HK2 cells was carried out,and the high glucose stimulation concentration was 10,20 mmol/L and the high glucose stimulation time was 24 h,36 h,48 h.Western blot was used to detect the expression changes of Slit2,ROBO1,?-SMA and Fibronectin,and the best high glucose was selected.The Slit2 plasmid was transfected into HK2 cell line by stimulation concentration and time.The cells were divided into normal group,control group,high glucose group,high glucose + vacant group,high glucose + Slit2 group.After 48 hours,the total protein of each group was collected.The expression of ?-SMA and Fibronectin was detected by Western blot.Part II: The effect of Gremlin1 on high glucose-induced renal tubular epithelial mesenchymal transition and its mechanismThe culture of HK2 cells was carried out,and the high glucose stimulation concentration was 10,20 mmol/L and the high glucose stimulation time was 24 h,36 h,48 h.The expression of Gremlin1 and BMP7 protein was detected by Western blot,and the optimal high glucose stimulation concentration and time were screened.Gremlin1 shRNA lentivirus stably transfected cell lines were divided into normal group,control group,high glucose group and high glucose +vacant group,high glucose + Gremlin1 shRNA group,total protein of each group was collected,and ?-SMA was detected by Western blot.Fibronectin expression changes.Part III: The effect of Slit2 and Gremlin1 negative feedback on high glucose-induced renal tubular epithelial mesenchymal transition and its mechanismStepsHK2 cells were divided into normal group,control group,high glucose group,high glucose +vacant group,high glucose + Slit2 group.After optimal high glucose stimulation time and concentration,the total protein of each group was collected and Western blot was used.The expression changes of Gremlin1,BMP7 and p-smad1/5/9 were detected.HK2 cells were divided into normal group,control group,high glucose group,high glucose + vacant group,high glucose + Gremlin1 shRNA group,and total protein of each group was collected.Western blot was used to detect the expression changes of Slit2 and ROBO1.Research Results:The first part:1.In the high glucose concentration gradient experiment,the expression of Slit2,ROBO1 decreased and the expression of Fibronectin and ?-SMA increased at the high glucose concentration of 20mmol/l compared with the normal group.2.In the high glucose time gradient experiment,the expression of Slit2,ROBO1 decreased and the expression of Fibronectin and ?-SMA increased at the point of 48 h after high glucose stimulation compared with the normal group.3.In the high glucose environment,after Slit2 overexpression and negative control plasmid transfection,the expression of Fibronectin in the high glucose group,high glucose + vacant group,high glucose + Slit2 group increased compared with the normal group,?-SMA expression levels increased;Fibronectin expression and ?-SMA expression decreased in the high glucose + Slit2 group compared to the high glucose + vacant group.the second part:1.In the high glucose concentration gradient experiment,the expression of Gremlin1 was increased and the expression of BMP7 was decreased at the high glucose concentration of 20 mmol/l compared with the normal group.2.In the high glucose time gradient experiment,the expression of BMP7 was decreased and the expression of Gremlin1 was increased at the point of 48 h after high glucose stimulation compared with the normal group.3.In the high glucose environment,after the successful transfection of Gremlin1 and negative control lentivirus,the expression of Fibronectin in the high glucose group,high glucose + vacant group,high glucose + Gremlin1 shRNA group was increased compared with the normal group.The expression level of ?-SMA was increased;the expression of Fibronectin and ?-SMA were decreased in the high glucose+Gremlin1 shRNA group compared with the high glucose+vacant group.the third part:1.In the high glucose environment,after Slit2 overexpression and negative control plasmid transfection,the expression of Gremlin1 in the high glucose group,high glucose + vacant group,high glucose + Slit2 group was increased compared with the normal group,BMP7? p-smad1/5/9 the expression level decreased;compared with the high glucose +vacant group,the expression level of Gremlin1 in the high glucose + Slit2 group decreased,p-smad1/5/9the expression level increased.2.In the high glucose environment,after the successful transfection of Gremlin1 and negative control lentivirus,the effect of Gremlin1 blockade on renal tubular EMT was observed.Compared with the normal group,high glucose group,high glucose + vacant group,the expression of Slit2 and ROBO1 in the high glucose+Gremlin1 shRNA group decreased.Compared with the high glucose+vacant group,the expression of Slit2 and ROBO1 in the high glucose+Gremlin1 group increased.Conclusion:1.High glucose induces renal tubular epithelial transdifferentiation;After high glucose treatment,the expression of Slit2 and ROBO1 decreased;Overexpression of Slit2 partially inhibited high glucose-induced renal tubular epithelial transdifferentiation.2.High glucose promotes the expression of Gremlin1 and decreases the expression of BMP7;Knockdown of Gremlin1 can significantly inhibit high glucose-induced renal tubular epithelial-mesenchymal transition.3.Slit2 and Gremlin1 are mutually regulated in high glucose-induced renal tubular epithelial transdifferentiation.