Chronic allograft dysfunction (CAD) with progressive fibrotic remodeling persists as a critical barrier to long-term renal graft survival, primarily driven by ischemia-reperfusion injury (IRI)-triggered acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Despite the widespread consumption of low-calorie sweeteners (LCSs), their pathophysiological impact on renal allograft outcomes remains poorly characterized. This study aimed to investigate the underexplored effects of LCS exposure on the molecular mechanisms underlying AKI-CKD progression in renal injury models.

Male C57BL/6 mice (7-8 weeks old) were randomly allocated to three treatment regimens: standard chow with plain water (Control), 0.02% rebaudioside A (RA), or 0.03% aspartame (ASP) administered via drinking water. Longitudinal monitoring of metabolic parameters (body weight, food/water intake) was conducted at 4-day intervals. Following a 14-day acclimatization period, animals underwent a standardized AKI-CKD transition protocol involving left renal pedicle clamping (30 min ischemia via midline laparotomy) with subsequent contralateral nephrectomy 24 hours prior to terminal sacrifice (4 weeks post-IRI). To further delineate the effects of phenylacetylglycine (PAGly), a downstream metabolite of LCSs, an independent therapeutic intervention cohort received daily intraperitoneal administration of PAGly (two dosage regimens: 20/100 mg/kg) initiated on post-operative day 4 following IRI. The PAGly-treated mice underwent harvesting at 2 weeks post-IRI. Mouse kidneys were collected for real time quantitative PCR (qRT-PCR), western blot (WB) detection, and Masson staining to evaluate the severity of renal fibrosis. Serum was also collected for measuring the level of blood urea nitrogen (BUN), serum creatinine (SCr) and PAGly levels.

No significant differences in BW, food/water intake were observed among groups during the experimental period (Fig 1A). Mice administered ASP and RA exhibited a reduced kidney-to-body (K/B) weight ratio compared to controls (Fig 1B). Histological analysis via Masson staining demonstrated enhanced collagen deposition in both ASP- and RA-treated groups (Fig 1C). Renal functional assessment revealed significantly elevated BUN and SCre levels in the ASP group, with RA exposure also increasing SCre concentrations (Fig 2A). Molecular analyses showed upregulated mRNA expression of Acta2, Col1a1, and Fn1 in the ASP group, whereas RA treatment selectively elevated Col1a1 expression (Fig 2C). WB confirmed increased collagen-I protein levels in both sweetener-treated groups (Fig 2B), accompanied by higher serum PAGly concentrations (Fig 2D). In PAGly-intervened cohorts, decreased K/B weight ratios and elevated expression of fibrosis-related markers were observed (Fig 3). Notably, PAGly administration suppressed mitochondrial fusion regulators Mfn2 and Opa1, suggesting mitochondrial dysfunction may mediate its pro-fibrotic effects.

Our findings demonstrate that prolonged exposure to LCSs exacerbates the pathological progression from IRI-induced AKI to chronic renal dysfunction and fibrotic remodeling. This effect appears mechanistically linked to mitochondrial fusion impairment mediated through the LCSs metabolite PAGly. These results suggest that regulating artificial sweetener intake may serve as a complementary therapeutic strategy for improving long-term graft outcomes in kidney transplant recipients.