Vasopressin-induced stimulation of the Na(+)-activated K(+) channels is responsible for maintaining the basolateral K(+) conductance of the thick ascending limb (TAL) in EAST/SeSAME syndrome

Abstract

The renal phenotype of EAST syndrome, a disease caused by the loss-of-function-mutations of Kcnj10 (Kir4.1), is a reminiscence of Gitelman's syndrome characterized by the defective function in the distal convoluted tubule (DCT). The aim of the present study is to test whether antidiuretic hormone (vasopressin)-induced stimulation of the Na(+)-activated 80-150pS K(+) channel is responsible for compensating the lost function of Kcnj10 in the thick ascending limb (TAL) of subjects with EAST syndrome. Immunostaining and western blot showed that the expression of aquaporin 2 (AQP2) was significantly higher in Kcnj10(-/-) mice than those of WT littermates, suggesting that the disruption of Kcnj10 stimulates vasopressin response in the kidney. The role of vasopressin in stimulating the basolateral K(+) conductance of the TAL was strongly indicated by the finding that the application of arginine-vasopressin (AVP) hyperpolarized the membrane in the TAL of Kcnj10(-/-) mice. Application of AVP significantly stimulated the 80-150pS K(+) channel in the TAL and this effect was blocked by tolvaptan (V2 receptor antagonist) or by inhibiting PKA. Moreover, the water restriction for 24h significantly increased the probability of finding the 80-150pS K(+) channel and the K(+) channel open probability in the TAL. The application of a membrane permeable cAMP analog also mimicked the effect of AVP and activated this K(+) channel, suggesting that cAMP-PKA pathway stimulates the 80-150pS K(+) channels. The role of the basolateral K(+) conductance in maintaining transcellular Cl(-) transport is further suggested by the finding that the inhibition of basolateral K(+) channels significantly diminished the AVP-induced stimulation of the basolateral 10pS Cl(-) channels. We conclude that vasopressin stimulates the 80-150pS K(+) channel in the TAL via a cAMP-dependent mechanism. The vasopressin-induced stimulation of K(+) channels is responsible for compensating lost function of Kcnj10 thereby rescuing the basolateral K(+) conductance which is essential for the transport function in the TAL.

Keywords: ClC-kb; Kcnj10; Kcnj16; NKCC2; PKA.

Fig 1. The disruption of Kcnj10 enhances AQP2 expression

Immunostaining of AQP2 in the kidney of WT (A) and Kcnj10^-/- mice (B). A dotted line indicates the marge of the kidney. kidney slices from WT and KO mice were placed in the same slide and treated identically. (C) A Western blot showing the AQP2 expression in the kidney of p9 Kcnj10^+/+ (WT), Kcnj10^+/-(Het) and Kcnj10^-/- (KO) mice. The normalized band density is shown in the bottom panel.

Fig 2. AVP hyperpolarizes the membrane of the TAL in Kcnj10^-/- mice

(A) A bar graph summarizes the results of experiments in which whole-cell Ba²⁺ -sensitive K⁺ currents of the TAL in Kcnj10^-/- mice at -60 mV were measured with the whole-cell recording with or without AVP. The pipette and bath solution contained symmetrical 140 mM K⁺. (B) A whole cell recording showing the effect of AVP on the K⁺ reversal potential in the TAL of p9 Kcnj10^-/- mice. For measurement of K⁺ reversal potential, the TAL was bathed in a solution containing 140 mM NaCl+ 5 mM KCl while the pipette solution has 140 mM KCl. The K⁺ currents were measured with RAMP protocol from -100 to 100 mV.

Fig 3. Two types of K⁺ channels are present in the basolateral membrane of the TAL

A channel recording demonstrates the presence of two types of K⁺ channels in the basolateral membrane of the cTAL (Top trace). A part of recording is extended to show the fast time resolution (lower trace). The 40-50 pS K⁺ channel and the 80-150 pS K⁺ channel are indicated by “*” and an arrow, respectively. The bath solution was composed of (in mM) 140 NaCl, 5 KCl, 1.8CaCl2, 1.8 MgCl2, and 10 HEPES (pH=7.4) whereas the pipette of solution contained (in mM) 140 KCl, 1.8 MgCl2, and 10 HEPES (pH=7.4). The experiments were performed in cell-attached patches and “C” indicates the channel close level.

