Blockade of Orai1 Store-Operated Calcium Entry Protects against Renal Fibrosis

Abstract

Evidence supports an important role of Ca release-activated Ca channel protein 1 (Orai1)-mediated Ca entry in the development of renal fibrosis, a common pathologic feature of CKDs that lead to ESRD, but the molecular mechanisms remain unclear. We determined the role of Orai1 calcium channel in renal fibrosis induced by high-fat diet and by unilateral ureteral obstruction. Mouse kidneys with fibrosis had higher levels of Orai1 protein expression than did kidneys without fibrosis. In vivo knockdown of Orai1 with adenovirus harboring Orai1-short hairpin RNA or inhibition of Orai1 with SKF96365 dramatically prevented renal fibrosis and significantly decreased protein expression of fibronectin, α‑smooth muscle actin, and TGF‑β1 in the kidney cortex of ApoE mice on a high-fat diet and in the obstructed kidneys of mice with unilateral ureteral obstruction. Compared with kidney biopsy specimens of patients with glomerular minimal change disease, those of patients with fibrotic nephropathy had higher expression levels of Orai1. In cultured human proximal tubule epithelial cells (HK2), knockdown of Orai1 Ca channel with adenovirus-Orai1-short hairpin RNA markedly inhibited TGF-β1-induced intracellular Ca influx and phosphorylation of smad2/3. Knockdown or blockade of the Orai1 Ca channel in HK2 cells also prevented epithelial-to-mesenchymal transition induced by TGF‑β1. In conclusion, blockade of the Orai1 Ca channel prevented progression of renal fibrosis in mice, likely by suppressing smad2/3 phosphorylation and TGF-β1-induced epithelial-to-mesenchymal transition. These results render the Orai1 Ca channel a potential therapeutic target against renal fibrosis.

Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil

“…Immunohistochemistry was performed using the streptavidin–biotin–peroxidase complex system as described previously . Briefly, paraformaldehyde‐fixed, paraffin‐embedded sections (8 μm) cleared of paraffin in Citroclear and rehydrated through graded industrial methylated spirit series.…”

“…[Ca 2+ ] i was measured as we previously described . Briefly, cells were suspended in Ca 2+ ‐free HBSS (130 mM NaCl, 4.8 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 25 mM NaHCO 3 , 10 mM glucose, 20 mM HEPES, 50 μM EGTA and 0.1% fatty acid free bovine serum albumin, pH 7.4) solution containing 2 μM fura2/AM for 45 min.…”

“…The siRNA duplexes against Orai1 (Qiagen, Valencia, CA, USA) were transiently transfected with Hiperfect Transfection Reagent (Qiagen) according to the manufacturer's instructions as previously described . The siRNA target sequence for Orai1 was 5′‐UUGCUCACCGCCUCGAUGUTG‐3′.…”

Orai1: CRACing the Th17 response in AKI

“…In a recent study, Ca 2+ signaling was found to control pathogenic Th17-driven inflammation by regulating mitochondrial function, suggesting additional mechanisms are involved in Th17 responses (21). Another recent study, by Mai et al, found a protective effect of Orai1 blockade in a model of high-fat dietinduced interstitial fibrosis in ApoE-deficient mice, as well as in a model of unilateral ureteral obstruction (UUO) kidney injury in mice (22). Thus, Th17 cell-specific blocking of Orai1 could provide a unique therapeutic opportunity for targeting AKI and ameliorating the effect of a high-salt diet in AKI-to-CKD transition in humans.…”

Plasma membrane ion channels and epithelial to mesenchymal transition in cancer cells

“…It should be noted that other studies have investigated ion channels in EMT in the context of other EMT relevant processes many of which intersect with disease states such as airway remodelling (Arthur et al 2015) and renal fibrosis (Mai et al 2016).…”