Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with “secretory”, “contractile” and “vascular” transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8, while the surviving proximal tubules (PTs) showed restored transcriptional signature. Furthermore, we found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

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 TGFb 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.