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Abstract
The aquaporin 2 (AQP2) plays a critical role in water reabsorption to maintain water homeostasis. AQP2 mutation leads to nephrogenic diabetes insipidus (NDI), characterized by polyuria, polydipsia, and hypernatremia. We previously reported that a novel AQP2 mutation (G215S) caused NDI in a boy. In this study, we aimed to elucidate the cell biological consequences of this mutation on AQP2 function and clarify the molecular pathogenic mechanism for NDI in this patient. First, we analyzed AQP2 expression in Madin-Darby canine kidney (MDCK) cells by AQP2-G215S or AQP2-WT plasmid transfection and found significantly decreased AQP2-G215S expression in cytoplasmic membrane compared with AQP2-WT, independent of forskolin treatment. Further, we found co-localization of endoplasmic reticulum (ER) marker (Calnexin) with AQP2-G215S rather than AQP2-WT in MDCK cells by immunocytochemistry. The functional analysis showed that MDCK cells transfected with AQP2-G215S displayed reduced water permeability compared with AQP2-WT. Visualization of AQP2 structure implied that AQP2-G215S mutation might interrupt the folding of the sixth transmembrane α-helix and/or the packing of α-helices, resulting in the misfolding of monomer and further impaired formation of tetramer. Taken together, these findings suggested that AQP2-G215S was misfolded and retained in the ER and could not be translocated to the apical membrane to function as a water channel, which revealed the molecular pathogenic mechanism of AQP2-G215S mutation and explained for the phenotype of NDI in this patient.
Highlights
Nephrogenic diabetes insipidus (NDI) is characterized by impaired arginine vasopressin (AVP)induced water reabsorption in the kidney, leading to polyuria, polydipsia, and hypernatremia
aquaporin 2 (AQP2) forms a homotetramer in the plasma membrane, and each monomer is composed of 271 amino acids, containing six transmembrane spanning regions with the intracellular COOH terminus, which is essential for correct routing of AQP2
AQP2 was decreased in the cell membrane of AQP2-G215S–transfected cells compared with AQP2-WT-transfected cells (p
Summary
Nephrogenic diabetes insipidus (NDI) is characterized by impaired arginine vasopressin (AVP)induced water reabsorption in the kidney, leading to polyuria, polydipsia, and hypernatremia. Molecular Characterization of Aquaporin-2 Mutation therapy) and electrolyte abnormalities, or caused by mutations in the vasopressin V2 receptor (AVPR2, OMIM#304800) or AQP2 (OMIM#125800, 107777) [4,5,6,7]. AVPR2 accounts for X-linked cases of NDI. When the plasma osmolality increases, antidiuretic hormone (AVP) is released from the pituitary gland and binds to AVPR2 in principal cells of the kidney collecting duct, resulting in the accumulation of AQP2 in the apical plasma membrane, which is responsible for water reabsorption [8, 9]. Most autosomal recessive cases had severe phenotypes in contrast to autosomal dominant NDI [20]
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