Cell-Biologic and Functional Analyses of Five New Aquaporin-2 Missense Mutations that Cause Recessive Nephrogenic Diabetes Insipidus

Abstract

Abstract. Mutations in the Aquaporin-2 gene, which encodes a renal water channel, have been shown to cause autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Most AQP2 missense mutants in recessive NDI are retained in the endoplasmic reticulum (ER), but AQP2-T125M and AQP2-G175R were reported to be nonfunctional channels unimpaired in their routing to the plasma membrane. In five families, seven novel AQP2 gene mutations were identified and their cell-biologic basis for causing recessive NDI was analyzed. The patients in four families were homozygous for mutations, encoding AQP2-L28P, AQP2-A47V, AQP2-V71M, or AQP2-P185A. Expression in oocytes revealed that all these mutants, and also AQP2-T125M and AQP2-G175R, conferred a reduced water permeability compared with wt-AQP2, which was due to ER retardation. The patient in the fifth family had a GϾA nucleotide substitution in the splice donor site of one allele that results in an out-of-frame protein.The other allele has a nucleotide deletion (c652delC) and a missense mutation (V194I). The routing and function of AQP2-V194I in oocytes was not different from wt-AQP2; it was therefore concluded that c652delC, which leads to an out-of-frame protein, is the NDI-causing mutation of the second allele. This study indicates that misfolding and ER retention is the main, and possibly only, cell-biologic basis for recessive NDI caused by missense AQP2 proteins. In addition, the reduced single channel water permeability of AQP2-A47V (40%) and AQP2-T125M (25%) might become of therapeutic value when chemical chaperones can be found that restore their routing to the plasma membrane.The aquaporin-2 (AQP2) water channel plays an important role in reabsorption of water in the kidney collecting duct and consequently in concentrating urine (1). Binding of arginine vasopressin (AVP) to its V2 receptor (AVPR2) at the basolateral side of principal cells of collecting ducts leads to an increase of intracellular cAMP levels, resulting in phosphorylation of AQP2 and possibly other proteins, by protein kinase A and subsequent redistribution of AQP2 from subapical storage vesicles to the apical plasma membrane. Driven by the interstitial hypertonicity, water reabsorption and urine concentration is thereby initiated. This process is reversed after dissociation of AVP from its receptor (2,3).Several mutations in the AVPR2 and AQP2 genes have been reported to cause congenital nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to AVP. Mutations in the AVPR2 gene result in NDI that is inherited as an X-linked recessive trait, whereas mutations in the AQP2 gene cause NDI that is inherited as either an autosomal recessive or a dominant trait (1,4 -6,7). Expression studies in oocytes showed that an AQP2 mutant in dominant NDI, AQP2-E258K, was a functional water channel but was retained in the region of the Golgi complex (7). In coexpression studies with wild-...

Lack of Arginine Vasopressin–Induced Phosphorylation of Aquaporin-2 Mutant AQP2-R254L Explains Dominant Nephrogenic Diabetes Insipidus

“…It is interesting that the total and plasma membrane expression as well as the Pf of AQP2-R254L and AQP2-S256A were similar, which indicated that the molecular cause for AQP2-R254L in dominant NDI indeed could be the lack of S256 phosphorylation. As shown for AQP2-S256A (5,20), functional AQP2 mutants in recessive NDI (13,22,23), a retained AQP2 mutant in dominant NDI (AQP2-E258K [7,22]), and also here for wt-AQP2 ( Figure 2), the conferred water permeability for AQP2-R25L was related directly to its expression level ( Figure 2C). …”

“…Selection of G418-resistant clones and immunocytochemistry of transfected MDCK cells were done as described (16). MDCK cell lines that stably expressed wt-AQP2 (wt-10), AQP2-S256A, or green fluorescent protein (GFP)-tagged AQP2 were as described (13,17,18).…”

“…principal cells of the patient, because of the following: Upon expression in oocytes and MDCK cells, AQP2-R254L was a functional water channel but was retained in the cell. However, it was not retained in the ER, as a 32-kD high-mannose glycosylation band, which is characteristic for ER-retained AQP2 mutants in recessive NDI (13,24,25), was not observed ( Figure 2). Also, AQP2-R254L formed heteroligomers with wt-AQP2 in oocytes ( Figure 2) and MDCK cells ( Figure 4C) and impaired the further trafficking of wt-AQP2 to the plasma membrane ( Figure 4B).…”

The laxative effect of bisacodyl is attributable to decreased aquaporin-3 expression in the colon induced by increased PGE2secretion from macrophages

“…However, even if the osmotic pressure in the colon increases via the administration of bisacodyl, as indicated in the above-mentioned mechanism, water transfer from the luminal side of the cells is decreased under the condition of decreased expression level of AQP3 in the colon. This occurs because the rate of water transfer via AQPs depends more on the expression level of AQPs on the cell surface than on the difference in the osmotic pressure between the inside and outside of the cells (21,36). Therefore, it is more appropriate to regard the mechanism of the laxative effect of bisacodyl as inhibited water absorption caused by a decrease in the expression of AQP3 in the colon than increased water secretion caused by an increase in the osmotic pressure in the intestinal tract.…”

Nephrogenic Diabetes Insipidus – The Novelly Potential Therapeutic Drugs

“…Interestingly, most mutations identified in the AQP2 gene in NDI are manifested as mis-routing errors rather than as structural defects that affect their ability in conducting water across the membrane. Some of these mutations, including L22V, L28P, A47V, V71M, T126M, A147T, V168M, P185A, R187C, E258K, and P262L have been characterized in Xenopus oocytes, yeast, and/or mammalian cells, and have been shown to have impaired folding, resulting in recognition by the endoplasmic reticulum (ER) quality control machinery, ubiquitination, and degradation by the 26S proteasome (Boccalandro et al, 2004;de Mattia et al, 2004;Deen et al, 1995;Kamsteeg et al, 2008;Kamsteeg et al, 2009;Levin et al, 2001;Marr et al, 2002;Mulders et al, 1997;Tamarappoo and Verkman, 1998). However, these AQP2 mutants are found to be intrinsically functional water channels when being characterized.…”