A Novel Signaling Pathway

The nicotinic acetylcholine receptor α1 (nAChRα1) was investigated as a potential fibrogenic molecule in the kidney, given reports that it may be an alternative urokinase (urokinase plasminogen activator; uPA) receptor in addition to the classical receptor uPAR. In a mouse obstructive uropathy model of chronic kidney disease, interstitial fibroblasts were identified as the primary cell type that bears nAChRα1 during fibrogenesis. Silencing of the nAChRα1 gene led to significantly fewer interstitial αSMA+ myofibroblasts (2.8 times decreased), reduced interstitial cell proliferation (2.6 times decreased), better tubular cell preservation (E-cadherin 14 times increased), and reduced fibrosis severity (24% decrease in total collagen). The myofibroblast-inhibiting effect of nAChRα1 silencing in uPA-sufficient mice disappeared in uPA-null mice, suggesting that a uPA-dependent fibroblastic nAChRα1 pathway promotes renal fibrosis. To further establish this possible ligand-receptor relationship and to identify downstream signaling pathways, in vitro studies were performed using primary cultures of renal fibroblasts. 35S-Labeled uPA bound to nAChRα1 with a Kd of 1.6 × 10−8 m, which was displaced by the specific nAChRα1 inhibitor d-tubocurarine in a dose-dependent manner. Pre-exposure of uPA to the fibroblasts inhibited [3H]nicotine binding. The uPA binding induced a cellular calcium influx and an inward membrane current that was entirely prevented by d-tubocurarine preincubation or nAChRα1 silencing. By mass spectrometry phosphoproteome analyses, uPA stimulation phosphorylated nAChRα1 and a complex of signaling proteins, including calcium-binding proteins, cytoskeletal proteins, and a nucleoprotein. This signaling pathway appears to regulate the expression of a group of genes that transform renal fibroblasts into more active myofibroblasts characterized by enhanced proliferation and contractility. This new fibrosis-promoting pathway may also be relevant to disorders that extend beyond chronic kidney disease.

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