Indoxyl Sulfate, a Uremic Toxin, Stimulates Reactive Oxygen Species Production and Erythrocyte Cell Death Supposedly by an Organic Anion Transporter 2 (OAT2) and NADPH Oxidase Activity-Dependent Pathways.

Gabriela Dias,Viktoriya Kuntsevich,Roberto Pecoits Filho,Silvia Rodrigues,Lia Nakao,Sara Tozoni,Natalia Bonan,Andréa Moreno-Amaral,Thiago Steiner,Peter Kotanko

doi:10.3390/toxins10070280

Abstract

It is hypothesized that the uremic toxin indoxyl sulfate (IS) plays a role in the pathogenesis of renal anemia. To further explore that hypothesis, we examined the effects of IS on reactive oxygen species (ROS) production, levels of reduced glutathione (GSH), and erythrocyte death (eryptosis) in red blood cells (RBC) from healthy controls (CON-RBC) and hemodialyzed patients (HD-RBC), respectively. RBC were incubated either in either TRIS-Glc-BSA buffer or IS at concentrations of 0.01, 0.09, and 0.17 mM, respectively. We measured ROS generation (expressed as % of DCFH-DA positive RBC), eryptosis (expressed as % of annexin-V positive RBC), and GSH levels after 6, 12, and 24 h. When incubated in buffer, ROS production was approximately seven-fold higher at all time points HD-RBC when compared to CON-RBC. Incubation with IS increased ROS production in CON-RBS dose-dependently up to 10-fold. Eryptosis in buffer-incubated HD-RBC was up to seven-fold higher as compared to COB-RBC. Incubation of CON-RBC with IS increased the eryptosis rate dose-dependently up to 6-fold. Pretreatment of CON-RBC with the organic anion transporter 2 (OAT2) specific inhibitor ketoprofen or with NADPH oxidase inhibitor diphenyleneiodonium-Cl blunted the IS effect on both ROS production and eryptosis induction. While GSH levels in HD-RBC were reduced when compared to CON-RBC, they were not affected by IS incubation. In summary, IS increases ROS generation and eryptosis in CON-RBC by an activity dependent of the IS influx through OAT2, and NADPH oxidase activity-dependent, and a GSH-independent mechanism. These findings lend support to a putative role of IS in the pathogenesis of renal anemia.

Highlights

Anemia is a common complication in chronic kidney disease (CKD)
We identified, for the first time, organic anion transporter 2 (OAT2) as important contributor to indoxyl sulfate (IS)-induced red blood cells (RBC) death in a dependent NADPH
We describe that the IS effects on reactive oxygen species (ROS) production and PS exposure by erythrocytes were abrogated by the presence of a specific inhibitor for organic anion transporter 2 (OAT2), suggesting internalization as a mechanism for IS uremic toxicity to RBCs

Summary

Introduction

Anemia is a common complication in chronic kidney disease (CKD). renal anemia is predominantly caused by an impaired renal erythropoietin synthesis, it can be worsened by other factors [1]. Erythropoiesis stimulating agents (ESA) have a limited capacity of raising hemoglobin levels in inflamed patients, suggesting the contribution of inflammation in erythropoietin hyporesponsiveness, in which shortened red blood cell life span due to premature erythrocyte death (eryptosis) is observed [2]. Many of these mechanisms are thought to induce oxidative stress or an altered ratio of pro-oxidative to antioxidant molecules within red blood cells (RBC). Both free indoxyl sulfate (IS) and total IS, were significantly associated with erythropoietin levels [9] in CKD patients; this correlation was demonstrated by IS suppressing erythropoietin (EPO) mRNA expression, via the disturbance in oxygen metabolism [10] and attenuating EPO-induced tyrosine phosphorylation of EPO receptor leading to an EPO resistance [11]

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