Free Light Chains, High Mobility Group Box 1, and Mortality in Hemodialysis Patients

Background: Uremic toxins are associated with immune dysfunction and inflammation. The inadequate removal by hemodialysis (HD) of serum free light chains (FLCs) determines their accumulation. This study evaluated FLCs in HD patients, analyzing their relations with other biomarkers, such as serum high mobility group box 1 (HMGB1). Methods: FLC and HMGB1 were evaluated in a cohort of 119 HD patients. κFLC and λFLC were summated to give a combined (c) FLC concentration. Patients were followed prospectively until the end of the observation period of four years, or until the endpoint: the patient’s death. Results: cFLC values in HD patients were 244.4 (197.9–273.5) mg/L. We detected a significant reduction in CD8+ cells and a decreased CD4+/CD8+ ratio. HMGB1 levels were 94.5 (55–302) pg/mL. After multivariate analysis, cFLCs correlated with β2-microglobulin and the CD4+/CD8+ ratio. Subjects with cFLC values above 263 mg/L and with sHMGB1 values < 80 pg/mL experienced a significantly faster evolution to the endpoint (mean follow-up time to progression of 27.5 and 28.5 months, respectively; p < 0.001). After an adjusted multivariate Cox analysis, cFLCs were associated with 11% increased risk of death, whereas low sHMGB1 increased this risk by 5%. Conclusions: cFLCs and HMGB1 reflect the inflammation and immune dysfunction in HD patients representing two strong and independent risk markers of mortality.


Introduction
Uremic toxins represent independent risk factors for mortality in end-stage renal disease (ESRD) [1,2]; these substances, poorly removed by diffusive hemodialysis (HD) techniques, are associated with the pathological features of uremia, such as immune dysfunction, inflammation, and adverse cardiovascular outcomes [3][4][5][6]. Whereas systemic inflammation contributes to atherosclerosis, cardiovascular disease, and anemia, immune deficiency leads to an impaired response to vaccination and an increased incidence and severity of microbial infections [7]. These two entities, not mutually exclusive, could represent two sides of the same coin [8]: uremic-associated inflammation is closely related to the activation of the innate immune system and the depletion and impaired activities of T and B lymphocytes [9,10].
High levels of serum HMGB1 characterized ESRD patients treated by chronic HD or peritoneal dialysis, positively correlating with pro-inflammatory cytokines and related with complications, such as heart failure and arteriovenous fistula occlusion [17]. light chain and positive serum protein electrophoresis, and immunofixation result [31]. Other exclusion criteria were cancer, active viral infections, history of transplantation, immunosuppressive treatments, or a recent infectious episode (<3 months).
Patients were included at least six months after the onset of renal replacement therapy, receiving three-weekly HD sessions lasting 4 h. The dialytic regimen and prescription were maintained stable for six months before the enrollment and during the entire study period.
All demographic, clinical, dialytic, and laboratory data were collected during the enrollment period by the nephrologists of the Centre.
The primary outcome was four years all-cause mortality. Study patients were followed until death or until the end of the study in March 2022. All patients were previously informed and gave their written consent. The University of Messina Ethics Committee approved the study (approval number n • 20/20), and all procedures were in accordance with the Declaration of Helsinki.

Laboratory Analyses
We collected blood samples before the start of the first dialysis session of the week. The serum was separated in a refrigerated centrifuge and then stored at −80 • C until analysis for κFLCs, λFLCs, and HMGB1. For data analysis, κFLC and λFLC were summated to give a combined (c) FLCs concentration. The reference range of normal cFLC levels was 9.3-43.3 mg/L [37]. Flow cytometry assessed CD3+, CD4+, and CD8+ lymphocyte subsets.

