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.
Highlights
Body Mg2+ content is rigorously regulated in the kidney
WT CLDN16 was colocalized with zonula occludens (ZO)-1, a scaffolding protein of the tight junctions (TJs), in the cell-cell border area (Fig. 2A)
The D97S mutant was partially colocalized with EEA1, an early endosome marker, and Rab[7], a late endosome marker, whereas it was not colocalized with lysotracker, a lysosome marker, BiP, an endoplasmic reticulum marker, and GM130, a Golgi apparatus marker (Fig. 2B,C)
Summary
Body Mg2+ content is rigorously regulated in the kidney. Approximately 80% of total plasma Mg2+ is daily filtered by the glomeruli followed by reabsorption from the renal tubules and only 5% is excreted into the urine[1,2]. The renal tubules are divided into several segments including the proximal tubule, thick ascending limb of Henle’s loop (TAL), and distal tubule, in which the reabsorption percentages are ~15%, ~60%, and ~5%, respectively. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is characterized by hypomagnesemia, advanced nephrocalcinosis, and progressive renal failure[7]. The causative genes of FHHNC are CLDN16 or CLDN198–10. Both CLDNs can form homo- or hetero-oligomeric complexes and make divalent cation-permeable pores. Over 30 different mutations of CLDN16 are reported in FHHNC patients[13,14,15,16]. The function of primaquine may be more complicated than has been appreciated previously, but we hypothesized that primaquine may recover the localization and function of CLDN16 mutants found in FHHNC patients
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
