Identification of Gene Transcripts Implicated in Peritoneal Membrane Alterations.

♦ BACKGROUND: Permanent stimulation of the peritoneum during peritoneal dialysis (PD) is likely to result in increased expression of genes encoding proteins involved in inflammation and tissue remodeling. Peritoneal fibrosis and neoangiogenesis may develop. ♦ OBJECTIVE: To assess highly expressed genes potentially in volved in peritoneal alterations during PD treatment using an animal model. ♦ METHODS: A PD catheter was implanted in 36 male Wistar rats after 70% nephrectomy. The rats were divided into 3 groups, exposed to dialysis solution for 8 weeks, and sacrificed 2 weeks later. Group B was exposed to a buffer, group D was exposed to a 3.86% glucose-based dialysis solution, and in group D+H, a second hit of intraperitoneal blood on top of the dialysis solution was given to induce the development of peritoneal sclerosis. Before sacrifice, peritoneal function was assessed. Omental tissue was obtained for analysis of gene expression using RT-qPCR. ♦ RESULTS: Fibrosis scores, vessel counts, and peritoneal function parameters were not different between the groups. Genes involved in the transforming growth factor beta signaling pathway, cell proliferation, angiogenesis, and inflammation were more expressed (p < 0.05) in the D+H group. Almost no differences were found between the control groups. We identified 4 genes that were related to peritoneal transport. ♦ CONCLUSION: Already a mid-term peritoneal exposure, when no microscopical and functional alterations are present, provokes activation of gene pathways of cell proliferation, fibrosis, neoangiogenesis, and inflammation.

Molecular diagnostics identifies risks for graft dysfunction despite borderline histologic changes.

The significance of borderline changes in kidney allograft biopsies is widely debated. To help resolve this, we studied differences in intrarenal gene expression patterns between early clinical and 3-month protocol biopsies, all of which had borderline histologic changes. The gene expression profiles in training set of patients by microarray analysis and data were validated in a larger cohort using RT-qPCR. There was greater expression of immunity- and inflammation-related genes in the early clinical biopsies compared to the 3-month protocol biopsies with borderline changes. In early clinically manifested borderline changes, graft deterioration within 24 months due to chronic rejection was associated with increased activation of immune, defense, and inflammatory processes. Regression modeling identified higher donor age and expression of macrophage receptor CLEC5A as risk factors for progression. In the 3-month protocol biopsies with borderline changes, graft dysfunction was associated with increased expression of fibrinogen complex transcripts. The discrimination power of fibrinogen was confirmed by cross-validation on two independent cohorts. Thus, our study highlights variations in gene expression between clinical and subclinical borderline changes despite similar histological findings. The data also support a recommendation for frequent patient monitoring, especially in those with borderline changes who received grafts from older donors.

Tubular atrophy and low netrin-1 gene expression are associated with delayed kidney allograft function.

BACKGROUND: Delayed graft function (DGF) caused by ischemia/reperfusion injury (I/RI) negatively influences the outcome of kidney transplantation. This prospective single-center study characterized the intrarenal transcriptome during I/RI as a means of identifying genes associated with DGF development. METHODS: Characterization of the intrarenal transcription profile associated with I/RI was carried out on three sequential graft biopsies from respective allografts before and during transplantation. The intragraft expression of 92 candidate genes was measured using quantitative real-time reverse transcriptase polymerase chain reaction (2) in delayed (n=9) and primary function allografts (n=26). RESULTS: Cold storage was not associated with significant changes to the expression profile of the target gene transcripts; however, up-regulation of 16 genes associated with enhanced activation of innate and adaptive immune responses and apoptosis was observed after reperfusion. Multivariate logistic regression analysis revealed that higher tubular atrophy scores (ct) together with a lower expression of Netrin-1 might predict DGF development (training area under the receiver operating curve=0.89, cross-validated area under the receiver operating curve=0.81). CONCLUSIONS: Poor baseline tubular cell quality (defined by a higher rate of tubular atrophy) combined with the reduced potential of apoptotic survival factors represented by decreased Netrin-1 gene expression were associated with delayed kidney graft function.

Molecular markers of rejection and tolerance: lessons from clinical research.

