Influence of Vasopressin on Vascular Smooth Muscle Cell Calcification

Abstract

Vascular calcification is one of the main risk factors of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Vascular calcification is an active process involving the upregulation of osteogenic transcription factors, including core-binding factor α-1 (CBFA1), msh homeobox 2 (MSX2), SRY-Box 9 (SOX9) and alkaline phosphatase (ALPL). Although vasopressin was originally believed to help prevent water loss, it could be a risk factor in several kidney diseases, including CKD. The present study was designed to explore the effect of vasopressin on osteogenic signaling and calcification, as well as its downstream mechanisms. In this study, human aortic smooth muscle cells (HAoSMCs) were cultured and used in all experiments from passages 4 to 10. Transcript levels were measured using qRT-PCR, protein abundance utilizing western blotting, reactive oxygen species (ROS) generation with 2’,7’-dichlorofluorescein diacetate (DCFDA) fluorescence. Cytosolic Ca2+ concentration ([Ca2+]i) was determined by Fura-2/AM fluorescence, SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin. Alizarin red S staining and calcium content assay were used to detect calcium deposition. ALP assay kit was used for the measurement of ALP activity. Vasopressin (100 nM) obviously enhanced the expressions of ORAI1 and STIM1, as well as SOCE, which were dramatically attenuated by SGK1 inhibitor GSK-650394 (1 µM). ORAI1 blocker MRS1845 (10 µM) counteracted vasopressin-induced upregulation of SOCE. Vasopressin also significantly enhanced the transcript levels of NHE1 and protein abundance of NHE1, an effect appreciably abolished by SGK1 inhibitor GSK-650394. Additional findings showed that vasopressin elevated ROS production in HAoSMCs, which was prevented by NHE1 inhibitor cariporide (10 µM). In turn, ROS scavenger N-acetyl-L-cysteine (NAC, 0.5 mM) downregulated vasopressin-stimulated NHE1 expressions in HAoSMCs. Vasopressin stimulated the upregulation of osteogenic transcription factors CBFA1, MSX2, SOX9 and ALPL, as well as ALP activity and Ca2+ content, which were significantly blunted by SGK1 inhibitor GSK-650394, ORAI1 blocker MRS1845, NHE1 inhibitor cariporide or ROS scavenger NAC. In summary, vasopressin stimulates at least partially via SGK1- SOCE and NHE1/ROS signaling, eventually leading to osteogenic signaling and vascular calcification.