Systematic analysis of GSK-3 signaling pathways in aging of cerebral tissue.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase, involved in regulation of multiple physiological processes. In brain, changes in GSK-3 signaling are related to neurodegenerative issues, including Alzheimer's disease. Due to the wide range of GSK-3 cellular targets, a therapeutic use of the enzyme inhibitors entails significant risk of side effects. Thus, altering the ratio of specific pool of GSK-3 or specific substrates instead of changing the global activity of GSK-3 in brains might be a more appropriate strategy. This paper provides a comprehensive data on abundances of proteins involved in GSK-3 signaling in three regions of young and old mouse brains. It might help to identify novel protein targets with the highest therapeutic potential for treatment of age-related neurodegenerative diseases.

Duration of brain death and cold ischemia time, but not warm ischemia time, increases expression of genes associated with apoptosis in transplanted kidneys from deceased donors.

Apoptosis is one of the most important mechanisms leading to kidney graft injury during transplantation. The aim of this study was to assess the expression of genes involved in apoptosis in transplanted kidneys derived from deceased donors (DD) at various stages of the transplant procedure, seventy eight transplanted kidneys procured from 43 DD were included in this study. As a baseline control for gene expressions we used six kidney allografts obtained from living donors (LD). Three core biopsies were performed: biopsy 1--5 minutes before organ perfusion in the donor; biopsy 2--at the end of cold ischemia before kidney implantation; and biopsy 3--30 minutes after reperfusion. Tumor protein p53 (TP53), caspase-3 (CASP3), B-cell lymphoma 2 protein (Bcl2), and heme oxygenase 1 (HO-1) gene expression levels were determined using custom-designed low-density arrays (TaqMan assay). Comparison of gene expression between DD and LD kidneys revealed greater expression of all genes in kidneys from DD in all biopsies; however, only CASP3 expression in biopsy 1 and TP53 expression in biopsy 3 were statistically significant. Prolongation duration of brain death beyond 10 hours in DD resulted in a significantly decreased CASP3 expression in biopsy 1. When the cold ischemia time (CIT) was longer than 24 hours, the expressions of Bcl2, TP53, and CASP3 were significantly higher compared to kidneys with ClT<24 hours. There was no correlation between warm ischemia time and gene expression in biopsy 3. CASP3 and TP53 expression only in biopsy 1 were significantly higher among kidney allografts with delayed (DGF) compared with immediate graft function. In conclusion expression of genes involved in apoptosis was more pronounced in kidney allografts from deceased donors. A prolonged donor brain-death period beyond 10 hours resulted in decreased CASP3 expression. CIT longer than 24 hours was associated with increased expressions of Bcl2, TP53, and CASP3. CASP3 and TP53 expressions were significantly higher among kidneys allografts displaying DGF.

Kidney ischemic injury genes expressed after donor brain death are predictive for the outcome of kidney transplantation.

The results of deceased donor kidney transplantation largely depend on the extent of organ injury induced by brain death and the transplantation procedure. In this study, we analyzed the preprocurement intragraft expression of 29 genes involved in apoptosis, tissue injury, immune cell migration, and activation. We also assessed their influence on allograft function. Before flushing with cold solution we obtained 50 kidney core biopsies of deceased donor kidneys immediately after organ retrieval. The control group included 18 biopsies obtained from living donors. Gene expression was analyzed with low-density arrays (Taqman). LCN2/lipocalin-2 is considered a biomarker of kidney epithelial ischemic injury with a renoprotective function. HAVCR1/KIM-1 is associated with acute tubular injury. Comparison of deceased donor kidneys to control organs revealed a significantly higher expression of LCN2 (8.0-fold P=.0006) and HAVCR1 (4.7-fold, P<.0001). Their expressions positively correlated with serum creatinine concentrations after 6 months after transplantation: LCN2 (r=.65, P<.0001), HAVCR1 (r=.44, P=.006). Kidneys displaying delayed graft function and/or an acute rejection episode in the first 6 months after showed higher LCN2 expression compared to event-free ones (1.7-fold, P=.027). A significantly higher increase in expression of TLR2 (5.2-fold), Interleukin (IL) 18 (4.6-fold), HMGB1 (4.1-fold), GUSB (2.4-fold), CASP3 (2.0-fold) FAS (1.8-fold), and TP53 (1.6-fold) was observed among deceased donor kidneys compared with the control group. Their expression levels were not related to clinical outcomes: however, they showed significant correlations with one another (r>.6, P<.0001). We also observed a slightly reduced expression of IL10 (0.6-fold, P=.004). Our data suggested that increased LCN2 and HAVCR1 expression observed in the kidneys after donor brain death were hallmarks of the organ injury process. LCN2 expression level in retrieved kidneys can predict kidney transplantation outcomes.

