Chronic kidney disease (CKD) increases the risk of cardiovascular disease (CVD) development. However, conventional treatments for CVD shows limited efficacy in patients with concurrent CKD, suggesting a different CVD mechanism. To better treat these patients, an improved understanding of how uremic toxins accumulate in the plasma during CKD is needed. Therefore, we aimed to characterise the plasma and urinary metabolic profiles of adenine-treated mice, with a focus on key renal drug transports, across various stages of renal injury to evaulate the fidelity of this model to study uremic toxicity.

8-week-old C57BL/6J male mice were exposed to a diet supplemented with 0.2% adenine to induce renal injury and divided into five groups: 1) control group (n=11), 2) two weeks of adenine diet (n=8), 3) four weeks of adenine diet (n=8), 4) four weeks of adenine diet and one week without (n=8), and 5) four weeks of adenine diet and two weeks without (n=7). The latter two groups allowed us to study recovery of the kidney disease. 24-h urine samples were collected the day before termination and blood and kidneys were harvested at termination. At termination, mice were anesthesised with 4% sevoflurane and the kidneys were harvested and a blood sample was collected from the heart. The mice were euthanasised using cervial dislocation. LC-MS/MS was employed for urinary and plasma metabolic profiling. Compound identification was performed using the mzLogic Data Analysis Algortihm (Compound Discover, Thermo Fischer), and untargeted data analysis was conducted using MetaboAnalyst 5.0. Gene expression of renal transporters in cortical kidney tissue was determined by qPCR.

Targeted metabolomics revealed a signifcant increased in mean plasma levels of the uremic toxins indoxyl sulfate, hippuric acid, and kynurenic acid after 2 and 4 weeks of adenine-feeding, which normalized after the 2-week washout period (p<0.05). This was accompanied by significantly reduced levels of urinary kynurenic acid, hippuric acid and indole-3-acetic. Importantly, these levels normalized after the washout period. Untargeted metabolomics demonstrated elevated plasma and reduced urinary levels of specifically uremic toxins after 2 weeks of adenine treatment. After 4 weeks, nucleic acids became the predominant metabolites in both plasma and urine. Notably, we found two previously unidentified metabolites among the top 20 upregulated metabolites in adenine-fed mice. mRNA expression analysis revealed a significant decreased of the proximal tubule influx transporters Slc22a6, Slc22a8, Slco4c1, Slc22a2 after 2 weeks, while only Slc22a6 and Slc22a2 was significantly reduced after 4 weeks. On the contrary, the proximal tubule efflux transporter Slc47a1 was significantly increased after 2 weeks, while the Abcg2 levels were decreased after both 2 and 4 weeks. This dysregulation of the proximal tubule drug transporters suggested proximal tubular stress.

In summary, adenine-feeding in mice induced a state of uremia characterised by increased levels of plasma uremic toxins and reduced levels in the urine probably due to proximal tubule transporter dysregulation. After 2 weeks of washout, renal transporter levels fully recovered, partially restoring the uremic state. We show that the adenine-model in mice is fit to investigate uremic toxicity.