Immunophenotypic Characterization of Citrate-Containing A Concentrates in Maintenance Hemodialysis: A Pre-Post Study

Introduction Due to chronic inflammation, maintenance hemodialysis (MHD) patients continue to show excess mortality. Acetate-free citrate-buffered A concentrates could be a way to improve the biocompatibility of the procedure, reduce chronic inflammation, and thus in the long term improve the prognosis of patients. Methods Using a pre-post design (3 months of acetate followed by 3 months of citrate-acidified A concentrates in standard bicarbonate-based dialysate hemodialysis, CiaHD) and linear mixed model analysis in 61 stable HD patients, we assessed the impact of CiaHD on counts and phenotypes of peripheral T cells and monocytes by flow cytometry. Results Switching to CiaHD left C-reactive protein (CRP) levels and leucocyte counts unaffected. However, CiaHD increased lymphocyte counts ex vivo. Furthermore, we found a decrease in total CD3+CD4+CD69+ ((109/L), mean ± SD: acetate, 0.04 ± 1.0 versus citrate, 0.02 ± 0.01; P = 0.02) activated cells, while the number of CD28+ T cells remained stable. No differences were noted regarding T-cell exhaustion marker expression, CD14+CD16+ monocyte counts, and PMN-MDSCs. Conclusion Compared with acetate, CiaHD has a minor impact on lymphocyte counts and CD4+T-cell activation, which was independent of systemic CRP and ionized magnesium, calcium levels, and other dialysis prescription modalities.


Introduction
Patients undergoing maintenance hemodialysis (MHD) have an adjusted mortality rate of 165 per 1000 patients/year [1].Tis is partly attributed to a residual uremic environment and chronic infammation [2,3].Although the pathophysiological mechanisms involved are not completely understood, immune dysfunction is regarded as a hallmark of progressive infammation in MHD patients [4].Chronic infammation is known to increase the risk of infectious and cardiovascular diseases [5].Immune dysfunction in MHD patients involves complex phenotypic changes in diverse immune cells [4,6].As a sign of aberrant T-cell activation, increased expression of the surface activation marker CD69 and loss of CD28 and a skewed CD4+/ CD8+ ratio have been described [7,8].Tese alterations have been linked to systemic infammation and atherosclerosis progression in MHD patients [9][10][11].Others have reported lymphopenia in MHD patients with overexpression of PD-1 and TIM-3 on peripheral lymphocytes, indicating T-cell exhaustion and vulnerability to chronic infections and viral diseases [12,13].Furthermore, altered expression and interaction of costimulatory receptors such as CD28/PD1 with their ligands CD86/PDL1 might impact monocyte activation, resulting in innate immune system dysfunction [14,15].In this regard, the aberrant expansion of CD14+CD16+ monocytes has been identifed as a predictor of mortality in HD patients [16,17].
Te spectrum of dysfunctional immune cells in MHD has recently been expanded for myeloid-derived suppressor cells (MDSCs).MDSCs can be divided into polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs).Interestingly, PMN-MDSCs share similar phenotypical and morphological features with neutrophils and are increased in chronic infectious or infammatory diseases [18,19].An expansion of PMN-MDSCs was reported for MHD patients and appeared to be associated with infections [20].
Acetate is widely adopted as an acidifying agent within the cation concentrate (A concentrate) in standard bicarbonatebased MHD.However, the intraindividual acetate concentration during HD procedures exceeds physiological levels, resulting in more intradialytic hypotensive episodes and associated adverse efects [21,22].In contrast, citrate-acidifed A concentrates (CiaHD) in standard bicarbonate-based HD are considered more biocompatible.Compared to the conventional acetate-bufered HD procedure, an improvement in dialysis efciency, a reduction of the systemic infammatory response, i.e., serum C-reactive protein (CRP), and a better metabolic state have already been described by others [23][24][25].Also, magnesium and calcium are essential cofactors required for immune cell activation [26].Herein, citrate as a potent chelator of calcium and magnesium ions could lower free extracellular concentrations and potentially reduce aberrant immune cell activation [27].However, the efect of citratebufered A concentrates in standard bicarbonate-based HD on the cellular immunophenotype is still unclear.Tis study was designed to investigate whether CiaHD impacts the cellular immunophenotypes in MHD patients.

