Serum trace metal association with response to erythropoiesis stimulating agents in incident and prevalent hemodialysis patients

Alterations in hemodialysis patients’ serum trace metals have been documented. Early studies addressing associations levels of serum trace metals with erythropoietic responses and/or hematocrit generated mixed results. These studies were conducted prior to current approaches for erythropoiesis stimulating agent (ESA) drug dosing guidelines or without consideration of inflammation markers (e.g. hepcidin) important for regulation of iron availability. This study sought to determine if the serum trace metal concentrations of incident or chronic hemodialysis patients associated with the observed ESA response variability and with consideration to ESA dose response, hepcidin, and high sensitivity C-reactive protein levels. Inductively-coupled plasma-mass spectrometry was used to measure 14 serum trace metals in 29 incident and 79 prevalent dialysis patients recruited prospectively. We compared these data to three measures of ESA dose response, sex, and dialysis incidence versus dialysis prevalence. Hemoglobin was negatively associated with ESA dose and cadmium while positively associated with antimony, arsenic and lead. ESA dose was negatively associated with achieved hemoglobin and vanadium while positively associated with arsenic. ESA response was positively associated with arsenic. Vanadium, nickel, cadmium, and tin were increased in prevalent patients. Manganese was increased in incident patients. Vanadium, nickel, and arsenic increased with time on dialysis while manganese decreased. Changes in vanadium and manganese were largest and appeared to have some effect on anemia. Incident and prevalent patients’ chromium and antimony levels exceeded established accepted upper limits of normal.

Hemodialysis (HD) is a blood purification method intended to remove metabolic waste products ('uremic toxins') by primarily equilibrating the composition between the patients sera (higher concentrations) and the dialysate (lower concentrations) across a semipermeable dialyzer membrane 1 . In practice the method removes both low and medium molecular weight compounds including normal biologics as well as uremic toxins. Many factors affect the equilibration of solutes across the dialyzer membrane including concentration, size, charge, and compartmentalization. Chronic hemodialysis as a medical tool has been used as supportive care for individuals who have lost the function of their kidneys. The largest fraction of dialysis care is provided to adults 65 years old or Scientific Reports | (2020) 10:20202 | https://doi.org/10.1038/s41598-020-77311-8 www.nature.com/scientificreports/ ESA response and trace metal concentration. Our secondary objectives were to examine the changes in trace metal concentration between incident and prevalent patients, and between male and female given known baseline sex-dependent differences in Hb levels [39][40][41] .

Results
A total of 100 prevalent and 33 incident subjects were enrolled and sufficient sample was available for analysis in 108 subjects (79 prevalent and 29 incident patients). The results for primary metals measurements have been uploaded into the metabolomics workbench of the NIH Common Fund Metabolomics Program Data Repository and Coordinating Center (DRCC) as study ST000565. The demographic information on the population are shown in Table 1. A Pearson bivariate correlation analysis is shown in Fig. 1. Significant positive correlations were observed between ER (erythropoietin response, dose/Hb achieved) and nickel, cadmium, tin; ESA dose and nickel, cadmium, tin; hemoglobin (Hb) and antimony; CRP and copper. Significant negative correlations were observed between ER and manganese, antimony; ESA dose and selenium; Hb and manganese, CRP and molybdenum, selenium; transferrin saturation (Tsat) and copper. Hepcidin was not found to be significantly correlated to any measured trace metal.
To determine if there were a linear relationship of trace metals and anemia metrics, a multivariate regression of prior month ESA dose, Hb concentration and 6-month average ER and all of the trace metal concentrations was conducted. Starting with all trace metals, the final regression model was determined after sequentially removing individual trace metals that did not contribute significantly. The model results are presented in Figs. 2, 3 and 4 and Table 2 showing the variables (final trace metals) with the standardized beta coefficient demonstrating the strength of the association. The final regression model for a trace metal association with the prior month ESA dose arrived at a four variable model. The model included positive associations with arsenic (approaching significance at p-value < 0.06) and lead, and negative associations with Hb concentration and vanadium. The regression model for trace metal association with Hb concentration arrived at a six variable model that included positive associations with antimony (approaching significance at p-value < 0.08), arsenic and lead and, negatively associations with manganese, cadmium, and ESA dose. The regression model for trace metal association with ER arrived at a three component model with positive associations with manganese and arsenic, and a negative associate with chromium.
