Prospective Analysis of B Lymphocyte Subtypes, before and after Initiation of Dialysis, in Patients with End-Stage Renal Disease.

End-stage renal disease (ESRD) is followed by alterations in adaptive immunity. The aim of this study was to evaluate B lymphocyte subtypes in ESRD patients before and after hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). PATIENTS AND METHODS: CD5, CD27, BAFF, IgM and annexin were evaluated by flow cytometry on CD19+ cells in ESRD patients (n = 40), at time of initiating HD or CAPD (T0) and 6 months later (T6). RESULTS: A significant reduction in ESRD-T0 compared to controls was noticed for CD19+, 70.8 (46.5) vs. 171 (249), p < 0.0001, CD19+CD5-, 68.6 (43) vs. 168.9 (106), p < 0.0001, CD19+CD27-, 31.2 (22.1) vs. 59.7 (88.4), p < 0.0001, CD19+CD27+, 42.1 (63.6) vs. 84.3 (78.1), p = 0.002, CD19+BAFF+, 59.7 (37.8) vs. 127.9 (123.7), p < 0.0001 and CD19+IgM+ cells, 48.9 (42.8) vs. 112.5 (81.7) (K/µL), p < 0.0001. The ratio of early/late apoptotic B lymphocytes was reduced (16.8 (10.9) vs. 110 (25.4), p = 0.03). CD19+CD5+ cells were the only cell type with an increased proportion in ESRD-T0 patients (2.7 (3.7) vs. 0.6 (1.1), p < 0.0001). After 6 months on CAPD or HD, CD19+CD27-(%) and early apoptotic lymphocytes were reduced further. The HD patients also showed a significant increase in late apoptotic lymphocytes, from 1.2 (5.7) to 4.2 (7.2) K/mL, p = 0.02. CONCLUSIONS: B cells and most of their subtypes were significantly reduced in ESRD-T0 patients compared to controls, the only exception being CD19+CD5+ cells. Apoptotic changes were prominent in ESRD-T0 patients and were exacerbated by HD.

Clustering of End Stage Renal Disease Patients by Dimensionality Reduction Algorithms According to Lymphocyte Senescence Markers.

End stage renal disease (ESRD) engenders detrimental effects in the Immune system, manifested as quantitative alterations of lymphocyte subpopulations, akin, albeit not identical to those observed during the ageing process. We performed dimensionality reduction of an extended lymphocyte phenotype panel of senescent and exhaustion related markers in ESRD patients and controls with Principal Component Analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP). The plane defined by the first two principal components of PCA showed two fuzzy clusters, for patients and controls, respectively, with loadings of non-senescent markers pointing towards the controls' centroid. Naive lymphocytes were reduced in ESRD patients compared to controls (CD4+CD45RA+CCR7+ 200(150-328) vs. 426(260-585cells/µl respectively, P = 0.001, CD19+IgD+CD27- 54(26-85) vs. 130(83-262)cells/µl respectively, P < 0.001). PCA projections of the multidimensional ESRD immune phenotype suggested a more senescent phenotype in hemodialysis compared to hemodiafiltration treated patients. Lastly, clustering based on UMAP revealed three distinct patient groups, exhibiting gradual changes for naive, senescent, and exhausted lymphocyte markers. Machine learning algorithms can distinguish ESRD patients from controls, based on their immune-phenotypes and also, unveil distinct immunological groups within patients' cohort, determined possibly by dialysis prescription.

End stage renal disease has an early and continuous detrimental effect on regulatory T cells.

End stage renal disease (ESRD) is followed by disturbed adaptive immunity, together with alterations in T cell subsets, including CD4+CD25+FoxP3+ cells (Tregs). In the present study, we assessed the effect of haemodialysis (HD) on the Treg population. CD3+CD4+, CD3+CD8+ and CD4+CD25+FoxP3+ cells were estimated by flow cytometry in 142 ESRD patients (45 ESRD-preHD, 97 on HD) and 30 healthy controls (HC). Patients on HD were classified into three groups according to time on dialysis (HD vintage - HDV): A < 2 years, B: 2-5 years and C: >5 years on HD. The mean age of patients on HD (M/F 53/44) was 54.8 ± 14 years and the median HDV 58 (78) months. We observed a significant progressive reduction in the percentage and count of lymphocytes (p < .001, p < .001, respectively), CD3+CD4+ (p = .003 and, p < .001, respectively) and Tregs (p = .001 and, p < .001, respectively), between HC, ESRD-preHD and HD patients. HDV had a significant inverse correlation with total lymphocyte, CD3+CD4+ and Treg cell counts (p = .001, p < .001, p < .001, respectively) and, the percentage of lymphocytes and CD3+CD4+ cells (p = .005, p = .01, respectively). Furthermore, we stratified patients on HD into three groups according to HDV: A < 2 years, B: 2-5 years and C: >5 years on HD. Total lymphocytes and Tregs were significantly different among the three vintage groups (Kruskal-Wallis H test, p < .001, p < .001 respectively). CD3+CD4+ and CD3+CD8+ cells were also significantly affected (p < .001 and p = .001, respectively), after at least 2 years of HD. Tregs show prompt and significant reduction at the pre-dialysis stage, and continue to decrease gradually even after long-term HD, in a context of total lymphocyte reduction.

