Albumin is an important contributor to indoxyl sulfate-mediated endothelial dysfunction

Background: Chronic kidney disease (CKD) is characterized by the accumulation of metabolic end-products in the blood, including Indoxyl sulfate (IxS). In CKD patients, elevated plasma levels of IxS are associated with an increased risk of cardiovascular disease (CVD) due to its harmful effects on endothelial cells. In plasma, albumin acts as a carrier for IxS that hinders its clearance via dialysis. However, whether albumin contributes IxS toxicity is currently unknown. Objective: This study investigates how albumin influences the pathophysiological effects of IxS on the vasculature. Hypothesis: Albumin facilitates IxS transport in endothelial cells and modulates its cellular toxicity. Methods: Endothelial cells (EA.hy926) were exposed to either IxS, 4% human serum albumin (HSA), or in combination for different time intervals. Fluorescence microscopy was used to visualize IxS and HSA localization, as well as the intracellular trafficking of endocytosed IxS in cells transfected with GFP-tagged markers for early (Rab5) and late endosomes (Rab7). Western blot analysis was performed to assess the induction of phosphorylation events by IxS and HSA. For the ex vivo studies, 11-13-week-old male C57BL/6J mice were fed a western diet (WD, n=6) or a control diet (n=9) for 6 weeks. The proximal abdominal aorta was dissected from these mice, and ~2 mm segments were mounted on a wire myograph. The aorta segments were treated with IxS and HSA or HSA alone, followed by exposure to noradrenalin (NA) and acetylcholine (Ach) to assess vessel function. Results: Microscopy revealed colocalization of IxS and HSA in vesicle-like structures. IxS was detected in Rab5-positive early- and Rab7-positive late endosomes, but only in the presence of HSA. Furthermore, IxS+HSA treatment increased ERK1/2 phosphorylation in endothelial cells after 30 compared to HSA alone, but not after 10 min. IxS treatment increased the vascular tonus of aorta segments in the control mice only. IxS pretreatment did not significantly alter responses to NA or Ach. Summary of results and conclusion: Our findings suggest that albumin facilitates IxS endocytosis into endothelial cells. In the presence of HSA, IxS was transported into early and late endosomes, potentially triggering ERK1/2 activation. Moreover, IxS induced a contractile phenotype in aortic segments. In conclusion, these results suggest that albumin may directly contribute to IxS-induced vascular dysfunction in CKD patients, exacerbating CVD risk. Ethics statement: Animal experiments were conducted in compliance with the Danish Animal Welfare Act and were approved by the Danish Animal Experiments Inspectorate. This study was funded by the Karen Elise-Jensen and Director Jacob Madsen’s and Wife Olga Madsen’s Foundation This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.