The results of the inductively coupled plasma-mass spectrometry (ICP-MS) analysis for each measured metal in prevalent and incident subjects are shown in Fig. 5. The differences with p-values less than 0.05 were observed for cadmium, manganese, tin, nickel, and vanadium. A linear regression of the metal concentration versus the time on dialysis showed an increase in vanadium (r2 = 0.15, p = 0.001), nickel (r2 = 0.06, p = 0.012), arsenic (r2 = 0.47, p = 0.019) and decreased in manganese (r2 = 0.07, p = 0.011). Population level differences in the Hb values are known 40 . Therefore an analysis of variance for trace metal concentrations by prevalence (incident versus prevalent patients) and sex (male versus female) was performed. As shown in Table 3 no metal by two-way ANOVA was significantly different considering sex alone. Manganese, nickel, cadmium and tin were significantly different as main effects and as previously described with simple t-test. Considering the combination of prevalence and sex, we observed a significant (p < 0.05) difference in the interaction p-value term for zinc; suggesting zinc increases in males and decreases in females. A comparison of mean fold differences for serum trace metal levels in incident and prevalent subject values compared to literature values for upper limit of normal [42][43][44][45][46] are shown in Fig. 6. As illustrated in Fig. 6, chromium and antimony serum levels were greater than twofold higher that upper limit of normal in both incident and prevalent patients. Significant excursions across the upper limit of normal with dialysis prevalence were observed for the mean vanadium levels (increased) and the mean manganese levels (decreased). www.nature.com/scientificreports/

Discussion
Anemia as well as trace metal derangements are common complications of advanced CKD and ESKD 17,47,48 . Since trace metals are important co-factors in many cellular processes including erythropoiesis and dialysis patients can have altered concentrations of trace metals we looked to see if ESA responsiveness was associated by differing concentrations of metals. We examined at three measures of ESA response: ESA dose, Hb achieved, and a factor that averages these measures over a 6 month period, the ER 49 . Of the trace metals tested, arsenic was the only one to be positively correlated within all three linear regression models for measures of ESA response. Arsenic was infrequently detected in the patient samples and at non-toxic concentrations that approached the maximum contaminant level for drinking water. Arsenic may be encountered in either inorganic or organic forms and with different valence states; arsenites/As(III) and arsenates/As(V) 50 . While our approach did not differentiate   In studies using CD34 + cells isolated from chronic myeloid leukemia patient bone marrow, sub-toxic levels of arsenic trioxide enhanced or primed erythroid colony forming activity. Additionally the same study demonstrated sub-toxic levels of arsenic trioxide inhibited globin gene expression in K562 cells, a chronic myelogenous leukemia patient derived erythroid-myeloid precursor cell line 53 . While the effect of arsenic appears biphasic in cell culture this cannot be assumed to be the case in the setting of chronic hemodialysis. Hence the positive correlation of arsenic with the anemia end-points (prior month ESA dose, Hb concentration, and ER) may reflect both the combined effects of sub-toxic levels of arsenic and increased erythroid colony formation with increased need for ESA dosing to achieve Hb target levels.
Lead was detected infrequently in patient samples and when observed it was noted the mean concentration decreased from incident to prevalent patients. Similar to arsenic the relative lead concentrations were below what  www.nature.com/scientificreports/ would be considered toxic. Lead was found to be a final component of the linear regression models associated with prior ESA dose and Hb concentration but not ER. Lead is a major public health issue and can cause adverse hematological effects in exposed workers 54 . Lead poisoning interferes with the heme synthesis pathway and can at high enough levels cause iron deficiency 55 . While there is no safe level of lead in the body, the maximum lead concentration observed in our study was 2.08 µg/dL. This is well below the action level of 5 µg/dL level if detect in children or the concentration of 10-25 µg/dL in adults indicating elevated exposure levels have been encountered. The positive correlation of lead with Hb may reflect ESA dosing overcoming lead impairment of erythropoiesis.
