The discovery of aquaporin water channels by Agre and coworkers answered a long-standing biophysical question of how the majority of water crosses biological membranes. The identification and study of aquaporins have provided insight, at the molecular level, into the fundamental physiology of water balance regulation and the pathophysiology of water balance disorders. In addition to the originally identified classical aquaporins, a second class of aquaporins has been identified. Aquaporins in this latter class, the so-called aquaglyceroporins, transport small uncharged molecules such as glycerol and urea as well as water. Aquaglyceroporins have a wide tissue distribution, and emerging data suggest that several of them may play previously unappreciated physiological or pathophysiological roles. Analyses of transgenic mice have revealed potential roles of aquaglyceroporins in skin elasticity, gastrointestinal function and metabolism, and metabolic diseases such as diabetes mellitus. This review comprehensively discusses the recent discoveries in the field of aquaglyceroporins, alongside a brief overview of the so-called unorthodox aquaporins.

Aquaporin-9 (AQP9) is an aquaglyceroporin membrane channel shown biophysically to conduct water, glycerol, and other small solutes. Because the physiological role/s of AQP9 remain undefined and the expression sites of AQP9 remain incomplete and conflicting, we generated AQP9 knockout mice. In the absence of physiological stress, knockout mice did not display any visible behavioral or severe physical abnormalities. Immunohistochemical analyses using multiple antibodies revealed AQP9 specific labeling in hepatocytes, epididymis, vas deferens, and in epidermis of wild type mice, but a complete absence of labeling in AQP9 ^−/− mice. In brain, no detectable labeling was observed. Compared with control mice, plasma levels of glycerol and triglycerides were markedly increased in AQP9 ^−/− mice, whereas glucose, urea, free fatty acids, alkaline phosphatase, and cholesterol were not significantly different. Oral administration of glycerol to fasted mice resulted in an acute rise in blood glucose levels in both AQP9 ^−/− and AQP9 ^+/− mice, revealing no defect in utilization of exogenous glycerol as a gluconeogenic substrate and indicating a high gluconeogenic capacity in nonhepatic organs. Obese Lepr ^db / Lepr ^db AQP9 ^−/− and obese Lepr ^db / Lepr ^db AQP9 ^+/− mice showed similar body weight, whereas the glycerol levels in obese Lepr ^db / Lepr ^db AQP9 ^−/− mice were dramatically increased. Consistent with a role of AQP9 in hepatic uptake of glycerol, blood glucose levels were significantly reduced in Lepr ^db / Lepr ^db AQP9 ^−/− mice compared with Lepr ^db / Lepr ^db AQP9 ^+/− in response to 3 h of fasting. Thus, AQP9 is important for hepatic glycerol metabolism and may play a role in glycerol and glucose metabolism in diabetes mellitus.

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser²⁵⁶, Ser²⁶¹, Ser²⁶⁴, and Ser²⁶⁹) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser²⁶⁹ and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)²⁶⁹-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser²⁵⁶ phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser²⁵⁶, but not Ser²⁶⁹. Phosphorylation of AQP2 at Ser²⁶⁹ did not occur when Ser²⁵⁶ was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser²⁶⁹ phosphorylation depends upon prior phosphorylation at Ser²⁵⁶. Immunogold electron microscopy localized Ser(P)²⁶⁹-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D “phosphomimic” AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser²⁶⁹:(a) depends on prior PKA-mediated phosphorylation of Ser²⁵⁶ and (b) enhances apical plasma membrane retention of AQP2.

To investigate the role of inner medullary collecting duct (IMCD) urea transporters in the renal concentrating mechanism, we deleted 3 kb of the UT-A urea transporter gene containing a single 140-bp exon (exon 10). Deletion of this segment selectively disrupted expression of the two known IMCD isoforms of UT-A, namely UT-A1 and UT-A3, producing UT-A1/3 ^-/- mice. In isolated perfused IMCDs from UT-A1/3 ^-/- mice, there was a complete absence of phloretin-sensitive or vasopressin-stimulated urea transport. On a normal protein intake (20% protein diet), UT-A1/3 ^-/- mice had significantly greater fluid consumption and urine flow and a reduced maximal urinary osmolality relative to wild-type controls. These differences in urinary concentrating capacity were nearly eliminated when urea excretion was decreased by dietary protein restriction (4% by weight), consistent with the 1958 Berliner hypothesis stating that the chief role of IMCD urea transport in the concentrating mechanism is the prevention of urea-induced osmotic diuresis. Analysis of inner medullary tissue after water restriction revealed marked depletion of urea in UT-A1/3 ^-/- mice, confirming the concept that phloretin-sensitive IMCD urea transporters play a central role in medullary urea accumulation. However, there were no significant differences in mean inner medullary Na ⁺ or Cl ^- concentrations between UT-A1/3 ^-/- mice and wild-type controls, indicating that the processes that concentrate NaCl were intact. Thus, these results do not corroborate the predictions of passive medullary concentrating models stating that NaCl accumulation in the inner medulla depends on rapid vasopressin-regulated urea transport across the IMCD epithelium.

The water channel aquaporin-2 (AQP2), expressed in the kidney collecting ducts, plays a pivotal role in maintaining body water balance. The channel is regulated by the peptide hormone arginine vasopressin (AVP), which exerts its effects through the type 2 vasopressin receptor (AVPR2). Disrupted function or regulation of AQP2 or the AVPR2 results in nephrogenic diabetes insipidus (NDI), a common clinical condition of renal origin characterized by polydipsia and polyuria. Over several years, major research efforts have advanced our understanding of NDI at the genetic, cellular, molecular, and biological levels. NDI is commonly characterized as hereditary (congenital) NDI, arising from genetic mutations in the AVPR2 or AQP2; or acquired NDI, due to for exmple medical treatment or electrolyte disturbances. In this article, we provide a comprehensive overview of the genetic, cell biological, and pathophysiological causes of NDI, with emphasis on the congenital forms and the acquired forms arising from lithium and other drug therapies, acute and chronic renal failure, and disturbed levels of calcium and potassium. Additionally, we provide an overview of the exciting new treatment strategies that have been recently proposed for alleviating the symptoms of some forms of the disease and for bypassing G protein-coupled receptor signaling.

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin–Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of ( i ) apical plasma membrane abundance of AQP2, ( ii ) internalization of AQP2, ( iii ) AQP2 protein–protein interactions, and ( iv ) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life ( t _1/2 = 5.1 h and t _1/2 = 4.4 h, respectively) compared to WT-AQP2 ( t _1/2 = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein–protein interactions.