Key wordsOver 10% of hemodialysis (HD) patients have prolonged intradialytic hypoxemia (PIH), a condition defined as arterial oxygen saturation below 90% for at least one third of the treatment time.1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis. This is matter of concern, as PIH is associated with significantly increased morbidity and mortality.1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis. In a large retrospective study on 983 HD patients , the risk of all-cause hospitalization was 2.2 fold higher in PIH patients compared to the non-hypoxemic group. Mortality risk was also significantly higher in HD patients with PIH, especially within individuals with no heart failure or chronic obstructive pulmonary disease (COPD).1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis.Intriguingly, patients with PIH also have higher requirement of erythropoietin (EPO) and other erythropoiesis stimulating agents (ESA), a finding that is counterintuitive, because hypoxia is the main stimulus for residual endogenous production of EPO.1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis. To start addressing this issue, we reasoned that hypoxemia has been shown to impair resilience against reactive oxygen species (ROS) and may increase erythrocyte apoptosis (eryptosis), characterized by reduced cell dimension, bleb formation and phosphatidylserine exposure.2Lang F. Lang E. Foller M. Physiology and pathophysiology of eryptosis. Therefore, we hypothesized that higher ESA requirement is the result of higher red blood cells (RBC) fragility and shorter half-life in PIH patients, due to an accumulation of intracellular ROS (iROS).To test this idea, we designed a cross sectional study to compare RBC phenotype and function in HD patients with or without PIH and in healthy controls. In HD patients, online intradialytic arterial oxygen saturation (from arteriovenous fistula) was monitored using the Crit-Line Monitor3Balter P. Artemyev M. Zabetakis P. Methods and challenges for the practical application of Crit-Line™ monitor utilization in patients on hemodialysis. (see Supplementary methods).

We collected blood samples and measured eryptosis and iROS production in RBCs from patients with or without PIH (see Supplementary methods). Finally, we measured reticulocytes (RC) and levels of the different RC subsets.

RESULTS Patients’ characteristicsFrom March 2019 to October 2020, we included 56 patients on chronic HD and 18 healthy controls. Within the 56 HD patients, 12 had PIH. Although PIH has been previously associated with older age and longer dialysis vintage,1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis. in our cohort demographic characteristics did not significantly differ between HD patients with or without PIH. Kt/V was also similar between the two groups.Consistent with prior reports,1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis. PIH patients had lower hematocrit levels, despite significantly higher use of ESA (Table 1). Healthy controls were significantly younger than HD patients (Table 1).

Table 1Patients’ characteristics and clinical parameters at sample collection.

ACEi: angiotensin converting enzyme inhibitors; BP: blood pressure; CHF: congestive heart failure; COPD: chronic obstructive pulmonary disease; ESA: erythropoiesis stimulating agents; HCT: hematocrit; HD, hemodialysis; HGB: hemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; PTH: parathyroid hormone; RBC, red blood cells; RC, reticulocytes; RDW: red cell distribution width; WBC: white blood cells.

Kt/V was calculated based on Daugirdas’ formula (J Am Soc Nephrol, 1993, vol. 4 (pg. 1205- 1213).

Data are represented as mean ± standard deviation or median (IQR). Bold fonts identify numbers that are significantly (*P<0.05) different between groups.

 HD patients with PIH have high levels of eryptosis and iROSWe started our analyses by measuring eryptosis and we found that HD individuals with PIH had significantly higher levels of annexin-V positive RBCs than healthy controls. In contrast, annexin-V positive RBCs trended to by higher in HD patients without PIH compared to healthy controls, but this difference did not reach statistical significance (Figure 1A-B).

Figure 1Red blood cells (RBC) phenotypic characterization. A-B) Representative plots (A) and data quantification (B) of annexin V+ RBCs; C-D) Representative plots (C) and quantification (D) of iROS+ RBCs; E-F) Representative plots of reticulocyte populations (E) and quantification (F) of total CD71+ reticulocytes; G) Quantification of CD71+ reticulocytes subpopulations. Quantifications of flow cytometry data are shown as average ± SEM. *P<0.05; n.s., not significant.

We next tested the hypothesis that increased eryptosis in HD patients with PIH is due to increased intracellular ROS production. The analyses were performed within 2 hours from withdrawal to prevent altered iROS production. As shown in Figure 1C-D, HD patients with PIH had significantly higher iROS levels than non-hypoxemic ones. Consistent with previously published results,S1 there was no significant difference in the iROS levels between healthy controls and non-hypoxemic HD patients, regardless from the hypoxic status.

Altogether, these data support the hypothesis that PIH increases iROS production in RBCs, leading to accelerated RBC eryptosis and loss. This mechanism explains, at least in part, the increased requirement for ESA in HD patients with PIH.