CD28null and Regulatory T Cells Are Substantially Disrupted in Patients with End-Stage Renal Disease Due to Diabetes Mellitus.

BACKGROUND: End-stage renal disease (ESRD) is associated with alterations in T-cell immunity, including increased CD28null and reduced regulatory T cells (Tregs). However, whether immune disturbances are due to ESRD or primary disease is not yet clear. As diabetes mellitus is the leading cause of ESRD, we evaluated its impact on the immune profile of ESRD patients. METHODS: CD28null, Tregs, and natural killer cells were initially analyzed by flow cytometry in 30 predialysis ESRD patients due to diabetes (DM), 30 non-DM (NDM), and 25 healthy controls. Measurements were repeated after 6 months on hemodialysis (HD) or peritoneal dialysis (CAPD). RESULTS: The percentage of CD4 + CD28null cells, CD8 + CD28null cells, and Tregs showed significant differences in DM, NDM, and controls; mean rank 33.71 vs. 25.68 vs. 18.88, p = 0.006, 37.79 vs. 28.82 vs. 17.08, p = 0.008, and 20.79 vs. 26.12 vs. 41.33, p = 0.001, respectively. DM vs. NDM had increased CD4 + CD28null and CD8 + CD28null cells, 11.5% (1.5%-24%) vs. 4.1% (0-42.3%), p = 0.02 and 61.3% (24%-76%) vs. 43% (5.7%-85%), p = 0.04, respectively. After 6 months on HD but not CAPD, DM showed a significant further increase in CD4 + CD28null cells, from 30 (14-100) to 52.7 (15-203), p = 0.02; and CD8 + CD28null cells, from 137 (56-275) to 266 (103-456), p = 0.01. CONCLUSIONS: Diabetes mellitus affects T-cell subtypes even at predialysis stage, though changes become more prominent after commencement on HD.

Systemic complement activation in anti-neutrophil cytoplasmic antibody-associated vasculitis and necrotizing glomerulonephritis.

AIM: Due to the accumulating evidence of complement activation in anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV), we decided to investigate the possibility of systemic complement activation in patients with Necrotizing Glomerulonephritis secondary to AAV. METHODS: Clinical, laboratory and histological findings, and serum levels of complement components, C3a, C5a and Bb fragment of Factor B and C4d, were estimated in patients with AAV and glomerulonephritis, at time of diagnosis, before any treatment had been applied. All patients were treated with the same immunosuppressive protocol and followed up for total 24 months. Twenty age and sex matched healthy individuals served as controls. RESULTS: Serum levels of all complement components were significantly increased in patients, compared to controls; C5a: 19.9(0.02-48) vs 9.06(2.1-16.3)pg/mL, P = .002, Bb: 7.3(0.02-31.4) vs 0.2(0.02-1.6)pg/mL, P < .0001, C3a: 4.7(0.4-7.2) vs 2.4(1.09-5)pg/mL, P = .05 and C4d: 11.6(0.07-70) vs 0.7(0.07-8.2)pg/mL, P = .001, respectively. There was strong correlation between serum Bb levels and eGFR and FFS2009 score at time of diagnosis (r = -.41, P = .002 and r = .41, P = .003 respectively). Also, serum Bb levels were increased in patients with severe interstitial infiltration (P = .04) and focal necrosis (P = .01) on renal biopsy. Serum Bb levels could also predict renal function outcome during the acute phase of disease, but not at the end of follow up. CONCLUSION: We provided strong evidence of systemic activation of complement alternative pathway in the development and progression of AAV and glomerulonephritis. Serum Bb seem to play a critical role in the induction, also predicting disease activity and outcome, yet activation of classical pathway cannot be excluded.

