Bradykinin Stimulates Renal Na+ and K+ Excretion by Inhibiting the K+ Channel (Kir4.1) in the Distal Convoluted Tubule

Abstract

Stimulation of BK2R (bradykinin [BK] B2 receptor) has been shown to increase renal Na⁺ excretion. The aim of the present study is to explore the role of BK2R in regulating Kir4.1 and NCC (NaCl cotransporter) in the distal convoluted tubule (DCT). Immunohistochemical studies demonstrated that BK2R was highly expressed in both apical and lateral membrane of Kir4.1-positive tubules, such as DCT. Patch-clamp experiments demonstrated that BK inhibited the basolateral 40-pS K⁺ channel (a Kir4.1/5.1 heterotetramer) in the DCT, and this effect was blocked by BK2R antagonist but not by BK1R (BK B1 receptor) antagonist. Whole-cell recordings also demonstrated that BK decreased the basolateral K⁺ conductance of the DCT and depolarized the membrane. Renal clearance experiments showed that BK increased urinary Na⁺ and K⁺ excretion. However, the BK-induced natriuretic effect was completely abolished in KS-Kir4.1 KO (kidney-specific conditional Kir4.1 knockout) mice, suggesting that Kir4.1 activity is required for BK-induced natriuresis. The continuous infusion of BK with osmotic pump for 3 days decreased the basolateral K⁺ conductance and the negativity of the DCT membrane. Western blot showed that infusion of BK decreased the expression of total NCC and phosphorylated NCC. Renal clearance experiments demonstrated that thiazide-induced natriuresis was blunted in the mice receiving BK infusion, suggesting that BK inhibited NCC function. Consequently, mice receiving BK infusion for 3 days were hypokalemic. We conclude that stimulation of BK2R inhibits NCC activity, increases urinary K⁺ excretion, and causes mice hypokalemia and that Kir4.1 is required for BK2R-mediated stimulation of urinary Na⁺ and K⁺ excretion.

Keywords: bradykinin B2, receptor; diuretics; hypertension; ion transport; sodium-chloride symporter.

Fig. 1. BK2R is expressed in Kir4.1-positive distal tubules

A double staining image shows the expression of Kir4.1 (brown) and BK2R (blue) with low magnification (A). Areas marked by two squares (B) are enlarged, demonstrating detailed view of BK2R staining in Fig. 1C and 1D, respectively. The distal convoluted tubules (DCT) are indicated by arrows. A red arrow indicates BK2R staining in the lateral membrane of the DCT.

Fig. 2. Bradykinin (BK) inhibits the basolateral 40 pS K⁺ channels in the DCT

(A) A single channel recording shows the effects of 1 and 10 μM BK on the basolateral K⁺ channels in the DCT. The top trace shows the time course of the experiments and 4 parts of the record indicated by numbers are extended to demonstrate the fast time resolution. The holding potential was 0 mV and the channel closed level is indicated by a dotted line and “C”. The experiments were performed in cell-attached patches with 140 mmol/L K⁺ in the pipette and 140 mmol/L Na⁺/5 mmol/L K⁺ in the bath solution. (B) A bar graph summarizes the results of experiments in which the effects of BK, PMA (10 μM), calphostin C (1μM) and BK+calphostin C on the basolateral 40 pS K⁺ channel were examined (n=6).

Fig. 3. BK decreases the basolateral K⁺ conductance of the DCT and depolarizes the membrane

A whole-cell recording shows Ba²⁺ -sensitive K⁺ currents in the DCT treated with 10 μM BK and the K⁺ currents were measured with a step protocol from −60 to 60 mV at a 20 mV step (A) or with Ramp protocol from −100 to 100 mV (B). Symmetrical 140 mmol/L KCl solution in the bath and pipette was used for the measurement. Results of six experiments are summarized in a bar graph (C). A whole-cell recording shows the effect of 10 μM BK on K⁺-current (I_K) reversal potential of the DCT (D). The bath solution contains (in mmol/L) 140 NaCl and 5 KCl while the pipette solution has 140 KCl. Results of five experiments are summarized in a bar graph (left panel of Fig. 3C).

Fig. 4. BK2R mediates BK-induced inhibition of the basolateral 40 pS K⁺ channels in the DCT

A single channel recording demonstrates the effect of 10 μM BK on the basolateral K⁺ channels in the DCT in the presence of 1 μM HOE (BK2R antagonist) (A) or in the presence of 1μM Lys-(des-Arg⁹, Leu⁸)-bradykinin (BK1R antagonist) (C). The holding potential was 0 mV and the channel closed level is indicated by a dotted line and “C”. (B) Bar graph summarizes the results of experiments in which the effects of HOE140 (1μM), BK (10 μM)+HOE140, BK1R antagonist (1μM) and BK+BK1R antagonist on the basolateral 40 pS K channel were examined (n=6).

Fig. 5. BK infusion stimulates renal Na⁺ and K⁺ excretion

(A) A line graph shows the results of each experiment in which urinary Na⁺ excretion (E_Na) was measured before and after BK infusion in WT mice and KS-Kir4.1 KO mice. (B) The mean value and statistical information are shown in a bar graph. The basal level of E_Na of KS-Kir4.1 KO mice is significantly different in comparison to WT mice. (C) A line graph shows the results of each experiment in which urinary K⁺ excretion (E_K) was measured before and after BK infusion in WT mice and KS-Kir4.1 KO mice. (D) The mean value of E_K and statistical information are shown in a bar graph. The basal level of E_K of KS-Kir4.1 KO mice is significantly different in comparison to WT mice.

Fig. 6. BK infusion inhibits Kir4.1 and NCC

(A) A whole-cell recording shows Ba²⁺ -sensitive K⁺ currents in the DCT of mice treated with vehicle (control) or BK infusion for 3 days. K⁺ currents were measured with a step protocol from −60 to 60 mV at a 20 mV step. (B) A whole-cell recording shows K⁺-current (I_K) reversal potential of the DCT in mice treated with vehicle (control) or BK infusion for 3 days. (C) A line graph shows the results of experiments in which urinary sodium excretion (E_Na) was measured before and after a single dose of HCTZ (25 mg/kg BW) in control and BK-treated mice. BK was delivered for three days through an osmatic pump. (D) A western blot shows the expression of pNCC and tNCC in control and BK-treated mice. (E) A bar graph summarizing the normalized band density of pNCC and tNCC from tissues obtained in the control and BK-treated mice (n=6). (F) A table shows the plasm Na⁺ and K⁺ concentrations in the control and BK-treated mice (n=7). Asterisk indicates a significant difference between two groups.