Animal models of chronic kidney disease (CKD) are crucial for understanding the disease, its pathogenesis, and for developing new treatments. The adenine-induced CKD model offers a non-surgical approach to studying CKD pathogenesis. At physiological concentrations, adenine is metabolised to AMP through adenine phosphoribosyl transferase, serving as a precursor for several adenine derivatives, and is ultimately metabolised to uric acid and excreted by the kidneys. However, at supraphysiological doses, adenine follows an alternative pathway, resulting in the formation of 2,8-dihydroxyadenine (2,8-DHA). Due to its limited solubility in urine, 2,8-DHA precipitates as crystals and deposits within the renal tubules, leading to tubulointerstitial injury. This study investigates kidney injury and fibrosis at 2 and 4 weeks of adenine-feeding, while also measuring plasma and urine levels of adenine metabolites to investigate the molecular fate of adenine.

To induce CKD, 8-week-old C57BL/6J mice were fed a diet enriched with 0.2% adenine. Mice were grouped randomly into three categories: control (n=11), 2-week adenine diet (n=8) and 4-week adenine diet (n=8). At sacrifice, anaesthesia was induced with 5% sevoflurane and maintained at 3–4%. Blood was collected and immediately after both kidneys were excised and cortical tissue was isolated for subsequent RNA analysis. Mice were euthanised by cervical dislocation. Plasma and urinary metabolites were analysed by LC-MS and compound identification was performed using the mzLogic Data Analysis Algorithm (Compound Discoverer Software, Thermo Scientific). Statistical analyses were performed using a one-way ANOVA, followed by Tukey’s multiple comparisons test. p<0.05 was considered significant.

Following 2 weeks of adenine feeding, kidney injury, as measured by plasma creatinine, blood urea nitrogen and cortical hepatitis A virus cellular receptor 1 (Havcr1) mRNA expression, was significantly increased compared with the control group. Additionally, fibrosis markers, fibronectin (Fn1), collagen type 1 (Col1a1) and transforming growth factor-β (Tgfb1) mRNA expressions were significantly increased compared with control. Unexpectedly, extending adenine feeding to 4 weeks resulted in significant reduction of both kidney injury and fibrosis markers compared to 2 weeks. These markers remained significantly elevated compared to control, except for Havcr1 expression, which had returned to control levels at 4 weeks.

Following 2 weeks of adenine feeding, plasma levels of adenine and 2,8-DHA were elevated compared to the control group, but at 4 weeks, these levels had returned to control levels. Interestingly, plasma concentrations of other adenine metabolites, such as inosine, hypoxanthine, guanine, and uric acid, were higher at 4 weeks compared to 2 weeks. In contrast, urinary levels of adenine and 2,8-DHA increased following 2 weeks of adenine feeding and remained elevated at 4 weeks compared to control levels.

The adenine-enriched diet resulted in significant increase in markers of renal injury and fibrosis within 2 weeks, which showed a noteworthy decrease at 4 weeks. Analysis of plasma metabolites revealed that adenine and 2,8-DHA levels were reduced at 4 weeks, suggesting an adaptive response in adenine metabolism over time, which could explain the less severe kidney injury and fibrosis observed at 4 weeks. We are currently further exploring these mechanisms by investigating the expression levels of enzymes involved in adenine metabolism.