Hemodialysis and erythrocyte epoxy fatty acids

Abstract Fatty acid products derived from cytochromes P450 (CYP) monooxygenase and lipoxygenase (LOX)/CYP ω/(ω‐1)‐hydroxylase pathways are a superclass of lipid mediators with potent bioactivities. Whether or not the chronic kidney disease (CKD) and hemodialysis treatments performed on end‐stage renal disease (ESRD) patients affect RBC epoxy fatty acids profiles remains unknown. Measuring the products solely in plasma is suboptimal. Since such determinations invariably ignore red blood cells (RBCs) that make up 3 kg of the circulating blood. RBCs are potential reservoirs for epoxy fatty acids that regulate cardiovascular function. We studied 15 healthy persons and 15 ESRD patients undergoing regular hemodialysis treatments. We measured epoxides derived from CYP monooxygenase and metabolites derived from LOX/CYP ω/(ω‐1)‐hydroxylase pathways in RBCs by LC–MS/MS tandem mass spectrometry. Our data demonstrate that various CYP epoxides and LOX/CYP ω/(ω‐1)‐hydroxylase products are increased in RBCs of ESRD patients, compared to control subjects, including dihydroxyeicosatrienoic acids (DHETs), epoxyeicosatetraenoic acids (EEQs), dihydroxydocosapentaenoic acids (DiHDPAs), and hydroxyeicosatetraenoic acids (HETEs). Hemodialysis treatment did not affect the majority of those metabolites. Nevertheless, we detected more pronounced changes in free metabolite levels in RBCs after dialysis, as compared with the total RBC compartment. These findings indicate that free RBC eicosanoids should be considered more dynamic or vulnerable in CKD.


| INTRODUCTION
Chronic kidney disease (CKD) is a risk factor for the composite outcome of all-cause mortality and cardiovascular disease (Weiner et al., 2004). Although mortality and cardiovascular disease burden have decreased for end-stage renal disease (ESRD) hemodialysis patients in the United States, the 5-year mortality is still ~50% (McGill et al., 2019), Most of these deaths are related to cardiovascular disease (CVD) (Felasa | Federation for Laboratory Animal Science Associations, 2012; Luft, 2000). Dietary omega-3 (n-3) fatty acid intake is associated with a reduced CVD risk (Harris et al., 2008;Huang et al., 2011;InterAct Consortium et al., 2011). Erythrocyte red-blood-cell (RBC) n-3 fatty-acid status is inversely related to cardiovascular events, such as cardiac arrhythmias, myocardial infarction, and sudden cardiac death (Bucher et al., 2002).

| METHODS
The Charité University Medicine Institutional Review Board approved this duly registered study (ClinicalTrials.gov,Identifier: NCT03857984). Recruitment was primarily via person-to-person interview. Prior to participation in the study, 15 healthy volunteers (6 male and 9 female) and 15 CKD patients (7 male and 8 female) undergoing regular hemodialysis treatment signed informed consent forms which outlined the treatments to be taken and the possible risks involved. All healthy control subjects were not taking medications. Venous blood was collected in each healthy subject by subcutaneous arm vein puncture in the sitting position. In the group of dialyzed patients (CKD group), all the blood samples were collected on the fistula arm right before beginning of the dialysis (starting of the HD, pre-HD) and at the end of the dialysis (5-15 min before termination, post-HD). Patients underwent thrice-weekly dialysis, which lasted from 3 hr 45 min to 5 hr, based on high flux AK 200 dialyzers (Gambro GmbH, Hechingen, Germany). All samples were analyzed for RBC lipids. All blood samples were obtained by 4°C precooled EDTA vacuum extraction tube systems. Cells were separated from plasma by centrifugation for 10 min at 1,000-2,000 g using a refrigerated centrifuge RBCs were separated from EDTA blood by centrifugation as previously described . RBC lipidomics was performed using LC-MS/MS tandem mass spectrometry as described in (Fischer et al., 2014;Gollasch et al., 2019;Gollasch et al., 2019). Concentrations are given in nanogram/g. Descriptive statistics were calculated and variables were examined for meeting assumptions of normal distribution without skewness and kurtosis. In order to determine statistical significance, t test or Mann-Whitney test was used to compare the values of CKD versus control groups. Paired ttest or paired Wilcoxon test were used to compare pre-HD versus post-HD values. In order to determine statistical significance between the four classes of epoxy-metabolites hydrolyzed to appear in the circulation, Friedman's test followed by applying Dunn's multiple comparison test was used. In order to determine statistical significance between the four classes of epoxy-metabolites hydrolyzed to appear in the circulation, Friedman's test followed by applying Dunn's multiple comparison test was used. The analysis included Mauchly's test of sphericity followed by applying the test of within-subjects effects with Greenhouse-Geisser correction to ensure sphericity assumption (Gollasch et al., 2019;Gollasch et al., 2019). The .05 level of significance (p) was chosen. All data are presented as mean ± SD. All statistical analyses were performed using SPSS Statistics software (IBM Corporation) or All-Therapy statistics beta (AICBT Ltd).

