Background <p>Approximately 20–40% of patients with diabetes mellitus (DM) develop diabetic kidney disease (DKD), a major microvascular complication of the condition. This study aimed to explore the potential diagnostic value of hsa-miR-5585-3p for DKD, and its regulatory effects, by targeting CHUK, on inflammatory factor expression and NLRP3 activation in HK-2 cells induced by high glucose (HG).</p> Methods <p>The expression profile of hsa-miR-5585-3p in the serum samples from patients with type 2 DM (T2DM) and DKD was analyzed by RT-qPCR. Correlation analysis between hsa-miR-5585-3p and UACR, eGFR, as well as inflammatory factors was investigated. Bioinformatics analysis predicted the downstream target genes of hsa-miR-5585-3p. The interaction between hsa-miR-5585-3p and CHUK was verified by dual luciferase reporter assay. An in vitro DKD model was established by exposing HK-2 cells to HG. The expression levels of inflammatory factors and the NLRP3 activation were detected by RT-qPCR, Western blot, or ELISA following modulation of the hsa-miR-5585-3p/CHUK axis.</p> Results <p>Hsa-miR-5585-3p was significantly downregulated in the serum of DKD patients and showed correlations with UACR, eGFR, and inflammatory factors. In HK-2 cells stimulated by HG, hsa-miR-5585-3p suppressed the expression of inflammatory factors as well as key molecules in the NLRP3 pathway. Data from the rescue experiment supported the partial role of CHUK overexpression in counteracting the inhibition mediated by hsa-miR-5585-3p.</p> Conclusions <p>Hsa-miR-5585-3p may serve as a potential diagnostic biomarker for DKD, pending further validation. Moreover, hsa-miR-5585-3p regulated the inflammatory response and activation of the NLRP3 in HK-2 cells induced by HG through CHUK.</p> Clinical trial number <p>Not applicable.</p>