In terms of finding specific molecular markers associated with graft outcome, attempts have been made to study whole genome transcripts using microarray assays or to study the effect of number of genes of interest using quantitative real-time polymerase chain reaction. Using these techniques, molecular phenotypes of rejection have been characterized, and the variability of the clinical outcome besides similar morphology explained in part. Recently, several specific transcripts including naïve B cell regulation have been identified in the peripheral blood of operationally tolerant kidney transplant recipients. The decrease in immature B cell-related transcripts in the peripheral blood in patients with immunosuppression was shown to be associated with acute rejection. Similarly, tolerance-associated antigen 1 transcripts were identified in biopsies and regulatory T cell transcripts in urine and biopsies in patients without rejection. Better understanding of molecular processes associated with allograft rejection or alloantigen hyporesponsiveness/tolerance may help to improve our knowledge about graft pathology and identify novel markers suitable for future monitoring and guided therapy and finally improve the outcome of kidney transplantation.

B-cell-related biomarkers of tolerance are up-regulated in rejection-free kidney transplant recipients.

BACKGROUND: Molecular signatures have recently been identified in operationally tolerant long-term kidney transplant patients; however, their expression in patients on immunosuppression remains unclear. METHODS: In this prospective study, the gene expression profiles of eight selected tolerance-associated genes (MS4A1, CD79B, TCL1A, TMEM176B, FOXP3, TOAG-1, MAN1A1, and TLR5) in the peripheral blood of 67 kidney transplant recipients at days 0, 7, 14, 21, 28, 60, 90, and at 6 and 12 months, and in graft biopsies were measured. Similarly, using flow cytometry, CD45CD19CD3 B-cell counts were evaluated in the follow-up. Expression patterns were compared among patients with biopsy-proven acute rejection, borderline changes, and in rejection-free patients. A generalized linear mixed model with gamma distribution for repeated measures adjusted for induction therapy was used for statistical analysis of longitudinal data and Kruskal-Wallis test for case biopsy data. RESULTS: Compared to patients with rejection, a significantly higher number of peripheral B cells were observed during follow-up in rejection-free patients and in patients with borderline changes. Gene expression patterns of MS4A1 (CD20), TCL1A, CD79B, TOAG-1, and FOXP3 genes were significantly higher in rejection-free patients as compared to rejection group with the highest differences during the first 3 months. In contrast, TMEM176B (TORID) was up-regulated in the rejection group. Similar trends were also observed between patients with borderline changes and acute rejection. Higher intragraft expression of TOAG-1 was observed in rejection-free patients. CONCLUSIONS: These observations suggest an association of B-cell signatures, seen also in drug-free tolerant patients, with controlled alloimmune response.

Molecular networks involved in the immune control of BK polyomavirus.

BK polyomavirus infection is the important cause of virus-related nephropathy following kidney transplantation. BK virus reactivates in 30%-80% of kidney transplant recipients resulting in BK virus-related nephropathy in 1%-10% of cases. Currently, the molecular processes associated with asymptomatic infections in transplant patients infected with BK virus remain unclear. In this study we evaluate intrarenal molecular processes during different stages of BKV infection. The gene expression profiles of 90 target genes known to be associated with immune response were evaluated in kidney graft biopsy material using TaqMan low density array. Three patient groups were examined: control patients with no evidence of BK virus reactivation (n = 11), infected asymptomatic patients (n = 9), and patients with BK virus nephropathy (n = 10). Analysis of biopsies from asymptomatic viruria patients resulted in the identification of 5 differentially expressed genes (CD3E, CD68, CCR2, ICAM-1, and SKI) (P < 0.05), and functional analysis showed a significantly heightened presence of costimulatory signals (e.g., CD40/CD40L; P < 0.05). Gene ontology analysis revealed several biological networks associated with BKV immune control in comparison to the control group. This study demonstrated that asymptomatic BK viruria is associated with a different intrarenal regulation of several genes implicating in antiviral immune response.

Cytokine gene expression profile in monocytic cells after a co-culture with epithelial cells.