Manganese is an abundant trace metal with many important extra-and intra-cellular functions 56 . Manganese and iron uptake shares a common transporter for dietary uptake (DMT1) and storage (transferrin) 27,57 . Blood manganese and Hb values both positively correlate with renal function 58,59 . Functional iron deficiency defined as insufficient iron incorporation into erythroid precursors despite adequate iron levels is a frequent cause of anemia and often may be associated with high manganese levels 27,60 . Hence the relationship between manganese concentration, Hb and anemia is complicated. Our data showed a significant reduction in manganese from incident (3.0 µg/L) to prevalent (1.1 µg/L) patients. Multivariate linear regression identified six features that best explained the relationship of Hb to observed trace metal concentrations and ESA dose includes manganese. It is difficult to extrapolate the effects of single variables from logistic regression models but these data may point to trace metal interactions that collectively associated with lower Hb values or diminished ER.
The final trace metals found significant in the tests on anemia were cadmium, antimony, vanadium, and chromium. All of these metals are found to be higher in multiple cohorts of dialysis patients 17,20,61 . Pan et al. report from the National Health and Nutrition Examination Survey (NHANES) data on the association between trace elements (iron, zinc, copper, cadmium, selenium, manganese) and Hb levels in patient with normal renal function or CKD 59 . Regardless of renal function status, Hb concentrations were associated positively with cadmium and negatively with copper. Bayhan et al. report that increased but sub-toxic levels of blood cadmium associate with ineffective erythropoiesis in thalassemia major, thalassemia intermedia and congenital dyserythropoietic anemia 62 . These data suggest that sub-toxic levels of blood cadmium in individuals with a chronic hypoxic state are susceptible to the contribution of cadmium toward ineffective erythropoiesis.
In our study, cadmium was observed in a majority of patient samples and the median concentrations were higher in prevalent patients. While the cadmium levels were detected below the threshold suggesting toxicity, a regression analysis identified a moderate negative association of cadmium with Hb concentrations ( Table 2). Published data would suggest that cadmium could contribute to decreased Hb concentrations either through the development of a state of functional iron deficiency 63 or direct effects to diminished bone marrow populations of cells in the erythroid lineage, larger circulating populations of immature erythrocytes, and/or smaller populations of mature erythrocytes concomitant with diminished oxygen carrying capacity 64,65 .
Vanadium showed the greatest accumulation between incident and prevalent patients and between patients on the lowest and highest ESA dose. Vanadium accumulates biologically through inhalation and ingestion as either the vanadate ion (V(V)) or vanadyl ion (V(IV)). Vanadium accumulation in dialysis patient blood has been demonstrated 17,20 . The biochemistry of vanadate and vanadyl ions is complex. The vanadium oxoanions can interfere with the functions of enzymes such as tyrosine kinases, cAMP-dependent kinases, ceramidases, and endothelial cell nitric oxide synthases 5,66-71 . As such, vanadate is commonly included in sample lysis buffer recipes to inhibit tyrosine phosphatases. Collectively the presence of vanadium compounds presence results in altered enzyme function and second messenger signaling [72][73][74] . It is noteworthy that vanadium compounds are actively taken up by red blood cells to compete with iron binding to hemoglobin 75,76 , influences HIF-1α signaling 68,77 and also can lead to increased erythrocyte death 78 with shorten red blood cell life span.. Significant differences exist in trace metal concentrations between incident and prevalent patients (Fig. 5). Vanadium, nickel, cadmium, and tin all appear to accumulate on dialysis while manganese are decreased. Regression analysis demonstrated several trace metals significantly associated with incident and prevalent patients many with low r2 values. These low r2 data suggest that time on dialysis is not the major contributor to the trace metals concentration. While encouraging these observations identify the need for prospective longitudinal studies to define specific trace metal association with dialysis initiation and dialysis vintage that addresses dietary and dialysis fluid trace metal contributions.