Influence of end stage renal disease on CD28 expression and T-cell immunity.

BACKGROUND: T-cell immunity is affected in end stage renal disease (ESRD). However, whether this happens at pre- or post-dialysis stage and what is the impact of different renal replacement methods, remains unclear. We investigated the alterations of T-cell subtypes in patients at pre-dialysis ESRD and their further changes during dialysis. METHODS: CD4+, CD8+, CD4 + CD28null and CD8 + CD28null T-cells were analysed in 40 ESRD patients at two different time points, (a) the day started on dialysis (ESRD-T0) and (b) 6 months later (ESRD-T6), while being on haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Twenty-five age matched healthy volunteers served as controls. RESULTS: CD4+ and CD8+ T-cells were significantly reduced in ESRD-T0 patients compared to controls, 604 (105-3551) vs 943 (584-1867)µ/L, P = .001, and 352 (103-1561) vs 422.4 (263-1453)µ/L, P = .05, respectively. However, proportions of CD4 + CD28null and CD8 + CD28null cells were significantly increased, 6.4 (0.3-30)% vs 2.7 (0.1-7.8)%, P = .04 and 58.2 (12.8-85.4)% vs 39 (7.8-57.1)%, P = .01, respectively. Proportion of CD4 + CD28null cells showed significant correlation with serum CRP (r = .4, P = .04) and albumin levels (r = -.5, P = .007) in ERSD patients. ESRD-T0 patients with cardiovascular disease (CVD) had increased CD4 + CD28null and CD8 + CD28null proportions, 8.6 (1-30)% vs 2.1 (0.1-19.8)%, P = .04 and 62.5 (12.8-85.4)% vs 45.5 (5.7-73.7)%, P = .02, respectively, compared to those without. Six months later, both CD4 + CD28null and CD8 + CD28null T-cells were increased in HD compared to CAPD patients, by +110.11 (-27.1 to 311.4)% vs -28.1 (-100 to 30)%, P = .003 and +55.23 (-29.06 to 197.93)% vs -8.34 (-54.99 to 66.72)%, P = .05, respectively. CONCLUSIONS: CD4 + CD28null and CD8 + CD28null T-cells are increased at pre-dialysis ESRD, and correlate with chronic inflammatory markers and the presence of CVD. Dialysis methods seem to have different impact on these subpopulations.

Impact of Τh1 and Τh2 cytokines in the progression of idiopathic nephrotic syndrome due to focal segmental glomerulosclerosis and minimal change disease.

BACKGROUND: Differential diagnosis between primary focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) is sometimes difficult as nephrotic syndrome is the main clinical symptom in both diseases. OBJECTIVES: This study has attempted to evaluate the urinary excretion of Th1 and Th2 cytokines as potential biomarkers in distinguishing the two types of nephrotic syndrome, and predicting outcome of renal function. PATIENTS AND METHODS: Thirty-six patients with FSGS (M/F 22/14, Age; 41.9 ± 17 years, SCr=1.7 ± 0.8 mg/dL, UProt=4.7 ± 5.5 g/24 h), and 21 with MCD (M/F 5/16, Age; 41.4 ± 15 years, SCr = 1 ± 0.4 mg/dL, UProt = 7.9 ± 9.3 g/24 h) were included in the study. Τh1 (IL-2, IL-12, GM-CSF, INF-γ, TNF-α) and Th2 cytokines (IL-4, IL-5, IL-10, IL-13) were measured by multiple cytokine assay, Luminex technology, in first morning urinary samples collected at the day of renal biopsy. RESULTS: No significant differences in urinary excretion of all cytokines were found between FSGS and MCD patients. In FSGS however, IL-12 urinary levels were independent factor correlated with both global sclerosis (R = 0.5, P = 0.009) and interstitial fibrosis (R = 0.5, P = 0.02). Th1 cytokines (IL-2 and GM-CSF) were significantly increased in FSGS patients who did not respond to treatment (P = 0.03 and P = 0.007, respectively). Th2 cytokines (IL-4, IL-5, IL-10, IL-13) were significantly increased in MCD patients with frequent relapses (P = 0.05, P = 0.001, P = 0.01, P = 0.03). CONCLUSIONS: Urinary excretion of Th1 and Th2 cytokines cannot discriminate FSGS from MCD. Th1 cytokines, especially IL-12, IL-2 and GM-CSF, may be involved in pathology and progression of FSGS, while Th2 cytokines are implicated in frequent relapses of nephrotic syndrome in MCD.