Aquaporin-2 Mutants Research Articles

Structural and functional analysis of aquaporin-2 mutants involved in nephrogenic diabetes insipidus

Aquaporins are water channels found in the cell membrane, where they allow the passage of water molecules in and out of the cells. In the kidney collecting duct, arginine vasopressin-dependent trafficking of aquaporin-2 (AQP2) fine-tunes reabsorption of water from pre-urine, allowing precise regulation of the final urine volume. Point mutations in the gene for AQP2 may disturb this process and lead to nephrogenic diabetes insipidus (NDI), whereby patients void large volumes of highly hypo-osmotic urine. In recessive NDI, mutants of AQP2 are retained in the endoplasmic reticulum due to misfolding. Here we describe the structural and functional characterization of three AQP2 mutations associated with recessive NDI: T125M and T126M, situated close to a glycosylation site and A147T in the transmembrane region. Using a proteoliposome assay, we show that all three mutants permit the transport of water. The crystal structures of T125M and T126M together with biophysical characterization of all three mutants support that they retain the native structure, but that there is a significant destabilization of A147T. Our work provides unique molecular insights into the mechanisms behind recessive NDI as well as deepens our understanding of how misfolded proteins are recognized by the ER quality control system.

Molecular Characterization of an Aquaporin-2 Mutation Causing Nephrogenic Diabetes Insipidus.

The aquaporin 2 (AQP2) plays a critical role in water reabsorption to maintain water homeostasis. AQP2 mutation leads to nephrogenic diabetes insipidus (NDI), characterized by polyuria, polydipsia, and hypernatremia. We previously reported that a novel AQP2 mutation (G215S) caused NDI in a boy. In this study, we aimed to elucidate the cell biological consequences of this mutation on AQP2 function and clarify the molecular pathogenic mechanism for NDI in this patient. First, we analyzed AQP2 expression in Madin-Darby canine kidney (MDCK) cells by AQP2-G215S or AQP2-WT plasmid transfection and found significantly decreased AQP2-G215S expression in cytoplasmic membrane compared with AQP2-WT, independent of forskolin treatment. Further, we found co-localization of endoplasmic reticulum (ER) marker (Calnexin) with AQP2-G215S rather than AQP2-WT in MDCK cells by immunocytochemistry. The functional analysis showed that MDCK cells transfected with AQP2-G215S displayed reduced water permeability compared with AQP2-WT. Visualization of AQP2 structure implied that AQP2-G215S mutation might interrupt the folding of the sixth transmembrane α-helix and/or the packing of α-helices, resulting in the misfolding of monomer and further impaired formation of tetramer. Taken together, these findings suggested that AQP2-G215S was misfolded and retained in the ER and could not be translocated to the apical membrane to function as a water channel, which revealed the molecular pathogenic mechanism of AQP2-G215S mutation and explained for the phenotype of NDI in this patient.

Novel AQP2 Mutations and Clinical Characteristics in Seven Chinese Families With Congenital Nephrogenic Diabetes Insipidus.

ObjectiveMutations in AQP2 (aquaporin-2) lead to rare congenital nephrogenic diabetes insipidus (NDI), which has been limitedly studied in Chinese population.MethodsTwenty-five subjects from seven families with NDI in a department (Beijing, PUMCH) were screened for AQP2 mutations. Clinical characteristics were described and genotype-phenotype correlation analysis was performed.ResultsWe identified 9 AQP2 mutations in 13 patients with NDI, including 3 novel AQP2 mutations (p.G165D, p.Q255RfsTer72 and IVS3-3delC). Missense mutations were the most common mutation type, followed by splicing mutations, and frameshift mutations caused by small deletion or insertion. The onset-age in our patients was younger than 1 year old. Common manifestations included polydipsia, polyuria (7/7) and intermittent fever (6/7). Less common presentations included short stature (3/7) and mental impairment (1/7). High osmotic hypernatremia and low osmotic urine were the main biochemical features. Dilation of the urinary tract was a common complication of NDI (3/6). Level of serum sodium in NDI patients with compound het AQP2 mutations was higher than non-compound het mutations.ConclusionIn the first and largest case series of NDI caused by AQP2 mutation in Chinese population, we identified 9 AQP2 mutations, including 3 novel mutations. Phenotype was found to correlate with genotypes, revealed by higher level of serum sodium in patients with compound het AQP2 mutations than non-compound het mutations. This knowledge broadens genotypic and phenotypic spectrum for rare congenital NDI and provided basis for studying molecular biology of AQP2.