Epithelial cells represent an important source of cytokines that may modulate the influx and functions of mononuclear phagocytes. The aim of our study was to characterize changes in the gene expression of selected cytokines in human macrophages co-cultured with respiratory epithelial cells. The A549 alveolar type II-like cell line was co-cultured with THP-1 cells (monocyte/macrophage cell line) in filter-separated mode to avoid their cell-cell contact. At different time-points (0, 4, 8, 12 and 24 h), the cells were harvested separately to evaluate their gene and protein expression (IL-1 beta, IL-6, IL-8, IL-10 and GM-CSF). Quantitative RT-PCR analysis showed prominent changes in the THP-1 cytokine gene expression induced by a co-culture with A549 cells. Fourfold upregulation of mRNA expression has been found in 12 genes and 4-fold downregulation in 5 genes as compared to the unstimulated control sample with a p value smaller than 0.05. The induction of inhibin beta A and IL-1 beta mRNA after 12 h and the expression of IL-1 alpha and GM-CSF mRNA after 24 h were the most prominent. When looking at the cytokine levels in culture supernatants, IL-1 beta and IL-8 were induced early (at 8 h) as compared to the release of IL-6 and GM-CSF (at 24 h). We conclude that respiratory epithelial cells constitutively regulate the cytokine gene expression of macrophages located in their environment and might further modulate the release of cytokines by posttranslational pathways.

Differential regulation of the nuclear factor-κB pathway by rabbit antithymocyte globulins in kidney transplantation.

BACKGROUND: Induction therapy is associated with excellent short-term kidney graft outcome. The aim of this study was to evaluate differences in the intragraft transcriptome after successful induction therapy using two rabbit antithymocyte globulins. METHODS: The expression of 376 target genes involved in tolerance, inflammation, T- and B-cell immune response, and apoptosis was evaluated using the quantitative real-time reverse-transcriptase polymerase chain reaction (2(-ΔΔCt)) method in kidney graft biopsies with normal histological findings and stable renal function, 3 months posttransplantation after induction therapy with Thymoglobulin, ATG-Fresenius S (ATG-F), and a control group without induction therapy. RESULTS: The transcriptional pattern induced by Thymoglobulin differed from ATG-F in 18 differentially expressed genes. Down-regulation of genes involved in the nuclear factor-κB pathway (TLR4, MYD88, and CD209), costimulation (CD80 and CTLA4), apoptosis (NLRP1), chemoattraction (CCR10), and dendritic cell function (CLEC4C) was observed in the biopsies from patients treated with Thymoglobulin. A hierarchical clustering analysis clearly separated the Thymoglobulin group from the ATG-F group, while the control group had a similar profile as the Thymoglobulin group. CONCLUSIONS: Despite normal morphology in graft biopsy taken 3 months posttransplantation, the intrarenal transcriptome differed in patients treated with induction therapy using different rATGs. In the Thymoglobulin high-risk group, the transcriptome profile was identical to the low-risk group. Therefore, the down-regulation of the nuclear factor-κB pathway after Thymoglobulin induction in vivo is likely to explain the clinical success of this biologic.

Regulatory T cells in kidney transplant recipients: the effect of induction immunosuppression therapy.

BACKGROUND: Regulatory T cells have been suggested to down-regulate the alloimmune response. The aim of this prospective open study was to evaluate the effects of different inductive agents on peripheral blood regulatory T cells in kidney transplant patients and to analyse their association with short-term graft outcome. METHODS: Regulatory and effector T cell numbers in peripheral blood were determined by flow cytometry in 71 prospectively followed kidney transplant recipients at postoperative day 0, 7, 14, 21, 28, 60 and 90. Patients were treated with a calcineurin inhibitor-based triple immunosuppression with polyclonal rabbit anti-thymocyte globulin (rATG, n = 28), basiliximab, the anti-CD25 monoclonal antibody (n = 18) or without induction (controls, n = 25). Flow cytometry data were correlated to rejection incidence. RESULTS: Compared to controls, CD4(+)CD25(+)FoxP3(+) regulatory T-cell expansion among CD4(+) T cells was noticed in the rATG group at all post-transplant time-points by Day 14 (P < 0.001). A significant decrease in Treg frequency (P < 0.001) and concurrently a transient increase of CD4(+)CD25(low/-)FoxP3(+) population were observed in basiliximab-treated patients 7-60 days post-transplantation. Biopsy-proven acute rejection occurred in 16.7% of controls, 10.7% of the rATG group and in 11.1% of the basiliximab group. Higher CD4(+)FoxP3(+)/CD8(+)CD45RA(+)CD62L(-) ratios were observed repeatedly in those patients after basiliximab induction who were rejection free (P < 0.01). CONCLUSIONS: In this study, the rATG induction therapy was associated with an expansion of regulatory cells. Sustained high CD4(+)FoxP3(+)/Teff ratios were associated with the absence of rejection after basiliximab induction.

