Nafamostat Mesylate Alleviates Renal Ischemia-Reperfusion Injury in a Rat Model Through HMGB1 Modulation: An Omics Analysis -Based Study of the Protective Effect and the Mechanisms Involved

Objective To investigate the organ protective role and the underlying mechanism of nafamostat mesylate (NM) in a renal ischemia-reperfusion injury (RIRI) model.

Methods A total of 21 healthy male Sprague-Dawley (SD) rats were randomly assigned to 3 groups (n = 7 in each group), including the sham operation group (Sham group), the RIRI group, and the NM intervention group (NM group). The RIRI and NM groups underwent ischemia-reperfusion injury (IRI) modeling. The NM group was given an intraperitoneal injection of NM at 0.75 mg/kg before modeling. Venous blood and renal tissue samples were then collected from the rats 24 hours after modeling. The levels of serum creatinine, cystatin C, and serum inflammatory factors were determined using the serum samples. Hematoxylin-eosin (HE) staining and TUNEL stainings were performed on the renal tissues to evaluate the damage of the renal tissues. The localization and expression of HMGB1 were analyzed by immunofluorescence and Western blotting, respectively. Single-cell RNA sequencing of the nuclei was performed to obtain the single-cell transcriptome of the kidneys from the rats in the RIRI and the NM groups and to acquire the RIRI cell profile. The cells were annotated according to the cell marker genes to explore the cell type composition in the disease model and the functional status of immune cells between the groups.

Results 1) Compared with those of the Sham group, the levels of cystatin C, creatinine, and inflammatory factors in the RIRI and NM groups were significantly increased, and the expression levels in the NM group were lower than those in the RIRI group (P < 0.05). Compared with those of the RIRI group, the tubular injury score and apoptosis rate in the NM group were significantly decreased (P < 0.05), but those of both the NM and RIRI groups were higher than those of the sham group. Compared with that in the RIRI group, the expression of HMGB1 in the NM group was significantly decreased (P < 0.05), but the expression levels in both the RIRI and NM groups were higher than that in the sham group. Immunofluorescence showed that there was increased cytoplasmic expression of HMGB1 in both the NM and RIRI groups, with the increase being more prominent in the RIRI group. 2) A total of 13 major cell populations were identified through the single-nucleus sequencing results. The proportion of tubular cells in the NM group was higher, with the HMGB1 gene being highly expressed in the damaged proximal convoluted tubular cells. The proportion of the polarized Macro3 cell subpopulation in the macrophages in the NM group was lower compared to that in the RIRI group.

Conclusion NM may play a protective role in a rat model of RIRI, and its underlying mechanisms may be related to the regulation of the functional abnormalities of HMGB1-mediated macrophages.