Abstract
BackgroundDiabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking.MethodsFourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ.ResultsThe number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model.ConclusionsUrinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.
Highlights
Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnos‐ tic biomarkers are still lacking
The results showed that CCL21 mRNA expression in urinary small extracellular vesicles (EVs) from DN patients was not diminished after RNase treatment, but was sharply decreased after membrane disruption when both Triton and RNase were applied (Fig. 1F)
CCL21 expression correlated with CD3 + T cell infiltration in DN we examined the expression of CCL21 in kidney biopsy in DN patients, normal kidney tissue from patients with renal carcinoma was used as the controls
Summary
Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnos‐ tic biomarkers are still lacking. Accumulating evidence points to the critical roles of the immune response and inflammation lately [6, 7]. Association between these inflammatory biomarkers and renal outcomes in DN has been reported [8]. Chemokines and cytokines are key players in various inflammatory processes, ranging from immune cell development, initiation of immune responses and recruitment of immune cells. A recent study found that urinary CXCL16 and endostatin could reflect the degree of interstitial fibrosis and tubular atrophy, which were served as independent risk factors for progression in patients with advanced DN [10]. The potential of urinary chemokines and cytokines as biomarkers of DN and the implication in the pathogenesis of DN remained to be explored
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