Atrial natriuretic peptide and nitric oxide signaling antagonizes

22 AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP 23 induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the 24 plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An 25 ANPand nitric oxide (NO)/soluble guanylate cyclase (sGC) induced insertion of 26 AQP2 into the plasma membrane is reported from different models. However, 27 functional data on the insertion of AQP2 is missing. We used primary cultured inner 28 medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein 29 quenching measurements and immunofluorescence and western blotting to analyze 30 the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression 31 and water permeability. While AVP led to acceleration in osmotically induced swelling, 32 ANP had no effect. However, in AVP pretreated cells ANP decreased significantly the 33 kinetic of cell swelling. This effect was mimicked by 8-bromo-cGMP (8-Br-cGMP) and 34 blunted by protein kinase G (PKG) inhibition. Stimulation of the NO/sGC pathway or 35 direct activation of sGC with BAY 58-2667 had similar effects like ANP. In cells treated 36 with AVP AQP2 was predominantly localized in the plasma membrane, after additional 37 incubation with atrial natriuretic peptide AQP2 was mostly localized in the cytosol 38 indicating an increased retrieval of AQP2 from the plasma membrane by ANP. 39 Western blot analysis showed that ANP was able to reduce AVP-induced 40 phosphorylation of AQP2 at position S256. 41 In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a 42 decreased localization of AQP2 in the plasma membrane which is mediated by cGMP 43 and PKG. 44 45