Impact of donor-dependent genetic factors on long-term renal graft function.

Our aim was to study the association of donor genetic features with long-term graft function as well as the impact of donor age, gender compatibility, cold ischemia time (CIT), and delayed graft function (DGF). We observed the outcomes of 125 kidney recipients for a minimum of 12 months (mean, 30.9 +/- 13.0 months). Grafts were obtained from 89 donors who underwent profiling for AHSG 1/2, MMP9 -1562C/T, IL6 -174G/C, IL1beta 3954C/T, MTHFR 677C/T, MTHFR 1298A/C, NOS3 -786C/T, and PAI1 4G/5G single-nucleotide polymorphisms (SNPs) using sequence-specific probe (SSP) polymerase chain reaction (PCR) and MPO -463G/A and CRP -390C/T/A with restriction fragment length polymorphism (RFLP) analysis. NOS3 IVa/b VNTR polymorphism was genotyped by gel electrophoresis of the respective PCR-generated DNA fragment. The presence of the aa eNOS genotype was connected with worse graft function. The aa genotype was also linked to acute rejection episodes. The lowest values of glomerular filtration rate (GFR) were displayed by recipients of grafts from donors with homozygotic PAI1 gene 5G polymorphism, linking paradoxically with lower PAI-1 synthesis suggesting that the intensity of proteolysis led to increased alloantigen specificity stimulating alloresponses. Graft function depended significantly on donor age with an influence of gender matching. GFR showed a significant dependence on DGF. Genetic features of the donor influenced long-term graft function. Variant eNOS gene polymorphism, which produced decreased eNOS activity, was linked to worse remote graft function. A similar negative impact was observed in the case of donor PAI1 polymorphism, with the functional consequence of lower gene product synthesis.

Pretransplantation cellular alloreactivity is predictive of acute graft rejection and 1-year graft function in kidney transplant recipients.

OBJECTIVE: To study cellular alloimmunity in kidney allograft recipients using an interferon-gamma enzyme-linked immunosorbent spot assay (ELISPOT). MATERIAL AND METHODS: Donor splenocyte peripheral blood mononuclear cells were obtained during kidney recovery in 53 kidney recipients including 11 with positive panel-reactive antibodies pretransplantation. For ELISPOT data analysis, the spot number, size, and intensity were calculated, reflecting the volume of cytokine secretion at the single-cell level. Results were recalculated as the ratio of the values observed for donor-stimulated to unstimulated recipient cells corrected for residual donor activity. RESULTS: Significantly greater pretransplantation donor-stimulated activity was observed in recipients who experienced an acute rejection episode (ARE) within 1 year (P < .05). Mean change in spot number, size, and intensity in patients without or with AREs was 0.99 vs 3.33, 1.60 vs 6.05, and 1.40 vs 6.31, respectively. The assessed parameters were prognostic of high risk of ARE: 1.5-fold increase in spot number (ARE incidence, 52% vs 9%), 2.5-fold increase in spot size (ARE incidence, 53% vs 13%), and 2.7-fold increase in spot intensity (ARE incidence, 52% vs 9%). The 3 parameters correlated with 1-year serum creatinine concentration (P < .05). In 14 recipients, AREs could have been predicted in 11 using pretransplantation ELISPOT results, and in only 2 on the basis of panel-reactive antibodies. CONCLUSION: The ELISPOT-determined capacity of donor-induced reactivity observed in recipient cells obtained just before transplantation is predictive of risk of graft rejection and 1-year allograft function.

Recipient genetic determinants of inflammatory process and nonstandard atherosclerosis risk factors affect kidney graft function early posttransplantation.

