#6158 uremic toxins are associated with immune-senescence and immune-exhaustion in hemodialysis patients

Abstract Background and Aims Immune-senescence and exhaustion have been described in hemodialysis (HD) patients, with changes in lymphocyte phenotype possibly stimulated by the uremic environment, independent of age and comorbid conditions. We evaluated the potential association of uremic toxin accumulation with changes in lymphocyte count and phenotype in HD patients. Method In the peripheral blood of 54 HD patients (23 female, mean age 51.3±16.91 years) and 31 age-matched healthy controls, lymphocytes surface molecules were determined by flow cytometry: T cells (CD4, CD8, CD31, CD45RA, CCR7, CD28, CD57, PD1) and B cells (CD27, IgD). Plasma levels of the protein bound uremic toxins [hippuric acid (HA), indoxyl sulfate (IxS) and p-cresyl sulfate (pCS)] were measured by ultra-performance liquid chromatography. Lymphocyte phenotypes were correlated with uremic toxins levels using Spearman's correlation coefficient test. Patients with recent or active infection, malignancy, autoimmune disease and/or diabetes mellitus were excluded. Results A higher concentration of uremic toxins was observed in HD patients, compared to healthy controls, namely: total HA 3.05 (1.66-5.37) vs 0.102 (0.04-0.2)mg/dl (p<0.001), free HA 1.482 (0.7-2.8) vs 0.029 (0.03-0.04)mg/dl (p<0.001); total IxS 2.207 (1.27-3.34) vs 0.063 (0.04-0.09)mg/dl (p<0.001), free IxS 0.146 (0.09-0.27) vs 0.0004 (0.0004-0.0004)mg/dl (p<0.001); total pCS 1.248 (0.84-1.66) vs 0.066 (0.04-0.13)mg/dl (p<0.001), free pCS 0.089 (0.06-0.13) vs 0.004 (0.004-0.005)mg/dl (p<0.001); respectively. There was a positive correlation between hemodialysis vintage and total as well as free HA (p = 0.005, p = 0.003, respectively). No significant differences in uremic toxin levels were found in HD patients according to sex, age, dialysis prescription and method. Patients with residual urine output had reduced total and free HA levels 1.739 (3.5) vs 3.347 (4.16)mg/dl (p = 0.03), and 0.717 (1.9) vs 1.901 (3.5)mg/dl (p = 0.04), respectively and free IxS levels, 0.117 (0.1) vs 0.177 (0.2)mg/dl (p = 0.02), respectively. Total lymphocyte count and absolute number of CD4 cells were negatively correlated with total (p = 0.018 and p = 0.02 respectively) and free HA levels (p = 0.024 and p = 0.017 respectively). Several naïve and less differentiated CD4 subpopulations also demonstrated a negative correlation with total and free HA levels, most importantly CD4CD31+ (p = 0.037 and p = 0.027 respectively), CD8CD28+CD57- (p = 0.018 and p = 0,014 respectively) CD4CD45+CD57- (p = 0.039 and p = 0.027 respectively) and naïve B cells (CD19+IgD+CD27-) (p = 0.042 and p = 0.032 respectively). Advanced differentiated lymphocyte subtypes, CD4CD28+CD57+ and CD4CD45-CD57+ had a positive correlation with free ΗΑ levels (p = 0.036 and p = 0.045 respectively). Exhausted CD4 cells, defined as CD4+PD1+ had positive correlations with total and free IxS levels (p = 0.004 and p = 0.011 respectively) and total and free pCS (p = 0.027 and p = 0.018 respectively), but not with HA levels. Moreover, further divided exhausted lymphocytes, according to CD45RA expression, CD4CD45RA-PD1+ cells correlated positively with total and free IxS levels (p = 0.004 and p = 0.01 respectively) and CD4CD45RA+PD1+ with pCS total and free levels (p = 0.039 and p = 0.045 respectively). Conclusion Uremic toxins are increased in hemodialysis patients and may influence adaptive immunity. HA is associated with an immune-senescent, while IxS and pCS to an immune-exhausted lymphocyte phenotype.