Claudin-16 is defective in familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Claudin-16 knockdown (CLDN16 KD) mice show reduced cation selectivity in the thick ascending limb. The defect leads to a collapse of the lumen positive diffusion voltage which drives Ca²⁺ and Mg²⁺ absorption. Based on the reduced tight junction permeability ratio for Na⁺ over Cl^-, we proposed a backleak of NaCl into the lumen. Systemic analysis had revealed lower blood pressure and a moderately increased plasma aldosterone concentration. In this study, we have measured the amiloride-sensitive equivalent short-circuit current in isolated perfused collecting ducts and found it increased by 5-fold in CLDN16 KD mice compared to wild-type mice (WT). Amiloride treatment unmasked renal Na⁺ loss in the thick ascending limb of the nephron. Under amiloride treatment, CLDN16 KD mice developed hyponatremia and the renal fractional excretion of Na⁺ was two-fold higher in CLDN16 KD mice compared to WT. The loss of claudin-16 also resulted in increased urinary flow, reduced HCO₃^- excretion and lower urine pH. We conclude that perturbation in salt- and acid/base metabolism in CLDN16 KD mice has its origin in the defective cation permselectivity of the thick ascending limb of the nephron. This study has contributed to the still incomplete understanding of the symptoms of FHHNC patients.