Mechanisms Of Regulator Of Calcineurin 1 Regulating Tubulointerstitial Fibrosis

Diabetic nephropathy is one of the important complications of diabetes mellitus,which will eventually lead to end-stage renal disease.In the progress of diabetic nephropathy,the function of glomeruli and tubules is impaired and fibrosis is induced.The main objective of this paper is to explore the molecular mechanism of regulator of calcineurin1（RCAN1）in regulating renal tubules fibrosis in the progress of diabetic nephropathy,and to provide a new target for the treatment of diabetic nephropathy.First,we explored the signaling pathways that RCAN1 subtypes RCAN1.1s or RCAN1.4 regulate the increase of extracellular matrix in renal tubular epithelial cells respectively.The eukaryotic expression vectors p LHCX-RCAN1.1s and p LHCX-RCAN1.4 were constructed and transient transfected into renal tubular epithelial cell line HK-2 respectively.The phosphorylation levels of protein kinase B（Akt）,mammals rapamycin target protein（m TOR）,ribosomal protein S6 kinase（S6K）,ribosomal protein S6（S6）,eukaryotic translation initiation factor 4E binding protein 1（4EBP1）and the expression levels of extracellular matrix fibronectin（FN）and type IV collagen（Col IV）were detected by protein immunoblotting（western blotting）.It was found that overexpressed RCAN1.1s or RCAN1.4 up-regulated FN and Col IV expression in HK-2 cells and activated the Akt/m TOR/S6K/S6 signaling pathway.To further confirm that RCAN1.1s and RCAN1.4 activate the Akt/m TOR/S6K/S6 signaling pathway,we used small interfering RNA to knockdown RCAN1 and then western blotting to verify the activation of Akt/m TOR/S6K/S6signaling pathway.The results showed that the Akt/m TOR/S6K/S6 signaling pathway was partially inhabited in HK-2 cells with RCAN1 knockdown.In addition,we pretreated HK-2 cells with rapamycin,an m TOR inhibitor,or LY294002,an Akt inhibitor,respectively,and then transfected with p LHCX-RCAN1.1s or p LHCX-RCAN1.4.The phosphorylation levels of S6K and S6 and the expression of extricellular matrix FN were detected by western blotting.The results showed that m TOR downstream pathway S6K/S6 was inhibited and the expression of FN was down-regulated.The above results demonstrate that RCAN1.1s and RCAN1.4 are involved in regulating the expression of extracellular matrix in HK-2 cells through the Akt/m TOR/S6K/S6 signaling pathway.Subsequently,to further explore the upstream molecular mechanism of Akt/m TOR signaling pathway in extracellular matrix expression of renal tubular epithelial cells regulated by RCAN1.1s and RCAN1.4.In our experiments,we found that cytoplasm and mitochondrial reactive oxygen species（ROS）were increased in HK-2cells with overexpressed RCAN1.1s or RCAN1.4,while mitochondrial membrane potential was decreased.The results showed that RCAN1.1s and RCAN1.4 caused increased oxidative stress and impaired mitochondrial function in HK-2 cells.Mitochondria morphology in HK-2 cells,which overexpressed RCAN1.1s or RCAN1.4,was observed by transmission electron microscopy and confocal microscopy,and it was found that mitochondria structure was damaged and fragmentation increased.Western blotting results showed that in mitochondria the protein level of the fission-related protein dynamin-related protein 1（Drp1）was up regulated and the protein level of the fusion-related protein optic atrophy1（Opa1）and mitochondrial fusion protein 2（Mfn2）was down regulated.The above results showed that overexpression of RCAN1.1s or RCAN1.4 promoted mitochondrial division and disrupted mitochondrial dynamic balance.Before transfected with p LHCX-RCAN1.4,HK-2 cells were pretreated with Mito-TEMPO,a specific inhibitor of mitochondrial ROS,or Mdivi1,an inhibitor of Drp1,respectively.The phosphorylation levels of Akt and m TOR and the expression levels of FN were detected by western blotting.The results showed that after inhibition of mitochondrial ROS production and Drp1expression in RCAN1.4 overexpressed HK-2 cells,the Akt/m TOR signaling pathway was inhibited,and the expression of extracellular matrix FN was down-regulated.The above results showed that RCAN1.4 and RCAN1.1s overexpression induced oxidative stress in HK-2 cells,destroyed mitochondrial structure and function,and then activated Akt/m TOR signaling pathway to regulate the expression of extracellular matrix.Finally,the regulation effect of RCAN1.1s on renal fibrosis in streptozocin（STZ）-induced diabetic nephropathy mice was investigated.Immunofluorescence was used to detect the localization of RCAN1.1s in the kidney and it was found that RCAN1.1s was highly expressed in the renal tubule.A mouse strain-RCAN1^{tec OX},specifically overexpressing RCAN1.1s in renal tubules was screened by Cre-Loxp recombinant enzyme system.Immunohistochemistry was used to detect the expression of RCAN1,FN andα-smooth muscle actin（α-SMA）in renal cortex of STZ model mice and RCAN1^{tec OX} mice.The kidney injury and fibrosis of mice were evaluated by Periodic Acid-Schiff stain,MASSON stain and Sirian red stain.These results showed that renal tubules of RCAN1^{tec OX} mice and STZ model mice were diseased,the lumen of renal tubules was wrinkled,the basement membrane was abnormally thickened,the FN and collagen protein were increased,and the myofibroblasts are activated.The phosphorylation levels of Akt and m TOR in the renal cortex of STZ model mice and RCAN1^{tec OX} mice were up-regulated,indicating that Akt/m TOR signaling pathway was activated in renal cortex of STZ model mice and RCAN1t^{ec OX} mice.In addition,it was also found that the expression of Drp1 was increased and the expression of Opa1 and Mfn2 were decreased.The above results preliminarily confirmed that overexpression of RCAN1.1s in mouse kidney promoted mitochondrial division,activated Akt/m TOR signaling pathway,and regulated renal tubular interstitial fibrosis.In conclusion,through inducing oxidative stress and mitochondrial fission,RCAN1.1s and RCAN1.4 activate Akt/m TOR/S6K/S6 signaling pathway to regulate the expression of extracellular matrix in HK-2 cells and mediate tubular interstitial fibrosis in diabetic nephropathy.