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

Abstract

Magnesium ion (Mg 2+ ) 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 Mg 2+ 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.

Mechanisms of paracellular transport of magnesium in intestinal and renal epithelia

“…When heterologously expressed in epithelial cells, CLDN16 has varied effects, 109–114 depending on the experimental model (see Ref. 71 for a review).…”

“…When heterologously expressed in epithelial cells, CLDN16 has varied effects, [109][110][111][112][113][114] affects Mg 2+ reabsorption is also unclear as a lower Mg permeability was reported when CLDN19 is expressed in MDCK II Tet-Off and LLC PK1 cells. 96,110 In analyses of recombinant proteins, CLDN16 and CLDN19 interact to form a cis heterodimer.…”

Claudins in Renal Physiology and Pathology

“…Blocking clathrin-mediated endocytosis increases surface expression of some CLDN16 mutants in vitro. Primaquine, an antimalarial agent increases also cell surface localization of a CLDN16 mutant in vitro [83].…”

“…Some investigators found that claudin 16 increases transepithelial transport of Ca 2+ [129] and of Mg 2+ [83,130,131], when heterogously expressed in epithelial cells (Table 6). Others found an effect of CLDN16 only on Mg 2+ (not Ca 2+ ) permeability [48].…”