Thick ascending limb claudins are altered to increase calciuria and magnesiuria in metabolic acidosis

Urinary calcium and magnesium wasting is a characteristic feature of metabolic acidosis, and this study focused on the role of the thick ascending limb of Henle's loop in metabolic acidosis-induced hypercalciuria and hypermagnesiuria because thick ascending limb is an important site of paracellular calcium and magnesium reabsorption. Male Sprague-Dawley rats were used to determine the effects of acid loading (by adding NH₄Cl, 7.2 mmol/220 g body wt/day to food slurry for 7 days) on renal expression of claudins and then to evaluate whether the results were reversed by antagonizing calcium-sensing receptor (using NPS-2143). At the end of each animal experiment, the kidneys were harvested for immunoblotting, immunofluorescence microscopy, and quantitative PCR (qPCR) analysis of claudins and the calcium-sensing receptor. As expected, NH₄Cl loading lowered urinary pH and increased excretion of urinary calcium and magnesium. In NH₄Cl-loaded rats, renal protein and mRNA expression of claudin-16, and claudin-19, were decreased compared with controls. However, claudin-14 protein and mRNA increased in NH₄Cl-loaded rats. Consistently, the calcium-sensing receptor protein and mRNA were up-regulated in NH₄Cl-loaded rats. All these changes were reversed by NPS-2143 coadministration and were confirmed using immunofluorescence microscopy. Hypercalciuria and hypermagnesiuria in NH₄Cl-loaded rats were significantly ameliorated by NPS-2143 coadministration as well. We conclude that in metabolic acidosis, claudin-16 and claudin-19 in the thick ascending limb are down-regulated to produce hypercalciuria and hypermagnesiuria via the calcium-sensing receptor. NEW & NOTEWORTHY This study found that the thick ascending limb of Henle's loop is involved in the mechanisms of hypercalciuria and hypermagnesiuria in metabolic acidosis. Specifically, expression of claudin-16/19 and claudin-14 was altered via up-regulation of calcium-sensing receptor in NH₄Cl-induced metabolic acidosis. Our novel findings contribute to understanding the regulatory role of paracellular tight junction proteins in the thick ascending limb.