Uremic Toxicity-Induced Eryptosis and Monocyte Modulation: The Erythrophagocytosis as a Novel Pathway to Renal Anemia.

BACKGROUND: We tested the effect of uremia on red blood cell (RBC) eryptosis, CD14++/CD16+ monocytes and erythrophagocytosis. DESIGN: RBC and monocytes from chronic kidney disease (CKD) stages 3/4 (P-CKD3/4) or hemodialysis (HD) patients and healthy controls (HCs) cells incubated with sera pools from patients with CKD stages 2/3 (S-CKD2/3) or 4/5 (S-CKD4/5) were evaluated to assess eryptosis, monocyte phenotypes and reactive oxygen species (ROS) by cytometer. Erythrophagocytosis was evaluated by subsequent co-incubation of preincubated HC-monocytes and autologous-RBC. RESULTS: HC-eryptosis (1.3 ± 0.9%) was lower than in HD (4.3 ± 0.5%) and HC-RBC incubated with S-CKD4/5 (5.6 ± 1%). CD14++/CD16+ were augmented in P-CKD3/4 (34.6 ± 8%) and HC-monocytes incubated with S-CKD4/5 (26.4 ± 7%) than in HC (5.4 ± 1%). In these cells, ROS was increased (44.5 ± 9%; control 9.6 ± 2%) and inhibited by N-acetylcysteine (25 ± 13%). Erythrophagocytosis was increased in CD14++/CD16+ (60.8 ± 10%) than in CD14++/CD16- (15.5 ± 2%). CONCLUSIONS: Sera pools from CKD patients increase eryptosis and promote a proinflammatory monocyte phenotype. Both processes increased erythrophagocytosis, thereby suggesting a novel pathway for renal anemia.

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.

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.

The lectin BJcuL induces apoptosis through TRAIL expression, caspase cascade activation and mitochondrial membrane permeability in a human colon adenocarcinoma cell line.

It has been demonstrated that the cytotoxic effect of BJcuL, the lectin isolated from Bothrops jararacussu venom, on human gastric carcinoma is accompanied by the inhibition of extracellular matrix adhesion, cytoskeleton disassembly and apoptosis induction. The present study aimed to evaluate the apoptosis mechanisms triggered by the BJcuL interaction with specific glycans on the surface of HT29 human colon adenocarcinoma cells. The results demonstrated that BJcuL interacts with glycoligands targets on the cell, which were inhibited in the presence of d-galactose. It shows a dose-dependently cytotoxic effect that is inhibited in the presence of d-galactose. A dose-dependent cell aggregation decrease was also observed for the HT29 cells. Analysis of cell proliferation inhibition was assessed by anti-PCNA and demonstrated that lectin diminishes PCNA expression when compared with untreated cells. Differences in apoptotic marker expression estimated by immunohistochemistry revealed that the lectin promotes an increase in TRAIL expression, leading to an increase in the expression of FADD, caspase-8 and Bax. Besides the increased expression of apoptosis-related proteins, our results revealed that the lectin promotes a mitochondrial respiration decrease and a 75% increase in the amount of cytochrome c released. Together these results suggest that the cytotoxicity of BJcuL can sensitize pro-apoptotic proteins in the cytoplasm and mitochondria, leading to the apoptotic cascade.