Influence of calcineurin inhibitors and genetic polymorphism of transporters on enterohepatic circulation and exposure of mycophenolic acid in Chinese adult renal allograft recipients

Aim: Study the influence of calcineurin inhibitors (CNI) and genetic polymorphisms of transporters on enterohepatic circulation (EHC) of mycophenolic acid (MPA) in Chinese adult renal allograft recipients and estimate the effect of various covariates on prediction performance of MPA AUC0-12h. Method: MPA concentrations of 125 Chinese patients were collected 0-12 hours after administration. Genotypes of transporters were determined in 64 patients. The influence of type of CNI and genetic polymorphisms on MPA exposure was studied. Shapley additive explanations method was used to study the impact of sampling times and covariates related to EHC on AUC0-12h. Extreme gradient boosting (XGboost) machine learning-based model was established to predict AUC0-12h. Results: Dn-AUC6-12h was significantly lower in patients co-administered with CsA (P<0.05). When co-administered with TAC, for SLCO1B1 T521C or ABCC C-24T, patients with wild-type genotype had significantly higher dn-AUC6-12h (P <0.05). Patients with SLCO1B3 334T/699G alleles had significantly lower dn-AUC6-12h than homozygotes (P=0.004). No significant difference was found in CsA subgroup. For estimating AUC0-12h, C0h, C2h, C8h, type of CNI, transporters genotypes and the difference between C0h and C2h were retained in the final model, which had good prediction performance (r2=0.9739). Conclusion: Patients co-administered with CsA had lower MPA EHC than those who received TAC. MPA EHC is affected by ABCC2 C-24T, SLCO1B3 T334G/G699A, and SLCO1B1 T521C genotypes in patients treated with TAC. Type of CNI and genetic polymorphisms of transporters can improve prediction performance of MPA AUC0-12h estimating model, developed using XGboost machine learning method.

Pre-transplant Transcriptional Signature in Peripheral Blood Mononuclear Cells of Acute Renal Allograft Rejection

Acute rejection (AR) is closely associated with renal allograft dysfunction. Here, we utilised RNA sequencing (RNA-Seq) and bioinformatic methods to characterise the peripheral blood mononuclear cells (PBMCs) of patients with acute renal allograft rejection. Pretransplant blood samples were collected from 32 kidney allograft donors and 42 corresponding recipients with biopsies classified as T cell-mediated rejection (TCMR, n = 18), antibody-mediated rejection (ABMR, n = 5), and normal/non-specific changes (non-AR, n = 19). The patients with TCMR and ABMR were assigned to the AR group, and the patients with normal/non-specific changes (n = 19) were assigned to the non-AR group. We analysed RNA-Seq data for identifying differentially expressed genes (DEGs), and then gene ontology (GO) analysis, Reactome, and ingenuity pathway analysis (IPA), protein—protein interaction (PPI) network, and cell-type enrichment analysis were utilised for bioinformatics analysis. We identified DEGs in the PBMCs of the non-AR group when compared with the AR, ABMR, and TCMR groups. Pathway and GO analysis showed significant inflammatory responses, complement activation, interleukin-10 (IL-10) signalling pathways, classical antibody-mediated complement activation pathways, etc., which were significantly enriched in the DEGs. PPI analysis showed that IL-10, VEGFA, CXCL8, MMP9, and several histone-related genes were the hub genes with the highest degree scores. Moreover, IPA analysis showed that several proinflammatory pathways were upregulated, whereas antiinflammatory pathways were downregulated. The combination of NFSF14+TANK+ANKRD 33 B +HSPA1B was able to discriminate between AR and non-AR with an AUC of 92.3% (95% CI 82.8–100). Characterisation of PBMCs by RNA-Seq and bioinformatics analysis demonstrated gene signatures and biological pathways associated with AR. Our study may provide the foundation for the discovery of biomarkers and an in-depth understanding of acute renal allograft rejection.

A Prospective Multicenter Trial to Evaluate Urinary Metabolomics for Non-invasive Detection of Renal Allograft Rejection (PARASOL): Study Protocol and Patient Recruitment

