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.

Red blood cell (RBC) death could contribute to anemia in chronic kidney disease (CKD) patients. Recent observational research has suggested a relationship between RBC death (eryptosis) and hypoxemia in hemodialysis patients. Thus, we studied the isolated and joint effects of a uremic toxin (indoxyl sulfate; IS) and hypoxia on RBC biology. We incubated RBC from healthy donors with IS at concentrations of 0.01mM, 0.09mM and 0.17mM under both normoxic (21% O2) and hypoxic (5% O2) conditions for 24 hours. Eryptosis was evaluated by RBC phosphatidylserine (PS) exposure, cell volume, and cytosolic calcium which were quantified by Annexin-V+, forward scatter, and Fluo-3AM+ binding, respectively. RBC redox balance was reported by reactive oxygen species (ROS) production and intracellular reduced glutathione (GSH). Analyses were performed by flow cytometry. Hypoxia induced a 2-fold ROS production compared to normoxia. PS exposure and cytosolic calcium increased, while cell volume decreased by hypoxia and likewise by IS. IS increased ROS production in a dose-dependent manner under conditions of both normoxia and hypoxia. The same conditions promoted a GSH decrease with IS intensifying the hypoxia-induced effects. In summary, our results indicate that the concurrent presence of hypoxia and uremia augments RBC death and may therefore, contribute to the genesis of anemia in CKD.

Key Points: HDF and MCO have shown greater clearance of middle-size uremic solutes in comparison with HF dialyzers; MCO has never been studied in HDF. MCO in HDF does not increase the clearance of B2M and results in a higher loss of albumin. Background: Middle molecule removal and albumin loss have been studied in medium cutoff (MCO) membranes on hemodialysis (HD). It is unknown whether hemodiafiltration (HDF) with MCO membranes provides additional benefit. We aimed to compare the removal of small solutes and β2-microglobulin (B2M), albumin, and total proteins between MCO and high-flux (HFX) membranes with both HD and HDF, respectively. Methods: The cross-over study comprised 4 weeks, one each with postdilutional HDF using HFX (HFX-HDF), MCO (MCO-HDF), HD with HFX (HFX-HD), and MCO (MCO-HD). MCO and HFX differ with respect to several characteristics, including membrane composition, pore size distribution, and surface area (HFX, 2.5 m²; MCO, 1.7 m²). There were two study treatments per week, one after the long interdialytic interval and another midweek. Reduction ratios of vitamin B12, B2M, phosphate, uric acid, and urea corrected for hemoconcentration were computed. Dialysis albumin and total protein loss during the treatment were quantified from dialysate samples. Results: Twelve anuric patients were studied (six female patients; 44±19 years; dialysis vintage 35.2±28 months). The blood flow was 369±23 ml/min, dialysate flow was 495±61 ml/min, and ultrafiltration volume was 2.8±0.74 L. No significant differences were found regarding the removal of B2M, vitamin B12, and water-soluble solutes between dialytic modalities and dialyzers. Albumin and total protein loss were significantly higher in MCO groups than HFX groups when compared with the same modality. HDF groups had significantly higher albumin and total protein loss than HD groups when compared with the same dialyzer. MCO-HDF showed the highest protein loss among all groups. Conclusions: MCO-HD is not superior to HFX-HD and HFX-HDF for both middle molecule and water-soluble solute removal. Protein loss was more pronounced with MCO when compared with HFX on both HD and HDF modalities. MCO-HDF has no additional benefits regarding better removal of B2M but resulted in greater protein loss than MCO-HD.

Bisphenol A (BPA)-based materials are used in the manufacturing of hemodialyzers, including their polycarbonate (PC) housings and polysulfone (PS) membranes. As concerns for BPA’s adverse health effects rise, the regulation on BPA exposure is becoming more rigorous. Therefore, BPA alternatives, such as Bisphenol S (BPS), are increasingly used. It is important to understand the patient risk of BPA and BPS exposure through dialyzer use during hemodialysis. Here, we report the bisphenol levels in extractables and leachables obtained from eight dialyzers currently on the market, including high-flux and medium cut-off membranes. A targeted liquid chromatography–mass spectrometry strategy utilizing stable isotope-labeled internal standards provided reliable data for quantitation with the standard addition method. BPA ranging from 0.43 to 32.82 µg/device and BPS ranging from 0.02 to 2.51 µg/device were detected in dialyzers made with BPA- and BPS-containing materials, except for the novel FX CorAL 120 dialyzer. BPA and BPS were also not detected in bloodline controls and cellulose-based membranes. Based on the currently established tolerable intake (6 µg/kg/day), the resulting margin of safety indicates that adverse effects are unlikely to occur in hemodialysis patients exposed to BPA and BPS quantified herein. With increasing availability of new data and information about the toxicity of BPA and BPS, the patient safety limits of BPA and BPS in those dialyzers may need a re-evaluation in the future.

Background: To date, it is unclear whether SARS-CoV-2 is present in spent dialysate from patients with COVID-19 on peritoneal dialysis (PD). Our aim was to assess the presence or absence of SARS-CoV-2 in spent dialysate from patients on chronic PD who had a confirmed diagnosis of COVID-19. Methods: Spent PD dialysate samples from patients on PD who were positive for COVID-19 were collected between March and August 2020. The multiplexed, real-time RT-PCR assay contained primer/probe sets specific to different SARS-CoV-2 genomic regions and to bacteriophage MS2 as an internal process control for nucleic acid extraction. Demographic and clinical data were obtained from patients’ electronic health records. Results: A total of 26 spent PD dialysate samples were collected from 11 patients from ten dialysis centers. Spent PD dialysate samples were collected, on average, 25±13 days (median, 20; range, 10–45) after the onset of symptoms. The temporal distance of PD effluent collection relative to the closest positive nasal-swab RT-PCR result was 15±11 days (median, 14; range, 1–41). All 26 PD effluent samples tested negative at three SARS-CoV-2 genomic regions. Conclusions: Our findings indicate the absence of SARS-CoV-2 in spent PD dialysate collected at ≥10 days after the onset of COVID-19 symptoms. We cannot rule out the presence of SARS-CoV-2 in spent PD dialysate in the early stage of COVID-19.