Fig 4. The activity of the 80-150 pS K⁺ channel is Na⁺-dependent

(A) A channel recording showing the 80-150 pS K⁺ channel in a cell-attached patch. The channel closed level is indicated by a dotted line and the holding potential is indicated on the top of each trace. (B) A current (I) and voltage (V) curve yielding the slope conductance of 100 pS at -60 mV. The K⁺ currents were measured at cell-attached patches which have only one type of K⁺ channels (140 mM Na⁺/5 mM K⁺ in the bath). (C) A single channel recording from an inside-out patch shows the effect of bath Na⁺ (facing cytoplasm side of the patch) on the activity of the 80-150 pS K channel. The pipette contains 145 mM KCl and the bath solution contains 140 mM LiCl +5 mM KCl (0 Na⁺ media) or 140 mM NaCl+5 mM KCl (140 mM Na⁺ media). The holding potential was 0 mV and “C” indicates the channel close level.

Fig 5. Water restriction increases the probability of finding the 80-150 pS K⁺ channel

(A) A bar graph showing the probability of finding 40-50 pS K channel and 80-150 pS K⁺ channel in the cTAL of rats under the control conditions and under water restriction conditions, respectively. The number of positive events is determined by the presence of K⁺ channels in the patches regardless NP_o. “*” indicates the significant difference (Chi Square test) (B) The effect of water restriction on the K channel activity (NP_o). All experiments were performed in cell-attached patches.

Fig 6. AVP stimulates the 80-150 pS K⁺ channel in the cTAL

(A) A channel recording shows the effect of 500 μM 8-Br-cAMP on the 80-150 pS K⁺ channel. The experiments were performed in a cell-attached patch and the channel closed level is indicated by “C”. The top trace shows the time course of the experiment and two parts of the trace are extended to show the fast time resolution. (B) A bar graph summarizes the results of experiments in which the 80-150 pS K⁺ channel activity was examined in the cTAL treated with 100 nM AVP, 1 μM tolvaptan (V2R antagonist), V2R antagonist+AVP, 500 μM 8-Br-cAMP, 10 μM H89 and AVP+H89. The experiments were performed at cell-attached patches and the holding potential was 0 mV.

Fig 7. Vasopressin and cAMP stimulates Cl^- channels in the cTAL

(A) A channel recording shows the effect of AVP (100 nM) on the 10 pS Cl^- channel. (B) A recording showing the effect of 200 μM dibutyryl-cAMP (db-cAMP) on the 10 pS Cl^- channels in the cTAL. The experiments were performed in a cell-attached patch and the channel closed level is indicated by a dotted line. (C) A bar graph summarizes the results of experiments in which the 10 pS Cl^- channel activity was examined in the cTAL treated with 100 nM AVP, 5 μM forskolin, 200 μM db-cAMP, 5 μM H89 and AVP+H89. The experiments were performed at cell-attached patches and the holding potential was -60 mV. The pipette solution contains (in mM) 140 NaCl, 1.8 MgCl2, and 10 HEPES (pH=7.4).

Fig 8. Inhibition of K⁺ channels diminishes the effect of AVP on the 10 pS Cl^- channels in the cTAL

(A) A channel recording shows the time course of the experiments in which Ba²⁺ was added in the presence of AVP (100 nM). Two parts of the trace are extended to show the fast time resolution. The experiments were performed in a cell-attached patch and the holding potential was -60 mV. The channel closed level is indicated by a dotted line. (B) A channel recording showing that adding Ba²⁺ into cytosolic side of an inside-out patch had no effect on the 10 pS Cl channel of the cTAL.