Statistical Analyses
Statistical analyses were performed with MedCalc and GraphPad Prism software. Data were presented as mean ± SD, median (range), or percentage frequency as appropriate. Differences between groups were established by unpaired t-test for normally distributed values and by Kruskal-Wallis analyses, followed by Dunn's test for nonparametric values. Pearson or Spearman correlation coefficients were used to test correlations between cFLCs and other variables. All non-normally distributed values were log-transformed to better approximate normal distributions. To find the best cut-off values for identifying the progression to the endpoint, receiver operating characteristics (ROC) analysis calculated the area under the curve (AUC) for cFLCs and other markers. Kaplan-Meier curves assessed survival in subjects with cFLCs and sHMGB1 values above and below the optimal ROCderived cut-off levels. Cox proportional hazard regression analyses calculated adjusted risk estimates for the progression to the endpoint. All results were considered significant if p was <0.05. Table 1 summarizes the baseline data. The population had a median age of 71 years (IQ = 55.2-76.7), with a mean dialysis vintage of 57.6 ± 16.8 months. The mean dialysis session length was 240 ± 0.11 min, and the mean values of single-pool KT/V were 1.4 ± 0.3.
We revealed a reduction in CD8+ cells at the end of a single session [493 (256-619 vs. 360 (219.5-505) count; p: 0.001], without variations in the percentage of CD3+ and CD cells.
We assessed an increased CD4+/CD8+ ratio immediately after the end of the dialy if compared with the pre-dialytic values There was no significant difference in CD3+, CD4+, CD8+ levels, and CD4+/CD ratio among different HD techniques (p > 0.05). We revealed variable levels of HMG [(94.5 (55-302) pg/mL], with a wide fluctuation of its values among HD patients.

Univariate/Multiple Cox Regression Analysis and Mortality Risk in HD Patients
To identify putative risk factors associated with death, we conducted a Cox regression analysis, inserting in the model all variables that were different at baseline in patients who reached the endpoint during the follow-up period. At univariate analysis, BNP, β2-MG, cFLCs, and sHMGB1 were significantly associated with the endpoint, whereas diabetes, ferritin, CRP, CD4+/CD8+ ratio, and age failed to reach statistical significance. We performed a multiple Cox regression, simultaneously inserting into the model all the variables significantly associated with the endpoint at univariate analysis. Age was also inserted in this model, although it was not associated with the endpoint. Results from this analysis indicated that both cFLCs and sHMGB1 predicted a higher risk of mortality independently from BNP and β2-MG. In detail, cFLCs were associated with an 11% increased risk of death (HR 1.11; 95% CI, 1.06-1.13; p: 0.02), whereas low sHMGB1 increased this risk by 5% (HR 0.95; 95%CI, 0.89-0.98; p: 0.03). Table 2 summarizes data from Cox analyses.