 PIH is not associated with higher levels of inflammatory markersAn alternative, additional mechanism responsible for higher ESA requirement in PIH patients could be an impaired bone marrow response to ESA, leading to lower reticulocyte production. One of the main reasons for resistance to ESA therapy is chronic inflammation, a condition that often associates with PIH.1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis. We analyzed the levels of serum ferritin and albumin, markers of systemic inflammation.4Kalantar-Zadeh K. Kalantar-Zadeh K. Lee G.H. The fascinating but deceptive ferritin: to measure it or not to measure it in chronic kidney disease?. Ferritin levels in HD patients were high overall, but did not significantly differ between patients with or without PIH. Similarly, we found no significant difference in serum albumin levels between the two HD groups (Table 1). No significant differences in reticulocyte counts between HD patients with or without PIHNext, we measured percentages of total reticulocytes and of those with high, intermediate, and low expression of CD71 (transferrin receptor), whose expression is lost during reticulocytes maturation5Borrione P. Spaccamiglio A. Parisi A. et al.A biparametric flow cytometry analysis for the study of reticulocyte patterns of maturation. (see Supplementary methods).We found no significant differences in the total reticulocyte percentages (Figure 1E-F), nor in any of the three subpopulations (Figure 1G), suggesting no impaired response to EPO in HD patients with PIH.DISCUSSIONMultiple causes have been implicated in the pathogenesis of PIH, including, amongst others, intradialytic hypotension, sleep apnea, congestive heart failure and COPD.6Campos I. Chan L. Zhang H. et al.Intradialytic Hypoxemia in Chronic Hemodialysis Patients. Regardless from its etiology, PIH is a serious medical condition associated with intradialytic complications, such as hypotension and cramps,6Campos I. Chan L. Zhang H. et al.Intradialytic Hypoxemia in Chronic Hemodialysis Patients. but also with poor clinical outcomes on the long term.1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis.HD patients with PIH have increased ESA requirement, and our data support the concept that this phenomenon is due to increased RBC eryptosis and turnover, induced by iROS accumulation. The levels of total reticulocytes and the percentages of reticulocytes in different stage of maturation were not significantly different in PIH patients over non-PIH controls, suggesting that their response to EPO is not impaired.1Meyring-Wösten A. Zhang H. Ye X. et al.Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis.

Although EPO deficiency represents a central mechanism in the pathogenesis of anemia in HD patients, the process is indeed multifactorial, and involves systemic inflammation that reduces response to EPO by erythroid cells in the bone marrow. Although HD patients in our study showed signs of inflammation compared to healthy controls, we did not detect significant differences in ferritin nor in serum albumin levels between PIH and non-PIH patients, suggesting that increased ESA requirement in PIH patients was not due more severe chronic inflammation.

Reduced RBC survival in HD patients can be promoted by different stimuli, including oxidative stress, various diseases (including diabetes, heart failure, and sepsis) and also uremic toxins7Triggers, inhibitors, mechanisms, and significance of eryptosis: the suicidal erythrocyte death. that promote eryptosis by increasing iROS production.8Dias G.F. Bonan N.B. Steiner T.M. et al.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. A recent study showed that uremic toxin indoxyl sulfate increases iROS production in a dose-dependent manner, both in hypoxic or not hypoxic condition, suggesting that hypoxia and uremic toxins promote eryptosis in an independent manner.9Tozoni S.S. Dias G.F. Bohnen G. et al.Uremia and Hypoxia Independently Induce Eryptosis and Erythrocyte Redox Imbalance. We did not have information on uremic toxins in our cohort, but this is a variable that it will be important considering in future studies.

The major limitation of our report is the relatively small sample size. However, the results of our comprehensive RBC phenotypic analyses will form the basis for the sample size estimation of larger investigations.

The pathophysiology of PIH is only partially understood and our findings are important as they support the concept that increased ESA requirement in PIH patients is due, at least in part, to increased RBC fragility. Our study provides the rationale for future studies testing the hypothesis that therapeutic strategies for anemia in PIH should aim at prolonging RBC survival (possibly by reducing ROS production) together with increasing erythrocyte production.

DISCLOSURES

The authors declare no conflicts of interest.

FUNDING

The project was supported by a grant from Renal Research Institute (RRI), New York, NY 10128, USA.

Supplementary MaterialREFERENCESMeyring-Wösten A. Zhang H. Ye X. et al.

Intradialytic Hypoxemia and Clinical Outcomes in Patients on Hemodialysis.

Clin J Am Soc Nephrol. 11: 616-625Lang F. Lang E. Foller M.

Physiology and pathophysiology of eryptosis.

Transfus Med Hemother. 39: 308-314Balter P. Artemyev M. Zabetakis P.

Methods and challenges for the practical application of Crit-Line™ monitor utilization in patients on hemodialysis.

Blood Purif. 39: 21-24Kalantar-Zadeh K. Kalantar-Zadeh K. Lee G.H.

The fascinating but deceptive ferritin: to measure it or not to measure it in chronic kidney disease?.

Clin J Am Soc Nephrol. 1: S9-18Borrione P. Spaccamiglio A. Parisi A. et al.

A biparametric flow cytometry analysis for the study of reticulocyte patterns of maturation.

Int J Lab Hematol. 32: 65-73Campos I. Chan L. Zhang H. et al.

Intradialytic Hypoxemia in Chronic Hemodialysis Patients.

Blood Purif. 41: 177-187

Triggers, inhibitors, mechanisms, and significance of eryptosis: the suicidal erythrocyte death.

Biomed Res Int. 2015: 513518Dias G.F. Bonan N.B. Steiner T.M. et al.

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.

Toxins (Basel). 10Tozoni S.S. Dias G.F. Bohnen G. et al.

Uremia and Hypoxia Independently Induce Eryptosis and Erythrocyte Redox Imbalance.

Cell Physiol Biochem. 53: 794-804Article InfoPublication History

Accepted: July 3, 2021

Received in revised form: June 30, 2021

Received: May 11, 2021

Publication stageIn Press Journal Pre-ProofIdentification

DOI: https://doi.org/10.1016/j.ekir.2021.07.005

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© 2021 Published by Elsevier Inc. on behalf of the International Society of Nephrology.

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