Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation

Abstract

The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways.

Trial registration: ClinicalTrials.gov NCT00497926.

Keywords: Immunology and pathology; kidney biopsy; kidney transplantation; tolerance; transcriptional profiling.

Copyright © 2018 by the American Society of Nephrology.

Figures

Figure 1.

Activation of the B cell receptor signaling canonical pathway in FCRx versus SIS: a schematic representation. Red indicates upregulation, and green indicates downregulation. Prediction of pathway activity is represented in blue (inhibition) and orange (activation). Color gradient intensities indicate magnitudes for expression and activation trends.

Figure 2.

Comparison analyses among DEGs stratifying FCRx as being in between D and SIS, and distinctly different from rejection (R) samples. (A) Venn diagram including differential probe sets from each pairwise comparison. (B) Principal component analysis using DEGs from FCRx versus R comparison. (C) Supervised hierarchical clustering analysis.

Figure 3.

Differential expression of miRNA demonstrating unique profile in FCRx, when compared to D, SIS and R groups. (A) Volcano plot showing differential expression of miRNA between FCRx and paired donor groups. (B) Volcano plot showing differential expression of miRNA between FCRx and SIS. (C) Volcano plot showing differential expression of miRNA between FCRx and R. (D) Figure showing miRNAs that are differentially expressed in FCRx biopsies versus other indicated groups (i.e., miR-31–5p, miR-9–5p, and miR125b-5p are uniquely upregulated in FCRx); other miRNAs shown in red are upregulated and those shown in green are downregulated in FCRx when compared with indicated groups.