Detection of infiltrating fibroblasts by single-cell transcriptomics in human kidney allografts

Abstract

We tested the hypothesis that single-cell RNA-sequencing (scRNA-seq) analysis of human kidney allograft biopsies will reveal distinct cell types and states and yield insights to decipher the complex heterogeneity of alloimmune injury. We selected 3 biopsies of kidney cortex from 3 individuals for scRNA-seq and processed them fresh using an identical protocol on the 10x Chromium platform; (i) HK: native kidney biopsy from a living donor, (ii) AK1: allograft kidney with transplant glomerulopathy, tubulointerstitial fibrosis, and worsening graft function, and (iii) AK2: allograft kidney after successful treatment of active antibody-mediated rejection. We did not study T-cell-mediated rejections. We generated 7217 high-quality single cell transcriptomes. Taking advantage of the recipient-donor sex mismatches revealed by X and Y chromosome autosomal gene expression, we determined that in AK1 with fibrosis, 42 months after transplantation, more than half of the kidney allograft fibroblasts were recipient-derived and therefore likely migratory and graft infiltrative, whereas in AK2 without fibrosis, 84 months after transplantation, most fibroblasts were donor-organ-derived. Furthermore, AK1 was enriched for tubular progenitor cells overexpressing profibrotic extracellular matrix genes. AK2, eight months after successful treatment of rejection, contained plasmablast cells with high expression of immunoglobulins, endothelial cell elaboration of T cell chemoattractant cytokines, and persistent presence of cytotoxic T cells. In addition to these key findings, our analysis revealed unique cell types and states in the kidney. Altogether, single-cell transcriptomics yielded novel mechanistic insights, which could pave the way for individualizing the care of transplant recipients.

The cellular landscape of the normal kidney allograft: Main players balancing the alloimmune response

“…Further proof is found through the ongoing alloimmune response, upregulation of proinflammatory signals by immune cells, and cellular damage exhibited by PT. Contrary to the normal native biopsies, a significant increase in COL1A and PDGFRA and slight increase in ACTA2, DCN, PDGFRB, POSTN, and VIM expression was present in normal allografts, consistent with previous observations of activation and replication of FB at wound sites (27,(67)(68)(69). The ongoing alloimmune response and FB activated at a low-level raises the possibility that these phenotypes necessitate balanced kidney repair with major cell types still retaining some level of normal function.…”

“…It is important to note, that donor kidney grafts were younger than native kidneys. Importantly, increased expression of the activated FB cell type, and upregulation of the Wnt signaling was observed in normal grafts compared to the normal native kidneys (15,16,27). Thus, the relative increase in the number of FB and gene expression alterations is likely a direct consequence of injury, healing, and adaptation.…”

“…Single cell transcriptome approaches applied to human kidney allograft samples are in its infancy, with a limited number of reports in the field. Limited published data include the evaluation of pathological conditions affecting the graft compared to normal native kidneys as controls (16, [24][25][26][27][28]. Nevertheless, comparative analysis utilizing the native kidney has many known limitations.…”

Transcriptional and spatial profiling of the kidney allograft unravels a central role for FcyRIII+ innate immune cells in rejection

“…2a) and one male-to-female transplantation (Supplementary Fig. 2b) were present in our cohort, of which the cells were assigned as donor-or recipient-derived by the expression of Y chromosome-encoded ZFY and DDX3Y and female-specific XIST and TSIX, involved in X chromosome inactivation 44 . The percentage of cells expressing sex-linked genes was heterogeneous among the cell types, which is in line with previous reports 44 .…”

“…2b) were present in our cohort, of which the cells were assigned as donor-or recipient-derived by the expression of Y chromosome-encoded ZFY and DDX3Y and female-specific XIST and TSIX, involved in X chromosome inactivation 44 . The percentage of cells expressing sex-linked genes was heterogeneous among the cell types, which is in line with previous reports 44 . Cell types composing the renal architecture were mostly donor-derived, but a fraction of glomerular endothelial cells (5%) expressed recipient-derived genes, which could reflect endothelial chimerism 45 .…”