1999 European Regional Conference Proceeding

May 12 - 16, 1999

Conference: 1999 European Regional Conference
Title: Routing and function of mutant AQP2 water channels in nephrogenic diabetes insipidus
Author: Deen, Peter M.T.
Institution: University of Nijmegen

In the kidney, which is the major organ in the regulation of water homeostasis, a daily volume of about 180 L of pro-urine is produced. Since our daily-expelled volume of urine is 1-1.5 L, nearly 180 L is reabsorbed and brought back into the blood circulation. This enormous task is accomplished by the basic renal units, the nephrons, of which about million are found in each kidney. Epithelial cells lining different parts of the nephron generate salt and urea gradients by active transport from the pro-urine to the interstitium and thereby establish the driving force for water re-absorption, which is a passive process (needs no energy). In the nephrons, 90% of the pro-urine water is re-gained in the proximal tubules and descending limbs of Henle's, which is attributed to the aquaporin-1 (AQP1) water channel. This protein is expressed in the apical and basolateral membranes of the epithelial cells lining these nephron sections. The re-absorption of the remaining ten percent of pro-urinary is regulated by the anti-diuretic hormone vasopressin (AVP). Upon need of water conservation, AVP is released from the pituitary, binds its vasopressin type-2 (V2) receptors in the basolateral membrane of collecting duct cells and initiates an intracellular cyclic adenosine mono-phosphate (cAMP) signalling cascade, which results in the fusion of AQP2-containing vesicles with the apical membrane. This insertion of AQP2 water channels in the apical membrane renders this membrane water permeable and initiates water re-absorption. A blockade of AVP release from the pituitary (e.g. because of drinking) induces retrieval of AQP2 from the membrane and restores the water-tight characteristic of the apical membrane and the consequent inhibition of further water re-absorption.


In patients suffering from Nephrogenic Diabetes Insipidus (NDI), a disease characterized by the inability of the kidney to concentrate urine in response to AVP, this process is disturbed. NDI can be acquired or inherited. The congenital form is inherited as an X-linked recessive or an autosomal trait, of which the latter can be divided in a recessive and a dominant form. It has been shown that the most prominent form of congenital NDI (X-linked) is caused by mutations in the V2-receptor, which is located at the X-chromosome. The autosomal form is caused by mutations in the AQP2 water channel, which has been localized at chromosome 12. To investigate the molecular defect of AQP2 mutants causing NDI, we studied the function and routing of AQP2 mutants in recessive and dominant NDI in oocytes and epithelial (Madin-Darby Canine Kidney; MDCK) cells. The AQP2 protein traverses the membrane six times and has its N- and C-terminus in the cytosol. Presently, more than 15 mutations in the AQP2 protein have been identified in families with recessive NDI and all these mutations are located in between the first and sixth transmembrane domain. Expression studies in oocytes and mammalian cells have shown that these mutants are sometimes unstable, but are always impaired in their transport from the Endoplasmic Reticulum (ER). In this cell organelle, proteins are synthesized, folded and post-translationally modified (e.g. glycosylation, isomerization), which occurs through binding with ER-resident chaperone proteins. If proteins are mis-folded, they are retained in the ER by the quality control (i.e. extended time of binding to certain chaperones), and are mostly subsequently degraded. Most likely, all AQP2 mutants in recessive NDI are retained in the ER because of mis-folding. With over-expression, however, some of these AQP2 mutants escaped the ER control system and were expressed in the plasma membrane of the cell. Subsequent functional analyses revealed that some mutants could function as water channels. Therefore, restoration of proper folding of these mutants or releave of their impaired transport by chemical chaperons or modulation of natural chaperones could provide a potential cure for these NDI patients.

After synthesis in the ER, wild-type (wt) AQP2 is routed via the Golgi compartment, in which additional post-translational modifications occur, to vesicles located just below the apical membrane. An AQP2 mutant (AQP2-E258K) found in a family with a dominant history of NDI appeared to be retained in the Golgi complex. This mutant was properly folded and therefore escaped the ER quality control, but contains a Golgi retention signal. Identification of this signal is presently under investigation. Expression studies in oocytes indeed revealed its dominant-negative effect on the function of wt-AQP2, in that the water transport (Pf) of oocytes co-expressing wt-AQP2 and AQP2-E258K was significantly lower than that of oocytes expressing an equal amount of wt-AQP2 only, whereas the Pf of oocytes co-expressing wt-AQP2 and AQP2-R187C (a mutant in recessive NDI) was not reduced. Since it was known that AQP1 is expressed as a homotetramer, in which every monomer is a functional unit, we speculated that AQP2-E258K mutant conferred its dominant-negative effect by forming heterotetramers with wt-AQP2, thereby inhibiting its further routing to the plasma membrane, because of its retention signal. Analyses of AQP2 in human and rat kidney revealed that AQP2 is also expressed as a homotetramer. Subsequent analyses of oocytes expressing wt-AQP2 together with AQP2-E258K or AQP2-R187C indeed revealed that AQP2-E258K, but not AQP2-R187C, formed heterotetramers. Therefore, heterotetramerization of the Golgi-retained AQP2 mutant AQP2-E258K with wt-AQP2 and subsequent impairment of further routing to the apical membrane of collecting duct cells likely explains dominant NDI in this particular family. Since the E258K mutant is a functional water channel, a medicine which releases its retainment in the Golgi might provide a means to relieve NDI in these particular patients.

Deen, et al., investigated the function and transport impairments of AQP2 mutants that result in nephrogenic diabetes insipidus (NDI). An AQP2 protein is like a beaded string (the beads are the amino acid residues that comprise the AQP2 protein). Some of the AQP2 protein is gathered within the cell membrane in six distinct coils known as transmembrane domains one through six. Part of the AQP2 protein snakes outside the membrane into the extracellular environment, forming three curves called extracellular loops A, C and E. Part of it snakes inside the cell to form two curves called intracellular loops B and D. Both ends of the AQP2 protein, the N- and C- terminus, are inside the cell with the extracellular loops.

More than 15 mutations in the AQP2 protein have been identified in families with autosomal recessive NDI, and all these mutations are located in between the first and sixth transmembrane domains. All of these mutants are unable to leave a structure within the cell called the endoplasmic reticulum (ER). The AQP2s are synthesized, folded and modified by binding with chaperon proteins within the ER. If the AQP2 is misfolded due to a mutation in its AQP2 gene, it is retained and degraded within the ER. Under some laboratory cell culture conditions, some of these mutants did escape the ER and were able to function as water channels. This led the researchers to suggest that synthetic chemical chaperones could be developed or natural chaperons modified to properly fold the mutant AQP2s. This would allow the AQP2s to leave the ER, reach their final destination and function as water channels.

One AQP2 mutant, AQP2-E258K, is inherited in an autosomal dominant fashion. It can leave the ER, but is retained in the Golgi complex. Deen, et al., discovered how this mutant exerts its dominant effect on normal AQP2s. The researchers' data revealed that AQP2-E258K forms heterotetramers with normal AQP2s thereby not allowing them to leave the Golgi complex. (A heterotetramer is a whole consisting of four parts, not all the same as each other.) Since the E258K mutant is a functional water channel, a medicine which would allow it to leave the Golgi complex might prove a cure for patients whose NDI is cause by this mutation.