Cells In Dialysis Patients Research Articles

Frequencies or Absolute Numbers? Cluster Analysis of Frequencies and Absolute Numbers of B-Cell Subsets in Dialysis Patients Who Are Candidates for Kidney Transplantation Reveals Different Profiles.

Background: Detailed characterization of B cells in dialysis patients who are candidates for kidney transplant is still lacking, with little information on how dialysis duration and modality impact B cell subsets. Methods: Cluster analysis of flow cytometry determined the frequencies and absolute numbers of B-cell subsets and divided the cohort of 78 candidates into two distinct clusters, one with shorter and one with longer dialysis duration. Results: The immune profiles of the clusters differed depending on whether frequencies or absolute counts were considered. In long-term dialysis patients, the frequency of total memory, double negative and marginal zone B cells increased, while the frequency of naive and regulatory B cells decreased. This pattern was reversed in short-term dialysis patients, with a decrease in memory and an increase in naive and regulatory populations. The B subset number decreased significantly in long-term dialysis patients, while it increased significantly in short-term dialysis patients. The dialysis modality affected the frequency-based subset immune profiles. Conclusions: It is important to determine whether the evaluation is based on frequencies or absolute numbers. The different distribution of B cell subsets in the clusters, in terms of frequencies and absolute numbers, was influenced by dialysis duration. Modality and age only influenced the frequencies.

Pleiotropic effect of erythropoiesis-stimulating agents on circulating endothelial progenitor cells in dialysis patients

Recent studies have suggested that erythropoiesis-stimulating agents (ESAs) may accelerate not only angiogenesis but also vasculogenesis, beyond erythropoiesis. We conducted a 12-week prospective study in 51 dialysis patients; 13 were treated with recombinant human erythropoietin (EPO, 5290.4 ± 586.9IU/week), 16 with darbepoetin (DA, 42.9 ± 4.3µg/week), 12 with epoetin β pegol (CERA, 40.5 ± 4.1µg/week) and 10 with no ESAs. Vascular mediators comprising endothelial progenitor cells (EPCs), vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), and high-sensitivity C-reactive protein (hs-CRP) were measured at 0 and 12weeks. EPCs were measured by flow cytometry as CD45lowCD34+CD133+ cells. The EPC count increased significantly to a greater extent in the EPO group than in the other three group, and increased significantly from 0 to 12weeks in a EPO dose-dependent manner. In both the DA and CERA groups, the EPC count did not change at 12weeks. Serum levels of VEGF, MMP-2 and hs-CRP were not affected by ESA treatment in all groups. In the CERA group, serum ferritin decreased significantly compared to the no-ESA group and correlated with CERA dose, although use of iron was permitted if required during the prospective study period of 12weeks. When patients on dialysis were treated with clinical doses of various ESAs, only EPO induced a significant increase of circulating EPCs from bone marrow, whereas, DA and CERA had no effect.

Evaluation of the Infection Risk in Dialysis and Chronic Kidney Disease Patients Using an ATP Monitoring Assay

The ATP monitoring assay is a useful biomarker for risk monitoring to detect infection and rejection episodes in transplant recipients. Hemodialysis patients have a higher rate of infectious mortality. Infections in hemodialysis patients are mainly caused by venous catheters, uremia, malnutrition and inflammation. However, the risk of infection episodes has not been evaluated using a lymphocyte ATP monitoring assay in hemodialysis and chronic kidney disease (CKD) patients. We measured the ATP amounts in the peripheral CD4+ cells of CKD (N=85) and dialysis patients (N=17) using an "Immuknow" assay kit. These CKD patients were divided, according to kidney disease stage, into G3a, G3b, G4, and G5 groups. The ATP amounts in CD4+ cells of the dialysis patients and each of the CKD groups were compared with healthy subjects. In both the dialysis and CKD patients, the ATP amounts in CD4+ cells were lower than in healthy subjects. Furthermore, there were significant differences in the ATP amounts between healthy subjects and each of the CKD-G3a, CKD-G3b, and CKD-G4 groups (P<0.05). Patients with CKD-G3a, CKD-G3b and CKD-G4 were evaluated as being at high risk for infection according to the lymphocyte ATP monitoring assay. However, the ATP amounts in the dialysis and CKD-G5 patients did not differ from those in healthy subjects to a statistically significant extent. These results suggest that the ATP amount in the CD4+ cells of these patients with serve renal failure are influenced by dialysis treatment, uremia and/or oxidative stress.