Discussion
To the best of our knowledge, this is the first prospective study revealing the association between cFLCs, HMGB1, and mortality in HD patients.
Elevated FLC levels characterized our cohort, with median values higher than those observed in CKD patients, due to abnormal production and an inadequate clearance by hemodialysis. Whereas Cohen demonstrated that bicarbonate dialysis and HDF were unable to normalize FLC values [39], in the last years, growing data highlighted a better clearance of medium molecules, including FLCs, through HDF with high convective volumes and HDx. Moreover, dialyzer performance significantly affected 3-year mortality, revealing that MCO filters improved mortality outcomes [40].
Whereas in CKD patients the impact of FLCs on mortality is still controversial [6,37], the excessive FLC endocytosis by proximal tubular cells and their accumulation at the distal tubule represent the main processes of the progression of the renal disease, with inflammation and pro-fibrotic effects [41][42][43].
However, FLCs are not only simple markers of inflammation such as CRP or PCT. Interestingly, FLCs are inversely correlated with CRP, PCT, and alpha-1 globulins. The kinetics of CRP and cFLC levels differ, with CRP levels more closely associated with acute, but not chronic, inflammation [44]. Similarly, PCT levels rise 3 to 6 h after a bacterial infection or sepsis, without significant variations in patients with non-infectious inflammation. Moreover, according to our ROC data, PCT and CRP revealed weaker diagnostic information about our endpoint than cFLCs, with low sensitivity and specificity. Another uremic toxin, β2-MG, had a better diagnostic profile and was positively correlated with cFLCs after multivariate analysis.
We assessed that this toxin represents an independent marker of mortality in our HD cohort, strengthening well-known data available in the literature [45,46] and considering β2-MG as another actor of inflammation and immune dysfunction in the uremic population.
Our data demonstrate the central role of these middle molecules, revealing a complex process growing during several years of pre-dialytic CKD, and achieving the peak during the dialysis period. This process has a common denominator: a vicious cycle between sub-clinical, chronic inflammation and quantitative and qualitative immune dysfunctions.
FLCs can modulate the qualitative functions of polymorphonuclear leukocytes by inhibiting spontaneous apoptosis and decreasing chemotaxis and glucose uptake [4]. The decreased granulocyte and monocyte/macrophage phagocytic function and the reduced capacity of antigen-presenting cells represent the main processes of natural immune dysfunction in these patients [25].
However, in clinical practice, few biomarkers adequately identify innate and acquired immunity dysfunction in HD patients.
We assessed the role of HMGB1 as one of the markers of the innate immune system, revealing higher values than those observed in CKD. Acute inflammatory and infective processes, such as acute kidney injury and sepsis, determine high HMGB1 levels [21]. Hypoxic, injured, or dying cells release DAMPs, activating the immune system and promoting inflammation [47,48]. According to these data, high HMGB1 values observed in our patients reflect the permanent, active inflammation and the consequent reactive response of the natural immune system. We assessed, interestingly, low levels of HMGB1 in patients who died during the follow-up period, demonstrating, after multivariate Cox analysis, that this alarmin represents an independent risk factor of mortality in our cohort.
Previous data revealed higher serum levels of this peptide in HD subjects if compared with CKD patients or those treated by peritoneal dialysis, with a time-dependent manner reduction [17]. Our data corroborate these findings, suggesting that reduced levels of HMGB1, characterizing our inflamed patients, are associated with a concomitant chronic depletion of innate immune cells, the leading source of this alarmin. If this process occurs, it has obvious consequences in terms of mortality risk.
In addition to the innate immune system, an altered acquired immunity characterizes HD patients. T lymphocyte dysfunction, found in ESRD, can be attributed to impaired innate immunity and dysfunction of Toll-like receptors, whose HMGB1 represents the leading ligand [49,50], associated with an almost linear decrease in the total B cell count, CD4+, and the CD8+ T cell compartment [51].
We detected a significant reduction in the percentage of CD8+ cells and a decreased CD4+/CD8+ ratio; the latter increased after a single dialysis session, suggesting that HD can temporarily improve this immune system. However, the accumulation of uremic toxins during the inter-dialytic period negatively and gradually acts on cellular function, as confirmed by the inverse relationship found in our cohort, after multivariate analysis, between FLCs and CD4+/CD8+ ratio. This datum links high inflammation to immune depression, such as the low CD4+/CD8+ ratio, characterizing patients who died during the follow-up and mirroring a suppressed acquired immunity. Our results were consistent with previous studies, indicating exhaustion of acquired immunity in HD patients due to a decrease in circulating naive T cells and age-related changes related to the pro-inflammatory environment, named in flammageing, observed in the uremic population [52].
However, all these markers could only partially highlight the immune dysfunction occurring in HD patients, with the necessity of further studies to corroborate these results and to create a panel of biomarkers evaluating all the immunologic pathways altered in these patients.
Nevertheless, if the subclinical inflammatory process involves all HD patients, the same patients could be widely heterogeneous from an immunological point of view, as suggested by the high variability of some immune markers, supposing different immune profiles among dialyzed patients. The clinical implications are that specific immune profiles may identify an increased risk of acute rejection, evaluable before transplantation, or may favor viral or bacterial infections, cause poor response to vaccinations, or increase the risk of malignancies. Betjes' results support our hypothesis, revealing that patients with a higher frequency of terminally differentiated CD8+ cells had a decreased risk of acute rejection [53].
The present study has some limitations. First, it was a single-center study, and the cohort of patients was relatively small. These limitations did not allow us, for example, to evaluate the influence of various dialytic techniques, the different causes of death, or immune profiles on FLCs or HMGB1. Confirmation in larger cohorts is indispensable to attribute general validity to our reports.
In the progressors group, the mean age was higher, with more patients with diabetes and in hemodialysis therapy for a longer time. One-third of the participants reached the endpoint during the follow-up, and the statistical model was powerful enough to establish independent relationships between cFLCs, HMGB1, and death.
Further in-depth examinations should verify whether these findings could be confirmed in a long-term observational period, determining if therapeutic measures targeting cFLCs and immune markers can improve HD patient survival. In clinical practice, our results might suggest stratifying HD patients according to FLCs and HMGB1 levels, personalizing the dialytic prescription with potential benefits from diffusive-convective methods and HDx techniques, and identifying patients with high mortality risk.

Conclusions
cFLCs and HMGB1 represent two independent risk markers of mortality in hemodialysis patients.
Author Contributions: A.L. and P.M. conceptualized and designed the study, drafted and revised the manuscript; R.G. analyzed and interpreted the data; S.C. collected data and revised the manuscript; G.F., D.C. and E.G. performed laboratory analyses. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The dataset generated and analyzed during the current study is available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.