Control (Mean ± SD) HD (Mean ± SD) p-value, Mann-Whitney test
Ratio (9,10-DiHOME+12,13-DiHOME)/ (9,10-EpOME+12,13-EpOME) T A B L E 1 (Continued) T A B L E 3 Effects of hemodialysis on epoxy-and hydroxy-metabolites in the CKD patients before (pre-HD) and at cessation (post-HD) of hemodialysis (n = 15 each)  ,compared to control subjects. Furthermore, hemodialysis treatment is insufficient to change the total concentrations of these and other LOX/CYP metabolites in RBCs of ESRD patients. Since the four subclasses of CYP epoxy metabolites increase in plasma after the dialysis treatment , we suggest that total CYP metabolites in RBCs are relatively invulnerable in CKD and hemodialysis (possibly due to slow exchange). Of note, ESRD is associated with increased levels of several free CYP epoxides and LOX/CYP ω/(ω-1)-hydroxylase metabolites in RBCs. Since several of those mediators are also increased by hemodialysis treatment itself, we suggest that free RBC eicosanoids constitute a fraction of lipid mediators, which are particularly vulnerable in CKD and hemodialysis. The extent to which the RBC eicosanoids exhibit beneficial or detrimental cardiovascular effects in CKD, possibly in comprehensive lipidomic (patho)physiological networks, remains to be explored. Nonetheless, our results indicate that RBCs could represent a reservoir for PUFA CYP epoxy-metabolites and LOX/ CYP hydroxy metabolites, which on release may act in a T A B L E 3 (Continued) T A B L E 4 Effects of hemodialysis on epoxide and their respective diol ratios in the CKD patients before (pre-HD) and at cessation (post-HD) of hemodialysis (n = 15 each). Ratios were estimated using total concentrations of epoxides and diols in RBCs

| EETs
RBCs are reservoir of EETs which on release may act in a vasoregulatory capacity (Jiang et al., 2010(Jiang et al., , 2011. In addition to serving as carriers of O 2 , RBCs are known to regulate the microvascular perfusion by liberating adenosine triphosphate (ATP) and EETs upon exposure to a low O 2 environment (Jiang et al., 2010;Sprague et al., 2010). The release of EETs is activated by P2X 7 receptor stimulation via ATP to cause the circulatory response (Jiang et al., 2007). RBCs are believed to serve as a source of plasma EETs, which are esterified to the phospholipids of lipoproteins. Therefore, levels of free EETs in plasma are found to be low (~3% of circulating EETs) (Jiang et al., 2010(Jiang et al., , 2011. Erythro-EETs are produced by direct oxidation of AA and the monooxygenase-like activity of hemoglobin (Jiang et al., 2010(Jiang et al., , 2011(Jiang et al., , 2012. On release, EETs and their diols (DHETs) produce vasodilation (Hercule et al., 2009;Lu et al., 2001), are profibrinolytic and reduce inflammation (Jiang et al., 2010(Jiang et al., , 2011(Jiang et al., , 2012. Exhaustive exercise increases the circulating levels of 5,6-DHET (Gollasch et al., 2019). In this study, we were able to demonstrate that RBCs of ESRD patients show increased accumulation of total DHETs. In particular, we observed increases in total concentrations of 8,9-DHET and 14,15-DHET in the RBCs. Hemodialysis did not affect this accumulation. It remains unknown whether RBCs are capable of liberating erythro-DHETs into the blood and/or tissues in kidney patients. Our results indicate that CKD affects the RBC reservoir for DHETs, but not EETs, which on release may affect the cardiovascular response.

| Other PUFA metabolites
We observed increases in total concentrations of EEQs  (Hercule et al., 2007;Lauterbach et al., 2002;Morin et al., 2011;Ulu et al., 2014). EDPs have antiangiogenic (McDougle et al., 2017), anti-fibrotic (Sharma et al., 2016) and protective effects in post-ischemic functional recovery, at least in particular by maintaining mitochondrial function and reducing inflammatory responses (Arnold et al., 2010;Darwesh et al., 2019). It is possible that their diols (DiHDPAs) are also biologically active and may exert beneficial effects in cardiac arrhythmias (Zhang et al., 2016). DiHDPAs dilate coronary microvessels with similar potency to EEQ isomers in canine and porcine models (Zhang et al., 2001) and inhibit human platelet aggregation with moderately lower potency to EDPs and EEQs (VanRollins, 1995). Specific 17,18-EEQ analogs are in development to serve as novel antiarrhythmic agents (Adebesin et al., 2019). HETEs are involved in many chronic diseases such as inflammation, obesity, cardiovascular disease, kidney disease, and cancer, for review see (Gabbs et al., 2015). Nonetheless, it remains unknown whether RBCs are capable of liberating EEQs, DiHDPAs, or HETEs into blood or tissues.
Our data indicate that both metabolite classes are novel candidates potentially released by RBCs to exhibit cardiovascular effects in health and CKD. Surprisingly, we did detect increases in various free CYP epoxides and LOX/CYP ω/(ω-1)-hydroxylase metabolites in RBCs in ESRD, which were augmented by hemodialysis. The mechanism by which CKD and hemodialysis raises the levels of those erythro-metabolites is not known. Since those metabolites cannot be synthesized endogenously in appreciable amounts, accelerated release into and uptake from plasma could be a possible explanation. The more pronounced changes observed in free metabolite levels within the RBCs, as compared with the total RBC compartment, indicate that free erythro-eicosanoids should be considered more dynamic or vulnerable with respect to metabolite flux. The design of our study does not differentiate between patient groups undergoing long-term dialysis therapy with regard to the specific underlying renal disease. Nevertheless, the impact of those epoxides and hydroxy metabolites has yet to be integrated into a (patho)physiological context.

| CONCLUSIONS
Our results show that CKD affects the levels of numerous CYP epoxides and hydroxy metabolites (DHETs, EEQs, DiHDPAs, and HETEs) in circulating RBCs compared to control subjects, which on release may act in a vasoregulatory capacity. Although hemodialysis treatment was insufficient to change the majority of those total metabolites, we detected pronounced changes in free metabolite levels within the ESRD RBCs and in response to hemodialysis, indicating that free erythro-epoxides could also contribute to the cardiovascular risk, for example, in diabetes or hypertension. More research is needed to determine the contribution of RBC epoxy-and hydroxy-metabolites to cardiac performance and blood pressure regulation in health, cardiovascular, and specific kidney diseases.