#3587 Beyond the kidney: unconventional roles of indoxyl sulfate in modulating purinergic receptors, transporters, and cytokines in mouse aorta

Abstract Background and Aims Indoxyl sulfate (IS) is a uremic toxin present in kidney diseases. It has hypertrophic effects on the heart, fibrotic effects on the kidney, and impairs the functioning of blood vessels through inflammatory processes. Purinergic signaling plays a role in the control of vascular tone and inflammatory processes. Chronic activation of purinergic receptors in the mouse aorta accelerates inflammation, which synergizes with the deleterious effects of IS. We evaluated the effects of IS on inflammatory and renal injury markers, P1 and P2 receptors, and ectonucleotidases. Method Fifty male C57BL/6 mice, 6 to 8 weeks old, weighing between 20 g and 28 g, were treated with saline solution (Control group-C, 21 days) and IS (intraperitoneal, 100 mg/kg/day) for 7 d, 14 d and 21 days. Total RNA was extracted from the aorta for cDNA synthesis and real-time PCR (qPCR). Serum samples were collected for renal marker analysis. Tibia was collected for morphometric analysis. Statistical analyses were performed using analysis of variance (ANOVA) followed by Tukey's post hoc test. Data presented as mean ± standard error of the mean. Results No significant variations were found in body weight, morphometric parameters, serum urea, or genes related to the transport of IS and ROS production. Creatinine levels were decreased at 14 and 21 days (respectively for C, 14 d, 21 d: 0.74 mg/dL, 0.59 mg/dL, 0.44 mg/dL). There was a decrease in the expression of cytokine transcripts. For IL-6-C, 7 d, 14 d, 21 d: 1 ± 0.25, 0.15 ± 0.04, 0.13 ± 0.02, 0.28 ± 0.6; IL-1β-7 d, 21 d: 1 ± 0.23, 0.15 ± 0.03, 0.26 ± 0.05; TNF-α-C, 7 d, 14 d, 21 d: 1 ± 0.14, 0.55 ± 0.11, 0.23 ± 0.02, 0.50 ± 0.08; TGF-β-7 d, 21 d: 1.28 ± 0.16, 0.38 ± 0.15; IL-13-C, 7 d, 14 d: 1 ± 0.27, 0.10 ± 0.016, 0.17 ± 0.04, IL-10-C, 7 d, 14 d, 21 d: 1 ± 0.28, 0.08 ± 0.01, 0.05 ± 0.01, 0.11 ± 0.03. The P2 and the P1 receptors had their transcript levels decreased in the aorta of IS-treated animals. For P2X4-C, 7 d, 14 d, 21 d: 1 ± 0.10, 0.17 ± 0.02, 0.63 ± 0.04, 0.61 ± 0.07; P2Y6-C, 7 d, 14 d: 1 ± 0.11, 0.61 ± 0.07, 0.66 ± 0.04; A1-C, 7 d, 14 d, 21 d: 1 ± 0.3, 0.28 ± 0.06, 0.31 ± 0.03, 0.23 ± 0.04; A2a-C, 21 d: 1 ± 0.27, 0.34 ± 0.06; A3-C, 7 d: 1 ± 0.36, 0.21 ± 0.07. No variations were found in the P2X7, P2Y1, P2Y2, A2b receptors, or the ectonucleotidases CD39, CD39L1, and CD73. Conclusion The results obtained suggest, initially, modulation of the inflammatory process by IS, both through decreased gene expression of cytokines and purinergic receptors involved in inflammation. Further experiments are necessary to confirm the results at the protein level and understand the mechanisms involved in the modulation.