The largest changes between incident and prevalent patients were in vanadium and manganese concentrations. The impact of vanadium accumulation on enzymatic processes and the reported relationship between manganese and anemia may explain the observation in the group of patients studied that a greater proportion of subjects requiring an ESA in the prevalent group and that anemia might worsen as dialysis vintage increases. We have observed the increase in ESA requirements in a separate population of patients that were followed over a 4 year period what ESA requirements increased by approximately 400 units/week/year as dialysis vintage increased from 4.6 to 7.3 years 79 .
The current study was developed to examine the association of trace metals and the anemia in hemodialysis patients. Prominent strengths of this study are the prospective recruitment of patients to identify individuals within the first months of dialysis initiation (incident population) as well as chronic dialysis patients. Additional strengths are the use high sensitivity clinical assays that phenotype for important markers of inflammation (hsCRP) and iron availability (hepcidin) and lastly a dialysis population using dynamic ESA dosing protocols delivered to maintain a target Hb value. Limitations of the current study included confounding factors that cannot be addressed due the cross-sectional nature of this study. These confounders significantly included a lack of dietary information for vitamin or mineral supplement usage that might have contributed to observed trace metals, lack of dialysate water sampling, point of source water measurements from patient homes, and due to the study vintage a loss of patients for follow-up due to the low life expectancy of hemodialysis patients. Also the trace metals analysis constrained to a subset of trace metals (n = 14) that did not include some (mercury, www.nature.com/scientificreports/ strontium, cesium or thallium) previously shown to be associated with death two years after dialysis initiation 3 . Our study examined total serum trace metal levels but not total blood trace metals that would be inclusive of the cellular blood compartment. Some metals exist as organo-metallic compounds (e.g. organo-mercury, lead or tins) such as Sn(II) or the more stable Sn(IV) whose toxicities are associated with the specific organic moiety, or as oxo-acids whose toxicity is associated with a specific oxidation state (e.g. arsenic or chromium) [80][81][82] . Therefore, absent sample handling to stabilize and separate these charge forms prior to ICP-MS (e.g. metal speciation) we cannot report if the observed associations are with a specific organo-metalloid or charge state (e.g. vanadate versus vanadyl ion). As shown in the Pearson Correlation table (Fig. 1) there are considerable correlations between the trace metals observed such as between nickel positively correlated with tin but negative correlated with copper, selenium, and antimony. Our data provide information for inferring an association of metals with anemia in ESKD but do not shed light on the toxicity mechanism (that of trace metal independent action or concentration addition) or trace metals not measured but correlating with the ones that were measured. Kobayashi et al. demonstrated in a 12-month study of 35 hemodialysis patients the use of zinc supplementation to decrease the need for EPO and decrease in the ER 83 . Lastly, while our study was not designed as an intervention study, our data raise the question of future trials used to examine the concentration addition effects of specific trace metals on ER in ESKD patients.
In conclusion, we report on 14 trace metals and their association with anemia treated with an ESA in both incident and prevalent dialysis patients. Linear regression modeling identified groups of metals that together were significantly associated Hb, ESA dose, and the Hb-dose averaged response of patients (ER). Hb was negatively associated with cadmium while positively associated with antimony arsenic and lead; (b) e amount of ESA dose was negatively associated with vanadium while positively associated with arsenic. The Hb-dose averaged response of patients ('ER') to ESAs most strongly positively associated with arsenic. No sex-dependent trace metal differences were observed in incident or prevalent populations. Other observations included significant accumulation of vanadium and reduction in manganese concentration as a function of incident versus prevalent status. These data support future studies for a broader comprehensive metabolomic study into anemia of ESKD and ESA hypo-response with a goal to define targets for interventional studies addressing ESA response and anemia management in hemodialysis populations.

