Uremia and Inadequate Oxygen Supply Induce Eryptosis and Intracellular Hypoxia in Red Blood Cells.

BACKGROUND/AIMS: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition. METHODS: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography. RESULTS: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis. CONCLUSION: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation.

Uremia and Hypoxia Independently Induce Eryptosis and Erythrocyte Redox Imbalance.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Serological testing for Hansen's disease diagnosis: Clinical significance and performance of IgA, IgM, and IgG antibodies against Mce1A protein.

Hansen's disease (HD) is an infectious, treatable, and chronic disease. It is the main cause of infectious peripheral neuropathy. Due to the current limitations of laboratory tests for the diagnosis of HD, early identification of infected contacts is an important factor that would allow us to control the magnitude of this disease in terms of world public health. Thus, a cross-sectional study was conducted in the Brazilian southeast with the objective of evaluating humoral immunity and describing the accuracy of the immunoassay based on IgA, IgM, and IgG antibodies against surface protein Mce1A of Mycobacterium, the predictive potential of these molecules, the clinical significance of positivity, and the ability to segregate new HD cases (NC; n = 200), contacts (HHC; n = 105), and healthy endemic controls (HEC; n = 100) as compared to α-PGL-I serology. α-Mce1A levels for all tested antibodies were significantly higher in NC and HHC than in HEC (p < 0.0001). The performance of the assay using IgA and IgM antibodies was rated as highly accurate (AUC > 0.85) for screening HD patients. Among HD patients (NC), positivity was 77.5% for IgA α-Mce1A ELISA, 76.5% for IgM, and 61.5% for IgG, while α-PGL-I serology showed only 28.0% positivity. Multivariate PLS-DA showed two defined clusters for the HEC and NC groups [accuracy = 0.95 (SD = 0.008)] and the HEC and HHC groups [accuracy = 0.93 (SD = 0.011)]. IgA was the antibody most responsible for clustering HHC as compared to NC and HEC, evidencing its usefulness for host mucosal immunity and as an immunological marker in laboratory tests. IgM is the key antibody for the clustering of NC patients. Positive results with high antibody levels indicate priority for screening, new clinical and laboratory evaluations, and monitoring of contacts, mainly with antibody indexes ≥2.0. In light of recent developments, the incorporation of new diagnostic technologies permits to eliminate the main gaps in the laboratory diagnosis of HD, with the implementation of tools of greater sensitivity and accuracy while maintaining satisfactory specificity.

SARS-CoV-2 in Spent Dialysate from Chronic Peritoneal Dialysis Patients with COVID-19.

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.

The Role of Eryptosis in the Pathogenesis of Renal Anemia: Insights From Basic Research and Mathematical Modeling.

Red blood cells (RBC) are the most abundant cells in the blood. Despite powerful defense systems against chemical and mechanical stressors, their life span is limited to about 120 days in healthy humans and further shortened in patients with kidney failure. Changes in the cell membrane potential and cation permeability trigger a cascade of events that lead to exposure of phosphatidylserine on the outer leaflet of the RBC membrane. The translocation of phosphatidylserine is an important step in a process that eventually results in eryptosis, the programmed death of an RBC. The regulation of eryptosis is complex and involves several cellular pathways, such as the regulation of non-selective cation channels. Increased cytosolic calcium concentration results in scramblase and floppase activation, exposing phosphatidylserine on the cell surface, leading to early clearance of RBCs from the circulation by phagocytic cells. While eryptosis is physiologically meaningful to recycle iron and other RBC constituents in healthy subjects, it is augmented under pathological conditions, such as kidney failure. In chronic kidney disease (CKD) patients, the number of eryptotic RBC is significantly increased, resulting in a shortened RBC life span that further compounds renal anemia. In CKD patients, uremic toxins, oxidative stress, hypoxemia, and inflammation contribute to the increased eryptosis rate. Eryptosis may have an impact on renal anemia, and depending on the degree of shortened RBC life span, the administration of erythropoiesis-stimulating agents is often insufficient to attain desired hemoglobin target levels. The goal of this review is to indicate the importance of eryptosis as a process closely related to life span reduction, aggravating 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.