Abstract
The kidney distal convoluted tubule (DCT) plays an essential role in maintaining body sodium balance and blood pressure. The major sodium reabsorption pathway in the DCT is the thiazide-sensitive NaCl cotransporter (NCC), whose functions can be modulated by the hormone vasopressin (VP) acting via uncharacterized signaling cascades. Here we use a systems biology approach centered on stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics of cultured mouse DCT cells to map global changes in protein phosphorylation upon acute treatment with a VP type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP). 6330 unique proteins, containing 12333 different phosphorylation sites were identified. 185 sites were altered in abundance following dDAVP. Basophilic motifs were preferential targets for upregulated sites upon dDAVP stimulation, whereas proline-directed motifs were prominent for downregulated sites. Kinase prediction indicated that dDAVP increased AGC and CAMK kinase families’ activities and decreased activity of CDK and MAPK families. Network analysis implicated phosphatidylinositol-4,5-bisphosphate 3-kinase or CAMKK dependent pathways in VP-mediated signaling; pharmacological inhibition of which significantly reduced dDAVP induced increases in phosphorylated NCC at an activating site. In conclusion, this study identifies unique VP signaling cascades in DCT cells that may be important for regulating blood pressure.
Highlights
The peptide hormone arginine vasopressin (VP) is essential for maintaining extracellular fluid homeostasis
The expression of ENaC (Scnn1) subunits at the mRNA and protein levels, coupled with the weak expression of parvalbumin (Pvalb) in mpkDCT cells is characteristic of the late distal convoluted tubule (DCT) (DCT2). mpkDCT cells responded to 15 min desamino-8-D-arginine vasopressin (dDAVP) stimulation from their basolateral side with significant increases in intracellular cAMP levels (Fig. 1C)
Similar to native DCT cells, mpkDCT cells have a gene specific molecular signature that is responsible for providing its specific transport characteristics, including players in transcellular Na+, K+ and Ca2+ transport. mpkDCT cells responded to V2 receptor stimulation with increased intracellular cAMP
Summary
The peptide hormone arginine vasopressin (VP) is essential for maintaining extracellular fluid homeostasis. VP actions to increase NCC phosphorylation and www.nature.com/scientificreports/ Both ENaC and NCC play a critical role in the maintenance of blood pressure. This is highlighted by gain-of-function in ENaC and NCC underlying the hypertensive Liddle and Gordon’s syndrome[15], and the role of both ENaC and NCC in the development of salt-sensitive hypertension[16,17] As both ENaC and NCC are activated by VP, and under certain pathological conditions VP actions may increase sodium retention and subsequently blood pressure[18], understanding of the complex signaling network that is activated in DCT cells following VP exposure is essential. Identification of the signaling network activated by VP and accounts for this increased Na transport via NCC and/or ENaC is essential and may be relevant for uncovering new mechanisms important for regulating blood pressure. Pathway analysis studies identified phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and CAMKK dependent pathways as important signaling components of the VP type II receptor; pharmacological inhibition of these pathways ex vivo using low concentrations of selective inhibitors resulted in decreased NCC phosphorylation at an activating site
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