Peter M. T. Deen

Autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by mutations in the Aquaporin-2 (AQP2) gene. Analysis of a new family with dominant NDI revealed a single nucleotide deletion (727ΔG) in one AQP2 allele, which encoded an AQP2 mutant with an altered and extended C-terminal tail. When expressed in oocytes, the tetrameric AQP2–727ΔG was retained within the cell. When co-expressed, AQP2–727ΔG, but not a mutant in recessive NDI (AQP2–R187C), formed hetero-oligomers with wild-type (wt) AQP2 and reduced the water permeability of these oocytes, because of a reduced plasma membrane expression of wt-AQP2. Expressed in renal epithelial cells, AQP2–727ΔG predominantly localized to the basolateral membrane and late endosomes/lysosomes, whereas wt-AQP2 was expressed in the apical membrane. Upon co-expressing in these cells, wt-AQP2 and AQP2–727ΔG mainly co-localized to late endosomes/lysosomes. In conclusion, hetero-oligomerization of AQP2–727ΔG with wt-AQP2 and consequent mistargeting of this complex to late endosomes/lysosomes results in absence of AQP2 in the apical membrane, which can explain dominant NDI in this family. Together with other mutants in dominant NDI, our data reveal that a misrouting, instead of a lack of function, is a general mechanism for the ‘loss of function’ phenotype in dominant NDI and visualizes for the first time a mislocalization of a wild-type protein to late endosomes/lysosomes in polarized cells after oligomerization with a mutant protein.

Nocturnal enuresis is one of the most common diagnoses in a pediatric clinic. Recently, linkage analysis revealed a 2-point lod score of 4.2 in 6 families with dominant primary nocturnal enuresis around the aquaporin-2 (AQP2) water channel locus. Since primary nocturnal enuresis is ameliorated by desmopressin, AQP2 expression is increased by desmopressin and AQP2 is essential for concentrating urine, we determined whether a mutation in the AQP2 gene could cause primary nocturnal enuresis in these families. Genomic DNAs of several patients from the 6 families were analyzed for disease causing mutations in the 4 exons of the AQP2 genes. In 1 family a G to A transition in the intron 1 splice donor site was found but it was also found in healthy subjects. In another family a C to T transition in the intron 1 splice acceptor region was identified but it was often found in splice acceptor sites. In 2 families a C to T transition was identified in the coding region of exon 3 but this mutation did not lead to a changed amino acid. Since no mutation in the AQP2 coding sequence was found, while this is essential for involvement in dominant primary nocturnal enuresis, the AQP2 gene is excluded as a candidate for autosomal dominant PNE in these families in which the disease co-segregates with chromosome 12q.

Renal regulation of mammalian water homeostasis is mediated by the aquaporin-1 (AQP1) water channel, which is expressed in the apical and basolateral membranes of proximal tubules and descending limbs of Henle, and aquaporin-2 (AQP2), which is redistributed from intracellular vesicles to the apical membrane (AM) of collecting duct cells with vasopressin. In transfected Madin-Darby canine kidney cells, AQP1 and AQP2 are regulated similarly, which indicates that routing elements reside in their primary sequences. We studied the role of the AQP2 COOH terminus in apical routing and AQP2 shuttling. An AQP1 chimera (AQP1 with an AQP2 tail: AQP1/2-N220) was located only in the AM independent of forskolin treatment. Forskolin increased the apical expression of AQP1 and AQP1/2-N220 less than twofold; that of AQP2 increased more than fourfold with concomitant changes in osmotic water permeabilities. The dimeric AQP2 tail coupled to placental alkaline phosphatase (AQP2-Plap) was retained in intracellular vesicles different from those of homotetrameric wild-type AQP2; the same protein without the AQP2 tail (TMR-Plap) was only expressed in the AM. The study shows that the AQP2 COOH tail is necessary but not sufficient for routing to the AM and suggests that other parts of AQP2 are needed for AQP2 accumulation in intracellular vesicles.

Aquaporin-2 (AQP2) missense mutants in recessive nephrogenic diabetes insipidus (NDI) are all retained in the endoplasmic reticulum (ER), but some could function as water channels. No conclusions could be drawn about the water permeability (P _f) of others, because there was no method for quantifying AQP2 expression in the plasma membrane. We recently developed such a method, which has allowed us to study the functionality of these AQP2 mutants. Immunoblot analysis of membranes of injected oocytes revealed that all mutants (AQP2-G64R, AQP2-N68S, AQP2-T126M, AQP2-A147T, AQP2-R187C, AQP2-S216P) are expressed as unglycosylated and high-mannose glycosylated AQP2. The level of the high-mannose form of AQP2-A147T in the plasma membranes was low, indicating that this mutation has a less severe effect on proper folding. Analysis of P _f values and plasma membrane expression levels reveals that AQP2-N68S, AQP2-R187C and AQP2-S216P are non-functional, AQP2-A147T is as functional as wt-AQP2, while AQP2-T126M and AQP2-G64R retain 20% of the permeability of wt-AQP2. Since G64 is highly conserved between AQPs and expected to form essential interactions with other amino acids within AQP1, the residual functionality of AQP2-G64R is surprising. Our data furthermore indicate that an eventual therapy with chemical chaperones that restores the routing of AQP2 mutants to the apical membrane of collecting ducts cells might relieve NDI in patients encoding AQP2-A147T, and to a lesser extent AQP2-T126M and AQP2-G64R, but not in patients encoding AQP2-N68S, AQP2-R187C or AQP2-S216P.

In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non–p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes.