THE RELATIONSHIP BETWEEN NLRP3 INFLAMMASOME AND CRESCENTIC GLOMERULONEPHRITIS REVEALED VIA DECONVOLUTION ANALYSIS WITH SINGLE-NUCLEUS RNA-SEQ–BASED SIGNATURE MATRIX AND ANIMAL EXPERIMENTS

Crescentic glomerulonephritis (CGN) often shows resistance to immunosuppressive therapy and leads patients to end-stage kidney disease. Therefore, better understanding of its pathogenesis and more effective therapeutic strategies are required. The nucleotide-binding domain leucine-rich repeat–containing receptor pyrin domain–containing 3 (NLRP3) is known as a key indicator of pyroptosis, a form of programmed cell death. Activation of the NLRP3/pyroptosis pathway results in the release of inflammatory cytokines, including IL-1β and IL-18. However, the relationship between CGN pathogenesis and the activation of the NLRP3 inflammasome/pyroptosis pathway in podocytes remains poorly understood.

We reanalyzed publicly available microarray data derived from glomerular mRNA samples of patients diagnosed with ANCA-associated vasculitis (AAV). Deconvolution analysis using a single-nucleus RNA-seq–based signature matrix was performed to estimate gene expression changes in podocytes. In addition to the bioinformatics analysis, we conducted animal experiments to evaluate the therapeutic effects of the NLRP3 inhibitor shikonin (SH) in a rat model of crescentic glomerulonephritis. A total of 32 eight-week-old female Wistar–Kyoto (WKY) rats were divided into three groups: Control (n = 10), nephrotoxic serum (NTS; n = 10, intravenous injection on Day 0), and NTS + SH (n = 12, intraperitoneal administration of 1 mg/kg daily from Day 0 to 9). All animals were sacrificed on Day 10, and the therapeutic effects of SH were evaluated. Finally, kidney biopsy specimens from patients with microscopic polyangiitis (MPA) were subjected to NLRP3 immunostaining.

Pathway enrichment analysis of the microarray data demonstrated significant activation of the NLRP3 inflammasome pathway in patients with AAV compared with controls (P < 0.05). Deconvolution analysis indicated higher NLRP3 expression in podocytes of AAV patients. In animal experiments, compared with the NTS group, the NTS + SH group showed significantly lower kidney weight (1.69 vs. 1.27 g/body, P < 0.0001) and markedly reduced urinary albumin excretion (229.8 vs. 118.7 mg/day, P < 0.0001). Serum creatinine levels were also significantly lower in the NTS + SH group (0.34 vs. 0.24 mg/dL, P < 0.0001). Histopathological evaluation using PAS staining revealed a significantly lower crescent score in the NTS + SH group than in the NTS group. Immunofluorescence analysis showed that the NTS + SH group had higher WT1-positive podocyte density and greater expression of the podocyte marker synaptopodin. Quantitative PCR of renal cortical tissue revealed significantly lower mRNA expression of pyroptosis-related markers (NLRP3, CASP1, GSDMD, IL18, and IL1B) in the NTS + SH group compared with the NTS group. Immunofluorescence staining for NLRP3 in human MPA samples demonstrated podocyte-specific NLRP3 activation.

Our findings from both bioinformatics and animal experiments suggest that activation of the NLRP3 inflammasome is associated with the pathogenesis of CGN. Regulation of the NLRP3 pathway may have therapeutic potential in CGN. However, further studies are required to confirm these findings.