Study Participants.
Between April and June 2016, we recruited 78 MHD patients, of which 61 completed the entire study period.Tey were aged ≥18 years from two local dialysis units in Munich, Germany.All of them required thrice-weekly dialysis sessions with an average duration of ≥4 hours and HD vintages ≥two months.Missing data were due to unwillingness to complete the study, death, and technical reasons.Missing data were not imputed and referenced below the tables.Exclusion criteria were pregnancy, ongoing severe infection, cancer, malignant hematologic diseases, and lack of written or informed consent.

Study Design and Intervention
. Tis post hoc pre-post design clinical trial was conducted in a subgroup of the "Substitution of Acetate by Citrate in Bicarbonate-Based-Hemodialysis" study (NCT02745340).Patients in two dialysis units from Munich were on acetate-containing A concentrates for 3 months (SelectBagOne; 3 mmol/L of acetate); they were then switched to three months of citrate-acidifed A concentrates (SelectBagCitrate; acidifed with citric acid, which is converted to 1 mmol/L citrate solution � CiaHD, supplied by Baxter).Te clinical data and blood specimens were collected before (three months of acetate-containing A concentrates) and after three months of CiaHD prior to a midweek dialysis session as predefned in the mother study NCT02745340.It should be noted that centers were allowed to adjust the membrane and dialysis prescription to achieve adequate Kt/V or due to supply difculties and fscal reasons.Te study protocol was approved by the local ethics commission.It was carried out in accordance with the Declaration of Helsinki, adhering to good clinical practice guidelines.

Clinical Data
Assessment.Characteristics such as patients' age, gender, body mass index (BMI), comorbidities, and medication were assessed as previously described [24].Immune suppressive mediation was recorded by chart review and included glucocorticoid, nonsteroidal antiinfammatory drugs (NSAIDs), calcineurin inhibitor (tacrolimus in the study), and mycophenolate mofetil (MMF).
Te cohort data set used to support the fndings of this study has not been made available because of legal and patient privacy reasons-it is available from the corresponding author upon reasonable request in an anonymized form.

Blood Specimen Collection and Experimental Methods.
Blood samples were collected prior to a midweek dialysis session prior and after three months of CiaHD and processed as previously described [24].Ionized magnesium and calcium levels were determined from frozen sera using the NOVA 8 analyzer and ion-selective electrodes for calcium and magnesium following the manufacturer's instructions (Nova Biomedical, Waltham, MA, US) [28].Te number of neutrophils and lymphocytes and the level of CRP were examined by an ISO-certifed clinical laboratory.
Peripheral blood mononuclear cells (PBMCs) were isolated using BD Vacutainer ® CPT tubes within 2 hours post collec- tion following the manufacturer's protocol.Cells were then stained and analyzed immediately using the antibodies provided in supplementary Table 1.FMOs were used to defne the gating for CD28, CD69, and TIM-3, respectively.T cells were stained using antibodies for CCR7, CD25, CD45RO, CD3, CD4, CD8, CD28, CD69, PD1, and TIM3.Most surface markers were stained for 30 mins at 4 °C.CCR7 was stained for 15 mins at 37 °C before the following "cold" incubation with the remaining cocktail, including an additional washing step in between.PI was used as a viability dye.FSC-Height versus FSC-Width blots were used for the exclusion of doublets.
Monocytes were stained using CD3 and CD56 to exclude NK cells and T cells.CD14, CD16, HLA-DR-APC, and PDL1 were used to identify monocyte subpopulations.FMO was used to defne gating for PDL1.Live/dead fxable blue stain (Termo Fisher Scientifc, Waltham, USA) was used as a viability dye.
PMN-MDCS was identifed using CD14, CD15, and CD11b.Gating ancestries can be retrieved from supplementary Figures 1-3   International Journal of Nephrology median, and interquartile range (IQR) as appropriate.Te independent samples t-test and Wilcoxon-Mann-Whitney test were used for comparing the baseline data from diferent units as necessary.A linear mixed model was built to analyze the alterations of immune phenotypes in acetate versus CiaHD (pre-post design).Absolute counts of cell populations were examined as dependent variables, with treatment, (citrate � 1, acetate � 0) as the main efect.In addition, to check for confounding of our results, linear mixed models were adjusted for dialysis prescription characteristics, i.e. type of membrane, dialysis modality, vascular access, and dialysis session duration (see results section and supplement for further details).To test for confounding by hemoconcentration, intake of immunosuppressive drugs including NSAIDs, and dialysis modality (HD vs. hemodiafltration (HDF)), an additional model was built and adjusted for these covariates (see supplementary Table 6).
Restricted maximum likelihood (REML) was used.P value <0.05 was considered signifcant.Since this was a post hoc exploratory study, no power calculation was performed.
With regards to diferences pre versus post CiaHD, it should be stated that one patient was switched from the AV fstula to the central venous catheter during the study course.Efective session duration was slightly shorter during CiaHD (acetate: 4.1 ± 0.2 vs. citrate: 3.9 ± 0.3 hours, P � 0.039, supplementary Table 3).In addition, diferent membranes were used over the course of the study in 46% of patients (supplementary Table 4), whereas other parameters of HD (F) prescription were kept constant as reported in supplementary Table 3.Nevertheless, our models were adjusted for these parameters to control for confounding (supplementary Table 5).

