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.

Breast cancer is a very heterogeneous disease. The intrinsic molecular subtypes can explain the intertumoral heterogeneity and the cancer stem cell (CSC) hypothesis can explain the intratumoral heterogeneity of this kind of tumor. CD44+/CD24- phenotype and ALDH1 expression are the major CSC markers described in invasive breast cancer. In the present study, 144 samples of invasive breast carcinoma, no special type were distributed in 15 tissue microarrays (TMA) and then evaluated for expression of the CD44+/CD24- phenotype and ALDH1 to understand the importance of these CSC markers and the clinical aspects of breast cancer. The samples were classified into four molecular subtypes according to clinicopathological criteria: Luminal A, Luminal B, HER2, and Basal-like. A statistical association was found between the molecular subtypes and the CSC markers, with HER2 the most frequent subtype for both markers. ALDH1 was also associated with other poor prognostic variables, such as a high histological grade and larger tumors, but it was not associated with the patients’ prognosis in this sample and nor was the CD44+/CD24- phenotype in a multivariate analysis. There are still many controversies about the role of these markers in breast cancer molecular subtypes. The identification of these populations of cells, through immunohistochemical markers, can help to better understand the CSC theory in clinical practice and, in the near future, contribute to developing new target therapies.

Uremic toxin (UT) retention in chronic kidney disease (CKD) affects biological systems. We aimed to identify the associations between UT, inflammatory biomarkers and biomarkers of the uremic cardiovascular response (BUCVR) and their impact on cardiovascular status as well as their roles as predictors of outcome in CKD patients. CKD patients stages 3, 4 and 5 (n = 67) were recruited and UT (indoxyl sulfate/IS, p-cresil sulfate/pCS and indole-3-acetic acid/IAA); inflammatory biomarkers [Interleukin-6 (IL-6), high sensitivity C reactive protein (hsCRP), monocyte chemoattractant protein-1 (MCP-1), soluble vascular adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble Fas (sFas)] and BUCVRs [soluble CD36 (sCD36), soluble receptor for advanced glycation end products (sRAGE), fractalkine] was measured. Patients were followed for 5.2 years and all causes of death was used as the primary outcome. Artery segments collected at the moment of transplantation were used for the immunohistochemistry analysis in a separate cohort. Estimated glomerular filtration rate (eGFR), circulating UT, plasma biomarkers of systemic and vascular inflammation and BUCVR were strongly interrelated. Patients with plaque presented higher signs of UT-induced inflammation and arteries from CKD patients presented higher fractalkine receptor (CX3CR1) tissue expression. Circulating IS (p = 0.03), pCS (p = 0.007), IL-6 (p = 0.026), sFas (p = 0.001), sCD36 (p = 0.01) and fractalkine (p = 0.02) were independent predictors of total mortality risk in CKD patients. Our results reinforce the important role of uremic toxicity in the pathogenesis of cardiovascular disease (CVD) in CKD patients through an inflammatory pathway.

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.