AQP2: Mutations Associated with Congenital Nephrogenic Diabetes Insipidus and Regulation by Post-Translational Modifications and Protein-Protein Interactions.

As a rare hereditary disease, congenital nephrogenic diabetes insipidus (NDI) is clinically characterized by polyuria with hyposthenuria and polydipsia. NDI results from collecting duct principal cell hyporesponsiveness or insensitivity to the antidiuretic action of arginine vasopressin (AVP). The principal cell-specific water channel aquaporin-2 (AQP2) plays an essential role in water reabsorption along osmotic gradients. The capacity to accumulate AQP2 in the apical plasma membrane in response to decreased fluid volume or increased plasma osmolality is critically regulated by the antidiuretic hormone AVP and its receptor 2 (AVPR2). Mutations in AVPR2 result in X-linked recessive NDI, the most common form of inherited NDI. Genetic defects in AQP2 cause autosomal recessive or dominant NDI. In this review, we provide an updated overview of the genetic and molecular mechanisms of congenital NDI, with a focus on the potential disease-causing mutations in AVPR2 and AQP2, the molecular defects in the AVPR2 and AQP2 mutants, post-translational modifications (i.e., phosphorylation, ubiquitination, and glycosylation) and various protein-protein interactions that regulate phosphorylation, ubiquitination, tetramerization, trafficking, stability, and degradation of AQP2.

Direct effect of protein kinase A on four putative phosphorylation sites of aquaporin 2 in vitro

The water channel aquaporin 2 (AQP2) has four phosphorylation sites at Ser256, Ser261, Ser264, and Ser269 in the C-terminus and these sites are important for AQP2 bioactivity. However, the exact role of each phosphorylation site still remains unclear. In this study, we generated unique AQP2 mutants in which we eliminated three phosphorylation sites but maintained only one site at the C-terminal end. The AQP2 phosphorylation of each single site by protein kinase A (PKA) was examined by in vitro translation and 32P incorporation. The ability of AQP2 trafficking to the cell membrane was evaluated by cell surface biotinylation. Among the four phosphorylation sites, AQP2 mutant with only S256 preserved the most ability of AQP2 to cell membrane expression. The AQP2 water permeability was measured in oocyte. Ser256 is the most important site for AQP2 function. Interestingly, Ser261 and Ser264 significantly inhibit AQP2 activity. Ser269 slightly but not statistically reduced AQP2 activity. Our data suggest that the four phosphorylation sites execute differential roles in concert in AQP2 functional regulation. AQP2 activity regulated by phosphorylation at Ser256 can be counterbalanced by phosphorylation at Ser261 and Ser264.

Molecular characterization of an aquaporin-2 mutation causing a severe form of nephrogenic diabetes insipidus.

The water channel aquaporin 2 (AQP2) is responsible for water reabsorption by kidney collecting duct cells. A substitution of amino acid leucine 137 to proline in AQP2 (AQP2-L137P) causes Nephrogenic Diabetes Insipidus (NDI). This study aimed to determine the cell biological consequences of this mutation on AQP2 function. Studies were performed in HEK293 and MDCK type I cells, transfected with wildtype (WT) AQP2 or an AQP2-L137P mutant. AQP2-L137P was predominantly detected as a high-mannose form of AQP2, whereas AQP2-WT was observed in both non-glycosylated and complex glycosylated forms. In contrast to AQP2-WT, the AQP2-L137P mutant did not accumulate on the apical plasma membrane following stimulation with forskolin. Ubiquitylation of AQP2-L137P was different from AQP2-WT, with predominance of non-distinct protein bands at various molecular weights. The AQP2-L137P mutant displayed reduced half-life compared to AQP2-WT. Treatment of cells with chloroquine increased abundance of AQP2-WT, but not AQP2-L137P. In contrast, treatment with MG132 increased abundance of AQP2-L137P but not AQP2-WT. Xenopus oocytes injected with AQP2-WT had increased osmotic water permeability when compared to AQP2-L137P, which correlated with lack of the mutant form in the plasma membrane. From the localization of the mutation and nature of the substitution it is likely that AQP2-L137P causes protein misfolding, which may be responsible for the observed functional defects. The data suggest that the L137P mutation results in altered AQP2 protein maturation, increased AQP2 degradation via the proteasomal pathway and limited plasma membrane expression. These combined mechanisms are likely responsible for the phenotype observed in this class of NDI patients.

