To maintain a proper environment for cells to communicate, the osmotic value of the extracellular fluid and blood plasma in human beings is kept between narrow limits. This homeostasis is mainly accomplished by the renal regulation of water reabsorption from pro-urine and is one of the few physiological processes that are prominent in everyday life. Daily, 180 l of pro-urine are formed in the renal nephrons, of which 90 % is constitutively reabsorbed in the proximal tubules and descending limbs of Henle through the water channel Aquaporin-1 (AQP1). The remaining 20 l of water can be reabsorbed in the renal collecting duct and is under control of the antidiuretic hormone arginine-vasopressin (AVP). In states of hypernatraemia/hypovolaemia, induction of osmo- or baroreceptors, respectively, in arteries and veins signals the pituitary to release AVP, which binds its vasopressin type-2 receptor (V2R) in the basolateral membrane of renal principal cells. This binding triggers a cAMP signalling cascade that leads to the activation of protein kinase A (PKA). In turn, PKA phosphorylates, among other proteins, AQP2 at Ser256, resulting in a redistribution of the homotetrameric water channel from intracellular vesicles to the apical membrane of the cell. Recent data indicate that the stoichiometry of S256 phosphorylated versus non-phosphorylated monomers in an AQP2 homotetramer plays a role in this redistribution process. Driven by the osmotic gradient over these cells, water then enters the cell through AQP2 and leaves the cells to the interstitium via AQP3 and AQP4, which are located in the basolateral membrane. Consequently, urine is concentrated. Removal of AVP reverses this process, restoring the water-impermeable state of the apical membrane.

This process is disturbed in nephrogenic diabetes insipidus (NDI), a disease characterized by the inability of the kidney to concentrate urine in response to AVP. Without medication, adult NDI patients consequently urinate 15-20 l daily. In congenital NDI, mutations have been found in the V2R gene, resulting in a sex-linked inheritance of NDI, whereas mutations in the AQP2 gene are causal to autosomal recessive and dominant NDI. To reveal the molecular cause underlying autosomal NDI, missense AQP2 mutants encoded in recessive NDI were expressed in Xenopus oocytes. Immunoblot and immunocytochemical analysis revealed that, although wild-type (wt) AQP2 was efficiently transported to the plasma membrane, all mutants were impaired in their export from the endoplasmic reticulum, presumably caused by misfolding. Recently, three mutations in the AQP2 gene of NDI families were found, which coded for a missense mutant (AQP2-E258K) and two mutants in which the encoded C-terminal tail was out of frame, caused by a guanosine deletion (AQP2-727delG) or an adenosine insertion (AQP2-779InsA). Since AQP2 is expressed as a homotetramer in which every monomer is a water channel, it was speculated that these mutants were also retained inside the cell, but were, in contrast to mutants in recessive NDI, able to form heterotetramers with wt-AQP2. Expression of all three mutants in oocytes indeed revealed that, though all were properly folded functional water channels, AQP2-E258K and AQP2-727delG were retained inside the cell. In addition, balanced co-expression studies in oocytes and polarized MDCK cells revealed that all mutants in dominant NDI formed heterotetramers with wt-AQP2, whereas a mutant in recessive NDI did not. The retention of AQP2-E258K in the Golgi region and its heterotetramerization with wt-AQP2 impaired the further routing of wt-AQP2 to the plasma membrane, resulting in a reduced transmembrane water permeability, thereby providing the molecular basis for dominant NDI in this particular family. AQP2-727delG, which was also retained inside the cell, also appeared to reduce the amount of wt-AQP2 in the plasma membrane. A similar effect was seen in double-transfected MDCK cells. The co-localisation of AQP2-727delG, and consequently wt-AQP2, with LAMP-1, however, indicated that NDI in this particular family was caused by the misrouting of wt-AQP2 to lysosomes instead of the Golgi complex. In contrast to the other mutants in dominant NDI, AQP2-779InsA was almost completely localised in the plasma membrane of oocytes. Since oocytes are not polarized, in contrast to collecting duct cells, it was hypothesized that this mutant might be mistargeted to the basolateral membrane. Indeed, upon expression in MDCK cells, AQP2-779InsA was localised in intracellular vesicles and redistributed to the basolateral membrane upon treatment with the adenylate cyclase activator, forskolin. Upon co-expression with wt-AQP2, both wt-AQP2 and AQP2-779InsA were routed to the basolateral membrane, clearly indicating that in this family mistargeting of wt-AQP2 to the basolateral membrane explained dominant NDI.

In conclusion, our expression studies clearly show that in autosomal recessive NDI, all mutations, which are found in between the first and last transmembrane domain of AQP2, result in misfolded proteins that are retained in the endoplasmic reticulum. In autosomal dominant NDI, however, all mutations, which are found in the C-terminal tail of AQP2, result in functional properly folded AQP2 mutants that pass the endoplasmic reticulum, but are targeted and retained in another subcellular location than the apical membrane. Their ability to form heterotetramers with wt-AQP2 and the consequent mistargeting of wt-AQP2 explains the dominant inheritance of NDI.

Erik-Jan Kamsteeg, Sabine Mulders, Irene Konings and Leo Monnens are acknowledged for their contributions to the research.