J.M Grinyó

Cyclosporine Sparing with Mycophenolate Mofetil, Daclizumab and Corticosteroids in Renal Allograft Recipients: The CAESAR Study

Although the calcineurin inhibitors (CNI) cyclosporine (CsA) and tacrolimus are highly effective immunosuppressants, they are associated with serious side effects. There is great interest in immunosuppressive regimens that permit reduction or elimination of CNIs, while maintaining adequate immunosuppression and acceptable acute rejection rates. Patients (n = 536) receiving their first renal allograft were randomized to one of three immunosuppressant regimens: daclizumab, mycophenolate mofetil (MMF), corticosteroids (CS) and low‐dose CsA (target trough levels of 50–100 ng/mL), weaned from month 4 and withdrawn by month 6; daclizumab, MMF, CS and low‐dose CsA; or MMF, CS and standard‐dose CsA. Mean GFR 12 months after transplantation (primary end point) was not statistically different in the CsA withdrawal and low‐dose CsA groups (both 50.9 mL/min/1.73 m2) vs. the standard‐dose CsA group (48.6 mL/min/1.73 m2). At 12 months, the incidence of biopsy‐proven acute rejection was significantly higher in the CsA withdrawal group (38%) vs. the low‐ or standard‐dose CsA groups (25.4% and 27.5%, respectively; p < 0.05). In summary, a regimen of continuous low‐dose CsA with MMF, CS and daclizumab induction is a clinically safe and effective immunosuppressive regimen in renal transplant recipients.

A good alternative to reduce the kidney shortage : Kidneys from nonheartbeating donors

BACKGROUNDnBecause of the shortage of kidneys available for transplantation, we began in 1985 to harvest kidneys from non-heartbeating (NHB) donors.nnnMETHODSnWe compared the results of a group of 66 kidney recipients from NHB donors (NHB group) with 122 kidney recipients from heartbeating donors (HB group). We analyzed, in the NHB group, the influence of ischemia times in graft survival and we tested the best cut-offs by receiver operating characteristic curves. We also studied, using a univariate and multivariate Cox hazard model, the capacity of different variables to predict graft loss.nnnRESULTSnPatient and graft survival were similar in both groups during the follow-up. The percentage of delayed graft function was the only significant difference between both groups (NHB group 62% vs. HB group 32%; P=0.0001). Delayed graft function, in the NHB group, is influenced by the warm ischemia time, which is directly related to the number of days to achieve a serum creatinine<300 mmol/L (P=0.0001). The best cut-off times in this group were 45 min for warm ischemia time and 22 hr for cold ischemia time. Recipients have a greater likelihood of losing the graft beyond those limits (P=0.017, relative risk: 7.3). The incidence of acute rejection was similar in both groups, and it was the only predictor factor of graft loss in the complete series of patients (P=0.0001), in the NHB group (P=0.007), and in the HB group (P=0.02).nnnCONCLUSIONSnReducing the incidence of acute rejection and shortening ischemia time are conditions needed to guarantee a long graft survival of kidneys from NHB donors.

Hepatocyte growth factor gene therapy enhances infiltration of macrophages and may induce kidney repair in db/db mice as a model of diabetes

Aims/hypothesisWe previously demonstrated hepatocyte growth factor (HGF) gene therapy was able to induce regression of glomerulosclerosis in diabetic nephropathy through local reparative mechanisms. The aim of this study was to test whether bone-marow-derived cells are also involved in this HGF-induced reparative process.MethodsWe have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells. We performed treatment with HGF gene therapy either alone or in combination with granulocyte-colony stimulating factor, in order to induce mobilisation of haematopoietic stem cells in these diabetic and chimeric animals.ResultsWe find HGF gene therapy enhances renal expression of stromal-cell-derived factor-1 and is subsequently associated with an increased number of bone-marrow-derived cells getting into the injured kidneys. These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model. Finally, HGF gene therapy is associated with the presence of a small number of Bowman’s capsule parietal epithelial cells producing EGFP, suggesting they are fused with bone-marrow-derived cells and are contributing to podocyte repopulation.Conclusions/interpretationAltogether, our findings provide new evidence about the therapeutic role of HGF and open new opportunities for inducing renal regeneration in diabetic nephropathy.