A versatile aquaporin-2 cell system for quantitative temporal expression and live cell imaging.

Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.

Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations.

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking.

Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd(2+)-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.

Structural characterization of mixed wt/mutant aquaporin‐2 tetramers using dual‐colored fluorescent subunit counting (1133.1)

Mutations in Aquaporin‐2 (AQP2) induce nephrogenic diabetes insipidus, a water reabsorption defect of the kidney. Using co‐immunoprecipitation and functional assays, we have recently shown that (at least) some mild recessive (rec) AQP2 mutants can adequately associate to its wild‐type (wt) counterpart to generate fully functional wt/rec heterotetramers. To better characterize actual subunit distributions within wt/rec AQP2 populations, we have developed a dual‐colored fluorescent subunit counting approach in TIRF condition, in order to identify and quantify individual monomers through bleaching of linked fluorophores. Assays performed on both Xenopus oocyte and transfected cell line have confirmed that mild mutations (K228E, D150E and V24A), similar to wt‐AQP2, adequately oligomerize in tetramer, unlike the severe R187C mutant which only displays monomeric structures. In dual transfections (sfGFP‐wt‐AQP2 + RFP‐rec‐AQP2), we also confirm the presence of mixed wt/rec tetramers for mild mutations, but not with R187C, which remains monomeric. Ultimately, we aim at describing subunit distributions within wt/rec tetramers in order to evaluate the properties, limitations and functional outcome of such heteromeric associations.

Novel AQP2 mutation causing congenital nephrogenic diabetes insipidus: challenges in management during infancy

Congenital nephrogenic diabetes insipidus (NDI) is a rare inherited disorder, mostly caused by AVPR2 mutations. Less than 10% of cases are due to mutations in the aquaporin-2 (AQP2) gene. Diagnosis and management of this condition remain challenging especially during infancy. Here, we report two unrelated patients, a 6-month-old Thai boy and a 5-year-old Emirati girl, with a history of failure to thrive, chronic fever, polydipsia, and polyuria presented in early infancy. The results of water deprivation test were compatible with a diagnosis of NDI. The entire coding regions of the AVPR2 and AQP2 gene were amplified by polymerase chain reaction and sequenced. Patient 1 was homozygous for a novel missense AQP2 mutation p.G96E, inherited from both parents. Patient 2 harbored a previously described homozygous p.T126M mutation in the AQP2 gene. Both patients were treated with a combination of thiazide diuretics and amiloride. Patient 1 developed paradoxical hyponatremia and severe dehydration 2 weeks after medical treatment began. In conclusion, we report a novel mutation of the AQP2 gene and highlight an important role of genetic testing for definite diagnosis. Vigilant monitoring of the fluid status and electrolytes after beginning the therapy is mandatory in infants with NDI.

