When subjected to stimulation, cells from the vascular compartment show a spontaneous collapse of the plasma membrane phospholipid asymmetry and phosphatidylserine is exposed at the external leaflet. Thus, phosphatidylserine externalization is essential for normal hemostasis and phagocytosis. The mechanism governing the migration of phosphatidylserine to the exoplasmic leaflet is not yet fully understood. We have proposed that store-operated calcium entry (SOCE) constitutes a key step of this process. Here, interaction of [Ca(2+)](i), cAMP and cGMP pathways and phosphatidylserine exposure was examined in human megakaryocytic cells. The membrane permeable cAMP and cGMP analogues, pCPT-cAMP and pCPT-cGMP, enhanced the Ca(2+) signal induced by ionophore and SOCE. Responses to pCPT-cAMP and pCPT-cGMP were independent of protein kinase A, protein kinase G (PKG) or ERK pathways. Inhibition of small G-proteins reduced or abolished the increase of [Ca(2+)](i) induced by pCPT-cAMP or pCPT-cGMP, respectively. pCPT-cGMP but not pCPT-cAMP enhanced the ability of cells to expose phosphatidylserine. This effect was not prevented by the inhibition of PKG or small G-proteins. These results show the differential role of cyclic nucleotides in the Ca(2+)-dependent membrane remodeling. Hence, pCPT-cGMP is another regulatory element for the completion of SOCE-induced phosphatidylserine transmembrane redistribution in HEL cells through a mechanism implicating small G-proteins.

Sirolimus (SRL) is suspected to induce proteinuria. We retrospectively studied proteinuria in a population of liver (n = 29) and kidney transplant (n = 30) recipients switched to SRL with progressive diminution or withdrawal of calcineurin inhibitors (CNI). We also observed estimated glomerular filtration rate (GFR), modification of treatment with antiproteinuric drugs, and changes in concentration of SRL. Collection of data started 3 months before SRL introduction at a mean follow-up of 21 months. Following SRL introduction, proteinuria was not detected in the 28 liver transplant patients, and was stable in the two others. In the kidney transplant group, proteinuria did not occur in 12 patients, remained stable in three, and was slightly increased in 14 (0.57 +/- 0.93 g/d vs 1.83 +/- 1.26 g/d). For all patients, eGFR remained stable; there was no difference in management of antiproteinuric drugs. As suspected, cyclosporin (CsA) and tacrolimus (FK) serum concentrations were decreased. We observed a significant correlation between the variation of proteinuria and the variation of serum concentration of CsA or FK (respectively, P =.001 and P =.007). On the other hand, we did not find any correlation between variation in proteinuria and concentration of SRL. This retrospective study suggests that in our cohort of liver transplant patients without previous renal damage, SRL did not provoke proteinuria. On the other hand, the slight aggravation of proteinuria in a subgroup of kidney transplant patients seems to be linked to the hemodynamic renal effects due to CNI withdrawal.

P264 Aims: Early diagnosis of acute rejection remains a pivotal problem in renal transplantation. Previous studies of urinary cell analysis have shown increased lymphocyturia (LU) during acute allograft rejection. On the other hand, LU was not observed durint calcineurin inhibitor toxicity nor acute tubular necrosis. Nevertheless, this diagnosis tool is still burdened with difficulties due to urine lymphocyte low viability. Cells undergoing apoptosis or necrosis show membrane redistribution of phospholipids. In particular transverse redistribution of plasma membrane phosphatidylserine (PS) is followed by the shedding of membrane particles (MPs). These particles carry the membrane antigens of the cells they issued from. We propose to assess measurement of MPs originated from activated lymphocyte (MP-CD4 and MP-CD8) as a novel non invasive marker of acute kidney allograft rejection. Methods: Totality of MPs are captured by immobilized annexin V (MPs-T). MPs originated from activated lymphocytes are captured by insolubilized anti-CD4 and anti-CD8 antibody. MPs are quantified through PS ability to promote the activation of prothrombin to thrombin which is revealed by a chromogenic substrate (prothrombinase assay). Results are expressed as nanomolar phosphatidylserine equivalent (nMEqPS) by reference to a standard curve constructed by using liposomes of defined composition. Twenty-three patients are included in the study. Nine patients have acute allograft rejection (« AR ») proven by graft biopsy (BANFF 97 classification). Four patients have calcineurin inhibitor toxicity (« Tox ») proven by graft biopsy. Ten patients have stable allograft kidney function (« Stable »). Results: The mean concentration of the MPs originated from activated lymphocytes (MP-CD4 + MP-CD8) is 1.31 ± 1.53 in the group « AR », 0 in the group « Tox » and 0,16 ± 0,17 in the group « Stable ». These differences are statistically significant (“AR” vs. “Stable”, p=0.02; “AR” vs. “Tox”, p=0.007). Conclusions: Measurement of urinary concentration of MPs originated from activated lymphocytes offers a noninvasive tool for diagnosis of acute kidney graft rejection, without difficulties concerning urine lymphocyte viability. This transversal study is actually associated with a longitudinal one: regular dosage of MPs concentration in urine of recent renal transplant patients in order to demonstrate that an increase in MP-CD4 and MP-CD8 precedes creatinine serum concentration changes during acute kidney graft rejection.

P346 Aims: glomerular filtration rate (GFR) can be determined exactly by measuring the clearance of an ideal filtration marker, such as inulin. The classic method of measuring inulin clearance includes constant intravenous infusion and timed collections of urine. In order to avoid the need for timed urine collections, iohexol has been used as a filtration marker. In non transplanted patients, good agreement has been shown for GFR determined by the classic inulin clearance and by the iohexol plasma clearance. The aim of this study was to validate iohexol clearance compared with inulin clearance in a population of kidney allograft recipients and to compare this approach with some published equations for determination of creatinine clearance. Patients and methods: thirty two kidney transplant recipients with stable graft function (creatininemia between 69 and 330 μmol/) were evaluated by both inulin and iohexol clearances. The patients underwent simultaneous measurements of renal clearance of inulin and plasma clearance of iohexol. Iohexol was given as a single IV dose (Omnipaque 300, 5 ml), and blood samples were drawn at 120, 150, 180, and 210 for patients with estimated clearance above 40 ml/min, and 240 and 270 minutes if estimated clearance was below 40 ml/min. Iohexol concentrations were analyzed by a one-compartment model corrected by the Brochner-Mortensen formula. Agreement between methods was evaluated by linear regression and according to Bland and Altman analysis. Results: We found excellent correlation (r=0.97; r2=0.95) and a good agreement between inulin clearance and iohexol clearance. Iohexol determination with four blood samplings (120, 150, 180 and 210 min) is sufficient in a large range of renal function. Moreover, predicted equations are inaccurate for precise estimation of DFG when compared to inulin clearance (Cockcroft and Gault: r2 = 0.82, Nankivell: r2 = 0.77, MDRD2: r2 = 0.60, Jellife: r2 = 0.40, Walser: r2 = 0.66). Conclusion: Simplified method using 4 points iohexol clearance and avoiding urine collection is a reliable method of determination of GFR in kidney transplanted patients and can be used for measuring GFR in multicenter clinical trials where GFR is required as a major endpoint even among patients with altered renal function.