Methods
Human subjects. The research protocol conformed to the Declaration of Helsinki and was approved by the Universities of Louisville and Washington Institutional Review Board. Subjects were enrolled following an informed consent process and all data were de-identified. One hundred prevalent (defined as > 6 months on dialysis) and 33 incident (defined as less than 3 months on dialysis) dialysis dependent hemodialysis subjects were enrolled at the University of Washington. Subjects donated 20 mL of blood, processed as both plasma and serum, and stored as 0.5 mL aliquots at − 70∘ until analyzed. These samples were collected using the same proto- Trace elemental analysis. Serum samples (0.5 mL) were digested with a mixture of concentrated nitric acid, hydrochloric acid, and hydrogen peroxide, then analyzed alongside calibration standards, blanks, and appropriate quality control samples to evaluate assay performance. Elements (antimony (Sb), arsenic (As), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), lead (Pb), manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se) , tin (Sn), vanadium (V) and zinc (Zn)) were monitored using a Thermo Element 2 high resolution sector field ICP-MS in multiple resolution modes to provide optimal analytical sensitivity and interference resolution for each element. See supplemental information for full method details.
Hepcidin assay. The hepcidin-25 EIA kit S-1337 was purchased from Peninsula Laboratories (San Carlos, CA). The hepcidin-25 standard was serially diluted. Serum samples were diluted 1:50. Samples and standards were brought to 50 μL and added to an antiserum pre-coated plated for competitive immunoassay according to kit instructions. A uniform volume of 25 μL biotinylated tracer was added to each well of the plate after 2 h and incubated for 1 additional. Captured biotinylated tracer was subsequently bound by streptavidin-conjugated horseradish peroxidase. A color correlated to the sample hepcidin concentrations was produced after TMB substrate was added. The absorbance was read at 450 nm and a four-parameter polynomial regression was used for the standard curve fitting. The coefficient of variation (CV) using a hepcidin-25 concentration of 1.56 ng/mL was 3.49% intra-assay and 3.43% inter-assay. All samples were measured in duplicates and the valid OD was kept correspondent to hepcidin standard between 0.78 ng/mL and 6.25 ng/mL for best reproducibility (lab validation not shown).
C-reactive protein (CRP) assay. CRP values were determined using the high sensitivity CRP (hsCRP) latex immunoturbidometric assay kit from Roche Diagnostics (Indianapolis, IN). Data were collected on an Roche Diagnostics Cobas Integra 800 using patient serum samples according to the manufacturer's instructions. Briefly human CRP agglutinates with latex particles coated with monoclonal anti-CRP antibodies. The precipitate is determined turbidimetrically at 552 nm. All samples were measured in duplicates and the valid OD was kept correspondent to the linear range of the hsCRP standards (lab validation not shown). www.nature.com/scientificreports/ Statistical analysis. Statistical analysis was performed using SAS version 9.4 and SPSS version 24. Measured serum concentrations of metals below the limit of detection (LOD) were imputed as ½ LOD if < 25% of the observations were below the LOD and then transformed using the log of the concentration base 2 to address non-normality in the dataset. For metals with a large number of missing values (> 25% of observations below LOD), comparisons of abundances were made based on detection and non-detection events using a chi-square test due to non-normal in the event distribution. Total Epoetin alfa dose (U) was categorized into three groups: 0; 1000 to 29,200; and 30,000 to 136,200. Erythropoietin response (ER) was normalized to achieved hemoglobin over 6 months as ESA Dose/1000 × Hb and categorized into three groups: 0; 0.02 to 2.55; and 2.60 to 11.81. Comparison of means between incident and prevalent hemodialysis groups was performed by t-test for metals < 25% LOD and chi-square for > 25% LOD (chromium, cobalt, lead, arsenic, vanadium). Metals were included in Pearson correlation calculations when observed in at least 75% of all samples. The effects sex and dialysis prevalence was tested using two-way ANOVA to determine main effects and interaction. The effect of metal concentration on erythropoietin response was tested using multivariate regression analysis using backward selection of the metal concentration vs. ESA dose, ER, and current Hb. Regression analysis was performed as linear regression. Post hoc analysis was done by one-way analysis of variance using Student-Newman-Keuls.

Data availability
Serum ICP-MS and associated data are available in the metabolomics workbench of the NIH Common Fund Metabolomics Program Data Repository and Coordinating Center (DRCC) as study ST000565. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.