Renal expression of exchange protein directly activated by cAMP (Epac) 1 and 2.

Contains fulltext : 71173.pdf (publisher's version ) (Open Access)In the kidney, many physiological processes of ion transport and cellular proliferation are mediated via cAMP, which classically activates protein kinase A (PKA). Recently, however, two new cAMP targets, the exchange protein directly activated by cAMP (Epac) 1 and 2, were identified, which mediate alternative pathways to PKA. To investigate their renal expression, antibodies specifically recognizing Epac1 and Epac2 were generated and used in rat immunohistochemistry with antibodies recognizing aquaporin-1 (AQP1), Tamm-Horsfall protein, Calbindin-D(28K), and AQP2 to mark proximal tubules (PT)/thin descending limbs of Henle's loop (tDLH), thick ascending limbs of Henle's loop (TAL), distal convoluted tubule/connecting tubule (DCT/CNT), and the collecting duct (CD) principal cells, respectively. Epac1 and Epac2 were expressed at the brush border of PT cells but were absent from tDLH cells. In the TAL, Epac1 and Epac2 were expressed throughout the cells with some confinement toward the apical membrane. In the DCT/CNT, Epac1 was confined to the apical region of the cells, whereas Epac2 was mainly expressed in the apical and basolateral regions. In the CD, a dispersed Epac1 expression was found in intercalated cells only (cortical CD), principal and intercalated cells [outer medullary CD (OMCD)], and mainly AQP2-negative cells in the inner medullary CD (IMCD). In contrast, Epac2 expression was at the apical and basolateral membrane of cortical principal cells, dispersed and apical in the OMCD, and in all cells of the IMCD. A similar distribution for Epac1/2 was found in the human kidney. The observed expression in different tubular segments suggests a major role for Epac 1/2 in tubular transport physiology and cellular proliferation

Lack of arginine vasopressin-induced phosphorylation of aquaporin-2 mutant AQP2-R254L explains dominant nephrogenic diabetes insipidus.

Contains fulltext : 48640.pdf (publisher's version ) (Open Access)Water homeostasis in humans is regulated by vasopressin, which induces the translocation of homotetrameric aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells. For this process, phosphorylation of AQP2 at S256 by cAMP-dependent protein kinase A is thought to be essential. Mutations in the AQP2 gene cause recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Here, a family in which dominant NDI was caused by an exchange of arginine 254 by leucine in the intracellular C terminus of AQP2 (AQP2-R254L), which destroys the protein kinase A consensus site, was identified. Expressed in oocytes, AQP2-R254L appeared to be a functional water channel but was impaired in its transport to the cell surface to the same degree as AQP2-S256A, which mimics nonphosphorylated AQP2. In polarized renal cells, AQP2-R254L was retained intracellularly and was distributed similarly as AQP2-S256A or wild-type AQP2 in unstimulated cells. Upon co-expression in MDCK cells, AQP2-R254L interacted with and retained wild-type AQP2 in intracellular vesicles. Furthermore, AQP2-R254L had a low basal phosphorylation level, which was not increased with forskolin, and mimicking constitutive phosphorylation in AQP2-R254L with the S256D mutation shifted its expression to the basolateral and apical membrane. These data indicate that dominant NDI in this family is due to a R254L mutation, resulting in the loss of arginine vasopressin-mediated phosphorylation of AQP2 at S256, and illustrates the in vivo importance of phosphorylation of AQP2 at S256 for the first time

p.R254Q mutation in the aquaporin-2 water channel causing dominant nephrogenic diabetes insipidus is due to a lack of arginine vasopressin-induced phosphorylation.

Contains fulltext : 79718.pdf (publisher's version ) (Closed access)Vasopressin regulates human water homeostasis by re-distributing homotetrameric aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells, a process in which phosphorylation of AQP2 at S256 by cAMP-dependent protein kinase A (PKA) is thought to be essential. Dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by AQP2 gene mutations. Here, we investigated a reported patient case of dominant NDI caused by a novel p.R254Q mutation. Expressed in oocytes, AQP2-p.R254Q appeared to be a functional water channel, but was impaired in its transport to the cell surface to the same degree as AQP2-p.S256A, which mimics non-phosphorylated AQP2. In polarized MDCK cells, AQP2-p.R254Q was retained and was distributed similarly to that of unstimulated wt-AQP2 or AQP2-p.S256A. Upon co-expression, AQP2-p.R254Q interacted with, and retained wt-AQP2 in intracellular vesicles. In contrast to wild-type AQP2, forskolin did not increase AQP2-p.R254Q phosphorylation at S256 or its translocation to the apical membrane. Mimicking constitutive phosphorylation in AQP2-p.R254Q with the p.S256D mutation, however, rescued its apical membrane expression. These date indicate that a lack of S256 phosphorylation is the sole cause of dominant NDI here, and thereby, p.R254Q is a loss of function instead of a gain of function mutation in dominant NDI

A novel mechanism in recessive nephrogenic diabetes insipidus: wild-type aquaporin-2 rescues the apical membrane expression of intracellularly retained AQP2-P262L.

Contains fulltext : 57229.pdf (publisher's version ) (Closed access)Vasopressin regulates water homeostasis through insertion of homotetrameric aquaporin-2 (AQP2) water channels in the apical plasma membrane of renal cells. AQP2 mutations cause recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Until now, all AQP2 mutants in recessive NDI were shown to be misfolded, retained in the endoplasmic reticulum (ER) and unable to interact with wild-type (wt)-AQP2, whereas AQP2 mutants in dominant NDI are properly folded and interact with wt-AQP2, but, due to the mutation, cause missorting of the wt-AQP2/mutant complex. Here, patients of two families with recessive NDI appeared compound heterozygotes for AQP2-A190T or AQP2-R187C mutants, together with AQP2-P262L. As mutations in the AQP2 C-tail, where P262 resides, usually cause dominant NDI, the underlying cell-biological mechanism was investigated. Upon expression in oocytes, AQP2-P262L was a properly folded and functional aquaporin in contrast to the classical mutants, AQP2-R187C and AQP2-A190T. Expressed in polarized cells, AQP2-P262L was retained in intracellular vesicles and did not localize to the ER. Upon co-expression, however, AQP2-P262L interacted with wt-AQP2, but not with AQP2-R187C, resulting in a rescued apical membrane expression of AQP2-P262L. In conclusion, our study reveals a novel cellular phenotype in recessive NDI in that AQP2-P262L acts as a mutant in dominant NDI, except for that its missorting is overruled by apical sorting of wt-AQP2. Also, it demonstrates for the first time that the recessive inheritance of a disease involving a channel can be due to two cell-biological mechanisms

Novel PTEN mutations in patients with Cowden disease: absence of clear genotype-phenotype correlations.

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Cell-biologic and functional analyses of five new Aquaporin-2 missense mutations that cause recessive nephrogenic diabetes insipidus.

Item does not contain fulltextMutations 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 G>A 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