Influence of MRP2 on MPA pharmacokinetics in renal transplant recipients–results of the Pharmacogenomic Substudy within the Symphony Study

BACKGROUNDnThe aim of this study was to determine the relationship between single-nucleotide polymorphisms (SNPs) in MRP2 genes and mycophenolic acid (MPA) pharmacokinetics in renal transplant recipients of the Symphony Pharmacogenomic substudy.nnnMETHODSnSixty-six renal transplant recipients of eight Spanish centres were randomized into four branches of immunosuppressive regimen: low dose of cyclosporine, standard dose of cyclosporine, tacrolimus and sirolimus, all in addition to mycophenolate mofetil and steroids. Fifty-five patients were genotyped for SNPs in MRP2, C24T and C3972T. Pharmacokinetic sampling was done before MPA administration and up to 12 h post-dose at Day 7, 1 month and 3 months post-transplant. Relationships of area under the curve (AUC) of MPA and MPAG plasma sampling with the presence of MRP2 SNPs and with the immunosuppressive regimens were studied.nnnRESULTSnAt steady-state conditions, MPA-reduced exposure was observed in C24T variant allele in MRP2 (CC: 68.73 ± 6.78; *T: 48.12 ± 4.90, P = 0.023); no significant differences linked to C3972T SNP were observed. Taking into account groups of treatment, lower MPA AUC in variant allele of C24T was only found under macrolides treatment with statistically significant differences at Month 3 (Tac and SRL, CC: 86.52 ± 10.98 versus *T: 41.99 ± 4.82, P = 0.001; CsA, CC: 52.31 ± 5.30 versus *T: 54.24 ± 8.30, P = 0.772); for C3972T, the same tendency was found but differences at steady state did not reach statistical significance.nnnCONCLUSIONSnRenal transplant recipients T carriers of C24T MRP2 with macrolides treatment were associated with reduced MPA AUC in steady-state conditions. Patients treated with cyclosporine lost the effect of this polymorphism.

Effect of a platelet-activating factor (PAF) receptor antagonist on hyperacute xenograft rejection; evaluation in a pig kidney-human blood xenoperfusion model

In pig to human discordant xenotransplantation, PAF may contribute to the pathogenesis of hyperacute xenograft rejection (HXR). We examined the release of PAF and the effect of a PAF receptor antagonist (BN 52021) on HXR in a pig kidney–human blood xenoperfusion model. Pig kidneys were perfused with porcine blood (AUTO group, nu2003=u20035), human blood (HETER group, nu2003=u20036) or human blood plus BN 52021 (BN group, nu2003=u20034), respectively. In contrast to HETER kidneys that never produced urine and were rejected in 15–30u2003min, the administration of BN 52021 induced a partial recovery of glomerular filtration rate and allowed kidneys to function until the end of the study. The release of PAF and soluble P‐selectin, as well as endothelial P‐selectin expression and tissue myeloperoxidase (MPO), were much higher in the HETER than in the AUTO group. HETER and BN kidneys displayed similar natural xenoantibody titres, CH50, PAF, soluble P‐selectin as well as renal immunoglobulin (IgM, IgG, IgA) and complement (C3, C1q) deposition. However, HETER kidneys displayed a full histologic picture of HXR (mainly interstitial haemorrhage and vascular microthrombi) and BN kidneys had only endothelial cell swelling. Also, BN 52021 administration attenuated glomerular and vascular P‐selectin expression and renal tissue MPO activity. We conclude that in the pig kidney–human blood xenoperfusion model, PAF is produced in higher amounts than in the pig kidney–pig blood autologous combination. The administration of BN 52021 exerts a protective effect by means of attenuating the acute inflammatory response and blocking vascular microthrombi formation.

