Urinary exosomes exacerbate diabetic kidney disease by promoting NLRP3 inflammasome activation via the microRNA-516b-5p/SIRT3/AMPK pathway

Abstract

Diabetic kidney disease (DKD) is a severe complication of diabetes mellitus. Urinary exosomal miRNAs play a prominent regulatory role in the pathogenesis of DKD, but the potential mechanisms remain largely unknown. Our research was designed to explain the pathogenesis of urine-derived exosomal microRNA-516b-5p (miR-516b-5p) in the DKD development. Urine-derived exosomes were identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Immunofluorescence staining was used to detect cellular internalization. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis was performed to measure the levels of miR-516b-5p and SIRT3. The secretion of inflammatory cytokines and Caspase-1 activity were evaluated via ELISA and flow cytometry, respectively. Expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome markers and genes associated with the SIRT3/AMPK signaling pathway were measured using Western blot. Bioinformatics tools and dual-luciferase reporter gene assay were used to confirm the correlation between miR-516b-5p and SIRT3. Blood glucose and renal function indexes were determined by the corresponding commercial kits. Hematoxylin and eosin (H&E) staining was exploited to examine the renal pathological changes. MiR-516b-5p was memorably upregulated in HKB-20 cells exposed to DKD-Exo. DKD-Exo introduction led to an increase in Caspase-1 activity, promoted inflammatory response and NLRP3 inflammasome activity, and inactivation of SIRT3/AMPK signaling pathway, which was partially reversed by silencing miR-516b-5p. SIRT3 was identified as a target gene of miR-516b-5p. SIRT3 overexpression reversed the influences of DKD-Exo and miR-516b-5p mimic. In the in vivo model, DKD-Exo exacerbated streptozotocin (STZ)-induced kidney injury through promoting inflammatory response and activating the NLRP3 inflammasome. Urinary exosomal miR-516b-5p plays a key role in DKD by promoting inflammatory response and activating the NLRP3 inflammasome through the SIRT3/AMPK pathway.NEW & NOTEWORTHY Urinary exosomal miR-516b-5p plays a key role in diabetic kidney disease (DKD) by promoting inflammatory response and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation through the SIRT3/AMPK pathway.

Keywords: NLRP3 inflammasome; SIRT3/AMPK pathway; diabetic kidney disease; microRNA-516b-5p; urinary exosomes.