Pretransplant Immune Status In Renal Transplant Recipients And Effect On Allograft Function

Part ⅠRelation between Pretransplant Peripheral Leucocyte Subsets and Allograft Function in Renal Transplant RecipientsObjectives. Renal transplant recipients are in the condition of T-cell dysregulation before transplantation, leading to a variable degree of lymphopenia and increased T-cell differentiation. This may cause a relevant reduction in T-cell immunity, yielding a lower risk for delay graft function or acute rejection of renal allograft, affecting the prognosis. Our study was conducted to explore the relation between pretransplant peripheral leucocyte subsets and allograft function in renal transplant recipients.Methods. Characteristics of end-stage renal disease patients who received renal transplantation in Zhongshan Hospital, Fudan University during December 1st,2012 to December 31st,2013 were collected in this study. Clinical and laboratory indexes were detected as well as lymphocyte subsets information was determined by flow cytometry. One-way analysis of variance and mono-factor linear regression were used to evaluate the relation between leucocyte and lymphocyte subsets and allograft function.Results. Patients were divided into three groups based on the tertiles of six-month posttransplant serum creatinine level (Group A:≤95μmol/L, Group B: 95～139μmol/L, Group C:>139μmol/L). Significant differences of leucocyte classification and lymphocyte subset compartment such as T cell count, CD4+ T cell count, CD8+ T cell count were shown among these three groups (all p value<0.05). Moreover, linear correlation analysis showed that leucocyte, lymphocyte, CD3+ T cell, CD3+CD4+ T cell, CD3+CD8+ T cell were positively associated with serum creatinine level of 6-month posttransplant (all p value<0.05).Conclusions. Pretransplant peripheral leucocyte, lymphocyte count and lymphocyte subsets were correlated with posttransplant allograft function. Recipients who had a higher level of peripheral lymphocyte especially T lymphocyte would tend to maintain a higher level of serum creatinine posttransplant, in order to affect allograft outcomes. This is associated with the important role which T cell immunity played in DGF and acute rejection.Part IIAlteration of Peripheral Leucocyte Classification and Related Factors of Lymphocyte Subsets in ESRD PatientsObjectives. Pretransplant recipients were affected by oxidative stress, uremic toxins retention, metabolic disturbance in the course of ESRD, and a severe alteration of leucocyte classification and lymphocyte subsets occurred, which was simultaneously presented as immune activation and immune deficiency. This disturbed pretransplant immune status was associated with short-time and long-term allograft outcomes. To explore risk factors of pretransplant immune status, we compared the leucocyte classification between ESRD patients and health volunteers, and performed lymphocyte subsets analysis.Methods. Characteristics of healthy volunteers and ESRD patients were collected in this study, respectively. Clinical and laboratory data were detected as well as lymphocyte subsets information was determined by flow cytometry. Patterns of immune cells between healthy volunteers and ESRD patients were compared with one-way analysis of variance. Correlation analysis was used to assess the relation between lymphocyte subsets and other clinical or laboratory indexes.Results. Peripheral lymphocyte count in ESRD group was significantly lower than which in healthy volunteers [(1.454±0.678)×109 vs. (1.875±0.417) ×109, p=0.007]. Peripheral neutrophil count in ESRD group was significantly higher than which in healthy volunteers [(4.353±1.709) ×109 vs. (3.279±1.263)×109, p=0.010]. Neutrophil/lymphocyte ratio (NLR) was significantly higher than which in healthy volunteers (3.50±1.81 vs.1.83±0.85,p=0.007). Peripheral neutrophil count was positively associated with history of DM, CVD, level of serum phosphate, NT-proBNP, hsCRP, procalcitonin and negatively associated with albumin, transferrin,25OH-vitD (all p value<0.05). Peripheral lymphocyte count was positively associated with eGFR, Hb, prealbumin, Chol, TG, LDL-C, CO2, serum calcium, transferrin and negatively associated with β2-microgobulin, BUN, Cr, NT-proBNP, hsCRP, iPTH, procalcitonin, erythropoietin (all p value <0.05). B cell count was positively associated with SBP, eGFR, HB, albumin, prealbumin, Chol, TG, LDL-C, transferrin and negatively associated with age, β2-microgobulin, BUN, Cr, NT-proBNP, hsCRP, procalcitonin, erythropoietin (all p value<0.05).Conclusions. Pretransplant recipients underwent an immunological disturbance, which presented as lower level in peripheral lymphocyte count, higher level in peripheral neutrophil count and higher NLR level than which in healthy volunteers. The fluctuation of immune cells was closely linked to lipid metabolism disorder, uremic toxins retention and other risk factors.Part IIIImpact of Indoxyl Sulfate and P-cresol Sulfate on Human Peripheral Lymphocyte Proliferation in vitroObjectives. Indoxyl sulfate and p-cresol sulfate, as two recently concerned uremic toxins, were associated to the alteration of peripheral leucocyte in ESRD patients, and related to the survival rate, incidence of infection and cardiovascular events. However, the impact of IS and PC on lymphocyte has not been reported. In this study, peripheral lymphocytes were co-cultured with different concentrations of IndS or PC, in order to explore the impact of IS or PC on lymphocyte proliferation or apoptosis.Methods. Blood samples of healthy volunteers were collected. PBMCs were extracted followed by isolation and culture of lymphocyte. Different concentrations of IndS (10μg/ml,25μg/ml,50μg/ml, respectively) or PC (0.10mmol/l,0.25mmol/l,0.50mmol/l, respectively) were added in the co-culture system. Cells were collected and counted after incubation for 12h,24h and 48h. CCK-8 kit was used to detect proliferation or apoptosis level of lymphocyte.Results. Given the right stimulus of PHA, after 24h co-culture with IndS, lymphocyte of IndS 50μg/ml group has higher proliferation rate than blank control group and IndS 10μg/ml group (both p value<0.05). After 24h coculture with PC, lymphocyte of PC 0.50mmol/1 group has higher proliferation rate than blank control group and PC 0.10mmol/l group (both p value<0.01). As to coculture 12h with IndS, lymphocyte of IndS 10μg/ml group has higher proliferation rate than blank control group (p value<0.05), while proliferation rate in higher IndS concentration group had no significant difference compared to blank control group. As to coculture 48h with IndS, lymphocyte of different concentration IndS group has higher proliferation rate than blank control group (all p value<0.01) while no significant difference was showed among them. As to coculture either 12h or 48h with PC, lymphocyte of different concentration PC group has higher proliferation rate than blank control group (all p value<0.01) while no significant difference was showed among them.Conclusions. Both indoxyl sulfate and p-cresol promoted lymphocyte proliferation under appropriate PHA stimulus in vitro. This promotion effect on lymphocyte was concentration-dependent only when coculture time lasted for 24h, which has not presented either short or long the co-culture lasted. Further studies were needed to clarify the specific mechanism.