Three-dimensional imaging and single-cell transcriptomics of the human kidney implicate perturbation of lymphatics in alloimmunity

Abstract

Studies of the structural and molecular features of the lymphatic vasculature, which clears fluid, macromolecules and leukocytes from the tissue microenvironment, have largely relied on animal models, with limited information in human organs beyond traditional immunohistochemical assessment. Here, we use three-dimensional imaging and single-cell RNA-sequencing to study lymphatics in the human kidney. We found a hierarchical arrangement of lymphatic vessels within human kidneys, initiating along specialised nephron epithelium in the renal cortex and displaying a distinct, kidney-specific transcriptional profile. In chronic transplant rejection we found kidney allograft lymphatic expansion alongside a loss of structural hierarchy, with human leukocyte antigen-expressing lymphatic vessels infiltrating the medulla, presenting a putative target for alloreactive antibodies. This occurred concurrently with lymphatic vessels invading and interconnecting tertiary lymphoid structures at early stages of lymphocyte colonisation. Analysis of intercellular signalling revealed upregulation of co-inhibitory molecule-mediated CD4+ T cell-lymphatic crosstalk in rejecting kidneys, potentially acting to limit local alloimmune responses. Overall, we delineate novel structural and molecular features of human kidney lymphatics and reveal perturbations to their phenotype and transcriptome in the context of alloimmunity.

A unique subset of pericystic endothelium associates with aberrant microvascular remodelling and impaired blood perfusion early in polycystic kidney disease

“…DEGs between healthy and ADPKD kidneys for each blood EC subset were visualised using the EnhancedVolcano package. Gene ontology (GO) analysis was performed using the PANTHER webtool for gene classification (42) as previously described (33). Marker genes were selected based on the following criteria: i) an adjusted p value > 0.05 from a Wilcoxon rank sum test; ii) a log 2 FC value greater than 1; iii) within the top ten DEGs when lists of genes were ordered by log 2 FC (iv) non-secreted proteins to be identifiable by subsequent immunofluorescence experiments; (v) specificity of the transcript for vascular ECs by visual inspection of feature plots and (vi) consistent expression across the same specialised EC subtype across three or more of the datasets used to generate the scRNA-seq atlas, examined using a dot plot.…”

“…We generated a transcriptomic atlas of the renal microvasculature using previously published single-cell RNA sequencing (scRNA-seq) data of healthy and diseased human kidneys (33). Healthy samples constituted tissues from donor kidneys which were not suitable for transplantation, non-rejection biopsies from kidney transplants or non-tumorous regions from tumour nephrectomies.…”

“…For validation of scRNA-seq data, healthy human adult kidney tissue was obtained from three deceased organ donors who were not for transplantation (47), retrieved by a UK National Organ Retrieval Services team. For validation of snRNA-seq data, tissue was acquired from two individuals with ADPKD derived by the transplant surgical team.…”

“…S1A) (47). These included 25,072 ECs from healthy kidneys and 16,474 ECs from kidneys with different aetiologies of chronic kidney disease (60) or chronic transplant rejection (47,61).…”

The use of tissue clearing to study renal transport mechanisms and kidney remodelling

“…Bo ¨hner et al [45] reported that renal denervation exacerbates LPS and antibody-induced acute kidney Injury and inflammation, but protects from pyelonephritis. Tissue clearing has provided more detailed insights into the development and structure of the lymphatic vascular system, which is required for the trafficking of immune cells [46][47][48][49][50]. Tissue clearing can be combined with topological data analysis to extract geometric features without prior definition of geometric information, as shown in the recent analysis of blood and lymphatic vessels of cleared tissue [48].…”