alphaENaC-mediated lithium absorption promotes nephrogenic diabetes insipidus

Abstract

Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.

Figure 1.

Li-treated KO mice do not exhibit higher water intake or urine output or lower urinary osmolality. (A through C) Water intake (A), urine output (B), and urine osmolality (C) are measured in Li-treated control mice (n = 12; black), Li-treated KO mice (n = 11; white), untreated control mice (n = 8; bricks), and untreated KO mice (n = 7; hexagons). After 4 days of Li treatment, water intake is significantly increased in the Li-treated control mice compared with the Li-treated KO mice; after 5 days, there is a highly significant difference between the Li-treated control mice and the three other groups (A). There is no difference in water intake among the Li-treated KO mice, the untreated control mice, and the untreated KO mice during the entire diet (A). At the end of the diet, the urine output is significantly increased and the urine osmolality significantly decreased in the Li-treated control mice compared with the three other groups (B and C), whereas no differences are observed among the Li-treated KO mice, the untreated control mice, and the untreated KO mice (B and C). At day 1, n = 6, 6, 3, and 3; at day 5 to 6 and 18 to 19, n = 9, 8, 8, and 7; at day 8 to 9, n = 11, 11, 8, and 7. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.

Li treatment decreases AQP2 expression in inner medulla but not in cortex of KO mice. (A through D) Western blot and corresponding densitometric analysis of AQP2 (A and B) and H⁺-ATPase expression (C and D) in cortex/OM (A and C) and IM (B and D) of untreated control mice (control), untreated CD-specific αENaC KO mice (ENaC KO), Li-treated control mice (Li-control), and Li-treated CD-specific αENaC KO mice (Li-ENaC KO). In cortex/OM, AQP2 protein is significantly reduced in Li-control compared with the other three groups, whereas AQP2 levels in the Li-ENaC KO is not different from ENaC KO (A). AQP2 protein in IM is significantly lower in Li-control compared with the two untreated groups but not compared with Li-ENaC KO (B). The expression of AQP2 in the IM of Li-ENaC KO is significantly lower compared with ENaC KO (B). No differences in H⁺-ATPase protein are observed between the Li-control and the other groups in cortex/OM (C). In the IM, H⁺-ATPase is significantly higher in the Li-control compared with the three other groups, whereas the expression in the Li-ENaC KO is not significantly different from the untreated groups (D). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Decreased AQP2 labeling in the CD of Li-treated control mice compared with Li-treated KO mice. (A through H) Immunohistochemistry using whole kidney sections from Li-treated CD-specific αENaC KO mice (A through D) and Li-treated control mice (E through H). Sections are incubated with anti-AQP2 antibody. In the IM (A and E), ISOM (B and F), and the CCD (C and G), AQP2 labeling is markedly reduced in the Li-treated control mice (E through G) compared with the Li-treated CD-specific αENaC KO mice (A through C). In the CNT, there are no changes in AQP2 labeling between the two groups (D and H).

Figure 4.

No increase in H⁺-ATPase–positive cells in Li-treated KO mice compared with Li-treated control mice. (A through H) Immunohistochemistry using whole kidney sections from Li-treated CD-specific αENaC KO mice (A through D) and Li-treated control mice (E through H). Sections were incubated with anti–H⁺-ATPase antibody. The density of H⁺-ATPase–positive cells was slightly increased in the IM and ISOM of the Li-treated control mice (E and F) compared with the Li-treated CD-specific αENaC KO mice (A and B). In the CCD (C and G) and in the CNT (D through H), there were no major changes in the density of H⁺-ATPase–positive cells.