Two-dimensional difference gel electrophoresis urinary proteomic profile in the search of nonimmune chronic allograft dysfunction biomarkers.

INTRODUCTION: Despite advances in therapeutics, graft loss associated with chronic allograft dysfunction (CAD) remains high. Urinary proteomic analysis is a noninvasive method that could be used to detect and evaluate CAD in renal transplant recipients. This study was aimed to establish the normal proteome map of stable transplant patients and to validate the utility of two-dimensional difference gel electrophoresis (2DE-DIGE) in identifying new candidates as urinary biomarkers of CAD. METHODS: Morning spot urine samples that were collected from kidney transplant recipients with biopsy-proven interstitial fibrosis and tubular atrophy (IFTA) stages 0-I-II/III (n=8/group) under immunosuppressive treatment with tacrolimus plus mycophenolate with or without prednisone. 2DE silver staining and mass spectrometry analyses were used to establish the normal proteome map, and 2DE-DIGE and mass spectrometry were used to identify proteins exhibiting differential abundance. RESULTS AND CONCLUSIONS: This study defines the normal proteome of stable renal transplant patients, which is composed of several plasma proteins, as well as of immunologic proteins that are probably specific to transplant recipients. The 2DE-DIGE study showed 19 proteins with differential concentrations, depending on the IFTA histologic score. These 19 proteins could be used as urinary biomarkers of the severity of IFTA in renal transplant recipients.

Thrombospondin-1 mRNA expression in experimental kidney transplantation with heart-beating and non-heart-beating donors.

BACKGROUND: Ischemia-reperfusion (IR) is a risk factor for delayed graft function, a clinical syndrome more frequently observed in non-heart-beating donors (NHBDs). Previous studies have reported that transforming growth factor-beta1 (TGF-beta1) and hypoxia-inducible factor-1alpha (HIF-1alpha) gene expression increase in the first few days after kidney transplant and that this increase in TGF-beta1 expression is lower in NHBD animals. The purpose of this study was to extend the gene profile analysis by characterizing TGF-beta1 activator thrombospondin-1 (TSP-1) and genes related to HIF-1alpha such as heme oxygenase-1 (HO-1), nitric oxide synthase-2 (NOS-2) and NOS-3. METHODS: The experimental pig model of kidney transplantation comprised heart-beating donors (HBDs, n=9) and NHBDs (n=22). Cortical biopsies were collected after anesthetic induction (baseline), after warm ischemia (WI), after cold ischemia (CI), after 1 hour of reperfusion (1R) and 5 days (5D) after transplant. TSP-1, HO-1, NOS-2 and NOS-3 mRNA expression was determined by real-time PCR. RESULTS: No change in expression of any of the genes analyzed was found during the transplant procedure (WI, CI, 1R) in HBD and NHBD cortical samples. TSP-1 mRNA was significantly increased at 5D in NHBD animals but unchanged in the HBD group. HO-1 was up-regulated in HBD (p<0.05) and NOS-2 mRNA was significantly increased in both groups (p<0.05). No difference in NOS-3 expression was observed at 5D. CONCLUSIONS: The increased TSP-1 expression in NHBDs may indicate a compensatory response to the reported diminished TGF-beta1 expression. The augmented NOS-2 and HO-1 expression in HBDs could have a positive effect on the recovery of kidney function.

Expression of transforming growth factor-beta1 and hypoxia-inducible factor-1alpha in an experimental model of kidney transplantation.

BACKGROUND: Ischemia-reperfusion syndrome has been recognized as an important pathogenic factor in renal transplantation, not only in the development of delayed graft function but also in the development of acute and chronic rejection. Hypoxia-inducible factor (HIF)-1 activates transcription of several genes implicated in cell survival, such as vascular endothelial growth factor (VEGF), and in tissue repair transforming growth factor (TGF)-beta. The purpose of this study was to characterize TGF-beta1, VEGF, and HIF-1alpha expression profiles during renal transplantation with heart-beating donors (HBD) and non-heart-beating donors (NHBD). METHODS: An experimental model of renal transplantation using 40 pairs of large, white, Landrace pigs and including HBD and NHBD was used. Cold-ischemia time was the same in all groups (6 hr), and three groups of NHBD (30, 45, and 90 min) were studied. Immunosuppressive therapy consisted of cyclosporine, except in one HBD group, which was treated with azathioprine. TGF-beta1, VEGF, and HIF-1alpha expression profiles were performed in renal biopsies obtained at different times: after anesthetic induction (basal); 30, 45, and 90 min after warm ischemia; after cold ischemia; 1 hr after reperfusion; and 5 days after transplantation. RESULTS: TGF-beta1 expression increased after cold ischemia in HBD and remained unaltered during the surgical process in all NHBD groups. HIF-1alpha and VEGF expression were not greatly modified during surgery in the HBD or NHBD groups. All groups showed a significant increase in TGF-beta1 and HIF-1alpha expression as well as down-regulation of VEGF 5 days after transplantation, and these effects were independent of immunosuppressive treatment. There were no statistically significant differences among the groups at 5 days after transplantation, although the increase in TGF-beta1 was more pronounced in the HBD groups, especially in azathioprine-treated animals. CONCLUSIONS: The initial up-regulation of TGF-beta1 observed in HBD immediately after cold ischemia could have a positive effect on epithelial-tubular regeneration. Warm ischemia has a detrimental effect on TGF-beta1 expression during the early phases of renal transplantation and has no effect on VEGF and HIF-1alpha expression. The up-regulation of TGF-beta1 and HIF-1alpha observed after transplantation could have a positive effect on tubular repair. TGF-beta1 expression was lower in animals treated with cyclosporine, probably because of cellular toxicity.

Effects of losartan and amlodipine on intrarenal hemodynamics and TGF-beta(1) plasma levels in a crossover trial in renal transplant recipients.

Hypertension and hyperfiltration are two important risk factors for the development of chronic allograft nephropathy. Transforming growth factor-beta(1) (TGF-beta(1)) is the main cytokine involved in the fibrotic process that is involved in chronic rejection. Angiotensin II upregulates TGF-beta(1) production. Angiotensin II receptor antagonists therefore could not only control BP but also reduce TGF-beta(1) production in renal transplant patients. The aim of this study was to compare the effects of losartan and amlodipine on renal hemodynamics, as well as TGF-beta(1) and endothelin-1 (ET-1) plasma levels in a group of renal transplant patients who had normal renal function and who were treated with cyclosporine. Seventeen renal transplant patients who were receiving cyclosporine and who had normal graft function were included in a random 2 x 2 crossover trial with amlodipine and losartan (6 wk with each therapy). Three studies were performed (at baseline and at the end of both treatment periods) to determine renal hemodynamics, TGF-beta(1), and ET-1. Both treatments controlled BP to a similar degree, but only amlodipine increased GFR through an increase in the estimated glomerular hydrostatic pressure and filtration fraction. In contrast, losartan maintained GFR and reduced estimated glomerular hydrostatic pressure and filtration fraction significantly. Losartan and amlodipine had opposite effects on TGF-beta(1). Amlodipine did not affect TGF-beta(1) concentrations. In contrast, losartan reduced the plasma levels of TGF-beta(1) by approximately by 50% (from baseline, 5.2 to 2.6 ng/ml; P: = 0.01); the majority of the patients reached normal levels of TGF-beta(1). ET-1 concentrations were significantly higher during amlodipine compared with losartan treatment. The present study documents that with similar control of BP, losartan and amlodipine have opposite effects on renal hemodynamics and on TGF-beta1 concentrations. These differences could be important for the management of chronic allograft nephropathy.