Effects of hemodialysis on plasma oxylipins

Abstract Chronic kidney disease (CKD) is an important risk factor for cardiovascular and all‐cause mortality. Survival rates among end‐stage renal disease (ESRD) hemodialysis patients are poor and most deaths are related to cardiovascular disease. Oxylipins constitute a family of oxygenated natural products, formed from fatty acid by pathways involving at least one step of dioxygen‐dependent oxidation. They are derived from polyunsaturated fatty acids (PUFAs) by cyclooxygenase (COX) enzymes, by lipoxygenases (LOX) enzymes, or by cytochrome P450 epoxygenase. Oxylipins have physiological significance and some could be of regulatory importance. The effects of decreased renal function and dialysis treatment on oxylipin metabolism are unknown. We studied 15 healthy persons and 15 CKD patients undergoing regular hemodialysis treatments and measured oxylipins (HPLC‐MS lipidomics) derived from cytochrome P450 (CYP) monooxygenase and lipoxygenase (LOX)/CYP ω/(ω‐1)‐hydroxylase pathways in circulating blood. We found that all four subclasses of CYP epoxy metabolites were increased after the dialysis treatment. Rather than resulting from altered soluble epoxide hydrolase (sEH) activity, the oxylipins were released and accumulated in the circulation. Furthermore, hemodialysis did not change the majority of LOX/CYP ω/(ω‐1)‐hydroxylase metabolites. Our data support the idea that oxylipin profiles discriminate ESRD patients from normal controls and are influenced by renal replacement therapies.


| METHODS
The Charité University Medicine Institutional Review Board approved this registered study (ClinicalTrials.gov, Identifier: NCT03857984). In total, 15 healthy volunteers (6 male and 9 female) and 15 ESRD patients (7 men and 8 women) participated in the study (Table 1). Inclusion criteria for the group of CKD patients were: history of renal failure requiring hemodialysis/hemofiltration therapy, age over 18 years, the ability to consent, and written consent of the study participant. The patients in the group CKD were diagnosed for the following conditions: diabetes mellitus (4 patients), hypertension (3 patients), membranous glomerulonephritis (2 patients), ADPKD (autosomal dominant polycystic kidney disease) (1 patient), other or unknown (5 patients). Exclusion criteria for healthy volunteers were: age under 18 years, chronic illness requiring any medication, pregnancy, inability to follow simple instructions, relevant or severe abnormalities in medical history, or physical examination . 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).
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 (pre-HD) and at the end of the dialysis (5-15 min before termination, post-HD). 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. Following centrifugation, supernatant plasma was immediately transferred into clean polypropylene tubes using an Eppendorf pipette. The samples were maintained at 2-8°C while handling. Aliquots (0.5 ml) were then stored at -80°C until further processing and extraction. Overall, the processing took no longer than 10 min. All samples were analyzed for free and total plasma oxylipins. Oxylipins were determined by high-performance liquid chromatography mass spectrometry (HPLC-MS) spectrometry described in (Fischer, 2014) (Gollasch, 2019a). Descriptive statistics were T A B L E 1 Characteristics of hemodialysis (HD) patients and control subjects (n = 15 each)

HD patients Controls
Age ( calculated and variables were examined for meeting assumptions of normal distribution without skewness and kurtosis. We used the Shapiro-Wilk test to determine if they were normally distributed. In order to determine statistical significance, a two-tailed t test or Mann-Whitney test were used to compare values of CKD versus control groups. Homogeneity of variances was asserted using Levene's test. Paired t test or paired Wilcoxon test were used to compare pre-HD versus post-HD values. In order to determine the statistical significance between the four classes of epoxy metabolites hydrolyzed to appear in the circulation, Friedman's test was used followed by applying Dunn's multiple comparison test (Gollasch, 2019a(Gollasch, , 2019b. 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, Armonk, NY, USA).

