Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na+ intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na+ diet and the ratio correlated with changes in aldosterone and fractional Na+ excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC in Xenopus oocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na+ intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.
Bibliografisk noteFunding Information:
The work was supported by grants from the Danish Diabetes Academy funded by the Novo Nordisk Foundation (OL 8201,ID phd0115); Faculty of Health Sciences, University of Southern Denmark; Odense University Hospital (815 (A)); The Beckett Foundation (49091); Bagermester August Jensen and Wife grants (95-102-71226); Grosserer L.F. Foght grants (21.265); Fru Ruth I. E. Kønig-Petersen Research Foundation (71623); Karen Elise Jensen Foundation (ReComiToCKD); the Novo Nordisk Foundation (NNF19OC0058780); The independent Research Fund-Denmark (1030-00330B); Danish Society of Nephrology, travel grants (71406); the Ph.D. school, Faculty of Health Sciences, University of Southern Denmark, travel grants (10-102-00000); Swiss National Science Foundation to EH, 31003A_182478/1.
Lab technicians Susanne Hansen, Mohamed Ahmed, Gitte Kitlen, Jesper K. Andresen and Camilla Enggaard, Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Anette Hansen, Institute of Biomedicine, University of Aarhus and Chloé Sergi, Department of Toxicology and Pharmacology, University of Lausanne are all thanked for skillful technical assistance. Prof. Olivier Staub is thanked for the kind gift of the HEK293 ENaC-expressing cells.