Nicotinic acetylcholine receptor α1 promotes calpain-1 activation and macrophage inflammation in hypercholesterolemic nephropathy.

The nicotinic acetylcholine receptor α1 (nAChRα1) was investigated as a potential proinflammatory molecule in the kidney, given a recent report that it is an alternative urokinase plasminogen activator (uPA) receptor, in addition to the classical receptor uPAR. Two animal models and in vitro monocyte studies were involved: (1) In an ApoE(-/-) mouse model of chronic kidney disease, glomerular-resident cells and monocytes/macrophages were identified as the primary cell types that express nAChRα1 during hypercholesterolemia/uninephrectomy-induced nephropathy. Silencing of the nAChRα1 gene for 4 months (6 months on Western diet) prevented the increases in renal monocyte chemoattractant protein-1 and osteopontin expression levels and F4/80+ macrophage infiltration compared with the nonsilenced mice. These changes were associated with significantly reduced transforming growth factor-ß1 mRNA (50% decrease) and α smooth muscle actin-positive (αSMA+) myofibroblasts (90% decrease), better glomerular and tubular basement membranes (GBM/TBM) preservation (threefold less disintegration), and better renal function preservation (serum creatinine 40% lower) in the nAChRα1-silenced mice. The nAChRα1 silencing was also associated with significantly reduced renal tissue calcium deposition (78% decrease) and calpain-1 (but not calpain-2) activation (70% decrease). (2) The nAChRα1 was expressed in vitro by mouse monocyte cell line WEHI-274.1. The silencing of nAChRα1 significantly reduced both calpain-1 and -2 activities, and reduced the degradation of the calpain substrate talin. (3) To further explore the role of calpain-1 activity in hypercholesterolemic nephropathy, disease severities were compared in CAST(-/-)ApoE(-/-) (calpain overactive) mice and ApoE(-/-) mice fed with Western diet for 10 months (n=12). Macrophages were the main cell type of renal calpain-1 production in the model. The number of renal F4/80+ macrophages was 10-fold higher in the CAST(-/-)ApoE(-/-) mice (P<0.05), and was associated with a significantly higher level of αSMA+ cells, increased GBM/TBM destruction, and higher serum creatinine levels. Our studies suggest that the receptor nAChRα1 is an important regulator of calpain-1 activation and inflammation in the chronic hypercholesterolemic nephropathy. This new proinflammatory pathway may also be relevant to other disorders beyond hyperlipidemic nephropathy.

In vivo knockdown of nicotinic acetylcholine receptor α1 diminishes aortic atherosclerosis.

OBJECTIVE: Nicotinic acetylcholine receptor α1 (nAChRα1) was recently identified as a functional cell receptor for urokinase, a potent atherogenic molecule. Here, we test the hypothesis that nAChRα1 plays a role in the pathogenesis of atherosclerosis. METHODS: Apolipoprotein E-deficient mice were initially fed a Western diet for 8 wks. Plasmid DNA encoding scramble RNA (pscr) or siRNA (psir2) for nAChRα1 was injected into the mice (n=16) using an aortic hydrodynamic gene transfer protocol. Four mice from each group were sacrificed 7 days after the DNA injection to confirm the nAChRα1 gene silencing. The remaining mice continued on a Western diet for an additional 16 wks. RESULTS: The nAChRα1 was up-regulated in aortic atherosclerotic lesions. A 78% knockdown of the nAChRα1 gene resulted in remarkably less severe aortic plaque growth and neovascularization at 16 wks (both P<0.05). In addition, significantly fewer macrophages (60% less) and myofibroblasts (80% less) presented in the atherosclerotic lesion of the psir2-treated mice. The protective mechanisms of the nAChRα1 knockdown may involve up-regulating interferon-γ/Y box protein-1 activity and down-regulating transforming growth factor-ß expression. CONCLUSIONS: The nAChRα1 gene plays a significant role at the artery wall, and reducing its expression decreases aortic plaque development.