Endothelin-1 Derived from Failed-Repair Proximal Tubular Cells Exacerbates Fibrosis after Kidney Injury

Background: Endothelin signaling contributes to the progression of chronic kidney disease across multiple etiology including acute kidney injury (AKI). Previously, single-cell transcriptomic analysis of mouse AKI kidney revealed that a distinct failed repair proximal tubular cell (FR-PTC) state drives fibrosis after injury and endothelin-1 (Edn1) signaling between FR-PTCs and fibroblasts were significantly upregulated (PNAS. 2020). However, the mechanistic link between FR-PTC derived Edn1 and renal fibrosis remains unclear. Methods: Tamoxifen-inducible proximal tubule-specific Edn1 knockout mice (Edn1flox/flox::Slc34a1CreERT2/+) underwent unilateral ischemia-reperfusion injury(IRI) surgery or administration of aristolochic acid (AA), following vehicle or tamoxifen treatment. Mice were sacrificed at 4 weeks after IRI or 2 weeks after AA-induced injury for phenotypic assessment. Immortalized tubular epithelial cells (NRK52E) and fibroblasts (NRK49F) were used in vitro. Furthermore, RNA sequencing (RNA-seq) was performed on Edn1-treated fibroblasts. Results: Proximal tubule-specific Edn1 deletion decreased the expression levels of profibrotic and proinflammatory genes (e.g. Tgfb1, Acta2, Ccl2 and Cd68) after injury. Moreover, immunofluorescence staining showed decrease of αSMA positive myofibroblasts around FR-PTCs. In vitro, RNA-seq revealed that profibrotic and proinflammatory pathways, also NFkB and ERK signaling were upregulated in Edn1-treated NRK49F. Western blotting revealed that Edn1 activated the phosphorylation of ERK in NRK49F. Edn1 expedited the proliferation and migration of NRK49F, which was attenuated by an ERK inhibitor. Conditioned medium (CM) from AA-treated NRK52E in which Edn1 was upregulated expedited the proliferation and migration of NRK49F. The enhanced fibroblast proliferation and migration was nullified by CM from AA-treated NRK52E with CRISPR-mediated Edn1 knockout. Conclusion: In this study, we clarified the important role of the proximal tubule-derived Edn1 on pERK-mediated fibroblast proliferation and migration in renal fibrosis. Our results also suggested that Edn1 induced NFkB-mediated profibrotic and proinflammatory signals on fibroblasts which may exacerbate renal fibrosis. Inhibition of Edn1 may be a therapeutic target for preventing renal fibrosis.