Extracellular Vesicles From Albumin Induced Tubular Epithelial Cells Promote The M1 Macrophage Phenotype By Targeting Klotho In The Development Of DKD

Objective:Proteinuria is an independent risk factor for the progression of diabetic kidney disease(DKD).It plays a central role in the initiation and maintenance of tubulointerstitial inflammation and is important in promoting fibrosis in the kidney Several studies have reported that proteinuria promotes tubulointerstitial inflammation through renal proximal tubule epithelial cells(PTECs).Macrophages are the most prominent and numerous inflammatory cells in the kidney,and their role there is dependent on their phenotypes.Classic M1 macrophages can promote kidneys inflammation thus accelerating renal fibrosis.However,whether albuminuria influences macrophage phenotypes is still unclear.In recent years,many researches have implied that PTECs can influence macrophage polarization,but the underline mechanism is unknown.Extracellular vesicles(EVs)act as messengers in the transfer of genetic information between cells.Because renal cells have been reported to interact with each other through EVs,we proposed that albumin-stimulated PTECs may influence macrophage polarization through EVs.In this study we aimed to discuss if albumin-stimulated PTECs may influence macrophage polarization through EVs and the underline mechanism.MethodsIn this study a human proximal tubule cell line(HK-2)and a human monocyte cell line(THP-1)were used.HK-2 cells were treated with human serum albumin(HSA)and THP-1 cells were differentiated into macrophages through phorbol myristate acetate(PMA).EVs were isolated using ExoQuick-TC and then were co-cultured with macrophages,RT-qPCR was used to detect the expression of miR-199a-5p and macrophage polarization-related genes.EVs from HK-2 cells transfected with an Inhibitor NC or Inhibitor 199a-5p then exposed to albumin were injected into the tail veins of db/db mice.EVs from HK-2 cells transfected with a miR-199a-5p mimic or mimic NC were injected into the tail veins of HFD/STZ mice.RT-qPCR and immunohistochemistry were used to detect renal macrophage polarization and renal fibrosis.Results:(1)mRNA expression of M1 macrophages related genes increased in the renal of db/db mice and HFD/STZ mice compared with the corresponding control group.(2)Co-cultured of albumin-stimulated HK-2 cells with LPS induced macrophages promoted macrophage M1 polarization.(3)HK-2 cell-derived EVs can be internalized by macrophages,and albumin-stimulated HK-2 cells promote LPS induced macrophage M1 polarization through EVs.(4)Through bioinformatics analysis,we proposed that miR-199a-5p in EVs may mediate macrophage polarization.We also found that miR-199a-5p was upregulated in urinary EVs from T2DM patients with macroalbuminuria and was positively correlated with AER.(5)Co-cultured with HSA-treated HK-2 cells derived EVs significantly upregulated miR-199a-5p expression in macrophages.Meanwhlie,HK-2 cells which was transfected with an miR-199a-5p inhibitor then exposed to albumin derived EVs impaired macrophage M1 polarization compared with HK-2 cells which was transfected with an NC inhibitor then exposed to albumin derived EVs.(6)Injection of Inhibitor NC transfected and HSA-treated HK-2-derived EVs into db/db mice promoted renal macrophage M1 polarization and promoted DKD progression.While EVs from HK-2 cells transfected with miR-199a-5p inhibitior and treated with HSA reversed renal macrophage M1 polarization and DKD progression.(7)Injection of HFD/STZ mice with miR-199a-5p overexpressed HK-2-derived EVs promoted renal macrophage M1 polarization and promoted DKD progression.(8)MiR-199a-5p could promote macrophage M1 polarization by targeting Klotho.Injecting Klotho plasmid into HFD/STZ mice through tail veins inhibited renal macrophage M1 polarization.ConclusionIn summary,miR-199a-5p from HSA-stimulated HK-2-cell-derived EVs induces Ml polarization by targeting the Klotho/TLR4 pathway and further accelerates the progression of DKD.