Elsevier

Kidney International

Volume 103, Issue 6, June 2023, Pages 1077-1092
Kidney International

Basic Research
Single nuclei transcriptomics delineates complex immune and kidney cell interactions contributing to kidney allograft fibrosis

https://doi.org/10.1016/j.kint.2023.02.018Get rights and content

Chronic allograft dysfunction (CAD), characterized histologically by interstitial fibrosis and tubular atrophy, is the major cause of kidney allograft loss. Here, using single nuclei RNA sequencing and transcriptome analysis, we identified the origin, functional heterogeneity, and regulation of fibrosis-forming cells in kidney allografts with CAD. A robust technique was used to isolate individual nuclei from kidney allograft biopsies and successfully profiled 23,980 nuclei from five kidney transplant recipients with CAD and 17,913 nuclei from three patients with normal allograft function. Our analysis revealed two distinct states of fibrosis in CAD; low and high extracellular matrix (ECM) with distinct kidney cell subclusters, immune cell types, and transcriptional profiles. Imaging mass cytometry analysis confirmed increased ECM deposition at the protein level. Proximal tubular cells transitioned to an injured mixed tubular (MT1) phenotype comprised of activated fibroblasts and myofibroblast markers, generated provisional ECM which recruited inflammatory cells, and served as the main driver of fibrosis. MT1 cells in the high ECM state achieved replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. MT1 in the low ECM state showed decreased apoptosis, decreased cycling tubular cells, and severe metabolic dysfunction, limiting the potential for repair. Activated B, T and plasma cells were increased in the high ECM state, while macrophage subtypes were increased in the low ECM state. Intercellular communication between kidney parenchymal cells and donor-derived macrophages, detected several years post-transplantation, played a key role in injury propagation. Thus, our study identified novel molecular targets for interventions aimed to ameliorate or prevent allograft fibrogenesis in kidney transplant recipients.

Section snippets

Patients and samples

Kidney graft biopsies from 8 kidney transplant recipients were studied; 5 patients with CAD whose biopsies were categorized as having IFTA, and 3 patients with normal/stable graft function, were categorized as normal/nonspecific. Independent histologic evaluations were performed by 2 kidney transplant pathologists. The institutional review board approved the study, and patients signed an informed consent form at the time of transplantation (HP-00091954). The clinical and research activities

Nuclei isolation using kidney allograft biopsies preserved in RNAlater

The feasibility of using kidney core biopsies preserved in RNAlater for single-nuclei RNA-sequencing was assessed (See the Supplementary Methods and Materials, and Supplementary Figure S1A–C). Eight biopsies passed quality control assessment (Supplementary Figure S2A–G). No significant differences in quality control were found when these biopsies were compared to snap-frozen kidney graft biopsies (samples included in the current study vs. publicly available data GEO: GSE13188220,21;

Discussion

The pathophysiology of fibrosis in the kidney graft is characterized by multidimensional regulation of complex networks influenced by various factors unique to the human transplant model, such as the alloimmune response and kidney parenchymal toxicity, resulting in impaired repair.

Our study identified high heterogeneity in the composition of fibrosis, diverse cell types, cell–cell interactions, and differential transcriptional profiles, providing deeper insight into the deleterious processes

Disclosure

All the authors declared no competing interests.

Data Statement

The data that support the findings of this study will be publicly available (GEO ID: GSE195718).

Acknowledgments

The research reported in this publication is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the: National Institutes of Health under awards numbers: R21DK100678 (DGM), and R01DK080074, R01DK109581, and R01DK122682 (VRM). The authors thank members of the oncology tissue service core at the Johns Hopkins University for their assistance with the immunostaining assays. The authors acknowledge the Shared Resource of the Georgetown Lombardi Comprehensive

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    Contributed equally to this work.

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