The FTO gene polymorphism is associated with end-stage renal disease: two large independent case-control studies in a general population.

BACKGROUND: Genome-wide association studies identified the FTO (fat mass and obesity gene) gene as an important determinant of body weight. More recently, the FTO gene was reported to be associated with other outcomes, including major risk factors for chronic kidney disease (CKD). We investigated the role of this gene in the risk of end-stage renal disease (ESRD) caused by CKD. METHODS: We conducted two large population-based case-control studies of ESRD. Study 1 compared 984 haemodialysed patients with ESRD with 2501 participants in the Czech post-MONICA study; Study 2 compared 1188 patients included in a kidney transplantation programme for ESRD with 6681 participants in the Czech HAPIEE study. The frequencies of the FTO rs17817449 single nucleotide polymorphism genotype were compared between cases and controls. RESULTS: The FTO rs17817449 genotype was significantly associated with CKD in both studies (P-values 0.00004 and 0.006, respectively). In the pooled data, the odds ratios of CKD for GG and GT, versus TT genotype, were 1.37 (95% confidence interval 1.20-1.56) and 1.17 (1.05-1.31), respectively (P for trend <0.0001). Among haemodialysed and kidney transplant patients, the onset of ESRD in GG homozygotes was 3.3 (P = 0.012) and 2.5 (P = 0.032) years, respectively, earlier than in TT homozygotes. CONCLUSIONS: These two large independent case-control studies in the general population found robust associations between the FTO rs17817449 polymorphism and the ESRD. The results suggest that the morbidities associated with the FTO gene include CKD.

A prospective longitudinal study of BK virus infection in 120 Czech renal transplant recipients.

Polyomavirus BK (BKV) is a common human polyomavirus that rarely causes clinical symptoms in immunocompetent individuals. However, BK virus reactivation occurs in 20-40% of kidney transplant patients and 1-10% of cases present with BK virus-associated nephropathy (BKVN) and reduced kidney allograft survival. In this study, 120 consecutive renal allograft recipients were monitored for BK virus replication by real-time PCR (qPCR) in the blood and urine during the first year post-transplantation and risk factors for BK viremia, viruria, and polyoma BKV-associated nephropathy were evaluated. Receiver operating characteristic curve analysis was used to determine the cutoff points for assessing the risk of developing BKVN. In total, 1,243 samples were tested. BK-DNAuria >10(7) copies/ml and BK-DNAemia >10(4) copies/ml were found in 25.8% and 5% of the samples screened, respectively, during the 12 month follow-up period. BKVN was confirmed histologically in 3/120 patients and viremic patients were treated with dialysis for longer time periods and had higher levels of panel [corrected] reactive antibodies. Patients with viruria were also treated longer with dialysis and had impaired graft function 12 months post-transplantation. Patients with sustained viruria exhibited more acute rejection episodes than patients with transient viruria. Using receiver operating characteristic curve analysis, the cutoff point for viremia and viruria was redefined to 10(3) copies/ml serum for BK viremia and a cutoff point of 6.7 × 10(7) copies/ml in urine. In conclusion, polyoma BK viremia and viruria are frequent findings in kidney transplant recipients that warrant intensive monitoring as a means of preventing graft failure [corrected].

Association of advanced vasculopathy and transforming growth factor-beta1 gene expression with immunoglobulin A nephropathy progression.