Do drug transporter (ABCB1) SNPs and P‐glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study

The function of the efflux pump P‐glycoprotein (Pgp) and ABCB1 single nucleotide polymorphisms (SNPs) should be considered as important tools to deepen knowledge of drug nephrotoxicity and disposition mechanisms. The aim of this study is to investigate the association of C3435T, G2677T, C1236T, and T129C ABCB1 SNPs with Pgp activity and exposure to different immunosuppressive drugs in renal transplant patients. Patients included in the Symphony Pharmacogenomic substudy were genotyped for ABCB1 SNPs. According to the design, patients were randomized into four immunosuppressive regimens: low and standard dose of cyclosporine (n = 30), tacrolimus (n = 13), and sirolimus (n = 23) concomitantly with mycophenolate and steroids. Pgp activity was evaluated in PBMC using the Rhodamine 123 efflux assay. TT carrier patients on C3435T, G2677T, and C1236T SNPs (Pgp‐low pumpers) showed lower Pgp activity than noncarriers. Pgp‐high pumpers treated with cyclosporine showed lower values of Pgp function than macrolides. There was a negative correlation between cyclosporine AUC and Pgp activity at 3 months. Results did not show any correlation between tacrolimus and sirolimus AUC and Pgp activity at 3 months. We found an important role of the ABCB1 SNPs Pgp function in CD3+ peripheral blood lymphocytes from renal transplant recipients. Pgp activity was influenced by cyclosporine but not macrolides exposure.

Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.

Background. Transforming growth factor (TGF)–β1 is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs). Methods. The contribution of SNPs at codons 10 and 25 of the TGF-β1 gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-β1 production. TGF-β1 genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-β1 messenger RNA (mRNA) was measured by real-time PCR and TGF-β1 plasma levels were assessed by enzyme-linked immunosorbent assay. Results. Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P=0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P=0.01) were independent predictors of subclinical rejection (SCR). TGF-β1 plasma levels, but not those of TGF-β1 mRNA, were increased in patients with SCR (2.59 ng/mL ± 0.91 [n=22] vs. 2.05 ng/mL ± 0.76 [n=43]; P=0.01). There was no association between allele T and TGF-β1 plasma or intragraft levels. Conclusions. Allele T at codon 10 of the TGF-β1 gene is associated with a higher incidence of SCR.

Tubular Epithelial Cells Transfected With hHGF Counteracts Monocyte Chemotactic Protein-1 Up-regulation After Hypoxia/Reoxygenation Insult

Acute kidney injury (AKI) which is mainly produced by nephrotoxic or ischemic insults is correlated with a high mortality and morbidity. Proximal tubular epithelial cells (PTEC) play a major role. They are the main target of ischemia/reperfusion injury. PTECs have also been proposed as the effectors of AKI reversibility, but also as the creator of the inflammatory milieu: cytokine, chemokine, and complement expression. An important chemokine implicated in this process is monocyte chemotactic protein-1 (MCP-1) due to its ability to recruit and activate monocytes. Hepatocyte growth factor (HGF) is a pleiotropic factor with mitogenic, anti-apoptotic, and proliferative effects which has recently been studied for its anti-inflammatory and antifibrogenic effects. Our aim was to evaluate the potential inflammatory effect of hypoxia and reoxygenation on rat PTECs. We created a stable human HGF (hHGF) expressing PTEC line that emulated in vivo transfection and analyzed the role of this cell type in the induction and reversibility of AKI. Our results showed the efficiency of transfection with the hHGF gene to promote sustained expression of the protein in the medium (7627.13 +/- 1144.078 to 8211.3 +/- 795.37 pg/mL). When rat PTECs were under a hypoxia/reoxygenation insult, MCP-1 was highly overexpressed (4479.3 +/- 154.3 pg/mL of protein and 5.099 +/- 1.23 times control gene expression). Transfected cells abrogated this effect (288.7 +/- 13.5 pg/mL and 1.169 +/- 0.0759 times control). In conclusion, we observed that the hypoxia/reoxygenation insult stimulated MCP-1 protein secretion in PTECs and that PTECs which were stably transfected and overexpressing hHGF abrogated the inflammatory reaction mediated by hypoxia/reoxygenation, being a suitable model for later studies.

