Reciprocal regulation of SIRT1 and AMPK by Ginsenoside compound K impedes the conversion from plasma cells to mitigate for podocyte injury in MRL/ lpr mice in a B cell-specific manner

Abstract

Background: Deposition of immune complexes drives podocyte injury acting in the initial phase of lupus nephritis (LN), a process mediated by B cell involvement. Accordingly, targeting B cell subsets represents a potential therapeutic approach for LN. Ginsenoside compound K (CK), a bioavailable component of ginseng, possesses nephritis benefits in lupus-prone mice; however, the underlying mechanisms involving B cell subpopulations remain elusive.

Methods: Female MRL/lpr mice were administered CK (40 mg/kg) intragastrically for 10 weeks, followed by measurements of anti-dsDNA antibodies, inflammatory chemokines, and metabolite profiles on renal samples. Podocyte function and ultrastructure were detected. Publicly available single-cell RNA sequencing data and flow cytometry analysis were employed to investigate B cell subpopulations. Metabolomics analysis was adopted. SIRT1 and AMPK expression were analyzed by immunoblotting and immunofluorescence assays.

Results: CK reduced proteinuria and protected podocyte ultrastructure in MRL/lpr mice by suppressing circulating anti-dsDNA antibodies and mitigating systemic inflammation. It activated B cell-specific SIRT1 and AMPK with Rhamnose accumulation, hindering the conversion of renal B cells into plasma cells. This cascade facilitated the resolution of local renal inflammation. CK facilitated the clearance of deposited immune complexes, thus reinstating podocyte morphology and mobility by normalizing the expression of nephrin and SYNPO.

Conclusions: Our study reveals the synergistic interplay between SIRT1 and AMPK, orchestrating the restoration of renal B cell subsets. This process effectively mitigates immune complex deposition and preserves podocyte function. Accordingly, CK emerges as a promising therapeutic agent, potentially alleviating the hyperactivity of renal B cell subsets during LN.

Keywords: Ginsenoside CK; Lupus nephritis; Plasma cell; Podocyte.

Graphical abstract

Fig. 1

CK alleviated lupus manifestations. (A) Chemical structure of CK. (B) Experimental scheme. (C) Overall survival curve. (D) Representative spleens and spleen indexes. (E) The levels of anti-dsDNA antibody. (F) The levels of TNF-α, IL-6, and IL-10. (G) The levels of CXCL12, CXCL13, and CCL19 in serum.

Fig. 2

CK re-established splenic B Cell equilibrium. (A) Quantification of splenic B cell subsets. (B) Splenic mRNA levels of B cell activation. (C) Splenic mRNA levels of inflammatory cytokines and chemokines.

Fig. 3

CK restores podocyte ultrastructure following renal damage. (A) Decreased urine protein and albumin levels were found in CK-treated MRL/lpr mice, BUN, and creatinine levels among groups, n = 6 per group. (B) Representative images of H&E, PAS, and Masson staining. Scale bars: 50 μm. And mean histological scores and collagen volume fraction. (C) C3 and IgG deposition in the glomeruli. (D) Detections of TGF-β1, Smad3, (E) nephrin, and SYNPO staining in the glomeruli, n = 3 per group. Scale bars: 20 μm. (F) The expression levels of nephrin, SYNPO, and GAPDH in renal cortex samples. (G) Electron microscopic images from fresh renal cortex samples from the indicated treatment groups. Original magnification, scale bars: 2 μm, × 7000; scale bars: 0.5 μm, × 20,000. (H) Wound healing assay of MPC-5 cells, n = 3 per group. (I) Results of TUNEL staining of MPC-5 cells after R848 treatment with or without CK, n = 3 per group. Scale bars: 50 μm.

Fig. 4

CK delays the renal conversion into plasma cells. (A) Each cell population was annotated and integrated with UMAP. (B) Quantified results of B cells from renal samples. (C) AUCell analysis of mean expression, dot plots, and clustering. (D) Gene expression of B cell activation-associated transcription factors, inflammatory cytokines, and chemokines. (E) The levels of CD19 (red) and CD138 (green) in the glomeruli. Scale bars: 20 μm.

Fig. 5

CK-mediated renal remission depends on Rhamnose. (A) PCA plots of metabolites in renal samples. (B) Heatmap and (C) profiles of differential metabolites. (D) Core metabolites were screened by effect size (Cohen's d). (E) Enrichment analysis of renal metabolites for metabolic pathways. (F) SCDE analysis of metabolic pathways in single-cell clusters.

Fig. 6

CK induces the B cell-specific activation of SIRT1 and AMPK to resist damage. (A) AUCell analysis of mean expression, dot plots, and clustering. (B) Molecular docking of SIRT1 and AMPK with CK. (C) Detections of SIRT1 and AMPK staining in the glomeruli. Scale bars: 20 μm. (D) and (E) SIRT1, AMPK, pAMPK, CPT1A, and GAPDH levels. (F) Diagram illustrating a proposed mechanism for this study.