VDR Expression on Circulating Endothelial Progenitor Cells in Dialysis Patients Is Modulated by 25(OH)D Serum Levels and Calcitriol Therapy

Background/Aims: Vitamin D deficiency is associated with endothelial dysfunction in uremic patients, possibly by the impairment in the number and function of endothelial progenitor cells (EPCs). In 89 hemodialysis patients, we investigated the factors associated with the number of circulating EPCs (CD34⁺/CD133⁺/KDR⁺ and CD34⁺/CD133^–/KDR⁺ cells), the presence of VDR and the determinants of VDR expression on EPCs, in particular in calcitriol therapy. Methods: EPC counts, percentages of VDR-positive EPCs and VDR expression were assessed by flow cytometry. Cells isolated from a subgroup of patients were cultured to analyze colony-forming units, specific markers expression and a capillary-like structure formation. Results/Conclusions: Our study demonstrates the presence of VDR on EPCs. In our dialysis patients, the parameters studied on both CD34⁺/CD133⁺/KDR⁺ and CD34⁺/CD133^–/KDR⁺ cells, in particular VDR expression, seem to be influenced by uremia-related factors, including anemia, inflammation, diabetes, 25(OH)D serum levels and calcitriol therapy.

Increased Binding of Beta-2-Microglobulin to Blood Cells in Dialysis Patients Treated with High-Flux Dialyzers Compared with Low-Flux Membranes Contributed to Reduced Beta-2-Microglobulin Concentrations

Background: Patients on long-term dialysis eventually develop amyloid deposits with β₂-microglobulin as a predominant component. Although several studies have suggested that high-flux membranes reduce β₂-microglobulin in plasma compared with low-flux dialyzers, the mechanisms underlying this observation are still discussed. Methods: We revisited this important subject and measured β₂-microglobulin in the plasma of healthy individuals (n = 8), and patients undergoing hemodialysis (n = 20) who for assigned periods of time were either treated with a low-flux membrane (cuprophan) or high-flux (polyamide) dialyzer with an ELISA. The number of blood cells was determined by FACS. β₂-Microglobulin was also measured on the surface of granulocytes, lymphocytes, and monocytes before, directly after, and 4 h after hemodialysis. Expression of β₂-microglobulin, c-fos, tumor necrosis factor-α (TNF-α), and interleukin-1 mRNA was determined in whole blood samples with quantitative RT-PCR using an internal standard in parallel. In the second part of the study, patients were assigned in a two-group cross-over design either to low- or high-flux dialyzers (n = 9 in each group), and dialyzer membranes were changed every 4 weeks for two consecutive periods. Serum β₂-microglobulin concentrations were measured at the end of each period. Results: Healthy controls had a low plasma β₂-microglobulin level of 1.2 ± 0.3 mg/l. Before hemodialysis, patients on low-flux dialyzers had a plasma β₂-microglobulin level of 42.0 ± 14.0 mg/l, patients treated with high-flux dialyzers 21.5 ± 10.8 mg/l (p < 0.05 vs. low-flux dialyzers). In contrast, there was no significant difference in plasma concentrations of active transforming growth factor-β1 with the two different membrane types. The difference in serum β₂-microglobulin between low- and high-flux membranes was more prominent directly after hemodialysis as well as 4 h after hemodialysis compared with the values directly before the start of treatment. At all studied time-points, leukocytes and platelets were significantly higher in patients on low-flux membranes. Healthy control persons exhibited a significantly higher amount of β₂-microglobulin bound to granulocytes, lymphocytes, and monocytes compared with dialysis patients. Interestingly, β₂-microglobulin bound to granulocytes, lymphocytes, and monocytes was significantly increased in patients treated with high-flux membranes compared with low-flux filters. Quantitative RT-PCR revealed no significant difference in β₂-microglobulin expression in whole blood before hemodialysis, directly after hemodialysis, and 4 h after hemodialysis. However, TNF-α and c-fos transcripts were significantly higher in whole blood obtained from patients treated with low-flux membranes compared to high-flux dialyzers. The two-group cross-over study over three periods of 4 weeks revealed that switching from low-flux to high-flux dialyzers significantly reduced serum β₂-microglobulin levels. Conclusion: Patients treated with a polyamide high-flux membranehad lower β₂-microglobulin concentrations compared with those patients on low-flux dialyzers. This difference might not be mediated by an increase in β₂-microglobulin mRNA, but may be caused by less β₂-microglobulin released from the blood cells in patients treated with high-flux dialyzers, in addition to a better β₂-microglobulin clearance.