Changes in renal hemodynamics and physiology after normothermic ischemia in animals supplemented with eicosapentaenoic acid.

Abstractu2002 In order to determine whether treatment of animals with an n‐3 fatty acid, eicosapentaenoic acid (EPA), could modify renal hemodynamics and physiology after normothermic ischemia, we studied 42 Spraque Dawley rats orally supplemented with either olive oil or a purified lysine salt of EPA for 4 weeks. Four experimental groups were established. Three groups were treated with increasing doses of EPA: 20 mg/kg per day (EPA 20), 40 mg/kg per day (EPA 40) and 80 mg/kg per day (EPA 80), and one group was supplemented with iso‐volumetric olive oil (OLI). A control group that received neither EPA nor ischemia was also studied. On day 28, right nephrectomy was performed, followed by 30 min of left renal warm ischemia. Basal arterial pressure and renal blood flow (RBF) were monitored in two kidneys before arterial occlusion and continuously thereafter throughout the experiment in one kidney using an electronic transducer and a flowmeter. From 60 to 120 min after the end of ischemia, urine output (μl/min), glomerular filtration rate (GFR, μl/ min), measured by inulin clearance, and fractional reabsortion of sodium (FRNa) were determined every 20 min. Renal plasma flow (RPF, ml/ min) and renal vascular resistance (VR, mm Hg/ml per min) were calculated. RPF was estimated as RBF (1‐hematocrit). Before ischemia, the mean RPF and RBF were higher in EPA‐fed than in olive oil‐fed animals and after ischemia showed a significantly greater increase in EPA‐fed animals than in olive oil‐fed animals. Mean VR was lower in EPA‐fed animals than in olive oil‐fed animals, both before arterial occlusion and after ischemia. Mean urine output was similar in the OLI and EPA 20 groups, and significantly higher in the EPA 40 and EPA 80 groups than in the control group. GFR was significantly lower in the OLI and EPA 20 groups than in the control group. Finally, the EPA 40 group showed a similar and the EPA 80 group a slightly higher GFR than the control group. We conclude that EPA supplementation provides protection from renal ischemic‐re‐perfusion injury, and this effect is more evident at higher EPA doses.

Contribution of anemia and hypertension to left ventricular hypertrophy during the initial 2 years after renal transplantation.

BACKGROUNDnCardiovascular disease is the main cause of mortality after renal transplantation. Left ventricular hypertrophy (LVH) is considered to be an independent predictor of cardiovascular events. The main risk factors for LVH after renal transplantation are anemia and hypertension. In hypertensive and renal transplant patients, ambulatory blood pressure monitoring (ABPM) has been demonstrated to be more closely related to LVH than office blood pressure. The aim of this study has to evaluate LVH after renal transplantation, particularly its association with measures derived from ABPM and cardiovascular risk factors.nnnPATIENTS AND METHODSnBetween March 2005 and October 2006, we recruited 101 consecutive kidney transplant patients to calculate left ventricular mass index (LVMI) by echocardiography at 3, 12, and 24 months. Hypertension was evaluated by office blood pressure measurements at 3, 12, and 24 months and also by ABPM at 3 months. Clinical and laboratory data were recorded during the study.nnnRESULTSnFrom 3 to 24 months LVMI was reduced from 129 ± 29 g/m(2) to 121 ± 34 g/m(2) (P = .0089). Multivariate stepwise regression analysis showed independent predictors of LVMI at 3 months to be hemoglobin at 1 month, day systolic blood pressure (SBP) derived from ABPM and donor age (R = .50, P < .001). The independent predictors of LVMI at 12 months were day SBP derived from ABPM, hemoglobin at 1 month, and proteinuria at 12 months (R = .55, P < .001). Office SBP at 12 months, proteinuria at 24 months, patient age and night diastolic blood pressure derived from ABPM at 3 months were independent predictors of LVMI at 24 months (R = .71, P < .001).nnnCONCLUSIONnWe observed a significant reduction in LVMI after renal transplantation. The main contributors to LVMI were anemia and elevated blood pressures measured by ABPM.