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Rescue of tight junctional localization of a claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells.

ABSTRACT: Magnesium ion (Mg²⁺) is paracellularly reabsorbed through claudin-16 (CLDN16) in the thick ascending limb (TAL) of Henle's loop in the kidney. Genetic disorders of CLDN16 cause mislocalization of CLDN16, resulting in hypomagnesemia. There is no effective treatment for hypomagnesemia except for magnesium administration. Here, we searched for a novel drug to restore tight junctional localization of a CLDN16 mutant. A D97S mutant, which has a mutation in the first extracellular loop (ECL) of CLDN16, was mainly colocalized with endosome marker, whereas wild-type (WT) CLDN16 was colocalized with ZO-1, an adaptor protein of tight junctions. The protein stability of the D97S mutant was lower than that of WT. The expression level of the D97S mutant was increased by lactacystin, a proteasomal inhibitor. Endocytosis inhibitors increased the tight junctional localization of the D97S mutant. We found that primaquine, an antimalarial agent, increased the protein stability and cell surface localization of the D97S mutant, but the localization of other mutants, which have mutations in the cytosolic domain or second ECL, was not affected. Transepithelial Mg²⁺ flux was increased by primaquine in D97S mutant-expressing cells. The expression of chaperon proteins, proteasome activity, and lactate dehydrogenase release were decreased by primaquine, and the proportion of viable cells increased. In contrast, these effects were not observed in WT CLDN16-expressing cells. These results suggested that primaquine increases the tight junctional localization of the D97S mutant, resulting in a reduction in ER stress and cellular injury. Primaquine may become an effective treatment drug for selected patients with mutant CLDN16.

SUBMITTER: Marunaka K

PROVIDER: S-EPMC6609605 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Rescue of tight junctional localization of a claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells.

Marunaka Kana K Fujii Naoko N Kimura Toru T Furuta Takumi T Hasegawa Hajime H Matsunaga Toshiyuki T Endo Satoshi S Ikari Akira A

Scientific reports 20190704 1

Magnesium ion (Mg<sup>2+</sup>) is paracellularly reabsorbed through claudin-16 (CLDN16) in the thick ascending limb (TAL) of Henle's loop in the kidney. Genetic disorders of CLDN16 cause mislocalization of CLDN16, resulting in hypomagnesemia. There is no effective treatment for hypomagnesemia except for magnesium administration. Here, we searched for a novel drug to restore tight junctional localization of a CLDN16 mutant. A D97S mutant, which has a mutation in the first extracellular loop (ECL ...[more]

PMID: 31273276

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Project description:Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.

Dataset Information

Rescue of tight junctional localization of a claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells.

Publications

Rescue of tight junctional localization of a claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells.

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