Exosome-mediated Tubulointerstitial Communication Promotes Renal Fibrosis In Diabetic Kidney Disease

Objective:Renal tubular injury initiates fibroblast activation and drives overproduction of extracellular matrix,leading to renal fibrosis which is a key pathological phenomenon of diabetic kidney disease（DKD）.Despite numerous efforts in dissecting the underlying mechanism involving DKD,it is still unclear how injured tubular epithelial cells relay paracrine signals to their neighboring fibroblasts and trigger fibroblasts differentiation to myofibroblasts.Recently,exosomes have been recognized as crucial mediators of intercellular communication.We hypothesized that exosome secretion from the injured tubular cells may be the underlying mechanism for the pathological development of DKD by mediating the activation and differentiation of fibroblasts.Hence,this study aims to verify this hypothesis,identify the characteristics of DKD exosomes,clarify whether exosomes mediate tubulointerstitial communication which promotes renal fibrosis in DKD,and initially explore its possible mechanism.Materials and Methods:In in vivo experiments,we firstly established a system for isolating and purifying exosomes from mouse kidney tissue using continuous ultracentrifugation.To identify the characterization of the mouse kidney cortical exosomes,we did a transmission electron microscope（TEM）to observe morphologic change;immunoelectron microscope CD63 gold staining to further identify exosomes;Western Blot to detect the expression levels of exosomal markers such as Alix,CD63,and CD9;nanoparticle tracking system（NTA）to examine the number and size distribution of exosomes.Then,we established a DKD in vivo mouse model,that is Akita mice.Blood glucose and ACR level were detected as well as PAS staining to identify DKD model.To evaluate the renal fibrosis level,we did the Sirius Red staining and q RT-PCR to measure the m RNA levels of fibrosis indicators such as FN,a-SMA,Col I,Col IV.Then,we isolated exosomes from Akita mice kidney cortical tissue.Compared with the WT mice,the characteristics of renal cortical exosomes in 11-week-old and 20-week-old Akita mice were analysed.In the in vitro part,we established four kinds of models which mimic the renal tubular epithelial cells under DKD condition,including the chronic hyperglycemia model,acute hyperglycemia model,hyperglycemia combined with hypoxia model,and TGF-b model.In the chronic hyperglycemia model,the glucose concentration was 30 m M for continuous culture of renal tubular epithelial cells for 8 days,while5.5m M glucose plus 24.5m M mannitol as control.After 8 days of culture,the last 24hours of the medium was harvest for exosome isolation.In the acute hyperglycemia model,tubular cells were cultured in 30 m M glucose for 24 hours and the medium was harvest for exosome isolation.In the hyperglycemia plus hypoxia model,the renal tubular epithelial cells were cultured at the condition of glucose 30 m M combined with 1%O₂ for 24 hours.In the TGF-b model,tubular cells were stimulated with TGF-b1 at 5μg/m L concentration for 24 hours.In these four models,the characterization of exosomes in the DKD group was compared with the control group by TEM,Western Blot,and NTA.To explore the mechanism of the decrease of exosome secretion in DKD,we detected the expression level of Rab27b in Akita mice and BUMPT cells under chronic hyperglycemia conditions by Western Blot and immunohistochemistry（IHC）.The Rab27b stable transfected BUMPT cell line was constructed by cell transfection technology to observe the effect of overexpression of Rab27b on the exosome secretion of renal tubular epithelial cells under hyperglycemia conditions.To clarify whether the exosomes derived from DKD renal tubular epithelial cells have an impact on the cell proliferation and fibrosis of renal fibroblasts,we choose the chronic hyperglycemia in vitro model which is highly consistent with the in vivo results for the further function study.The exosomes isolated from high-glucose cultured renal tubular epithelial cells were co-cultured with renal fibroblasts NRK-49F for 48 hours.The morphological changes were detected under a light microscope,cellular protein concentrations were measured using the BCA way and cell numbers were detected by cell counter.To further evaluate the fibrosis level,we used Western Blot to show the protein level of fibrosis-related protein markers such as fibronectin,collagen I,and a-SMA.To study the mechanism of how tubular exosomes promote fibrosis on renal fibroblasts,we used liquid chromatography-tandem mass spectrometry（LC-MS/MS）for proteomics and analyzed the differentially expressed proteins（DEPs）of tubular exosomes under chronic hyperglycemia condition.Hub proteins and protein-protein interaction（PPI）networks were revealed by String-Cytoscape analysis.literature reviews were performed to show the association among hub proteins and DKD and/or fibrosis.The GO/GEGG enrichment analysis was done by DAVID to show the function and signaling pathway in which DEPs were involved.Also,the Nephroseq platform was used to study the association between DEPs and clinical DKD.Results:Through cutting,grinding,digestion,and differential centrifugation,the kidney cortical exosome isolation system was established successfully.Compared with WT mice,renal fibrosis was significantly increased in Akita mice,and the production of exosomes in the kidney tissue decreased significantly in Akita mice.Among the four in vitro models which mimic the DKD condition of renal tubular epithelial cells,the chronic hyperglycemia model showed a significant decrease in exosome production,which was highly consistent with the results of the in vivo model.On the contrary,the secretion of exosomes by tubular cells was significantly increased in the acute hyperglycemia model,hyperglycemia combined hypoxia model,and TGF-b model.Rab27b is down-regulated in Akita mice and renal tubular epithelial cells under chronic hyperglycemia condition.Overexpression of Rab27b promotes an increase in exosome secretion of renal tubular epithelial cells.Renal fibroblasts with the stimulation of tubular exosomes under chronic hyperglycemia conditions showed a higher level of renal fibrosis.Proteomics analysis revealed 22 DEPs in tubular exosomes under chronic hyperglycemia conditions.The String-Cytoscape analysis revealed 16 hub proteins and 2 protein-protein interaction（PPI）networks with 11 hub proteins.While one of the PPI networks comprised of Eno1,Hspa8,Txn1,Ppia,Pgk1,Top2b,and Actb,the other had the family proteins of Ywhag,Ywhae,Ywhaq,and Ywhaz.Further analysis among the hub protein and DKD and/or fibrosis was performed through kinds of literature review that showed that Eno1,Pgk1,Hspa8,Actb,Ywhaz may be related to DKD,and Eno1,Pgk1,Hspa8 may be related to fibrosis.The GO/KEGG enrichment analysis using David showed DEPs have a strong correlation with signal pathways such as Hippo,PI3K-Akt,Rap1 and cell cycle,suggesting an involvement of DEPs in the regulation of renal fibrosis in DKD.Further analysis on the Nephroseq platform found that the expression of Eno1 in DKD patients was increased,and was highly correlated with tubulointerstitium enrichment,GFR,gender,BMI,and weight.Conclusion:This study successfully established a system for exosome isolation from mouse kidney tissue.We firstly showed that DKD is associated with a decrease in exosome secretion in renal tubular cells which is regulated by Rab27b.Exosomes from hyperglycemia conditioned tubular cells may regulate the proliferation and activation of fibroblasts,contributing to the paracrine signaling mechanism responsible for the pathological onset of renal interstitial fibrosis in DKD.Exosomal Eno1 may regulate renal fibrosis,and Hippo,PI3K-Akt and Rap1 signaling pathways may be the involved mechanisms of exosome-mediated tubulointerstitial communication in DKD.