Decreased B cells and IgA-secreting B cells partially explain the high prevalence of IgA deficiency in dialysis patients

We previously reported 2 hemodialysis (HD) patients with recurrent infections and selective immunoglobulin A deficiency (IgAD). We further demonstrated that serum IgA levels were lower and the prevalence of IgAD was higher in uremic patients. The exact mechanisms of IgAD in uremic patients largely remained unclear. In some patients, it was caused by anti-IgA antibody neutralization and subsequent destruction. We performed the present study to survey if there is any defect in IgA production. 288 patients were initially included for examination of serum immunoglobulins. 16 normal persons, 16 dialysis patients without IgAD, and 12 dialysis patients with IgAD were enrolled after the initial examination. Blood was drawn into heparinized tubes. WBC counts and lymphocyte percentage were examined by a CBC counter. Lymphocytes were separated by the Ficoll-Paque method. Flow cytometry was utilized to isolate the B cell and IgA-secreting B cell after staining with CD 19 phycoerythrin and FITC-conjugated rabbit anti-human IgA antibody. There is no significant difference between WBC counts or total lymphocyte counts of these 3 groups. However, we found a lower percentage of total lymphocyte counts in dialysis patients, either with or without IgAD. The total B cell numbers were lower in dialysis patients with IgAD. In addition, there were fewer IgA-secreting B cells in dialysis patients with IgAD. Decreased B cell and IgA-secreting B cell counts are seen in uremic patients with IgAD. This, in turn, indicates that there might be a defect of IgA production in some patients, rather than IgA destruction by anti-IgA antibodies as seen in some other patients. Further study is needed to investigate the mechanisms of decreased B cells and IgA-secreting B cells.

Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in dialysis patients, renal transplant recipients and healthy controls

It has been suggested that infection with Chlamydia pneumoniae plays a role in the development and maintenance of atherosclerosis based on differences in the prevalence of antibodies against Chlamydia pneumoniae in patients with and without atherosclerotic lesions and on the presence of bacteria in atherosclerotic lesions. It is well known that patients undergoing chronic dialysis treatment and renal transplant recipients have a considerably increased risk of cardiovascular disease. In this study it is hypothesized that patients with these conditions have a higher prevalence of Chlamydia pneumoniae DNA in the white cells of the peripheral blood. Blood samples from 196 dialysis patients, 114 renal transplant recipients and 342 healthy controls were analysed with an in-house nested polymerase chain reaction (nPCR) and tested for the presence of Chlamydia pneumoniae DNA. The prevalence of Chlamydia pneumoniae DNA was significantly higher in dialysis patients (16.3%) than in healthy controls (8.5%, p<0.01), whereas no significant difference was found between the prevalence in renal transplant recipients (9.6%) and healthy controls. The prevalence was not related to gender or age in either group, and it was the same in diabetics and non-diabetics. Dialysis patients have a higher prevalence of Chlamydia pneumoniae DNA than healthy controls. The lower prevalence of Chlamydia pneumoniae DNA in renal transplant recipients than in dialysis patients may be due to selection of dialysis patients with few or no cardiovascular complications for renal transplantation. Our results are consistent with the hypothesis that Chlamydia pneumoniae is associated with the pathogenesis of atherosclerosis.

Inhibition of immunoglobulin production by parathyroid hormone. Implications in chronic renal failure

Available data indicate that B cell proliferation is inhibited in chronic renal failure and this is due to excess blood levels of PTH. This defect may also affect immunoglobulin production. We examined production of IgG, IgM and IgA by B cells stimulated with Staphylococcus aureus Cowan I (SAC) or with pokeweed mitogen (PWM) after eight days of culture and evaluated the effect of PTH on this process in 34 hemodialysis patients and 44 normal subjects. IgG, IgM and IgA production by B cells from patients was lower (P less than 0.01) than by B cells from normal subjects. Both 1-34 and 1-84 PTH inhibited (P less than 0.01) immunoglobulin production by B cells from normal subjects and dialysis patients. However, this inhibitory effect was evident in dialysis patients only with the higher dose of PTH. The inhibition of immunoglobulin production by PTH occurred only when the hormone was added at the initiation of the B cell culture. Inactivation of PTH abolished its inhibitory effect on immunoglobulin production. Agents that stimulate cAMP production (forskolin, cholera toxin) and the cAMP analogue, 8-bromoadenosine 3',5' cyclic monophosphate inhibited immunoglobulin production by B cells from both normal and dialysis patients, and the degree of inhibition was not different between the two groups. The calcium inophore A23187 also inhibited IgG, IgA and IgM production by B cells from normal subjects and dialysis patients; there was no significant difference in the degree of inhibition between the two groups. The resting levels of cytosolic calcium in B cells of dialysis patients was significantly (P less than 0.01) higher than that of B cells from normal subjects. The data show that: (1) immunoglobulin production is impaired in dialysis patients; (2) B cells of dialysis patients have elevated resting levels of cytosolic calcium; (3) PTH inhibits IgG, IgA and IgM production and this effect is at least partly mediated by PTH-induced cAMP production and alterations in cytosolic calcium into B cells; (4) this inhibitory effect is mediated by events that affect initial stages of B cell proliferation and maturation; (5) the requirement for high dose of PTH for its inhibitory effect on B cells from dialysis patients is probably due to desensitization and/or down-regulation of PTH receptors on B cells. The results are consistent with the proposition that impaired immunoglobulin production by B cells from dialysis patients is at least partly due to the state of secondary hyperparathyroidism in these patients.