To investigate whether the high-glucose environment in the distal renal tubule induced by empagliflozin triggers senescence of distal tubular cells as well its potential signaling pathways.

Nine-week-old male C57BL/6 mice were selected and randomly divided into an empagliflozin group [intragastric administration of empagliflozin at 10 mg/(kg·d) and a control group (intragastric administration of an equal volume of normal saline), with six mice in each group. After 4 weeks of consecutive intervention, tissues were harvested, and blood glucose and urine glucose were measured. Immunofluorescence staining and Western blot were performed to detect the expression of senescence-related markers p16, p21, and p53 in distal renal tubules. Transcriptome sequencing was used to screen potential signaling pathways, and gene set enrichment analysis was carried out subsequently. Mouse distal renal tubular epithelial cells were divided into a control group (untreated), a mannitol group (30 mmol/L mannitol), and a high-glucose group (30 mmol/L glucose). After 24 h of incubation, cellular senescence was assessed by senescence-associated β-galactosidase staining. The mRNA expression of the senescence marker p21 and ferroptosis-related genes (Alox15, Ncoa4, Slc11a2, Gpx4) was determined by RT-qPCR. All data were analyzed using GraphPad Prism software.

Compared with the control group, urinary glucose was significantly increased in the empagliflozin group (P<0.05). Immunofluorescence and Western blot analysis showed that the expression levels of p21 and p53 were markedly upregulated in the renal distal tubules of empagliflozin-treated mice (all P<0.05). Transcriptome sequencing and gene set enrichment analysis revealed that ferroptosis-related pathways were significantly activated in the empagliflozin group (adjusted q<0.05). The expression of ferroptosis-related genes Alox15, Ncoa4, and Slc11a2 was upregulated, while Gpx4 expression was downregulated in both mouse renal tissues and cultured mouse distal renal tubular epithelial cells (all P<0.05).

Empagliflozin induced elevation of urinary glucose in mouse renal distal tubules promoted cellular senescence, which may be mediated by the activation of ferroptosis-related pathways.