Novel transcriptomic signatures associated with premature kidney allograft failure.

BACKGROUND: The power to predict kidney allograft outcomes based on non-invasive assays is limited. Assessment of operational tolerance (OT) patients allows us to identify transcriptomic signatures of true non-responders for construction of predictive models. METHODS: In this observational retrospective study, RNA sequencing of peripheral blood was used in a derivation cohort to identify a protective set of transcripts by comparing 15 OT patients (40% females), from the TOMOGRAM Study (NCT05124444), 14 chronic active antibody-mediated rejection (CABMR) and 23 stable graft function patients ≥15 years (STA). The selected differentially expressed transcripts between OT and CABMR were used in a validation cohort (n = 396) to predict 3-year kidney allograft loss at 3 time-points using RT-qPCR. FINDINGS: Archetypal analysis and classifier performance of RNA sequencing data showed that OT is clearly distinguishable from CABMR, but similar to STA. Based on significant transcripts from the validation cohort in univariable analysis, 2 multivariable Cox models were created. A 3-transcript (ADGRG3, ATG2A, and GNLY) model from POD 7 predicted graft loss with C-statistics (C) 0.727 (95% CI, 0.638-0.820). Another 3-transcript (IGHM, CD5, GNLY) model from M3 predicted graft loss with C 0.786 (95% CI, 0.785-0.865). Combining 3-transcripts models with eGFR at POD 7 and M3 improved C-statistics to 0.860 (95% CI, 0.778-0.944) and 0.868 (95% CI, 0.790-0.944), respectively. INTERPRETATION: Identification of transcripts distinguishing OT from CABMR allowed us to construct models predicting premature graft loss. Identified transcripts reflect mechanisms of injury/repair and alloimmune response when assessed at day 7 or with a loss of protective phenotype when assessed at month 3. FUNDING: Supported by the Ministry of Health of the Czech Republic under grant NV19-06-00031.

ELISpot assay and prediction of organ transplant rejection.

The ELISpot assay is a sensitive technique applied to assess cytokine-producing memory/effector T cells and human leukocyte antigens (HLA)-specific IgG-producing B cells. Besides the fact that the method is laborious and is difficult to standardise between laboratories, it may provide valuable information on the immune response of recipients before and after organ transplantation. In this article, we briefly review the recent literature and discuss the clinical significance of the ELISpot assay in predicting the risk and incidence of allograft rejection and survival.

Steroid free immunosuppression is associated with enhanced Th1 transcripts in kidney transplantation.

BACKGROUND: Steroid avoidance in immunosuppression in kidney transplantation offers several metabolic advantages, however it is associated with higher early acute rejection rate. Cellular and molecular mechanisms of this phenomenon remain poorly understood. METHODS: In this single center observational study, low-risk kidney transplant recipients randomized into large multicenter prospective ADVANCE trial with steroid avoidance/early withdrawal and center standard of care treated patients were monitored for 12months. The expressions of 28 transcripts, associated with alloimmune response and operational tolerance, were evaluated in the peripheral blood using RT-qPCR at 0, 7, 14, 90 and 365 postoperative days (POD) and in the protocol graft biopsy at 3months while lymphocyte subpopulations were analyzed by flow-cytometry within the follow-up. RESULTS: Both steroid avoidance and withdrawal regimens were associated with significantly higher granzyme B (GZMB) transcript at POD 14 and perforin 1 (PRF1) transcript at POD 7. The higher interleukin 2 (IL-2) expression at POD 7 was detected only in the steroid avoidance group. Initial steroids decreased the expression SH2D1B transcript at POD14 and there were no further differences in other operational tolerance transcripts among groups. The statistically significant decrease in absolute numbers of peripheral NK cells in the first 14days was observed in the standard of care group only. There were no differences in analyzed intrarenal transcripts in 3-month biopsies among groups. CONCLUSIONS: The enhanced expression of some of Th1 associated transcripts and limited effects on NK cells of steroid avoidance immunosuppression suggest higher susceptibility for early acute rejection.

Molecular patterns of diffuse and nodular parathyroid hyperplasia in long-term hemodialysis.

Secondary hyperparathyroidism is a well-known complication of end-stage renal disease (ESRD). Both nodular and diffuse parathyroid hyperplasia occur in ESRD patients. However, their distinct molecular mechanisms remain poorly understood. Parathyroid tissue obtained from ESRD patients who had undergone parathyroidectomy was used for Illumina transcriptome screening and subsequently for discriminatory gene analysis, pathway mapping, and gene annotation enrichment analysis. Results were further validated using quantitative RT-PCR on the independent larger cohort. Microarray screening proved homogeneity of gene transcripts in hemodialysis patients compared with the transplant cohort and primary hyperparathyroidism; therefore, further experiments were performed in hemodialysis patients only. Enrichment analysis conducted on 485 differentially expressed genes between nodular and diffuse parathyroid hyperplasia revealed highly significant differences in Gene Ontology terms and the Kyoto Encyclopedia of Genes and Genomes database in ribosome structure (P = 3.70 × 10-18). Next, quantitative RT-PCR validation of the top differently expressed genes from microarray analysis proved higher expression of RAN guanine nucleotide release factor (RANGRF; P < 0.001), calcyclin-binding protein (CACYBP; P < 0.05), and exocyst complex component 8 (EXOC8; P < 0.05) and lower expression of peptidylprolyl cis/trans-isomerase and NIMA-interacting 1 (PIN1; P < 0.01) mRNA in nodular hyperplasia. Multivariate analysis revealed higher RANGRF and lower PIN1 expression along with parathyroid weight to be associated with nodular hyperplasia. In conclusion, our study suggests the RANGRF transcript, which controls RNA metabolism, to be likely involved in pathways associated with the switch to nodular parathyroid growth. This transcript, along with PIN1 transcript, which influences parathyroid hormone secretion, may represent new therapeutical targets to cure secondary hyperparathyroidism.

Circulating biomarkers of tolerance.

On the basis of reviewed literature here we describe models of tolerance and summarize the evidence of circulating biomarkers suitable for the assessment of immunological risk in organ transplantation. We focused on results of evaluation of specific peripheral immune cell populations and transcripts in peripheral blood of operationally tolerant liver and kidney transplant recipients. Validation of described markers to define potentially tolerant patients before their use in clinical trials is critical.

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.

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.

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].