Hereditary nephrogenic diabetes insipidus in Japanese patients: analysis of 78 families and report of 22 new mutations in AVPR2 and AQP2

Familial form of nephrogenic diabetes insipidus (NDI) is a rare hereditary disease caused by arginine vasopressin type 2 receptor (AVPR2) or water channel aquaporin 2 (AQP2) gene mutations. It is speculated that 90% of NDI families carry disease-causing mutations in AVPR2 and 10% carry the mutations in AQP2; however, these percentages have not been supported by actual data. It is also unknown whether these percentages vary in different ethnic groups. Gene mutation analyses were performed for 78 Japanese NDI families. All exons and intron-exon boundaries of the AVPR2 and AQP2 genes were directly sequenced. A total of 62 families (79%) carried disease-causing mutations in AVPR2, while nine families (12%) carried mutations in AQP2. We identified 22 novel putatively disease-causing mutations (19 in AVPR2 and 3 in AQP2). Regarding AVPR2, 52 disease-causing mutations were identified. Among them, missense mutations were most common (54%), followed by deletion mutations. In the 64 women who had monoallelic disease-causing AVPR2 mutations, 16 (25%) had NDI symptoms, including 4 complete NDI subjects. Regarding AQP2, 9 disease-causing mutations were identified in nine families. Two missense mutations and one deletion mutation showed a recessive inheritance, while one missense mutation and five small deletion mutations in the C-terminus of AQP2 showed a dominant inheritance. Most Japanese NDI families carry disease-causing mutations in AVPR2 and 12% carry mutations in AQP2. A total of 22 novel putatively disease-causing mutations were identified. The relatively high occurrence of symptomatic carriers of AVPR2 mutations needs attention.

Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus

Mutations in the aquaporin-2 (AQP2) gene cause nephrogenic diabetes insipidus (NDI), a renal disorder characterized by polyuria due to a lacking antidiuretic response to vasopressin. While most AQP2 mutants in recessive NDI are misfolded and retained in the endoplasmic reticulum, AQP2-P262L in NDI was impaired in its vasopressin-dependent translocation from vesicles to the plasma membrane. Vasopressin-induced translocation of AQP2 coincides with AQP2 phosphorylation at S256, S264 and T269 and dephosphorylation at S261. Since P262 lies adjacent to S261, we tested whether a changed phosphorylation could underlie AQP-P262L missorting in NDI. In polarized cells, AQP2-P262L expressed as a double 29/30 kDa band, whereas wt-AQP2 expressed only as a 29 kDa band. Phosphatase treatment revealed that the 30 kDa AQP2-P262L band was due to changed phosphorylation. The use of newly developed phospho-specific antibodies showed that forskolin not only increased pS256 and pT269, but, in contrast to wt-AQP2, also pS261 in AQP2-P262L. The expression of AQP2-P262L proteins in which S261 phosphorylation was prevented (S261A), however, was still missorted to vesicles/basolateral membrane, despite the absence of the 30 kDa band. Together, our data reveal that vasopressin induces instead of reduces the phosphorylation of S261 in AQP2-P262L, but it remains to be established whether the changed phosphorylation causes its missorting in NDI.

Mutations in the AVPR2, AVP-NPII, and AQP2 genes in Turkish patients with diabetes insipidus

The aim of this study was to identify mutations in three different genes, the arginine-vasopressin-neurophysin II (AVP-NPII) gene, the arginine-vasopressin receptor 2 (AVPR2) gene, and the vasopressin-sensitive water channel aquaporin-2 (AQP2) gene in Turkish patients affected by central diabetes insipidus or nephrogenic diabetes insipidus. This study included 15 patients from unrelated families. Prospective clinical data were collected for all patients including the patients underwent a water deprivation-desmopressin test. The coding regions of the AVPR2, AQP2, and AVP-NPII genes were amplified by polymerase chain reaction and submitted to direct sequence analysis. Of the 15 patients with diabetes insipidus referred to Gulhane Military Medical Academy, Department of Endocrinology and Metabolism, eight patients have AVPR2 mutations, five patients have AQP2 mutations and two patients have AVP-NPII mutations. Of the patients, which have AVPR2 mutations, one is compound heterozygous for AVPR2 gene. Seven of these mutations are novel. Comparison of the clinical outcomes of these mutations may facilitate in understanding the functions of AVP-NPII, AQP2, and AVPR2 genes in future studies.

