Single-cell transcriptomic profiling of kidney fibrosis identifies a novel specific fibroblast marker and putative disease target

Background Persistent kidney fibroblast activation and tubular epithelial cell (TEC) injury are key contributors to CKD. However, transcriptional and cellular identities of advanced kidney disease, along with renal fibroblast specific markers and molecular targets contributing to persistent tubular injury, remain elusive. Methods We performed single-cell RNA sequencing with two clinically relevant murine kidney fibrosis models. Day 28 post-injury was chosen to ensure advanced fibrotic disease. Identified gene expression signatures were validated using multiple quantitative molecular analyses. Results We revealed comprehensive single cell transcriptomic profiles of two independent kidney fibrosis models compared to normal control. Both models exhibited key CKD characteristics including renal blood flow decline, inflammatory expansion and proximal tubular loss. We identified novel populations including “secretory”, “migratory” and “contractile” activated fibroblasts, specifically labelled by newly identified fibroblast-specific Gucy1a3 expression. Fibrotic kidneys elicited elevated embryonic and pro-fibrotic signaling, including separate “Embryonic” and “Pro-fibrotic” TEC clusters. Also, fibrosis caused enhanced cell-to-cell crosstalk, particularly between activated fibroblasts and pro-fibrotic TECs. Analysis of factors mediating mesenchymal phenotype in the injured epithelium identified persistent elevation of Ahnak, previously reported in AKI, in both CKD models. AHNAK knockdown in primary human renal proximal tubular epithelial cells induced a pro-fibrotic phenotype and exacerbated TGFβ response via p38, p42/44, pAKT, BMP and MMP signaling. Conclusions Our study comprehensively examined kidney fibrosis in two independent models at the singe-cell resolution, providing a valuable resource for the field. Moreover, we newly identified Gucy1a3 as a kidney activated fibroblast specific marker and validated AHNAK as a putative disease target. Significance Statement Mechanistic understanding of kidney fibrosis is principal for mechanistic understanding and developing targeted strategies against CKD. However, specific markers and molecular targets of key effector cells - activated kidney fibroblasts and injured tubular epithelial cells - remain elusive. Here, we created comprehensive single cell transcriptomic profiles of two clinically relevant kidney fibrosis models. We revealed “secretory”, “contractile” and “migratory” fibroblasts and identified Gucy1a3 as a novel marker selectively labelling all three populations. We revealed that kidney fibrosis elicited remarkable epithelial-to-stromal crosstalk and pro-fibrotic signaling in the tubular cells. Moreover, we mechanistically validated AHNAK as a putative novel kidney injury target in a primary human in vitro model of epithelial-to-mesenchymal transition. Our findings advance understanding of and targeted intervention in fibrotic kidney disease. ### Competing Interest Statement P.D. is a co-inventor on patents for the use of NGAL as a biomarker of kidney injury.