Role Of Exosomes In Proteinuria Induced Tubulointerstitial Inflammation And Diagnostic Potential As Biomarker Of Kidney Disease

Background: Tubulointerstitial inflammation is the common pathological change in both acute and chronic kidney disease and plays a crucial role in the prognosis of renal disease.Proteinuria is not only the clinical manifestation of renal injury,but also a key factor in tubulointerstitial inflammation.Previous studies demonstrated that tubular epithelial cells(TECs)secrete inflammatory cytokines and chemokines and subsequently activate inflammatory cells after albumin exposure.However,the mechanisms by which albumin overload contributes to tubular inflammation and transfers to tubulointerstitium remain unclear.Exosomes are nano-sized extracellular vesicles,packaging m RNA and mi RNA selectively.Exosomes are vital carriers in cell-to-cell communication,as well as the potential biomarkers of a variety of diseases.Therefore,the present study aimed to investigate the role of exosomes in TECs and macrophages communication in proteinuria induced tubulointerstitial inflammation in vivo and in vitro,and further to explore the potential underlying mechanism.Methods: In the first part,we established LPS-induced acute kidney injury(AKI)model and 5/6 subtotoal nephrectomy chronic kidney injury model and examined the pathological changes and characteristics of renal exosomes.Besides,the features of TEC-derived exosomes after albumin exposure were also studied.In the second part,the mechanisms of exosome biogenesis and release were explored in ADR-injected mice model and in vitro experiments.Moreover,the role of exosome production in albumin handling in TECs were detected.In the third part,the expression profiles of mi RNAs in kidney exosomes from LPS-induced AKI model were examined by Exiqon microarray.Exosomes purified from TECs were added to macrophages or injected to mice to determine their effects in vivo and in vitro.In the fourth part,urinary exosomes were isolated and purified and RNA were extracted from Ig A nephropathy patients to search for the novel and invasive biomarkers.The correlation among clinical data,kidney histological damage and urinary exosomes and exosomal m RNAs were analyzed.Results: Compared with the control group,the urinary protein and the number of infiltrated macrophages in tubulointerstitium in model group were increased significantly,accompanying enhanced renal exosomes production.Consistent with in vivo experiments,the gene expression of inflammatory cytokines and exosome production were found to be markedly elevated in TECs under albumin exposure.The increasing uptake of BSA-treated TEC-derived exosomes by macrophages were observed and contributed to tubulointerstitial inflammation.Markedly up-regulation of Rab27 a m RNA and protein was detected both in ADR-induced model and BSA-treated TECs.Lentivirus Rab27 a sh RNA intrarenal injection downregulated the renal exosomes production.IRF-1/Rab27a-dependent exosome secretion mediated albumin handling in TECs.Global mi RNA expression profiling on renal exosomes was examined in LPS-induced AKI model and mi R-19b-3p was identified as the most remarkable mi RNA increased in TEC-derived exosomes compared with controls.Interestingly,once released,TEC-derived exosomal mi R-19b-3p was internalized by macrophages,leading to M1 phenotype polarization through targeting NF-?B/SOCS-1.Moreover,exosome excretion in urine was increased with increasing severity of proteinuria and NGAL in Ig AN patients.Urinary exosomal CCL2 m RNA was significantly upregulated in Ig AN compared with controls,and correlated with the deterioration of renal function as determined by e GFR.Exosomal CCL2 m RNA also increased in patients with high scores of tubular atrophy and interstitial fibrosis.Conclusion: Exosomes released from TECs with albumin exposure contributed to the development of tubulointerstitial inflammation.IRF-1/Rab27 a mediated exosomes production in TECs after albumin exposure,participating cellular albumin handling and formation of kidney inflammation.Exosome/mi R-19b-3p/SOCS1 axis played a critical pathologic role in tubulointerstitial inflammation that might represent a new therapeutic target for kidney disease.Besides,urinary exosomes production and the CCL2 m RNA might be the promising biomarkers of Ig AN reflecting the deterioration of renal function and pathologic damage.