Clinical And Experimental Research On Platelet Apoptosis Of End Stage Renal Disease And DNA Damage Of Autosomal Dominant Polycystic Kidney Disease

Part I Clinical and experimental research on platelet apoptosis in EndStage Renal Disease patientsObjective: To explore the risk of clinical bleeding, changes of platelet parameter andinfluencing factors in end-stage renal disease (ESRD) patients and to analyse plateletsapoptosis in uremic patients.Methods: In clinical study, a retrospective analysis was carried out among278patients with ESRD. Clinical bleeding manifestations, treatment methods and such clinicalparameters as serum creatinine (SCr), parathyroid hormone (PTH) and platelet (PLT) wereanalysed as to disclose the effection of renal function, primary diseases and treatmentmodalities on bleeding and platelet parameters. In the experimental study,16patients withuremia underwent hemodialysis (HD), peritoneal dialysis (PD) or non-dialysis (ND)treatments were studied. Platelet-rich plasma (PRP) was detected by Flow Cytometry forapoptotic events including depolarization of mitochondrial inner membrane potential(ΔΨm), phosphatidylserine (PS) exposure and for activation events with P-Slectin.Platelets function was assessed by ristocetin induced aggregative function test.Then,washed normal platelets were incubated with normal or uremic platelet poor plasma (PPP)and then were detected apoptotic events including ΔΨm depolarization, PS exposure byFlow Cytometry and activation of caspases-3and Bcl-2family proteins variations byWestern-Blot. Finally, washed normal platelets were stimulated by such uremic toxins ascreatinine, urea and parathyroid hormone withdifferent concentration and were detectedapoptosis events. Results: The results of clinical study showed that the incidence of bleeding in ESRDpatients was25%. Among them, patients with lupus nephritis, Hepertensive Nephropathy,Diabetic Nephropathy and chronic glomerulonephritis accounted for92.86%of bleedingpatients. There were significant differences in Hb, PLT, MPV and HCT parameters amongdifferent primary diseases. Patients in stage4and stage5of chronic kidney disease (CKD)had higher risk for bleeding (26.47%and28.85%, respectively). There were significantdifferences in Hb, PLT, PDW and HCT parameters among stage3-5groups of CKD. PLTwas negatively related with the serum creatinine. Bleeding rate were32.61%(HD),30.95%(PD) and15.09%(ND) respectively. HD and PD group had lower PLT than ND group.The results of experimental study showed that uremic platelets demonstrated greaterapoptosis for the ΔΨm depolarization (43.48±9.58versus52.76±15.36, P=0.005) as wellas PS exposure (1.36±0.51versus0.99±0.27, P<0.001) when compared with controls.There was no significant difference among different treatment groups. Ristocetin inducedplatelet aggregation was markedly diminished in uremic patients.Platelets exposed touremic plasma exhibited distinct apoptosis phenomena for ΔΨm depolarization P=0.0001,for PS exposure P=0.002and for P-Slectin P=0.14. Western Blot analyses showed a17kdfragment which exhibited caspase-3activation and decreased levels of anti-apoptotic Bcl-2,Bcl-xl and enhanced level of pro-apoptotic Bax expression. Creatinine, urea andparathyroid hormone failed to induce ΔΨm depolarization in normal platelets.Conclusions: Patients with ESRD have relatively high bleeding tendency. Thebleeding tendency is associated with primary disease and renal function. Platelet count isreduced with the renal function deterioration. Platelet reduction has relationship with highbleeding risk of patients on hemodialysis and peritoneal dialysis. Enhanced plateletapoptosis are incurred in uremia patients. It is a result of continous retention of varioustoxins and cannot be corrected by dialysis. Uremic platelets apoptosis has no relationshipwith dialysis modality. Apoptosis may cause thrombocytopenia and platelet dysfunction inuremia. Part Ⅱ Genomic instability in patients with autosomal dominantpolycystic kidney diseaseObjective: To detect DNA damage of peripheral blood lymphocytes in patients andfamily members of autosomal dominant polycystic kidney disease(ADPKD), and to studythe effect of irradiation on genomic stability of lymphocytes．Methods: Before and after0.5Gy radiation dose, single-cell gel eleetrophoresis(SCGE)was employed to analyze DNA damage of lymphocytes in10ADPKDpatients(group A),3members without clinical symptoms of a ADPKD family(group B)and20healthy control people(group C). The damage was estimated based on the contentof DNA in tail (TDNA％) with comet analysis software(CASP)．Results: Both before and after irradiation，the TDNA％(8.85%±0.14%，14.84±0.77%)and the TDNA％value-added (5.99%±0.77%) of group A were significantly higher thanthose of group C (7.50%±0.37%,12.46%±0.26%,4.96%±0.44%) respectively. Group Bhad no clinical symptoms, but2cases of them showed similar TDNA%changes beforeand after irradiation to those of group C, while another case had similar TDNA%changesto those of group A.Conclusions: The lymphocytes of ADPKD patients may more sensitive to ionizingradiation as compared to healthy people. The genomic instability in ADPKD patients maytrigger cystic formation in multi-organs when exposing to environmental agents. SCGEmay provide a new approach to elucidate the pathogenesis and prognosis of ADPKD．...