Uremic Toxin-Induced Pathological Signaling in Sinus Node Cells Is Attenuated by Probenecid.

introductionUremic toxins, including indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are well-recognized contributors to cardiac fibrosis and maladaptive remodeling in chronic kidney disease (CKD). Although their roles in ventricular and vascular pathology have been extensively studied, their effects on sinus node cells and the associated molecular alterations remain poorly defined. Understanding how the uremic milieu affects sinus node cell homeostasis may provide insight into CKD-associated conduction abnormalities.

methodsTo investigate uremic toxin-induced stress responses, H9C2 cardiomyocytes were used as a comparative cardiac stress-responsive model, whereas primary sinus node cells served as the principal pacemaker-relevant model. Both cell types were treated with IS and PCS to compare shared cardiac stress signaling with sinus node cell-associated remodeling responses. Profibrotic, pro-apoptotic, and stress-related signaling pathways were assessed using protein and mRNA analyses. Activation of stress kinases, including p38 and ERK, was evaluated to define downstream signaling mechanisms. In parallel, a CKD mouse model was generated by 2-week adenine feeding, and sinus node cells were isolated for molecular analysis. The protective effects of probenecid were examined in cultured cells and ex vivo using primary sinus node cells isolated from adenine-fed mice, with particular attention to remodeling-associated molecular markers and pacemaker identity marker expression.

resultsIS and PCS induced profibrotic, pro-apoptotic, and remodeling-associated signaling in both H9C2 cardiomyocytes and primary sinus node cells, indicating activation of shared cardiac stress pathways. Conclusions regarding pacemaker-relevant molecular alterations were based primarily on findings obtained from primary sinus node cells, including cells isolated from 2-week adenine-fed mice. In primary sinus node cells, uremic toxin exposure increased fibronectin accumulation, altered the Bax/Bcl-2 balance, and activated p38 and ERK signaling. TBX3 expression was preserved, suggesting that these molecular changes occurred without overt loss of this pacemaker identity marker. These findings indicate activation of apoptosis-associated, profibrotic, and stress-responsive molecular alterations rather than direct evidence of functional pacemaker impairment. Consistent with in vitro findings, sinus node cells isolated from 2-week adenine-fed mice exhibited increased expression of remodeling-associated molecular markers. Probenecid treatment attenuated toxin-associated fibronectin accumulation, Bax/Bcl-2 imbalance, and stress kinase activation in primary sinus node cells. Similar attenuation of these remodeling-associated molecular marker changes was observed in sinus node cells isolated from adenine-fed mice following ex vivo treatment with probenecid.

conclusionThese findings suggest that uremic toxin-driven stress signaling is associated with molecular remodeling signatures in sinus node cells under CKD-related conditions. However, the functional consequences of these molecular alterations were not assessed. Probenecid attenuated uremic toxin-associated profibrotic, pro-apoptotic, and stress kinase signaling, suggesting that targeting uremic toxin-driven molecular stress pathways may represent a potential strategy to preserve sinus node cell homeostasis in CKD.