Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA137

Poster Communications

Endocytosis of NKCC2 is impaired in renal tubule in moesin knockout mice

K. Kawaguchi1, S. Asano1

1. College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan.

Tubular reabsorption of electrolytes in the kidney is an essential function in regulating fluid balance in the body. In the thick ascending limb of Henle (TAL), 20-40% Na+ filtered by the glomeruli are reabsorbed by Na+-K+-2Cl- cotransporter type 2 (NKCC2). Mutations in the human gene coding for NKCC2 were identified in patients of Bartter syndrome type I, which is characterized by severe salt losing tubulopathy. Despite of the physiological importance of NKCC2 in NaCl homeostasis, the molecular mechanisms for its membrane trafficking have not been fully elucidated. An actin-binding protein, moesin was reported to play an important role in the apical recycling of NKCC2 by in vitro experiments (Carmosino et al. 2012). However, the physiological roles of moesin in the kidney remain unclear. Here, we examined the physiological roles of moesin in the regulation of renal function in vivo by using male moesin-null (Msn-/y) mice. Mice were anesthetized by isoflurane inhalation. Statistical analysis was performed by student T test or one way ANOVA. To evaluate the cell surface NKCC2 expressions and endocytosis of NKCC2 in mouse TAL, TAL suspensions were prepared by collagenase treatment and filtering with nylon mesh. We measured surface expression of NKCC2 using protein biotinylation and found that apical surface expression of NKCC2 was significantly increased in Msn-/y TAL (n = 11, respectively. P < 0.05 vs. WT). We performed endocytosis assay with TAL suspensions. The endocytosis of NKCC2 was observed in both WT and Msn-/y tubules within 60 min incubation at 37 °C. However, internalized NKCC2 was significantly reduced in the Msn-/y TAL (n = 3, respectively. WT; 25.2±3.0 % internalized, Msn-/y; 10.5±3.8 %, values are means ± S.E.M. P < 0.05). We further investigated the distribution of NKCC2 in lipid raft fractions by the 10-40 % Optiprep-based density gradient centrifugation using renal medullary lysate, since lipid raft distribution of NKCC2 is important for the endocytosis (Ares et al. 2010). In this result, lipid raft expression was significantly decreased in Msn-/y mice (n = 3, respectively. P < 0.05 vs. WT). To examine the renal physiological roles related to the NKCC2 in Msn-/y mice, we performed biochemical analysis of plasma and urine (WT: n = 12; Msn-/y: n = 13). Significant increase of plasma Cl- concentration (PCl) concomitant with slight increase of plasma Na+ concentration (PNa) was observed in Msn-/y mice (PCl: 112.4±0.7 vs. 115.5±1.0 mM, P < 0.05; PNa: 150.6±0.7 vs. 151.9±0.5 mM). Urinary absolute excretions of Na+ and Cl- (ENa and ECl) in Msn-/y mice were lower than those of WT mice (ENa: 716.1±163.8 vs. 652.7±97.5 μmol/100 g BW/day; ECl: 505.9±91.1 vs. 452.1±58.4 μmol/100 g BW/day). These results suggest that moesin regulates the apical surface expression level of NKCC2 by targeting NKCC2 to lipid raft and plays important roles in the renal electrolyte handling.

Where applicable, experiments conform with Society ethical requirements