Citrate-Containing A Concentrates Are Associated with
Reduced Activation of CD4+ T Cells.Still, due to various reports by others [13][14][15][16], the initial hypothesis was that CiaHD would benefcially impact the chronic infammatory milieu and alter the T-cellular infammatory phenotypes, which could still occur in the absence of measurable alterations in systemic CRP levels.
It should be noted that there was no signifcant interaction efect of CRP treatment (citrate � 1) on CD3+CD4+CD69+ cells.Furthermore, calcium and magnesium levels were inversely associated with these cells and did not signifcantly improve the regression model (Table 4).
Analysis of exhaustion markers, PD-1 and TIM-3, on peripheral lymphocytes did not reveal altered frequencies of PD1+ T cells.Yet, a trend of slightly lower numbers of TIM3+CD3+CD8+ T cells was associated with 3 months of citrate-bufered A concentrates ((10 9 /L), mean ± SD: acetate, International Journal of Nephrology 0.007 ± 0.005 versus citrate, 0.006 ± 0.005; P � 0.21) (Table 3, supplementary Figure 4(A)-4(D)).Terefore, we conclude that citrate might impact T-cell activation and exhaustion status in MHD patients in the absence of measurable alterations in systemic CRP levels.
International Journal of Nephrology   6 International Journal of Nephrology adjusted for change in membranes, efective session duration, vascular access type, and HD modality (P value � 0.2; supplementary Table 5).

