| Background and Purpose:Glomerular hyperpermeability is a critical pathophysiologic change of diabetic nephropathy(DN).However,the mechanism is still not fully understood.Some factors,such as high glucose,AGEs(Advanced glycation end products),decreased endogenous NO(nitric oxide),inflammation,and oxidative stress,can promote the endothelial permeability of glomerular.Still,these factors ultimately work by altering the barrier function of endothelial cells.Therefore,we must study the critical mechanism of regulating and stabilizing the endothelial barrier to find the therapeutic target of DN.ADP ribosylation factors(ARFs),the members of Ras superfamily,are highly conserved and widely expressed GTP binding proteins in eukaryotic cells.As a special subtype of ARFs,Arf6 is located on plasma membrane and endosomal membrane.Like other small molecule GTP binding proteins,Arf6 is converted between two binding conformations of active GTP and inactive GDP.The conversion of Arf6-GDP to Arf6-GTP requires the catalysis of guanine nucleotide exchange factors(GEFs).GTPase-activating proteins(GAPs)hydrolysis Arf6-GTP into Arf6-GDP to deactivate it.The biological functions of Arf6 mainly include the regulation of cytomembrane transport,the rearrangement of cytomembrane and cytoskeleton,the change of cell permeability and other physiological activities of cytomembrane.ARNO(ARF nucleotide-binding-site opener)is the most widely reported GEF protein.High expression of ARNO can up-regulate the activity of Arf6 and promote the transfer of vascular endothelial cadherin(VE-Cadherin)and other connective proteins on cytomembrane surface into intracellular environment,thereby damaging intercellular connections and increasing endothelial permeability.Many inflammatory factors can destroy endothelial permeability via ARNO/Arf6 signaling.For example,hyperpermeability of endotheliocyte stimulated by interleukin-1(IL-1)is associated with the activation of ARNO/Arf6 signaling.Endothelin-1(ET-1)promotes the migration and tubulogenesis of endotheliocyte,which is related to the activity of Arf6.Endotheliocyte migration and percolation induced by VEGF165 and fibronectin are also associated with increased Arf6 activity,which can be reversed with ARNO inhibitor.In the microenvironment of diabetes mellitus,a large number of inflammatory cells are activated and aggregated,and inflammatory factors are highly expressed,so we suspect that the microenvironment of DN activates ARNO/Arf6 signaling,which further damages the stability of glomerular endothelial barrier,leading to the leakage of protein and the production of proteinuria.Therefore,it’s necessary to study the role of ARNO/Arf6 signaling in DN.We treated human renal glomerular endothelial cells(HRGECs)and HRGECs transfected by ARNO siRNA or Arf6 siRNA with AGEs,then detecting ARNO expression,Arf6 activity and endothelial permeability induced by AGEs.We try to explore the role of ARNO/Arf6 signaling in AGEs-induced endothelial hyperpermeability,to put forward the mechanism of the AGEs-induced unstability of glomerular endothelial barrier,and provide a theoretical basis for the future selection of drugs to treat DN.Method:The experiment is divided into three parts:Part I: Effects of AGEs on ARNO/Arf6 signaling and permeability of HRGECsHRGECs were cultured in vitro for AGEs concentration(20,40,60 and 80μg/ml AGEs stimulation for 24h)and time(80μg/ml AGEs stimulation for 6,12,24,48h)gradient experiments,respectively.Real-time RCR and Western blotting were used to detect ARNO m RNA transcription and protein expression.Pulldown assay was used to detect Arf6activity(Arf6-GTP).FITC-dextran(40kDa)was used to detect endothelial permeability.Part Ⅱ: Effects of ARNO silencing on the permeability of HRGECs induced by AGEsHRGECs were transfected by recombinant lentivirus carrying ARNO siRNA and stimulated by AGEs.Real-time RCR was used to detect ARNO mRNA transcription.Western blotting was used to detect the expressions of ARNO and membrane VE-Cadherin.Pulldown assay was used to detect Arf6 activity(Arf6-GTP)and Rac1 activity(Rac1-GTP).FITC-dextran(40kDa)was used to detect endothelial permeability.Part Ⅲ: Effects of Arf6 silencing on the permeability of HRGECs induced by AGEsHRGECs were transfected by recombinant lentivirus carrying Arf6 siRNA and stimulated by AGEs.Real-time RCR was used to detect ARNO and Arf6 m RNA transcriptions.Western blotting was used to detect the expressions of ARNO,Arf6 and membrane VE-Cadherin.Pulldown assay was used to detect Rac1 activity(Rac1-GTP).FITC-dextran(40kDa)was used to detect endothelial permeability.Result:Part I: In the AGEs concentration gradient experiment,ARNO mRNA transcription and protein expression in HRGECs were increased in a concentration-dependent manner(P<0.05).Arf6 activity and endothelial permeability were also increased in a similar manner(P<0.05).In the AGEs time gradient experiment,ARNO mRNA transcription and protein expression in HRGECs were increased in a time-dependent manner(P<0.05).Arf6 activity and endothelial permeability were also increased in a similar manner(P<0.05).Part Ⅱ: After ARNO gene silencing by recombinant lentivirus carrying ARNO siRNA,Arf6 activity,Rac1 activity and endothelial permeability induced by AGEs were significantly decreased,and the expression of VE-Cadherin on cytomembrane was significantly increased(P<0.05).Part Ⅲ:After Arf6 gene silencing by recombinant lentivirus carrying Arf6 siRNA,Rac1 activity and endothelial permeability induced by AGEs were significantly decreased,and the expression of VE-Cadherin on cytomembrane was significantly increased(P<0.05),while ARNO expression was unchanged.Conclusion:1.Hyperpermeability of HRGECs induced by AGEs is associated with the activation of ARNO/Arf6 signaling.2.Inhibition of ARNO/Arf6 signaling is expected to be a new direction for the treatment of glomerular endothelial hyperpermeability in diabetic nephropathy. |