BACKGROUND: The mechanism of IgA nephropathy (IgAN) progression remains ill-defined. In this prospective study, the prognostic role of clinical, histological and molecular markers over a 2-year follow-up was evaluated. METHODS: Fifty-one patients with biopsy-proven IgAN were followed for 24 months. Besides routine histology, the intrarenal gene expressions of cytokines and chemokines were quantified by reverse transcription quantitative real-time polymerase chain reaction, and the presence of lymphocytes and macrophages were immunohistochemically examined. RESULTS: Higher transforming growth factor-ß1 and severe chronic vasculopathy (but not glomerulosclerosis, interstitial fibrosis or lymphocyte infiltrate) were associated with the IgAN progression 24 months after biopsy. The gene expression of chemokine (C-C motif) ligands 2 and 5, hepatocyte growth factor, bone morphogenic protein-7 and transforming growth factor-ß1 and the interstitial infiltrate of T and B lymphocytes and macrophages were significantly associated with serum creatinine and glomerular filtration rate at the time of biopsy. The intrarenal chemokine (C-C motif) ligand 2 and hepatocyte growth factor gene expression were associated with the proteinuria. CONCLUSIONS: Besides the known risk factors for chronic kidney disease, advanced vasculopathy and molecular signatures of fibrogenesis were associated with the IgAN progression.

Potential predictive markers in protocol biopsies for premature renal graft loss.

BACKGROUND/AIMS: Protocol biopsies offer new possibilities to predict kidney allograft outcome. The aim of this study was to find clinical, laboratory, morphological and molecular predictors of short-term renal graft survival. METHODS: Three-month protocol kidney graft biopsy was carried out on 257 patients. The real-time RT-PCR was used to identify intragraft mRNA expression of several cytokines and chemokines and predictive statistics was performed to find markers connected with the risk of premature graft failure. RESULTS: Compared to patients with normal morphology at 3 months, patients with subclinical rejection including borderline changes had experienced more frequent (p < 0.001) acute rejections before 3-month biopsy, serum creatinine >or=170 micromol/l (p < 0.01), and higher intrarenal expression of RANTES, IP-10 (p < 0.001), C3, CD3, IgJ (p < 0.01) and CD20 (p < 0.05). There was a significant correlation between subclinical rejection and the occurrence of late acute rejection and graft failure at the first year after transplantation. Moreover, higher RANTES and IP-10 expressions in subclinical rejection predicted graft loss at one year after transplantation in the univariate analysis. CONCLUSIONS: Patients with subclinical rejection including borderline changes in 3-month biopsy and particularly those with higher intrarenal expression of RANTES and IP-10 mRNA were found to be at risk for premature kidney graft loss.

RAGE polymorphisms, renal function and histological finding at 12 months after renal transplantation.

OBJECTIVES: Rage (receptor for advanced glycation end products) is involved in pathogenesis of many diseases. The aim of the study was to test whether polymorphisms of RAGE gene are associated with the outcome of kidney transplantation. DESIGN AND METHODS: Four polymorphisms of the RAGE gene (-429T/C, -374T/A, Gly82Ser and 2184A/G) were assessed in 145 renal transplant recipients and their relationship to histological changes in 12 months protocol kidney graft biopsy and renal function was examined. RESULTS: Genotype frequencies of each polymorphism corresponded to expected frequencies according to Hardy-Weinberg equilibrium. No differences between allelic and genotype frequencies among patients with normal histological findings, chronic allograft nephropathy and subclinical rejection were observed. CONCLUSION: This is the first study on polymorphisms of the RAGE gene in patients with the transplanted kidney. No association of RAGE selected gene polymorphisms with 12-months outcome of renal transplants was shown in study.

Haplotypic structure of ABCB1/MDR1 gene modifies the risk of the acute allograft rejection in renal transplant recipients.

