Nephrol Dial Transplant (2001) 16: 361-367
© 2001 European Renal Association-European Dialysis and Transplant Association
Plasma transforming growth factor ß1 and platelet activation: implications for studies in transplant recipients
Renal Research Laboratories, Manchester Institute of Nephrology and Transplantation, The Royal Infirmary, Manchester, and 1 Laboratory Medicine Academic Group, University of Manchester, UK
Background. Evidence from animal models supports the hypothesis that dysregulated transforming growth factor ß1 (TGFß1) expression plays a role in chronic allograft rejection, the progression of diabetic nephropathy and fibrotic glomerulopathies. However, more evidence is required to support this hypothesis in man, and the current literature concerning blood TGFß1 levels in clinical studies is highly confused. We have investigated: (i) the hypothesis that the widespread practice of activating clinical samples prior to measurement of TGFß1 is detecting the platelet-released pool of TGFß1, artefactually generated on venepuncture and unrepresentative of the real circulating in vivo TGFß1 pool; and (ii) the effect of different immunosuppressive drugs on apparent TGFß1 plasma levels.
Methods. The effect of two different venepuncture procedures on plasma TGFß1 was compared in 10 healthy volunteers, one procedure designed to minimize platelet activation and the other representing standard venepuncture practice in a clinic situation. Blood samples from 52 renal transplant recipients on either cyclosporine or tacrolimus immunosuppression were taken by standard venepuncture to investigate the effect of immunosuppressive drugs on plasma TGFß1. Plasma TGFß1 and ß thromboglobulin were measured by ELISA.
Results. Among 10 healthy volunteers who underwent two different methods of venepuncture, eight of 10 had undetectable levels of TGFß1 (<100 pg/ml) under conditions that minimize platelet activation. In contrast, all 10 paired plasma samples collected by vacutainer had measurable TGFß1 (median 7.70 ng/ml, interquartile range 5.87–13.64 ng/ml) following acid/ urea activation. The median ßTG level (a measure of platelet degranulation) was 0.71 µg/ml (interquartile range 0.53–1.19 µg/ml) in the special collections compared with 3.39 µg/ml (interquartile range 2.27–4.33 µg/ml) in the vacutainer samples (P=0.0029).
Among 52 allograft recipients there was a significantly higher mean TGFß1 level in plasma from patients on cyclosporine therapy compared with patients on tacrolimus (28 090±26 860 pg/ml vs 7173±10 610 pg/ml, respectively; P<0.002). Mean plasma ßTG levels were also significantly higher during cyclosporine therapy compared with tacrolimus (8.14±5.54 µg/ml vs 3.66±3.32 µg/ml, respectively; P<0.002). However, when TGFß1 values were corrected for the degree of platelet activation (by factoring with ßTG) there was no significant difference between TGFß1 levels on cyclosporine or tacrolimus (4117±2993 pg/µg ßTG vs 2971±658 pg/µg ßTG, respectively; P=0.294).
Conclusions. To avoid erroneous hypotheses concerning TGFß1 and perpetuating confusion in the literature over levels in health and disease, it is imperative that proper internal controls for platelet activation are used. The effects of experimental treatments and drugs on platelet biology must be rigorously controlled when attempting to measure and interpret plasma levels of TGFß1 in clinical practice.
Keywords: immunosuppression; platelets; renal allografts; TGFß1; immunoassay
Correspondence and offprint requests to: Dr PEC Brenchley, Renal Research Labs, Manchester Institute of Nephrology and Transplantation, The Royal Infirmary, Oxford Road, Manchester M13 9WL, UK.
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