Indoxyl Sulfate Counteracts Endothelial Effects of Erythropoietin Through Suppression of Akt Phosphorylation

Background: Erythropoietin (EPO) is used to treat anemia in patients with chronic kidney disease (CKD). A wide variation in individual response to EPO, however, is often observed, causing EPO resistance. EPO exhibits not only hematopoietic but also extra-hematopoietic functions such as endothelial effects. Indoxyl sulfate, a uremic toxin, is involved in endothelial dysfunction, and consequently, the pathogenesis of CKD-associated cardiovascular disease. The aim of the present study was to determine the effect of indoxyl sulfate on the extra-hematopoietic functions of EPO in human umbilical vein endothelial cells (HUVECs). Methods and Results: HUVECs were incubated with or without indoxyl sulfate or an Akt inhibitor, and then stimulated with or without EPO. Indoxyl sulfate suppressed EPO-induced survival/proliferation, anti-apoptosis function, phosphorylation of endothelial nitric oxide synthase, and the expression of thrombospondin-1, an erythroid-stimulating factor, in HUVECs. Although EPO induced phosphorylation of both Akt and extracellular signal-regulated kinases (ERK) in HUVECs, indoxyl sulfate suppressed phosphorylation of Akt but not ERK. An Akt kinase inhibitor or Akt small interfering RNA suppressed all the EPO-induced cellular effects in HUVECs. As a site of action of indoxyl sulfate on EPO signaling, indoxyl sulfate attenuated EPO-induced tyrosine phosphorylation of EPO receptor (EPOR) in HUVECs. Conclusions: Indoxyl sulfate negatively regulates the EPOR-Akt pathway in endothelial cells, and might contribute to EPO resistance and endothelial dysfunction in patients with CKD. (Circ J 2013; 77: 1326–1336)