We analyzed the connections between recipient genetic features and 12-month graft function. The gene polymorphisms of myeloperoxidase (MPO), interleukin (IL)-1beta, IL-6, C-reactive protein (CRP), fetuin A, and homocysteine and their gene product concentrations were correlated with 12-month kidney transplant function. The 125 kidney recipients had at least 12 months of follow-up (average, 30.9 +/- 13.0 months). IL6-174G/C, IL1beta 3954C/T, MTHFR 677C/T, MTHFR 1298A/C, AHSG 1/2 SNPs were determined using SSP-polymerase chain reaction (PCR) and MPO-463G/A and CRP- 390C/T/A with RLFP analysis. Enzyme-linked immunosorbent assay (ELISA) was applied to estimate MPO, fetuin A, IL-6, and IL-1beta; FPIA was applied for L-homocysteine concentrations. The highest CRP values were linked to the presence of the TT genotype. We observed a positive correlation of CRP concentrations and GFR. Lower fetuin A concentrations were linked to the 256Ser allele, and higher levels to better graft function. Worse graft function was inversely associated with serum homocysteine concentrations. Two polymorphisms (CRP and fetuin A) showed functional consequences in recipients. None of the examined genetic determinations influenced long-term graft function. Higher values, although still within the normal range of CRP concentrations on the day of transplantation and 3 months thereafter, were related to greater values of eGFR at 12 months, suggesting that the higher intensity of the inflammatory reaction may be a manifestation of more effective healing of an ischemia reperfusion injury. Both homocysteine and fetuin A showed long-term prognostic importance.

Variability in donor-specific alloantibody production after transplantation.

The role of de novo donor-specific alloantibodies (DSAs) in renal allograft injury is still unclear. The aims of this study were as follows: to assess the development of DSAs during the first year after transplantation, to determine the cause of DSA production, and to evaluate the association of DSA with allograft function. The study included 78 consecutive transplant recipients with negative cross-matches before transplantation. Recipient serum samples were assayed for DSA at 2 weeks as well as at 1, 3, 6, 9, and 12 months using a complement-dependent lymphocytotoxic (CDC) cross-match technique with donor lymphocytes. Among 545 cross-match tests performed after transplantation, there were 79 positive results. DSA appeared de novo in 44.8% of recipients: in 20 patients at 2 weeks; in 23 patients at 1 month; in 14 patients at 3 months; in 9 patients at 6 months; in 5 patients at 9 months; and in 8 patients at 12 months. Between month 3 and 9 after transplantation, DSA disappeared in 22 patients and appeared in 11 others. In 20 patients (57.1%) the appearance of DSA was associated with an acute rejection episode. In 11 of these, C4d deposition was found. In comparison with 43 patients without DSA, the serum creatinine levels during the first year after transplantation were significantly higher among patients with DSA. Transplant recipients produce antidonor alloantibodies. The highest rate occurs during the first month with the incidence diminishing at 3 months after transplantation. The development of DSAs in more than half of the patients was associated with rejection episodes. Patients with antidonor alloreactivity showed worse renal function.

C4D deposition and positive posttransplant crossmatch are not necessarily markers of antibody-mediated rejection in renal allograft recipients.

UNLABELLED: The aim of the study was to search for serologic, immunopathologic, and morphologic evidence of antibody-mediated rejection (AMR) among patients with acute renal allograft dysfunction. The study included 19 patients with episodes of acute rejection (ARE) within the first year after transplantation. All patients had negative crossmatch tests before transplantation. Patients underwent biopsy for histologic and C4d examinations. All patients were monitored for donor-specific HLA alloantibodies during the first posttransplant year. Complement-dependent cytotoxic crossmatches were performed with donor lymphocytes. In eight patients, the crossmatch test results changed to positive during ARE. In all biopsies except one with cortical infarction, we observed C4d staining (group 1). The biopsies of four patients showed histologic changes of AMR, and all of their grafts were lost. In one patient, cellular and vascular rejection (Banff II) were present; in two, Banff I; and in one, borderline lesions. These results were compared with 11 patients with ARE but negative posttransplant crossmatches and negative staining for C4d (group 2). The histologic findings in the biopsies of these patients were cellular interstitial and vascular rejection (Banff I and Banff II). With no features suggestive of AMR. During the first year after transplantation, the creatinine levels of group 1 patients, were significantly higher than group 2 patients. One-year graft survival was 50% in group 1 and 91% in group 2. CONCLUSIONS: C4d and a positive posttransplant crossmatch were not associated with histologic features of AMR in half of the ARE. Nevertheless, C4d deposition and positive posttransplant crossmatches correlated with allograft injury among renal transplant patients.