Association between mitophagy and NLRP3 inflammasome in uric acid nephropathy

Abstract

Objective: This study was recruited to investigate the role of mitophagy in activating NLRP3 inflammasome in the kidney of uric acid (UA) nephropathy (UAN) rats.

Methods: This study developed a uric acid nephropathy (UAN) rat model divided into five groups: Negative control (NC), UAN model (M), UAN + autophagy inhibitor (3-MA), UAN + lysosome inhibitor (CQ), and ROS scavenger (N-acetylcysteine, N). H&E staining assessed renal structure, ROS levels were measured with 2, 7-dichlorofluorescin diacetate, and ELISA measured serum markers (creatinine, UA, cystatin C, NGAL, IL-1β, IL-18). Western blot and qRT-PCR evaluated autophagy and inflammation-related protein (LC3 II/I, p62, Pink1, Parkin, NLRP3, Caspase1, IL-1β) expression. NRK-52E cells treated with uric acid and shRNA were analyzed by western blot.

Results: Renal injury in UAN rats was aggravated by ROS accumulation, which promoted mitophagy and activated the NLRP3 inflammasome. Eliminating ROS reduced mitophagy, inhibited NLRP3 activation, lowered IL-1β and IL-18 levels, and alleviated renal injury. Notably, inhibiting mitophagy increased ROS accumulation, up-regulated NLRP3, Caspase1, and IL-1β expression, further worsening renal injury. In vitro, uric acid treatment of NRK-52E cells altered autophagy-related protein and pro-inflammatory cytokine levels, highlighting the interplay between mitophagy and inflammation in uric acid nephropathy.

Conclusion: Mitophagy influences renal injury in uric acid nephropathy (UAN) by regulating ROS accumulation and NLRP3 inflammasome activation, suggesting that mitophagy may serve as a potential therapeutic target for UAN.

Keywords: NLRP3 inflammasome; Uric acid nephropathy; hyperuricemia; inflammation; mitophagy; reactive oxygen species.

Figure 1.

Relative level of ROS (ratio of the NC group) of the renal tissue in each group. The ROS level in the NC group was normalized to 1. Data were expressed as mean ± SD (n = 6). **p < 0.01; ^##p < 0.01. ROS, reactive oxygen species; NC, negative control. NC, negative control group; M, uric acid nephropathy (UAN) model group; CQ, UAN+lysosome inhibitor chloroquine group; 3-MA, UAN+autophagy inhibitor 3-methyladenine group; N, UAN+reactive oxygen species (ROS) scavenger N-acetylcysteine group.

Figure 2.

Mitophagy inhibitors CQ and 3-MA aggravate uric acid nephropathy in Sprague Dawley rats. The levels of (A) SCr, (B) UA, (C) Cys-C, and (D) NGAL in the serum were detected by ELISA. Data were expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01; ^## p < 0.01. CQ, chloroquine; 3-MA, 3-methyladenine; SCr, serum creatinine; UA, uric acid; Cys-C, cystatin-C; NGAL, neutrophil gelatinase-associated lipocalin; ELISA, enzyme-linked immunosorbent assay.

Figure 3.

Effect of mitophagy inhibition on the histopathology of uric acid nephropathy. H&E staining detected the pathological changes of renal in each group (× 400). (A) NC (negative control); (B) UAN (uric acid nephropathy); (C) CQ (chloroquine); (D) 3-MA (3-methyladenine); (E) N (N-acetylcysteine). Arrows in the figure indicate the presence of inflammatory cells and urate crystals.

Figure 4.

Inhibition of mitophagy promotes the release of reactive oxygen species and inflammatory factors in uric acid nephropathy. Level of serum inflammatory factors in each group detected by ELISA. Data were expressed as mean ± SD (n = 6). ** p < 0.01, ^## p < 0.01. ELISA, enzyme-linked immunosorbent assay.

Figure 5.

Changes of mitophagy-associated and NLRP3 inflammasome pathway mRNA levels in uric acid nephropathy. (A–B) The mRNA levels of mitophagy-related protein LC3 II/I, p62, Pink1, Parkin, Caspase1, NLRP3, and IL-1β in renal tissues were examined by qRT-PCR. Data were expressed as mean ± SD (n = 6). *p < 0.05, ** p < 0.01; ^# p < 0.05, ^## p < 0.01. LC3II/I; qRT-PCR, quantitative real-time PCR.

Figure 6.

Crosstalk between mitophagy and NLRP3 inflammasome pathway in uric acid nephropathy. (A–B) Detection and quantification for the expression of NLRP3-related and mitophagy-related protein in renal tissues by Western blot. Data were expressed as mean ± SD (n = 6). ** p < 0.01; ^# p < 0.05, ^## p < 0.01. IL, interleukin.

Figure 7.

Immunofluorescence expression of Pink1 and Parkin in renal tissue of rats with uric acid nephropathy. (A–B) Immunofluorescent expression and quantitative analysis of Pink1 and Parkin in rat renal tissues. Scale bar = 50 µm. Data were expressed as mean ± SD (n = 3). ** p < 0.01; ^## p < 0.01.

Figure 8.

Effects of Pink1 inhibitor on autophagy-related proteins and inflammatory factors in NRK-52E cells induced by uric acid nephropathy. (A–B) Detection and quantification of NLRP3 pathway-related and mitophagy-related protein expression in NRK-52E cells by Western blot. Data were expressed as mean ± SD (n = 3). ** p < 0.01; ^## p < 0.01.