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      Characterization of Two Novel Missense Mutations in the AQP2 Gene Causing Nephrogenic Diabetes Insipidus

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          Here, we report the aquaporin 2 (AQP2) mutational analysis of a patient with nephrogenic diabetes insipidus heterozygote due to two novel missense mutations. Direct sequencing of DNA in the male patient revealed that he was compound heterozygote for two mutations in the AQP2 gene: a thymine-to-adenine transversion at position 450 (c.450T>A) in exon 2 and a guanine-to-thymine at nucleotide position 643 (c.643G>T) in exon 4. The double heterozygous 450T>A and 643G>T transversion causes the amino acid substitution D150E and G215C. Direct sequencing of exons 2 and 4 of the AQP2 gene from each of the parents revealed that the c.450T>A mutation was inherited from the father while the c.643G>T mutation was inherited from the mother. Analysis of AQP2 excretion demonstrated that no AQP2 was detectable in the urine of the proband, whereas normal AQP2 levels were measured in both parents. When expressed in renal cells, both proteins were retarded in the endoplasmic reticulum and no redistribution was observed after forskolin stimulation. Of note, homology modeling revealed that the two mutations involve two highly conserved residues providing important clues about the role of the wt residues in AQP2 stability and function.

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          Most cited references 21

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          Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine.

          Concentration of urine in mammals is regulated by the antidiuretic hormone vasopressin. Binding of vasopressin to its V2 receptor leads to the insertion of water channels in apical membranes of principal cells in collecting ducts. In nephrogenic diabetes insipidus (NDI), the kidney fails to concentrate urine in response to vasopressin. A male patient with an autosomal recessive form of NDI was found to be a compound heterozygote for two mutations in the gene encoding aquaporin-2, a water channel. Functional expression studies in Xenopus oocytes revealed that each mutation resulted in nonfunctional water channel proteins. Thus, aquaporin-2 is essential for vasopressin-dependent concentration of urine.
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            Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones.

            Five single-point aquaporin-2 (AQP2) mutations that cause non-X-linked nephrogenic diabetes insipidus (NDI) were characterized to establish the cellular defect and to develop therapeutic strategies. In Xenopus oocytes expressing AQP2 cRNAs, single-channel water permeabilities of mutants L22V, T126M, and A147T were similar to that of wild-type AQP2, whereas R187C and C181W were nonfunctional. In [35S]methionine pulse-chase experiments in transiently transfected CHO cells, half-times for AQP2 degradation were approximately 4 h for wild-type AQP2 and L22V, and mildly decreased for T126M (2.7 h), C181W (2.4 h), R187C (2.0 h), and A147T (1.8 h). Immunofluorescence showed three distinct AQP2-staining patterns: plasma membrane and endosomal staining (wild-type, L22V), endoplasmic reticulum (ER) staining (T126M > A147T approximately R187C), or a mixed pattern of reticular and perinuclear vesicular staining. Immunoblot of fractionated vesicles confirmed primary ER localization of T126M, R187C, and A147T. To determine if the AQP2-trafficking defect is correctable, cells were incubated with the "chemical chaperone" glycerol for 48 h. Immunoblot showed that glycerol produced a nearly complete redistribution of AQP2 (T126M, A147T, and R187C) from ER to membrane/endosome fractions. Immunofluorescence confirmed the cellular redistribution. Redistribution of AQP2 mutants was also demonstrated in transfected MDCK cells, and using the chaperones TMAO and DMSO in place of glycerol in CHO cells. Water permeability measurements indicated that functional correction was achieved. These results indicate defective mammalian cell processing of mutant AQP2 water channels in NDI, and provide evidence for pharmacological correction of the processing defect by chemical chaperones.
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              Molecular biology of hereditary diabetes insipidus.

              The identification, characterization, and mutational analysis of three different genes-the arginine vasopressin gene (AVP), the arginine vasopressin receptor 2 gene (AVPR2), and the vasopressin-sensitive water channel gene (aquaporin 2 [AQP2])-provide the basis for understanding of three different hereditary forms of "pure" diabetes insipidus: Neurohypophyseal diabetes insipidus, X-linked nephrogenic diabetes insipidus (NDI), and non-X-linked NDI, respectively. It is clinically useful to distinguish two types of hereditary NDI: A "pure" type characterized by loss of water only and a complex type characterized by loss of water and ions. Patients who have congenital NDI and bear mutations in the AVPR2 or AQP2 genes have a "pure" NDI phenotype with loss of water but normal conservation of sodium, potassium, chloride, and calcium. Patients who bear inactivating mutations in genes (SLC12A1, KCNJ1, CLCNKB, CLCNKA and CLCNKB in combination, or BSND) that encode the membrane proteins of the thick ascending limb of the loop of Henle have a complex polyuro-polydipsic syndrome with loss of water, sodium, chloride, calcium, magnesium, and potassium. These advances provide diagnostic and clinical tools for physicians who care for these patients.

                Author and article information

                Nephron Physiol
                Nephron Physiology
                S. Karger AG
                February 2007
                28 December 2006
                : 105
                : 3
                : p33-p41
                aDepartment of Biochemistry and Biomedical Technologies, University Federico II – CEINGE, Naples, bDepartment of General and Environmental Physiology, cCentro di Eccellenza in Genomica Comparata, University of Bari, Bari, dClinica Pediatrica, University of Padua, Padua, eDepartment of Biomedical Science, Division of Nephrology, University of Foggia, Foggia, Italy; fDepartment of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, Calif., USA
                98136 Nephron Physiol 2007;105:p33–p41
                © 2007 S. Karger AG, Basel

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                Page count
                Figures: 5, Tables: 1, References: 33, Pages: 1
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                Original Paper

                Cardiovascular Medicine, Nephrology

                Polyuria, Nephrogenic diabetes insipidus, Aquaporin


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