Extracorporeal photopheresis and the cellular mechanisms: Effects of 8-methoxypsoralen and UVA treatment on red blood cells, platelets and reactive oxygen species.

BACKGROUND AND OBJECTIVES: Extracorporeal photopheresis (ECP) is a widespread cellular therapy for graft-versus-host disease, autoimmune diseases and Sézary disease. One of the main effects of ECP is the apoptosis of leukocytes, but the therapeutic mechanisms are not completely known. The aim of this study was to investigate the effects on red blood cells, platelets and the induction of reactive oxygen species. MATERIALS AND METHODS: We used human cells from healthy blood donors to simulate in vitro the composition in an apheresis bag. Cells were treated with 8-methoxypsoralen (8-MOP) and UVA. Red blood cell stability, platelet activity and induction of reactive oxygen species were analysed. RESULTS: After 8-MOP and UVA treatment, the red blood cells showed high cell integrity with low levels of eryptosis and no increase of free haemoglobin or red blood cell distribution width (RDW). Red blood cell immune-associated antigens CD59 and CD147 were hardly affected by the treatment. Platelet glycoproteins CD41, CD62P and CD63 indicated strong platelet activation after 8-MOP and UVA treatment. Reactive oxygen species were slightly but not significantly induced by the treatment. CONCLUSION: The effect of the ECP therapy is probably not exclusively mediated by leukocytes. Platelet activation is another striking effect caused by the treatment of the apheresis product with 8-MOP/UVA. However, since we could hardly identify any evidence for eryptosis or haemolysis, it is unlikely that red blood cell eryptosis is part of the therapeutic mechanism. Further research on this topic seems to be promising.

Ratio of Urinary Proteins to Albumin Excretion Shifts Substantially during Progression of the Podocytopathy Alport Syndrome, and Spot Urine Is a Reliable Method to Detect These Pathologic Changes.

The urinary albumin- and protein-to-creatinine ratios (UACR and UPCR, respectively) are key endpoints in most clinical trials assessing risk of progression of chronic kidney disease (CKD). For the first time, the current study compares the UACR versus the UPCR head-to-head at early stages of CKD, taking use of the hereditary podocytopathy Alport syndrome (AS) as a model disease for any CKD. Urine samples originated from the prospective randomized, controlled EARLY PRO-TECT Alport trial (NCT01485978). Urine samples from 47 children with confirmed diagnoses of AS at very early stages of CKD were divided according to the current stage of AS: stage 0 (UACR < 30 mg/g), stage 1 (30-300 mg/g) or stage 2 (>300 mg/g). The range of estimated glomerular filtration rate was 75-187.6 mL/min. The mean age was 10.4 ± 4.5 years. In children at stage 0, proteinuria in spot urine, confirmed in 24 h urine, was almost ten times higher than albuminuria (106.4 ± 42.2 vs. 12.5 ± 9.7; p < 0.05); it was "only" about three times higher in stage 1 (328.5 ± 210.1 vs. 132.3 ± 80.5; p < 0.05) and almost equal in stage 2 (1481.9 ± 983.4 vs. 1109.7 ± 873.6; p = 0.36). In 17 children, UACRs and UPCRs were measured simultaneously in 24 h urine and spot urine in the same study visit. Interestingly, the UACR (and UPCR) in 24 h urine vs. in spot urine varied by less than 10% (266.8 ± 426.4 vs. 291.2 ± 530.2). In conclusion, our study provides the first evidence that in patients with normal glomerular filtration rate (GFR) and low amounts of albuminuria, especially in children with podocytopathies such as AS, measuring the UACR and UPCR in spot urine is a reliable and convenient alternative to 24 h urine collection. Our study advocates both the UACR and the UPCR as relevant diagnostic biomarkers in future clinical trials in children with glomerular diseases because the UPCR seems to be a very significant parameter at very early stages of podocytopathies. The German Federal Ministry of Education and Research funded this trial (01KG1104).