Arginine vasopressin (AVP) leads to an increase of water permeability by insertion of aquaporin-2 (AQP2) into the plasma membrane in the collecting duct (CD). In contrast, atrial natriuretic peptide (ANP) is known as a diuretic and natriuretic factor acting through natriuretic peptide receptor type A (NPR-A) by elevating cyclic guanylate monophosphate (cGMP). The nitric oxide (NO)/soluble guanylate cyclase (sGC) pathway also generates cGMP in the CD.

Primary cultured inner medullary collecting duct cells (IMCD) express all components of the AQP2 regulatory pathway. We used this model to analyze the mechanism of ANP/NO on AQP2 regulation. Digital holographic microscopy and a calcein fluorescence quenching method were used to detect cell volume kinetics after osmotically induced cell swelling. We quantified AQP2 immunofluorescence as ratio of membrane inserted versus cytosolic signals to detect trafficking of AQP2 to the plasma membrane.

AVP led to a significant increase in membrane expression of AQP2 compared to untreated and ANP stimulated cells. But in AVP pretreated cells ANP decreased membrane expression of AQP2, indicating a retrieval of AQP2 from the plasma membrane. The immunofluorescence quantification correlates with cell volume kinetics. ANP alone had no influence on water permeability of IMCD cells. In AVP pretreated cells ANP was able to reverse AVP enhanced water permeability almost to control levels. The natriuretic peptide uroguanylin had no influence on AQP2 regulation.

The effect of ANP on water permeability was mimicked by 8-Bromo-cGMP and could be inhibited by the specific protein kinase G (PKG) inhibitor KT5823, indicating a cGMP/PKG mediated decrease of water permeability in IMCD cells upon ANP. Another cGMP generating pathway is the NO/soluble guanylate cyclase system. NO synthesis was stimulated via sodium nitroprusside. This led to decreased water permeability in AVP-pretreated cells. The direct stimulation of sGC with Bay 58–2667 had the same effect on water permeability, suggesting that cGMP synthesized by this pathway had the same effect on AQP2 as cGMP elevations by ANP.

In conclusion, we provide evidence that the diuretic effect of ANP is in part due to a cGMP/PKG dependent decrease of water permeability mediated by increased endocytosis of AQP2. The NO/sGC signaling pathway had the same effect on regulation of AQP2.