| Diol/epoxide ratios
As shown in Figure 1, the main pathway of EET, EpOME, EEQ, and EDP metabolism in many cells is conversion into DHETs, DiHOMEs, DiHETEs, and DiHDPAs by the soluble epoxide hydrolase enzyme (sEH), even though epoxy-polyunsaturated fatty acids (EpPUFA) can also be nonenzymatically hydrolyzed into dihydroxymetabolites (DiHPUFA) (Spector, 2004). Since ESRD might have caused EET, EpOME, EEQ, and EDP production rapidly degraded to their diols, we next analyzed the sums of the individual CYP epoxy metabolites and their diols (Table 3A). We found that ESRD was associated with increased levels of the majority of those CYP metabolites (Table 3A). To provide insights into possible mechanisms underlying this increase, we calculated diol/epoxide ratios of the epoxy metabolites (Table 3B and C). We found that the four classes of epoxy metabolites are unequally hydrolyzed to appear in the circulation (Friedman's test, p < .05). We found that EpOMEs and EDPs are better metabolized into their diols (ratio of DiHOMEs/EPOMEs and DiHDPAs/EDPs; 0.156 ± 0.301 and 0.114 ± 0.141, respectively; Dunn's multiple comparison test, p > .05) than EETs and EEQs (ratios of those diols/epoxy metabolites, T A B L E 2 Comparison of oxylipins between control subjects versus. CKD patients before hemodialysis (HD) (n = 15)

Amount ng/mL Control (Mean ± SD) HD (mean ± SD) p-value t-Test ( # Mann-Whitney Test) Data grouping effect
A. Total oxylipins in plasma.  (Table 3B). In fact, the following order of ratios was identified: DiHOMEs/ EpOMEs = DiHDPA/EDPs > DHETs/EETs = DiHETEs/ EEQs (Dunn's multiple comparison test, p < .05). This pattern was also found for the individual metabolites in vivo, as shown (Table 3C). Together, the findings indicate that CYP epoxy metabolites are released and accumulated in the circulation of ESRD HD patients, compared to controls, with epoxy metabolite substrate classes unequally hydrolyzed by sEH in vivo.

| DISCUSSION
Our data demonstrate that all four subclasses of CYP epoxy metabolites and several LOX/CYP ω/(ω-1)-hydroxylase metabolites are increased by the hemodialysis treatment. We found that these changes are unlikely related to altered sEH activity. The data are also not related to alterations of plasma or red blood cell (RBC) fatty acid levels, in particular RBC n-3 fatty acid status, which we demonstrated in our previous study . Despite significant changes in fatty acids signatures between healthy persons and CKD patients, we observed that hemodialysis does not alter plasma or RBC fatty acid levels to potentially explain the observed changes of oxylipins in the present study . Our data support the idea that 9,10-EpOME, 12,13-EpOME, 5,6-DHET, T A B L E 3 (Continued) T A B L E 4 Effects of hemodialysis on oxylipins in the CKD patients before (pre-HD) and at cessation (post-HD) of hemodialysis (n = 15 each) and 5-HETE are key markers to discriminate ESRD patients from healthy controls (Hu, 2018). It is unlikely that these changes occurred in response to chronic dialysis treatment since altered levels in 9,10-EpOME, 12,13-EpOME, 5,6-DHET, and 5-HETE levels were observed in patients with CKD before starting renal replacement therapy (Hu, 2018). While the ESRD patients in (Hu, 2018) were uremic Asians (eGFR 5-6 ml/min/1.73 m 2 ) and recruited before beginning renal replacement therapy for fistula construction surgery, our patients were Caucasian ESRD patients undergoing regular, thrice weekly dialysis. Nonetheless, we observed similar changes in 9,10-EpOME, 12,13-EpOME, 5,6-DHET, and 5-HETE levels. However, our study revealed also other oxylipins that is, specific signature, which are up-or down-regulated in plasma of the ESRD patients. The extent to which they exhibit beneficial or detrimental cardiovascular effects, T A B L E 5 Effects of hemodialysis on oxylipins and their ratios in the CKD patients before (pre-HD) and at cessation (post-HD) of hemodialysis (n = 15)