Differential, Phosphorylation Dependent Trafficking of AQP2 in LLC-PK1 Cells

The kidney maintains water homeostasis by modulating aquaporin 2 (AQP2) on the plasma membrane of collecting duct principal cells in response to vasopressin (VP). VP mediated phosphorylation of AQP2 at serine 256 is critical for this effect. However, the role of phosphorylation of other serine residues in the AQP2 C-terminus is less well understood. Here, we examined the effect of phosphorylation of S256, S261 and S269 on AQP2 trafficking and association with recycling pathway markers. We used LLC-PK1 cells expressing AQP2(S-D) or (S-A) phospho mutants and a 20°C cold block, which allows endocytosis to continue, but prevents protein exit from the trans Golgi network (TGN), inducing formation of a perinuclear AQP2 patch. AQP2-S256D persists on the plasma membrane during cold block, while wild type AQP2, AQP2-S256A, S261A, S269A and S269D are internalized and accumulate in the patch. Development of this patch, a measure of AQP2 internalization, was most rapid with AQP2-S256A, and slowest with S261A and S269D. AQP2-S269D exhibited a biphasic internalization profile with a significant amount not internalized until 150 minutes of cold block. After rewarming to 37°C, wt AQP2, AQP2-S261A and AQP2-S269D rapidly redistributed throughout the cytoplasm within 20 minutes, whereas AQP2-S256A dissipated more slowly. Colocalization of AQP2 mutants with several key vesicular markers including clathrin, HSP70/HSC70, EEA, GM130 and Rab11 revealed no major differences. Overall, our data provide evidence supporting the role of S256 and S269 in the maintenance of AQP2 at the cell surface and reveal the dynamics of internalization and recycling of differentially phosphorylated AQP2 in cell culture.

Stimulating Effect of External Myo-Inositol on the Expression of Mutant Forms of Aquaporin 2

Myo-inositol (MI; hexahydroxycyclohexane, C(6)H(6)O(12)) is a small neutral molecule used as a compatible osmolyte in the kidney medulla. At high concentrations, MI appears to act as a chemical chaperone and was shown to promote plasma membrane expression of the impaired cystic fibrosis chloride channel (Delta508-CFTR). In the present study, we measured whether MI could increase expression of two human aquaporin 2 (AQP2) mutants which were recently identified as causing nephrogenic diabetes insipidus (NDI). Both proteins (D150E and G196D) were expressed in Xenopus laevis oocytes, but only D150E displayed an increase in oocyte water permeability (P (f)). Adding 5 mM MI to the bathing solution for 24 h produced a 50% increase in the D150E-associated P (f), while it had no effect on noninjected oocytes or on oocytes expressing wt-AQP2 or G196D. Western blots performed on purified plasma membrane preparations confirmed that MI increased the amount of D150E present at the plasma membrane, while G196D was always undetectable. X. laevis oocytes are remarkably impermeable to MI, and the effect of MI on D150E expression does not require the presence of intracellular MI. The effect of external MI was dose-dependent (K (0.5) was 130 microM) and specific with respect to other forms of inositols. Further studies on a second group of AQP2 mutants causing NDI showed that K228E activity was similarly stimulated by MI, while V71M, A70D and S256L were not. It is concluded that physiological concentrations of extracellular MI can stimulate the expression of a specific subgroup of AQP2 mutants.

Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin-Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein-protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t(1/2) = 5.1 h and t(1/2) = 4.4 h, respectively) compared to WT-AQP2 (t(1/2) = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein-protein interactions.

Repulsion between Lys258 and upstream arginines explains the missorting of the AQP2 mutant p.Glu258Lys in nephrogenic diabetes insipidus

Regulation of body water homeostasis occurs by the vasopressin-dependent sorting of aquaporin-2 (AQP2) water channels to and from the apical membrane of renal principal cells. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease that renders the kidney unresponsive to vasopressin, resulting in polyuria and polydipsia. The AQP2 mutant c.772G>A; p.Glu258Lys (AQP2-E258K) causes dominant NDI by oligomerizing with wild-type AQP2 and missorting of this AQP2 complex to multivesicular bodies instead of the apical membrane. The motif causing this missorting of AQP2-E258K was identified here. Functional analyses and plasma membrane expression studies of truncation mutants in oocytes revealed that AQP2-E258K shortened to Leu259 is still intracellular retained. Alanine scanning and glutamic acid to arginine exchanges revealed increased function and plasma membrane expression for AQP2-E258K mutants with the following additional changes: Leu259Ala, Arg252Glu, Arg253Glu, or Arg252Ala-Arg254Ala, or for the AQP2 mutant p.Glu258Ala, indicating that the motif RRRxxxK(258)L confers AQP2-E258K retention. Fusion of this motif to aquaporin-1 also resulted in missorting of that water channel, indicating that this retention motif is transferable. In conclusion, our data reveal that the RRRxxxKL motif and repulsion between K258 and the arginine-triplet within this motif are the primary cause of missorting of AQP2-E258K in NDI.

