NMN Alleviates Renal Fibrosis in Chronic Kidney Disease by Improving Mitochondrial Function and Inhibiting NF-κB Signaling Pathway-Mediated Apoptosis

Objective To investigate the therapeutic effect and potential mechanism of nicotinamide mononucleotide (NMN), a synthetic precursor of nicotinamide adenine dinucleotide (NAD+), on renal fibrosis in mice with chronic kidney disease (CKD).Methods In in vivo experiments, CKD mouse models were first induced by multiple low-dose intraperitoneal injections of cisplatin, and the mice were randomly divided into control (Ctrl) group and cisplatin (Cis) group. Serum creatinine (SCr) and blood urea nitrogen (BUN) were determined to assess renal function. H&E and Masson's trichrome staining were applied to observe renal pathological changes and collagen deposition. Microarray sequencing was performed to analyze the expression of related genes and pathway enrichment. NAD+ levels in renal tissues were detected. Secondly, to explore the potential role of NMN in CKD, the above CKD mouse models were replicated and treated with NMN, with the mice randomly divided into Ctrl group, Cis group, and Cis+NMN group. NAD+, SCr, and BUN levels were detected in each group. H&E and Masson's trichrome staining were applied to observe renal pathological changes and collagen deposition. Western blot was used to examine the expression levels of the fibrosis-related proteins, nuclear factor-κB p65 (NF-κB p65), its phosphorylated form (NF-κB p-p65), and apoptosis-related proteins. Immunohistochemical staining was used to observe the expression of Bcl-2-associated X protein (BAX). TUNEL staining was performed to analyze cell apoptosis. In in vitro experiments, human renal tubular epithelial HK-2 cells were treated with cisplatin and NMN, and divided into Ctrl group, NMN group, Cis group, and Cis+NMN group. NAD+ levels in HK-2 cells were detected. Fluorescent probes were utilized to determine mitochondrial membrane potential and reactive oxygen species (ROS) . Flow cytometry and TUNEL staining were employed to detect cell apoptosis. Immunofluorescence was used to observe the expression and distribution of NF-κB p-p65 in HK-2 cells. Western blot was performed to detect the expression levels of NF-κB p65, NF-κB p-p65, and apoptosis-related proteins.Results Compared with the Ctrl group, the Cis group demonstrates impaired renal function and collagen deposition in renal tissues (P<0.001). Microarray sequencing analysis demonstrated that genes with differential expression were predominantly enriched in mitochondrial dysfunction and cell apoptosis, and NAD+ levels in renal tissues were decreased (P<0.0001). After NMN treatment, compared with the Cis group, NAD+ levels in renal tissues of mice were increased (P<0.0001), renal function and renal interstitial collagen deposition were significantly improved (P<0.01), the expression levels of fibrosis-related proteins and apoptosis-related proteins were reduced, the activation of the NF-κB signaling pathway was inhibited (P<0.05), and renal tissue cell apoptosis was significantly decreased. In vitro experiments confirmed that compared with the Cis group, NMN treatment increased NAD+ levels in HK-2 cells (P<0.05), alleviated mitochondrial dysfunction and reduced apoptosis of HK-2 cells, decreased the expression levels of apoptosis-related proteins, and inhibited the activation of the NF-κB signaling pathway (P<0.05).Conclusion NMN may mitigate CKD kidney injuries and fibrosis by alleviating mitochondrial dysfunction in renal tubular epithelial cells and Inhibiting NF-κB signaling pathway-mediated apoptosis.