Discussion
Chronic infammation and aberrant immune cell activation are well described characteristics of MHD patients.All of them have been linked to adverse outcomes in this population [30][31][32].Compared with acetic-acid-bufered A concentrates in standard bicarbonate HD, CiaHD has been reported to enhance HD efciency and reduce systemic infammation and vascular smooth muscular cell (VSMC) dysfunction [33,34].Furthermore, CiaHD has been one attempt to improve the HD-procedure's biocompatibility and restore immunity in end-stage renal disease (ESRD) [23,35].Nevertheless, and in contrast to similar studies [33,36], in our pre-post study, CiaHD did not benefcially infuence systemic CRP or IL-6 levels.However, our data are not unique in fnding stable or increasing systemic CRP and IL-6 levels [35], which might be related to patient individual factors, diferently tuned dialysate mixtures, or cohort size.Despite that, we found a signifcant reduction of circulating activated CD3+CD4+CD69+ cells after 3 months of  International Journal of Nephrology CiaHD, indicating some impact on cellular immunity.Still, the post hoc and exploratory study design must be considered when interpreting these data.In our cohort, reduction of CD3+CD4+CD69+ cells during CiaHD treatment occurred independently of changes in CRP and systemic magnesium and calcium levels, which in contrast to CiaHD were not signifcantly associated with CD3+CD4+CD69+ cells (Table 4).Tis association was further independent of diferent HD-membranes used in 46% of study participants and other parameter prescription.Tus, citrate per se (independent of its function as a chelator) or the lack of supra-physiological acetate concentrations might have reduced CD4+ T-cell activation.In fact, acetate has been shown to impact T-cell efector function and promote interferon production upon chronic infammatory conditions such as tumor environments [37].Tese considerations however remain speculative and require further study.
In addition, and consistent with previous studies [38], we found increased lymphocyte counts after 3 months of CiaHD.T-cell lymphopenia in hemodialysis is documented to increase the risk of infectious episodes and is considered a marker of impaired immunity [13,[39][40][41].Taken together, it is tempting to speculate that CiaHD could reverse some aspects of aberrant immunity in MHD, namely, CD4+ T-cell activation and lymphopenia.Yet, the overall impact of CiaHD on immunophenotypic changes appeared small, as CD14+CD16+ monocyte counts and the number of PMN-MDSCs, which are known to suppress T-cell activation and function during infectious conditions [42], were unafected, and no benefcial serologic changes were noted.Lastly, it should be mentioned that diferent membrane types matched by dialysis centers were also independently associated with a reduction in CD4+CD69+ T cells (see supplementary Table 5).Tus, cumulatively, further optimization of MHD biocompatibility seems feasible and reasonable by several approaches.
Taken together, our study is merely a characterization of immune-phenotypic changes related to acetate-free citrate-acidifed A concentrates in standard bicarbonate MHD.Several limitations must be mentioned.Since this was an exploratory and descriptive study with only a decent sample size, we cannot exclude residual confounding including nonobserved changes in dialysis prescription by the participating units.In addition, the immunephenotypic changes observed were relatively minor on citrate versus acetate-containing dialysates with no efect on the systemic proinfammatory mediator milieu.Furthermore, we lacked prospective data on clinical endpoints to defne the clinical implications of the observed immunephenotypic changes.
In conclusion, compared with acetate-containing A concentrates in standard bicarbonate MHD, CiaHD had a mild impact on the cellular immunophenotype: increased lymphocyte count and reduced CD3+CD4+ T-cell activation, indicating that CiaHD could revert some disorders of cellular immunity independently of systemic CRP levels.Investigating the implications of these changes requires more extensive studies. .

2. 5 .
Statistical Analysis.SPSS Statistics 23 (IBM, Armonk, NY, USA) was used for statistical analysis.We report the percentage of total, mean ± standard deviation (SD),

Table 1 :
Baseline characteristics of the study population.
Body mass index (BMI); Carlson comorbidity index (CCI); chronic obstructive pulmonary disease (COPD); nonsteroidal anti-infammatory drugs (NSAIDs).Values are given as either mean ± standard deviation or number (percentage) or median (IQR).Te independent-samples t-test and the Wilcoxon-Mann-Whitney U test were used for comparison of dialysis units at baseline.P < 0.05 was considered signifcant.a One patient was prescribed combined immunosuppressive treatment.b 1 missing value.

Table 2 :
Changes in clinical parameters after switching to citrate-acidifed A concentrates.

Table 3 :
Changes in leucocyte and T-cell phenotypes after switching to citrate-acidifed A concentrates.

Table 5 :
Changes in monocyte phenotypes and PMN-MDSCs after switching to citrate-acidifed A concentrates.linearmixedmodel was built to analyze the parameters before and after switching to citrate dialysate (with treatment as the main efect: citrate � 1, acetate � 0).To test for robustness of our results and to rule out confounding as far as possible, another model was built and adjusted for: HD-membranes used during acetate versus citrate, vascular access type, HD vs. HD (F), and session duration (see supplementary Table4).Similar results as depicted above were obtained for treatment (citrate � 1).P < 0.05 was considered signifcant.a 1 missing values in the citrate treatment group.b 9 missing values of PMN-MDSCs in acetate treatment groups.Figure3: Te alterations of monocytes and the subset PMN-MDSC and PDL1+ cells before and after switching to citrate-acidifed A concentrate dialysis for three months.Absolute cell numbers are presented as histograms (mean with the standard error of mean).� 61; citrate, n � 60).P � 0.05 was considered signifcant due to the exploratory design of the study. A