Characterization of D150E and G196D aquaporin-2 mutations responsible for nephrogenic diabetes insipidus: importance of a mild phenotype

Aquaporin-2 (AQP2) is a water channel responsible for the final water reabsorption in renal collecting ducts. Alterations in AQP2 function induce nephrogenic diabetes insipidus (NDI), a condition characterized by severe polyuria and polydipsia. Three patients affected with severe NDI, who were compound heterozygous for the AQP2 mutations D150E and G196D, are presented here along with a mildly affected D150E homozygous patient from another family. Using Xenopus oocytes as an expression system, these two mutations (G196D and D150E) were compared with the wild-type protein (AQP2-wt) for functional activity (water flux analysis), protein maturation, and plasma membrane targeting. AQP2-wt induces a major increase in water permeability (P(f) = 47.4 +/- 12.2 x 10(-4) cm/s) whereas D150E displays intermediate P(f) values (P(f) = 12.5 +/- 3.0 x 10(-4) cm/s) and G196D presents no specific water flux, similar to controls (P(f) = 2.1 +/- 0.8 x 10(-4) cm/s and 2.2 +/- 0.7 x 10(-4) cm/s, respectively). Western blot and immunocytochemical evaluations show protein targeting that parallels activity levels with AQP2-wt adequately targeted to the plasma membrane, partial targeting for D150E, and complete sequestration of G196D within intracellular compartments. When coinjecting AQP2-wt with mutants, no (AQP2-wt + D150E) or partial (AQP2-wt + G196D) reduction of water flux were observed compared with AQP2-wt alone, whereas complete loss of function was found when both mutants were coinjected. These results essentially recapitulate the clinical profiles of the family members, showing a typical dominant negative effect when G196D is coinjected with either AQP2-wt or D150E but not between AQP2-wt and D150E mutant.

A cell culture system for the induction of Mallory bodies: Mallory bodies and aggresomes represent different types of inclusion bodies

Mallory bodies (MBs) represent keratin-rich inclusion bodies observed in human alcoholic liver disease and in several chronic non-alcoholic liver diseases. The mechanism of their formation and their relationship to other inclusion bodies such as aggresomes is incompletely understood. We could induce keratin aggregates typical of MBs in cultured clone 9 rat hepatocytes by transgenic expression of wild-type and mutant aquaporin2 or alpha1-antitrypsin and under various forms of other cellular stress. By immunocytochemical analysis, p62 and poly-ubiquitin, components of classical MBs, could be demonstrated in the keratin aggregates of clone 9 hepatocytes. In addition, histone deacetylase 6, a microtubule-associated deacetylase, was identified as a novel component of the keratin aggregates. Thus, together with their ultrastructural appearance as randomly oriented, organelle-free aggregates of keratin filaments, the keratin aggregates in clone 9 hepatocytes correspond to MBs. An imbalance in keratin 8 to 18 with very low levels of keratin 18 appears to be the underlying cause for their formation. The formation of MBs was microtubule-dependent although not depending on the activity of histone deacetylase 6. Forskolin-induced MBs in clone 9 hepatocytes were reversible structures which disappeared upon drug withdrawal. The MBs were not related to aggresomes since overexpressed misfolded transgenic proteins were undetectable in the keratin aggregates and no vimentin fiber cage was detectable, both of which represent hallmarks of aggresomes. Thus, cultured clone 9 hepatocytes are a useful system to study further aspects of the pathobiology of MBs.