BACKGROUND: Bioavailability of tacrolimus (Tac) and cyclosporine is determined by cytochrome P450IIIA and by P-glycoprotein encoded by the CYP3A4/CYP3A5 and ABCB1 genes. Polymorphisms in these genes have been suggested to influence acute rejection and pharmacokinetics in renal transplantation. We aimed to validate these findings in a haplotype analysis. METHODS: A total of 832 renal transplant recipients were genotyped for the CYP3A4 -288A>G, CYP3A5 +6986G>A, ABCB1 +1236C>T, +2677G>T>A, and +3435C>T polymorphisms. Their association with acute rejection and with pharmacokinetic parameters was analyzed in haplotype models. RESULTS: Apart from human leukocyte antigen-DR mismatches, delayed graft function and age at renal transplantation, acute rejection was also predicted by the [ABCB1 +1236C; +2677G; +3435T] haplotype. Allograft survival was determined by donor age, age at renal transplantation, delayed graft function, cold ischemia, and history of more than two acute rejections. Homozygotes for the [CYP3A4 -288A; CYP3A5 +6986G] haplotype achieved earlier therapeutic concentrations of Tac and a higher concentration to dose ratio at week 1. ABCB1 haplotypes did not influence pharmacokinetic parameters. CONCLUSIONS: ABCB1 haplotypes modify the risk of acute rejection, suggesting that ABCB1 allelic arrangement is a stronger regulator of P-glycoprotein activity than single polymorphisms. The risk of acute rejection determined by ABCB1 is independent of pharmacokinetic parameters. CYP3A haplotypes control the bioavailability of Tac, but do not modify the risk of acute rejection.

Genetic variability of major inflammatory mediators has no impact on the outcome of kidney transplantation.

BACKGROUND: Functionally relevant polymorphisms in genes of the Th1 and Th2-inflammatory pathway influence the susceptibility to acute rejection (AR), chronic allograft nephropathy (CAN), and subclinical rejection (SR) as well as graft survival after renal transplantation. Because these findings have not been validated, we sought confirmatory evidence of these associations in a larger group of renal transplant recipients. METHODS: A total of 436 kidney transplant recipients were genotyped for 9 single nucleotide polymorphisms (TNF-alpha-308G/A, MCP-1-2518A/G, RANTES-403G/A, -109T/C and -28C/G, CCR2+190G/A, IFN-gamma+874A/T, TGF-beta+869T/C and +915G/C) and for the 32-bp indel polymorphism in CCR5. The effects of these polymorphisms on the incidence of AR, SR, CAN and graft survival were analyzed in single locus and haplotype models. RESULTS: Single locus analysis revealed that there was no significant difference in the distribution of the genotype frequencies between patients with and without AR, and between patients with CAN or SR, and individuals without CAN. Furthermore, no influence of any of the polymorphisms on the long-term graft survival was observed. Haplotype [TGF-beta +869G; TGF-beta +915C] seemed to be associated with the presence of SR (odds ratio: 3.45, 95% confidence interval: 1.19 - 9.99, P=0.023), but the association was nonsignificant due to the insufficient power. CONCLUSION: In contrast to previous allelic association studies, neither of the polymorphisms has been associated with the outcome of kidney transplantation in the single locus analysis nor in the haplotype model. Our findings reinforce the need for more rigorous research compliant with the currently accepted standards for polymorphism-disease association studies.

Intrarenal cytokine and chemokine gene expression and kidney graft outcome.

AIMS: Proinflammatory cytokines are thought to play an important role in various kidney graft diseases resulting in interstitial fibrosis and tubular atrophy frequently found in case biopsies. To explore the role of various cytokines and chemokines in the long-term graft outcome, the transcription patterns of their genes in kidney allograft biopsies were evaluated. METHODS: The real-time RT-PCR was used to identify intragraft mRNA expression of cytokines and chemokines in 74 kidney graft recipients and the results were correlated with histological and clinical parameters and long-term graft outcome. RESULTS: We observed up-regulated IL-10 (p < 0.001), TGF-beta1, IL-6, MCP-1, RANTES (p < 0.01) and TNF-alpha (p < 0.05) mRNA expression in patients with chronic allograft nephropathy (CAN) as compared to controls. There were positive correlations between the mRNA expression of IL-6 (p < 0.001), IL-10 (p < 0.01), TNF-alpha, MCP-1 (p < 0.05) and the proteinuria. The up-regulation of intrarenal MCP-1 in patients with CAN increased the risk for the graft failure within the next 42 months (OR 5.1, p < 0.05). Kaplan-Meier survival analysis revealed that proteinuria and higher intragraft expression of TGF-beta1 and MCP-1 predict a poor kidney graft outcome. CONCLUSION: Expression patterns of intrarenal proinflammatory genes might discriminate patients at a higher risk for the earlier allograft failure.