Data grouping effect
A. Concentrations of individual total epoxides plus their respective diols in plasma.
9,10-EpOME + 9,10-DiHOME possibly in metabolite-interacting networks, remains to be explored. Furthermore, future studies can clarify whether specific underlying renal diseases may have specific oxylipin profiles to discriminate between ESRD patients.

| EETs/DHETs
We demonstrated that hemodialysis increased EETs/DHETs levels, as detected for 5,6-EET, 14,15-EET, 8,9-DHET, and 11,12-DHET. Endothelial cells are reservoirs of EETs and the primary source of plasma EETs (Jiang, Anderson, & McGiff, 2010Jiang, 2011;Schunck, 2017), which produce profibrinolysis and reduce inflammation, vascular tone, and blood pressure (Jiang, Anderson, & McGiff, 2010Jiang, 2011). 5,6-DHET as like 5,6-EET can produce vasodilation (Hercule, 2009;Lu, 2001), which could contribute to the cardiovascular response during maximal exercise (Gollasch, 2019a). The mechanisms of how epoxides and diols are released from the tissues and eventually become constituents of circulating lipoproteins are largely unknown, making it difficult to explain our findings. Cells preferentially release DHETs while storing the EETs (Roman, 2002), suggesting that certain diols might be overrepresented in the circulating blood compared with the respective diol/epoxide ratios (Fischer, 2014). Our data support the idea that DHETs/ EETs are attractive signaling molecules for cardiovascular effects in ESRD because they are potent vasodilators (Campbell & Fleming, 2010), which could counteract circulating vasoconstrictor substances during dialysis. Therapeutic sEH inhibition is considered a novel approach for enhancing the beneficial biological activity of EETs (Spector & Kim, 2015). However, presumably higher levels of EETs in blood and tissue in vivo may have also detrimental cardiovascular side effects (Gschwendtner, 2008;Hutchens, 2008;Wutzler, 2013). Of note, levels of all four EETs (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) were high in the ESRD patients compared to the control. The extent to which this increase has beneficial or detrimental cardiovascular effects remains to be explored.

| EEQs/DiHETEs
We observed increases in 8, 11,14,17,14,and 17,18-DiHETE during dialysis. While the putative biological functions of EEQs/DiHETEs have not received much attention, 17,18-EEQ has been identified as a potent vasodilator, which seems to be even more potent than EETs (Hercule, 2007;Lauterbach, 2002). Their diols could contribute to the cardiovascular response during maximal exercise (Gollasch, 2019a). The mechanisms of how EEQs/DiHETEs are released from the tissues are largely unknown, making it difficult to explain our findings. Based on our calculations of diol/epoxide ratios, we have no evidence that the higher levels of 14,15-DiHETE, 17,18-DiHETE result from in vivo sEH enzyme activation. Nevertheless, the role of circulating EEQs/DiHETEs has yet to be integrated into a physiological and pathophysiological context. This is particularly important since drugs that mimic 17,18-EEQ are viewed as novel promising drug candidates to overcome limitations of dietary EPA/DHA (C20:5 n-3/22:6 n-3) supplementation for cardiovascular health benefits (Schunck, 2017).
Our data indicate that both EDPs and DiHPAs metabolites are novel candidates for vasoactive substances potentially released by dialysis to affect hemodynamics in these conditions.

| CONCLUSIONS
To our knowledge, this is the first study to assess the impact of single hemodialysis treatment oxylipins in plasma using large-scale lipidomics. We confirmed our hypothesis that the oxylipins status is influenced by hemodialysis treatment.
Our data demonstrate that all four subclasses of CYP epoxy metabolites and a number of LOX/CYP ω/(ω-1)-hydroxylase metabolites are increased by the treatment. Moreover, ESRD patients undergoing regular dialysis show marked differences in plasma oxylipin profiles, that is, specific signatures, compared to control subjects. Future research is required to determine the contribution of the identified oxylipins in reducing the risk from CVD in patients with kidney disease.