Background: In an earlier monocentric study, we have developed a novel non-invasive test system for the prediction of renal allograft rejection, based on the detection of a specific urine metabolite constellation. To further validate our results in a large real-world patient cohort, we designed a multicentric observational prospective study (PARASOL) including six independent European transplant centers. This article describes the study protocol and characteristics of recruited better patients as subjects.Methods: Within the PARASOL study, urine samples were taken from renal transplant recipients when kidney biopsies were performed. According to the Banff classification, urine samples were assigned to a case group (renal allograft rejection), a control group (normal renal histology), or an additional group (kidney damage other than rejection).Results: Between June 2017 and March 2020, 972 transplant recipients were included in the trial (1,230 urine samples and matched biopsies, respectively). Overall, 237 samples (19.3%) were assigned to the case group, 541 (44.0%) to the control group, and 452 (36.7%) samples to the additional group. About 65.9% were obtained from male patients, the mean age of transplant recipients participating in the study was 53.7 ± 13.8 years. The most frequently used immunosuppressive drugs were tacrolimus (92.8%), mycophenolate mofetil (88.0%), and steroids (79.3%). Antihypertensives and antidiabetics were used in 88.0 and 27.4% of the patients, respectively. Approximately 20.9% of patients showed the presence of circulating donor-specific anti-HLA IgG antibodies at time of biopsy. Most of the samples (51.1%) were collected within the first 6 months after transplantation, 48.0% were protocol biopsies, followed by event-driven (43.6%), and follow-up biopsies (8.5%). Over time the proportion of biopsies classified into the categories Banff 4 (T-cell-mediated rejection [TCMR]) and Banff 1 (normal tissue) decreased whereas Banff 2 (antibody-mediated rejection [ABMR]) and Banff 5I (mild interstitial fibrosis and tubular atrophy) increased to 84.2 and 74.5%, respectively, after 4 years post transplantation. Patients with rejection showed worse kidney function than patients without rejection.Conclusion: The clinical characteristics of subjects recruited indicate a patient cohort typical for routine renal transplantation all over Europe. A typical shift from T-cellular early rejections episodes to later antibody mediated allograft damage over time after renal transplantation further strengthens the usefulness of our cohort for the evaluation of novel biomarkers for allograft damage.

Poor Long-Term Renal Allograft Survival in Patients with Chronic Antibody-Mediated Rejection, Irrespective of Treatment—A Single Center Retrospective Study

The Banff 2017 report permits the diagnosis of pure chronic antibody-mediated rejection (cAMR) in absence of microcirculation inflammation. We retrospectively investigated renal allograft function and long-term outcomes of 67 patients with cAMR, and compared patients who received antihumoral therapy (cAMR-AHT, n = 21) with patients without treatment (cAMRwo, n = 46). At baseline, the cAMR-AHT group had more concomitant T-cell-mediated rejection (9/46 (19.2%) vs. 10/21 (47.6%); p = 0.04), a higher g-lesion score (0.4 ± 0.5 versus 0.1 ± 0.3; p = 0.01) and a higher median eGFR decline in the six months prior to biopsy (6.6 vs. 3.0 mL/min; p = 0.04). The median eGFR decline six months after biopsy was comparable (2.6 vs. 4.9 mL/min, p = 0.61) between both groups, and three-year graft survival after biopsy was statistically lower in the cAMR-AHT group (35.0% vs. 61.0%, p = 0.03). Patients who received AHT had more infections (0.38 vs. 0.20 infections/patient; p = 0.04). Currently, antihumoral therapy is more often administered to patients with cAMR and rapidly deteriorating renal function or concomitant TCMR. However, long-term graft outcomes remain poor, despite treatment.

Single Nucleotide Polymorphisms of IL-33 Gene Correlated with Renal Allograft Fibrosis in Kidney Transplant Recipients

Background. Nowadays, renal allograft survival is confined by the development of allograft fibrosis. Previous studies have reported interleukin-33 (IL-33) upregulated significantly in patients with chronic renal allograft dysfunction, and it could induce renal tubular epithelial to mesenchymal transition (EMT), which eventually contributed to renal allograft fibrosis. Our study intended to detect the underlying association between single nucleotide polymorphisms (SNPs) of IL-33 gene and renal allograft fibrosis in kidney transplant recipients. Methods. We collected blood samples from 200 renal transplant recipients for the identification of SNPs and transplanted kidney tissue samples for identifying differentially expressed genes (DEGs). Intersection of SNP-related genes and DEGs was conducted for further analysis. Relationships between these SNPs and renal allograft fibrosis were evaluated by the inheritance models. Immunohistochemical (IHC) staining and western blotting (WB) were used to detect the expression of IL-33 and the markers of EMT in human kidney tissues obtained from control and chronic renal allograft dysfunction (CAD) patients. In vitro, we detected the progressions of EMT-related markers and the levels of MAPK signaling pathway mediators after transfecting IL-33 mutant plasmids in HK2 cells. Results. Three intersected genes including IL-33 genes were significantly expressed. IL-33 expression was validated in kidney tissues by IHC and WB. Thirty-nine IL-33-related SNPs were identified in targeted sequencing, in which 26 tagger SNPs were found by linkage disequilibrium analysis for further analysis. General linear models indicated sirolimus administration significantly influenced renal allograft fibrosis ( P < 0.05 ), adjustment of which was conducted in the following analysis. By multiple inheritance model analyses, SNP rs10975519 of IL-33 gene was found closely related to renal allograft fibrosis ( P < 0.005 ). Furthermore, HK2 cells transfected with mutated plasmid of rs10975519 showed stronger mobility and migration ability. Moreover, IL-33 mutant plasmids could promote the IL-33-induced EMT through the sustained activation of p38 MAPK signaling pathway in HK2 cells. Conclusion. In our study, rs10975519 on the IL-33 gene was found to be statistically associated with the development of renal allograft fibrosis in kidney transplant recipients. This process may be related to the IL-33-induced EMT and sustained activation of p38 MAPK signaling pathway.