WCN26-7279 Association Between AI-Derived Retinal Aging Speed and eGFR slope in Japanese Health Checkup Participants

Abstract

Introduction: water channels in collecting duct principal cells are essential for water reabsorption and body fluid homeostasis.Arginine vasopressin (AVP) binds to its receptor V2R on the basolateral membrane, activating cAMP signaling and promoting AQP2 translocation to the apical membrane.Dysregulation of the AVP-V2R-AQP2 axis contributes to disorders of water balance: loss of AQP2 function causes nephrogenic diabetes insipidus, whereas excessive AQP2 activation results in water retention in conditions such as heart failure, liver cirrhosis, and SIADH.Although the V2R antagonist tolvaptan induces aquaresis and is used clinically, it has not demonstrated significant survival benefits.This may reflect cross-reactivity with other receptors and alternative signaling pathways independent of V2R.Therefore, the development of direct AQP2 inhibitors with higher selectivity is desirable.However, no clinically applicable direct AQP2 inhibitors exist due to the narrow pore structure (30 ) hindering drug access, and the lack of a robust high-throughput screening system.Mercury is the only known AQP2 blocker, but its toxicity precludes therapeutic use.Methods: The objective of this study is to establish a reproducible, high-throughput cell-based assay for evaluating AQP2 water permeability and identifying direct inhibitors.We utilized mouse collecting duct-derived mpkCCD cells, known to preserve functional characteristics of principal cells.Endogenous AQP2 expression and its upregulation by the vasopressin analog dDAVP were verified by Western blot and immunofluorescence.Phosphorylated AQP2 at Ser269 appeared only with dDAVP stimulation, indicating functional activation and apical membrane accumulation.For water transport assays, mpkCCD cells were cultured on permeable membranes in Transwell plates (ultimately 96-well format).After epithelial polarization, sucrose was added to the basolateral compartment to create an osmotic gradient.Water movement across the cell layer was quantified by measuring volume changes in the apical

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