PTK7 knockout suppresses TGF-β1-induced renal tubular epithelial cell fibrosis by inhibiting Smad2/3 phosphorylation

Renal fibrosis is a common endpoint for various chronic kidney diseases, leading to irreversible kidney damage and end-stage renal failure. A key driver of this process is the activation of renal tubular epithelial cells, which undergo epithelial-mesenchymal transition (EMT), contributing significantly to extracellular matrix accumulation. The TGF-β1/Smad signaling pathway is a central mediator of renal fibrosis and EMT. Current therapies often fall short in effectively halting fibrosis progression, highlighting an urgent need for novel therapeutic targets. This study investigates protein tyrosine kinase-like 7 (PTK7), a transmembrane receptor, as a potential new target.

Researchers investigated PTK7's role in renal fibrosis using a mouse model and a PTK7-knockout transformed C3H mouse kidney-1 (TCMK-1) cell line generated via CRISPR/Cas9. They examined PTK7 expression in mouse kidney tissue using immunohistochemistry, Western blotting, and qRT-PCR. Public single-cell transcriptomic datasets were analyzed for PTK7 distribution. In vitro, PTK7-knockout TCMK-1 cells were stimulated with transforming growth factor-beta 1 (TGF-β1). Key endpoints included cellular morphological changes, expression of EMT-related proteins (E-cadherin, vimentin) and fibrosis-related proteins (α-SMA, fibronectin, collagen I), assessed by Western blotting. Cell proliferation was measured by CCK-8, and migratory capacity by wound healing assays. Finally, Western blotting was used to analyze phosphorylation levels of Smad2/Smad3 to elucidate mechanisms.

PTK7 expression was significantly upregulated in the kidney tissue of the mouse model of renal fibrosis and was highly expressed in renal tubular epithelial cells. Single-cell transcriptomic data further confirmed its distribution in these critical renal cell types. In vitro, PTK7 knockout dramatically altered the cellular response to fibrotic stimuli.

PTK7 knockout suppressed TGF-β1-induced EMT progression and the expression of fibrosis-related proteins (E-cadherin, vimentin, α-SMA, fibronectin, collagen I) with a statistical significance of P<0.001. This deficiency also attenuated the migratory ability of renal tubular epithelial cells under TGF-β1 stimulation, indicating a reduction in their pro-fibrotic behavior. Mechanistically, PTK7 deficiency effectively inhibited TGF-β1-triggered activation of Smad2/Smad3 phosphorylation, a crucial step in the TGF-β1/Smad signaling pathway that drives fibrosis.