Deletion of claudin-10 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis

Abstract

In the kidney, tight junction proteins contribute to segment specific selectivity and permeability of paracellular ion transport. In the thick ascending limb (TAL) of Henle's loop, chloride is reabsorbed transcellularly, whereas sodium reabsorption takes transcellular and paracellular routes. TAL salt transport maintains the concentrating ability of the kidney and generates a transepithelial voltage that drives the reabsorption of calcium and magnesium. Thus, functionality of TAL ion transport depends strongly on the properties of the paracellular pathway. To elucidate the role of the tight junction protein claudin-10 in TAL function, we generated mice with a deletion of Cldn10 in this segment. We show that claudin-10 determines paracellular sodium permeability, and that its loss leads to hypermagnesemia and nephrocalcinosis. In isolated perfused TAL tubules of claudin-10-deficient mice, paracellular permeability of sodium is decreased, and the relative permeability of calcium and magnesium is increased. Moreover, furosemide-inhibitable transepithelial voltage is increased, leading to a shift from paracellular sodium transport to paracellular hyperabsorption of calcium and magnesium. These data identify claudin-10 as a key factor in control of cation selectivity and transport in the TAL, and deficiency in this pathway as a cause of nephrocalcinosis.

Fig. 1.

Analysis of claudin-10 expression in the kidney. (A) Western blot analysis of kidney membrane extracts from control (ctr) and cKO mice. A dramatic reduction in claudin-10 protein can be seen in kidneys of cKO mice. Levels of the TJ marker occludin are unchanged. (B) Gene expression analysis of Cldn10 variants on cDNA from isolated segments of the nephron. (C) Immunohistological detection of claudin-10 and markers for PCT (NHE3) and TAL (NKCC2) on sections from control mice (ctr) and cKO mice demonstrates no difference in the signal for claudin-10 in the PCT between WT and cKO. Claudin-10 is expressed in TAL tubules positive for NKCC2. No specific claudin-10 staining is evident in the TAL of cKO mice. Claudin-10 is detected in TJs positive for ZO-1. This signal is absent in cKO mice, whereas ZO-1 staining is unchanged. (Scale bar: 25 μm.)

Fig. 2.

Histological analysis of kidneys revealing nephrocalcinosis in claudin-10–deficient kidneys (A, B, and D) compared with kidneys from control mice (C and E). Von Kossa (A–C) and alizarin red S (D and E) staining show a characteristic calcification pattern in both stripes of the outer medulla in cKO kidney sections. (Scale bars: 1 mm in A–C; 0.4 mm in D and E.)

Fig. 3.

Electrophysiological properties and permeability ratios of isolated perfused TAL in control and cKO mice. (A) Original chart recordings of furosemide-inhibitable lumen-positive V_te (continuous line). Voltage deflections are caused by current injections for the measurement of R_te. In the presence of luminal furosemide (furo), V_te drops to virtually 0 mV (dashed line). The respective change in V_te is larger in cKO TAL compared with control TAL. (B) Summarized data of V_te, R_te, and equivalent I′_sc under control conditions and in the presence of furosemide. cKO tubules exhibit higher voltage, higher resistance, and equal transport current. (C) Original traces of NaCl dilution potentials (Left) and bi-ionic diffusion potentials (Right), shown by the continuous line. Voltage deflections are caused by current injections. (Box) Schematic drawing indicating ion gradients across the tubular epithelium (luminal and basolateral ion activities, in mmol/L). The dashed line represents V_te at full inhibition of transcellular transport without paracellular ion gradients. cKO tubules show lower diffusion voltage and a strong shift of V_te to more positive values compared with control. (D) Relative ion permeabilities calculated from diffusion voltages. Absence of claudin-10 reduces Na⁺ permeability in favor of Mg²⁺ and Ca²⁺ permeability. Data are shown as mean ± SEM. n = 22/15. *P < 0.05; ***P < 0.001.

Fig. 4.

Gene expression analysis of renal claudins (A) and representative renal ion transporters and channels (B) by real-time PCR. Cldn10 deficiency results in differential gene expression of several genes. Values from cKO animals are shown relative to control mice (mean ± SEM). Wnk1, Wnk1-KS, Kcnj1, and Trpm6, n = 5/4; all other genes, n = 10/10. *P < 0.05; **P < 0.01; ***P < 0.001.