Biological pathways and comparison with biopsy signals and cellular origin of peripheral blood transcriptomic profiles during kidney allograft pathology

Abstract

Kidney transplant injury processes are associated with molecular changes in kidney tissue, primarily related to immune cell activation and infiltration. How these processes are reflected in the circulating immune cells, whose activation is targeted by strong immunosuppressants, is poorly understood. To study this, we analyzed the molecular alterations in 384 peripheral blood samples from four European transplant centers, taken at the time of a kidney allograft biopsy, selected for their phenotype, using RNA-sequencing. In peripheral blood, differentially expressed genes in 136 rejection and 248 no rejection samples demonstrated upregulation of glucocorticoid receptor and nucleotide oligomerization domain-like receptor signaling pathways. Pathways enriched in antibody-mediated rejection (ABMR) were strongly immune-specific, whereas pathways enriched in T cell-mediated rejection were less immune related. In polyomavirus infection, upregulation of mitochondrial dysfunction and interferon signaling pathways was seen. Next, we integrated the blood results with transcriptomics of 224 kidney allograft biopsies which showed consistently upregulated genes per phenotype in both blood and biopsy. In single-cell RNASeq (scRNASeq) analysis of seven kidney allograft biopsies, the consistently overexpressed genes in ABMR were mostly expressed by infiltrating leukocytes in the allograft. Similarly, in peripheral blood scRNASeq analysis, these genes were overexpressed in ABMR in immune cell subtypes. Furthermore, overexpression of these genes in ABMR was confirmed in independent cohorts in blood and biopsy. Thus, our results highlight the immune activation pathways in peripheral blood leukocytes at the time of kidney allograft pathology, despite the use of current strong immunosuppressants, and provide a framework for future therapeutic interventions.

Keywords: BK virus; gene expression; immune cells; kidney transplantation rejection.

Graphical abstract

Figure 1

Differentially expressed genes in the peripheral blood for different phenotype comparisons. Red line indicates the false discovery rate (FDR) 0.05 significance level. The most significant features are denoted by name. ABMRh, histology of antibody-mediated rejection; HLA–DSA, human leukocyte antigen–donor-specific antibody; PVAN, polyoma-virus associated nephropathy; TCMR, T cell–mediated rejection.

Figure 2

Top 10 canonical pathways for the different phenotype comparisons obtained using Ingenuity Pathway Analysis. The orange line denotes the false discovery rate (FDR) P value 0.2 level of significance. The top 5 contributing features are shown per pathway. ABMRh, histology of antibody-mediated rejection; ARE, antioxidant response element; CXCR, C-X-C C-X-C chemokine receptor; fMLP, N-Formylmethionyl-leucyl-phenylalanine; GM-CSF, Granulocyte-macrophage colony-stimulating factor; HLA–DSA, human leukocyte antigen–donor-specific antibody; IL, interleukin; ILK, Integrin-linked kinase; iNOS, Inducible nitric oxide synthase; LXR, Liver X Receptor; NF, nuclear factor; MSP-RON, macrophage-stimulating protein - recepteur d'origine nantais; NFAT, Nuclear factor of activated T cells; PKR, protein kinase R; PPAR, Peroxisome proliferator-activated receptor; PVAN, polyoma-virus associated nephropathy; RXR, retinoid X receptor; STAT, signal transducer and activator of transcription; TCMR, T cell–mediated rejection; Th, T helper; VDR, vitamin D receptor.

Figure 3

The top 10 overexpressed features across biopsy microarray data and peripheral blood RNAseq data for any rejection, histology of antibody-mediated rejection (ABMRh), and T cell–mediated rejection (TCMR). The scatter plot shows the distribution of the individual genes, supplemented with a violin plot demonstrating overall distribution with included median and interquartile range.

Figure 4

Cell enrichment scores in the different phenotypes of kidney allograft pathology as obtained from blood (upper panel) and biopsy transcriptomic data. Heatmaps of the cell enrichment scores, ordered by hierarchical clustering. In the blood samples, cell enrichment scores did not discriminate among the different phenotypes, whereas in the biopsy samples, discrimination of rejection versus no-rejection phenotypes based on their cell enrichment scores in immune cells is seen. ABMRh, histology of antibody-mediated rejection; CD, cluster of differentiation; DC, dendritic cell; HLA–DSA, human leukocyte antigen–donor-specific antibody; NK, natural killer cell; NKT, natural killer T cell; PVAN, polyoma-virus associated nephropathy; Tcm, Tem, TCMR, T cell–mediated rejection; Th, T helper cell.

Figure 5

Overview of the single-cell RNA-sequencing analysis on 12 peripheral blood samples with and without antibody-mediated rejection (ABMR) to determine the cellular origin of the ABMR signals in the blood. Briefly, scRNAseq was performed on 6 peripheral blood samples from kidney transplant recipients with a concomitant diagnosis of ABMR, and 6 stable patients without ABMR. After quality control and filtering were completed, along with removal of 2 clusters containing only doublets, 69,127 cells were detected. (a–c) Unsupervised clustering revealed 15 clusters corresponding to the main myeloid and lymphoid cells and granulocytes/platelets. (d) We evaluated the expression of genes corresponding to the top 10 consistently overexpressed genes in ABMR from both blood and biopsy (GBP5, CCL4, GBP1, C1QA, CRTAM, FCGR1B, C1QB, GBP4, AIM2, and SLAMF7) across the peripheral blood cells. (e) When performing differential expression for ABMR versus no ABMR per cell type, significantly differentially expressed genes in ABMR in nonclassical monocytes included C1QA, C1QB, GBP5, GBP1, GBP4, and FCGR1B; in T cells, GBP5, GBP1, and GBP4; and in B cells, AIM2. CD, cluster of differentiation; cDC, conventional dendritic cell; IL, interleukin; MT, mitochondrial transcripts; NK, natural killer cell; NKT, natural killer T cell; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell.