Inhibition of AT2R and Bradykinin Type II Receptor (BK2R) Compromises High K+ Intake-Induced Renal K+ Excretion

Supplemental Digital Content is available in the text. The inhibition of Type II angiotensin II receptor (AT2R) or BK2R (bradykinin type II receptor) stimulates basolateral Kir4.1/Kir5.1 in the distal convoluted tubule (DCT) and activates thiazide-sensitive NCC (Na-Cl cotransporter). The aim of the present study is to examine the role of AT2R and BK2R in mediating the effect of HK (high dietary K+) intake on the basolateral K+ channels, NCC, and renal K+ excretion. Feeding mice (male and female) with HK diet for overnight significantly decreased the basolateral K+ conductance, depolarized the DCT membrane, diminished the expression of pNCC (phosphorylated NCC) and tNCC (total NCC), and decreased thiazide-sensitive natriuresis. Overnight HK intake also increased the expression of cleaved ENaC-α and -γ subunits but had no effect on NKCC2 expression. Pretreatment of the mice (male and female) with PD123319 and HOE140 stimulated the expression of tNCC and pNCC, augmented hydrochlorothiazide-induced natriuresis, and increased the negativity of the DCT membrane. The deletion of Kir4.1 not only decreased the NCC activity but also abolished the stimulatory effect of PD123319 and HOE140 perfusion on NCC activity. Moreover, the effect of overnight HK loading on Kir4.1/Kir5.1 in the DCT and NCC expression/activity was compromised in the mice treated with AT2R/BK2R antagonists. Renal clearance study showed that inhibition of AT2R and BK2R impairs renal K+ excretion in response to overnight HK loading, and the mice pretreated with PD123319 and HOE140 were hyperkalemic during HK intake. We conclude that synergistic activation of AT2R and BK2R is required for the effect of overnight HK diet on Kir4.1/Kir5.1 in the DCT and NCC activity.

P revious studies have demonstrated that both AT2R and BK2R (bradykinin type II receptor) play a role in stimulating renal K + excretion, 1,2 the effect has been achieved, in part, by modulating the basolateral K + channels in the distal convoluted tubule (DCT) and NCC (Na-Cl cotransporter) activity. We have demonstrated that the stimulation of either AT2R or BK2R inhibits whereas the inhibition of AT2R or BK2R increases the basolateral K + channel activity in the DCT. The basolateral K + channel activity in the DCT is known to regulate NCC activity such that the inhibition of the basolateral K + channels decreases whereas the stimulation of the K + channels in the DCT increases NCC activity. [3][4][5] Since NCC plays an important role in controlling Na + and volume delivery to the late aldosterone-sensitive distal nephron (ASDN) for K + excretion, [6][7][8][9] changes in NCC function have a profound effect on renal K + excretion and K + homeostasis. The role of NCC in regulating renal K + excretion is demonstrated in patients with Gitelman's syndrome and with PHAII (pseudohypoaldosteronism type 2) syndrome or familial hyperkalemic hypertension.
A low NCC activity in Gitelman's syndrome is associated with hypokalemia and metabolic alkalosis 7 whereas a high NCC activity in PHAII causes hypertension, metabolic acidosis, and hyperkalemia. 10 Thus, NCC and the basolateral K + channel in the DCT are critically involved in the renal K + excretion.
The basolateral K + channel in the DCT is composed of Kir4.1 and Kir5.1 and Kir4.1/Kir5.1 heterotetramer has been suggested to be a key player for K + -sensor mechanism because this K + channel is essential for the inhibitory effect of HK (high dietary K + ) intake on NCC activity. 3,5 Previous studies have demonstrated that potassium loading increased the expression of AT2R and BK2R. [11][12][13] Also, increased dietary K + intake augmented urinary excretion of kallikrein which converted kininogen to bradykinin. 11,14 Furthermore, Jin et al 11 have demonstrated that adding 1% KCl in the drinking water increased the expression of BK2R mRNA and the density of renal BK2R thereby lowing systolic blood pressure of spontaneous hypertension rats. Thus, it is conceivable that AT2R and BK2R may be involved in stimulating renal K + excretion during increasing dietary K + intake by modulating the basolateral K + channel in the DCT and NCC. Indeed, our previous finding that the inhibition of AT2R or BK2R increased NCC expression/activity only in wild-type mice but not in renal tubule-specific Kir4.1 deficient mice has strongly suggested the role of basolateral Kir4.1 in mediating effect of AT2R or BK2R on NCC. 1,2 Although the role of AT2R and BK2R in regulating Kir4.1/Kir5.1 in the DCT and NCC has been demonstrated, it is not known whether these 2 receptors are involved in mediating the effect of high dietary K + intake on the basolateral K + channels of the DCT and NCC. Thus, the aim of the present study is to examine whether the inhibition of AT2R and BK2R impairs the effect of HK intake on the basolateral K + channels, NCC activity, and renal K + excretion.

Methods
The authors declare that all supporting data and detailed methods including animal preparation, electrophysiology, Western blot, and renal clearance method are available within the article (and in the online-only Data Supplement).

Animals
All animal studies were approved by the Institutional Animal Care and Use Committees of Harbin Medical University and New York Medical College. Male and female C57BL/6 mice (4-8 weeks old) were purchased from the Second Hospital of Harbin Medical University. Kidney-specific Kir4.1 knockout (Ks-Kir4.1 Knockout) mice were generated in New York Medical College. The detailed method for generating Ks-Kir4.1 KO mice has been described previously and also in the online-only Data Supplement. 4,5

Whole-Cell Patch-Clamp Recording
An Axon 200B amplifier was used for the measurement of K + currents (I K ) reversal potential and Ba 2+ -sensitive whole-cell I K . We used a Narishige electrode puller (Narishige, Japan) for pulling the patchclamp pipettes with borosilicate glass (1.70 mm outer diameter). The electric resistance of the pipette was 2 MΩ (for whole-cell recording) when it was filled with 140 mmol/L KCl solution. For the measurement of I K reversal potential, the tip of the pipette was filled with a solution containing (in mmol/L) 140 KCl, 1.8 MgCl 2 , and 10 HEPES (titrated with KOH to pH 7.4). The pipette was then back-filled with the same pipette solution containing amphotericin B (20 μg/0.1 mL). The bath solution was composed of (in mmol/L) 140 NaCl, 5 KCl, 2 MgCl 2 , 1.8 CaCl 2 , and 10 HEPES (titrated with NaOH to pH 7.4). For the measurement of whole-cell Ba 2+ -sensitive I K , the bath and pipette solutions contained (in mmol/L), 125 K + -gluconate, 15 KCl, 2 MgCl 2 , 1 EGTA, and 10 HEPES (pH=7.4). After forming a high resistance seal (>2 GΩ), the membrane capacitance was monitored until the whole-cell patch configuration was formed. The currents were low-pass filtered at 1 KHz, digitized by an Axon interface with 4 KHz sampling rate (Digidata 1332). Data were analyzed using the pClamp software system 9.0 (Axon).

Results
Previous study has demonstrated that feeding the mice with HK diet for 7 days inhibited the basolateral K + channel in the DCT, depolarized the DCT membrane, and decreased the expression of NCC. 5 Now, we tested whether overnight-feeding the mice with HK (2% or 10%) was also able to inhibit the basolateral K + channel activity in the DCT. The male and female mice were free to access the water but without food from 7 am to 7 pm at the day before the experiment and then were fed with NK (normal K + ; 1% K + ) and 2% HK or 10% HK diets from 7 pm to 7 am of the next day. The mice were euthanized after overnight HK loading to examine Ba 2+ -sensitive wholecell I K in the early part of the DCT (DCT1). Figure 1A and 1B show a set of the traces of Ba 2+ -sensitive whole-cell I K measured with a step-protocol from −60 to 60 mV in the DCT1 of male or female mice, respectively. Figure 1C and 1D are scatter plots summarizing each individual data point and mean value of the experiments in which Ba 2+ -sensitive whole-cell I K were measured at −60 mV in male and female mice, respectively. We observed that overnight K + loading decreased Ba 2+ -sensitive whole-cell I K in both male and female mice. Figure 1E is a bar graph showing the mean value and statistical information of the experiments. Overnight K + loading decreased the Ba 2+ -sensitive whole-cell I K from 1170±60 to 800±40 pA (2% HK) or 590±47 pA (10% HK) in male mice (n=6 for each group). Similar results were obtained in female mice (n=6 for each group), and Figure 1E shows that overnight K + loading decreased the Ba 2+ -sensitive wholecell I K from 1250±50 to 890±40 pA (2% HK) or 630±35 pA (10% HK). This suggests that overnight K + loading was sufficient to inhibit the basolateral Kir4.1/Kir5.1 activity in the DCT1, and the difference between male and female mice was not significant.
We then measured the I K reversal potential (an index for the membrane potential) in the DCT1 using whole-cell recording. Figure 2A and 2B are a set of traces showing I K reversal potentials measured with ramp protocol from −80 to 80 mV in the DCT1 of male and female mice on NK and HK (2%) or HK (10%), respectively. The results of the above experiments are summarized in a scatter plot showing the individual data point and mean value for male ( Figure 2C) and female mice ( Figure 2D), respectively. It is apparent that overnight HK intake depolarized the DCT membrane of both male and female mice. Figure 2E is a bar graph demonstrating the mean value and statistic information of the experiments in which I K reversal potential was measured in the DCT1 of the mice on NK and HK diets. Overnight K + loading depolarized the DCT membrane from 64±1 to 58±1 mV (2% HK) or 53±1 mV (10% HK) in male mice (n=6 for each group) and from 65±1 to 59±1 mV (2% HK) or 54±1 mV (10% HK) in female mice (n=6 for each group). Thus, overnight K + loading was able to depolarize the DCT membrane in both male and female mice.
We next examined the effect of overnight K + loading on the expression of pNCC and tNCC in male and female mice (n=8 for each group). Overnight K + loading also decreased the expression of pNCC and tNCC. Figure 3A is a Western blot showing the expression of pNCC and tNCC in female and male mice on NK, HK (2%), and HK (10%), respectively (full-page gel of the western blot is shown in Figure S1 in the online-only Data Supplement). We confirmed the previous report that the expression of pNCC and tNCC was higher in female mice than in male mice. 15 However, overnight K + loading inhibited the expression of pNCC and tNCC of female and male mice in a similar way. Figure 3B and 3C are bar graphs summarizing the normalized band density of pNCC and tNCC, respectively. The overnight HK loading significantly decreased the expression of pNCC to 79±6% (2% HK) and 40±6% (10% HK) in comparison to NK in female mice and to 74±3% (2% HK) and 37±2% (10% HK) in comparison to NK in male mice. Also, the overnight HK loading significantly decreased the expression of tNCC to 80±7% (2% HK) and 70±5% (10% HK) in comparison to NK in female mice and to 83±7% (2% HK) and 75±5% (10% HK) in comparison to NK in male mice.
The effect of overnight K + loading on NCC expression was specific because the expression of NKCC2 was not altered in the mice on HK ( Figure 4A). Figure 4B is a bar graph summarizing the results of normalized NKCC2 expression for male mice (2% HK, 100±6% of the control; 10% HK, 95±8% of the control) and for female mice (2% HK, 94±7% of the control; 10% HK, 101±10% of the control), respectively (n=8 for each group). We have also examined the effect of overnight K + loading on the expression of ENaCα. Figure 4C is a Western blot showing the effect of overnight K + loading on the expression of α-ENaC-f (full-length ENaCα) and α-ENaC-c (cleaved ENaCα) in female and in male mice (n=8 for each group), respectively. It is apparent that the overnight K + loading increased the expression of cleaved ENaCα. Figure 4D is a bar graph summarizing the normalized band density of α-ENaC-f (left panel) and α-ENaC-c (right panel). Overnight K + loading increased the expression of α-ENaC-c to 210±15% (2% HK) and 185±12% (10% HK) of the control value (NK) in male mice and to 200±10% (2% HK) and 170±5% (10% HK) of the control value (NK) in female mice. However, the overnight K + loading did not significantly alter the expression of α-ENaC-f. Overnight K + loading (10%) also increased the expression of ENaCβ in male (190±6%) and in female mice (193±7 % of the control value); and cleaves ENaCγ to 152±6% (male) and 149±5% (female) of the control value (n=5), although it had no significant effect on the expression of fulllength ENaCγ ( Figure S2). The effect of overnight K + loading on ENaC was also confirmed by measurement of amiloride (10 μm)-sensitive Na + currents in the DCT2. Figure S2C is a bar graph summarizing Na + currents measured at −60 mV with the perforated whole-cell recording. Overnight K + loading increased amiloride-sensitive Na + currents from 195±12 pA (n=5) to 300±30 pA (n=3) in the DCT2. Thus, the data strongly suggest that like long term of K + loading, overnight K + loading inhibited the basolateral Kir4.1/Kir5.1 in the DCT and suppressed NCC expression. Previous studies have showed that AT2R and BK2R are involved in the regulation of the basolateral Kir4.1/Kir5.1 activity in the DCT. 1,2 Thus, we next examined the role of AT2R and BK2R in mediating the effect of overnight HK loading on the basolateral K + channel activity in the DCT. Male and female mice were treated with vehicle or with PD123319 (AT2R antagonist, 4 μg min −1 kg −1 ) and HOE140 (BK2R antagonist, 0.9 μg min −1 kg −1 ) for 3 days (antagonists were applied through an osmotic pump installed subcutaneously). We then conducted whole-cell recording to measure I K reversal potential in the DCT1, and Figure 5A is a scatter plot showing the mean value and each individual data point of experiments (n=5 for each group). The inhibition of AT2R and BK2R hyperpolarized DCT membrane from 62.5±1 to 71.6±1 mV in male mice and from 63.0±1 to 72.2±1.2 mV in female mice. Since the treatment of PD123319 and HOE140 caused the DCT hyperpolarization, we expected the hyperpolarization should stimulate NCC activity. Thus, we used renal clearance method to examine the HCTZ-induced renal Na + excretion in both male and female mice (3 male and 3 female mice for each group). Figure 5B is a bar graph summarizing the results of the experiment in which net renal Na + excretion was measured before and after a single dose of HCTZ (25 mg/kg body weight [BW]) in male and female mice (data are pooled) with or without PD123319 and HOE140 treatment. It is apparent that the inhibition of AT2R and BK2R increased HCTZ-induced net renal Na + excretion from 1.56±0.22 to 3.83±0.25 μEq/min per 100 gram BW, suggesting that the inhibition of AT2R and BK2R stimulated NCC. The stimulatory effect of PD123319 and HOE140 on NCC function was dependent on the basolateral Kir4.1 since the inhibition of AT2R and BK2R had no effect on NCC activity in kidney-specific-Kir4.1 knockout mice (vehicle: 0.13±0.04; PD123319+HOE140: 0.22 ±0.08 μEq/min per 100 gram body weight; 3 male and 3 female mice for each group). This suggests the role of Kir4.1/Kir5.1 of the DCT in mediating the effect of AT2R and BK2R on NCC activity.
After demonstrating that the inhibition of AT2R and BK2R stimulated the basolateral Kir4.1/Kir5.1 in the DCT and NCC activity, we examined the effect of overnight HK loading on I K reversal potentials of the DCT in vehicle-and PD123319+HOE140 treated mice (3 male and 3 female mice for each group). Figure 5C is a bar graph summarizing the results of experiments in which I K reversal potentials of the DCT were measured in male and female mice (data are pooled). It is apparent that I K reversal potential of the DCT (62.5±1 mV) was significantly higher in PD123319/ HOE140 treated mice on HK than those of untreated mice on HK (53.5±0.5 mV). Since the basolateral K + channel activity regulates the NCC activity, it is conceivable that the inhibition of AT2R and BK2R may also compromise the effect of HK intake on NCC. Thus, we used renal clearance method to examine the effect of the overnight K + (10%) loading on NCC function in control mice (vehicle) and in the mice treated with PD123319 and HOE140 for 3 days (3 male and 3 female mice for each group). The results are summarized in a scatter plot showing the mean value, and each data point of the experiment in which HCTZ-induced net renal Na + excretion was examined ( Figure 5D). The overnight HK (10%) loading significantly decreased net Na + excretion from 1.56±0.25 to 0.85±0.11 μEq/min per 100 gram BW in the vehicle-treated mice. Treatment of the mice with PD123319 and HOE140 not only increased HCTZ-induced natriuresis (3.88±0.25 μEq/ min per 100 gram BW) but also significantly attenuated the inhibitory effect of overnight HK loading on NCC function (net renal Na + excretion, 2.86±0.25 μEq/min per 100 gram BW).
The notion that the inhibition of AT2R and BK2R attenuates the effect of HK diet on NCC was also confirmed by Western blot analysis. Figure 6 are western blots showing the effect of overnight HK (10%) on the expression of NCC in female (n=6) and male mice (n=6), respectively. The fullsize gel has been demonstrated in Figure S3. The normalized band density of pNCC and tNCC are summarized in a scatter plot for female ( Figure S4A) and male mice ( Figure S4B). It  Overnight K + loading increases the expression of cleaved ENaCα subunit but has no effect on Type 2 Na-Cl-cotransporter (NKCC2). A, A Western blot shows the expression of NKCC2 in female and male mice on NK (normal K + ), 2% K + and 10% K + , respectively. B, A bar graph showing the normalized band density of NKCC2 in male and female mice. C, A Western blot shows the expression of full-length (f) and cleaved (C) epithelial Na+ channel-alpha subunit (indicated by an arrow). D, A bar graph showing the normalized band density of α-ENaC-f and α-ENaC-c in female and male mice on NK, 2% K + and 10% K + , respectively. * and # indicate that P value is <0.05 and 0.01, respectively. is apparent that the overnight HK (10%) decreased the expression of pNCC (39±3% of control value for female and 37±3% of the control for the male mice) and tNCC (70±5% of control value for female and 75±5% of the control for the male mice). The inhibition of AT2R and BK2R significantly increased the expression of pNCC (150±10% of corresponding control value for female and 140±10% of the corresponding control for the male mice) and tNCC (150±10% of control value for female and 155±10% of the control for the male mice). The overnight HK loading still decreased the expression of pNCC (95±10% of control value for female and 90±10% of control value for male mice) and tNCC (110±10% of control value for female and 115±10% of control value for male mice) in the mice treated with PD123319 and HOE140. However, the inhibitory effect of HK diet on pNCC in the mice treated with PD123319 and HOE140 was significantly attenuated. Figure S4C is a bar graph showing the normalized ratio of pNCC between HK diet and NK diet. While the overnight HK loading decreased the expression of pNCC by 61% in untreated female and to 63% in untreated male mice, HK only decreased the expression of pNCC by 35% in antagonists treated female and by 33% in antagonists treated male mice. In contrast, Figure S4C shows that the overnight HK loading similarly inhibited the expression of tNCC in vehicle-or PD123319+HOE140-treated female or male mice. Thus, the Western blot analysis is consistent with the results obtained with renal clearance, suggesting that the inhibition of AT2R and BK2R impairs the effect of HK intake on NCC activity. The compromised effect of HK on NCC was due to the fact that the inhibition of AT2R and BK2R impaired the effect of HK on ste20-proline-alanine rich kinasea. Figure 6B is a Western blot showing the effect of overnight K + loading (10%) on pSPAK and tSPAK (total-SPAK). Overnight K + loading decreased pSPAK to 70±5% (n=4) of the control value (NK) but had no significant effect on tSPAK ( Figure S5). The inhibition of AT2R and BK2R increased pSPAK to 125±6% (n=4) of the control value (NK with no inhibitors) but had no effect on tSPAK. Moreover, while overnight K + loading decreased pSPAK in the mice treated with PD123319+HOE140, the expression of pSPAK was still significantly higher (110±6%) than the corresponding control value (HK with no inhibitors).
Since NCC plays an important role in regulating renal K + excretion by determining the Na + and volume delivery to the ASDN, it is conceivable that renal K + excretion should be compromised in the mice treated with PD123319+HOE140. Thus, we used renal clearance method to examine the renal K + excretion rate at basal conditions. Figure 6C is a scatter plot showing the mean value and each data point of the experiment in which the net renal K + excretion rates at steady state were measured in the male and female mice (data are pooled) on NK and overnight HK loading (n=6 for each group). It is apparent that HK intake increased the net renal K + excretion from 0.43±0.03 to 1.80±0.14 μEq/min per 100 gram BW. Although the inhibition of AT2R and BK2R did not significantly affect the basal level of net renal K + excretion (0.51±0.06 μEq/min per 100 gram BW), it significantly diminished the net renal K + excretion rate (0.90±0.08 μEq/ min per 100 gram BW) in comparison to untreated mice during overnight HK loading. The notion that the inhibition of AT2R and BK2R impairs K + homeostasis is also supported by the finding that the overnight HK loading caused hyperkalemia in the mice treated with PD123319+HOE140 (NK, 4.35±0.18 and HK, 5.49±0.27 mmol/L; Figure 6D). In contrast, overnight HK loading did not significantly change the plasma K + level in vehicle-treated mice (NK, 3.76±0.09 and HK 3.83±0.10 mmol/L). However, plasma Na + levels were the same between treated and untreated mice on NK or HK diet.

Discussion
It is well established that increased dietary K + intake inhibits the basolateral K + channel in the DCT, a Kir4.1 and Kir5.1 heterotetramer, [3][4][5] and that HK-intake-induced inhibition of the K + channels in the DCT is essential for the inhibitory effect of HK on NCC expression and activity. 3,5,16 Now, not like the previous experiments examining the effect of HK on the basolateral K + channels in the DCT of the animals on HK diet for several days, we have shown that overnight HK loading was sufficient to inhibit the basolateral K + channels in the DCT. Moreover, our study also demonstrated that overnight HK loading not only inhibited basolateral Kir4.1/Kir5.1 but also decreased NCC expression and activity. The mechanism by which Kir4.1/Kir5.1 in the DCT regulates NCC is most likely mediated by Cl − -sensitive WNK (with-no-lysine kinase) pathway. 17 Since Kir4.1/Kir5.1 is the only type of K + channel expressed in the basolateral membrane of the DCT, [18][19][20][21][22][23] the activity of Kir4.1/Kir5.1 determines the membrane potential which is the driving force for controlling Cl − movement through electrogenic Cl − channels across the basolateral membrane. Thus, HK-intake-induced inhibition of basolateral Kir4.1/Kir5.1 activity should increase the intracellular Cl − concentrations thereby inhibiting WNK activity. Because WNK activity is an upstream protein kinase for SPAK and OSR (oxidation-sensitive response kinase), 2 kinases which stimulate NCC activity, [24][25][26][27] increased intracellular Cl − concentration should indirectly suppress SPAK/OSR activity thereby downregulating NCC phosphorylation/activity during increasing dietary K + intake. Thus, Cl − -sensitive WNK activity links the basolateral Kir4.1/Kir5.1 to NCC activity. The possibility that AT2R and BK2R may regulate SPAK was supported by the finding that the inhibition of AT2R and BK2R not only increased the expression of pSPAK but also partially blocked the inhibitory effect of HK on pSPAK. This strongly suggests the role of AT2R and BK2R in mediating the effect of HK on SPAK activity.
We confirmed previous finding that the expression of pNCC and tNCC was higher in female mice than in male mice. 15,28 However, it is unlikely that different Kir4.1/Kir5.1 activity in the DCT between male and female mice is responsible for higher NCC expression/activity in female mice than in male mice because there is no significant difference regarding the K + conductance in the DCT and membrane potential between male and female mice. Moreover, we demonstrated that HK intake inhibited Kir4.1/Kir5.1 activity in the Figure 6. Inhibition of type II angiotensin II receptor and BK2R (bradykinin type II receptor) compromises the effect of overnight K + loading on pNCC (phosphorylated NCC). A, A Western blot shows the expression of pNCC and tNCC (total NCC) in vehicle or PD123319+HOE140 treated female mice or male mice on NK (normal K + ) and 10% K + diets. B, A Western blot shows the expression of pSPAK (phosphor-SPAK) and tSPAK (total-SPAK) in vehicle or PD123319+HOE140 treated mice on NK and 10% K + diets. The mice were perfused with PD123319 and HOE140 continuously through an osmatic pump installed subcutaneously. C, A scatter graph summarizes the results of experiments in which renal K + excretion at steady-state level was measured in the mice treated with vehicle or AT2R/BK2R antagonists during NK and HK (high dietary K + ; 10%) loading for overnight. D, A table shows the plasma K + and Na + level in the vehicle-treated or PD123319+HOE140-treated mice on NK and HK for overnight. BW indicates body weight.
DCT, depolarized the DCT membrane, and inhibited NCC expression by a similar way in both male and female mice, suggesting a similar role of Kir4.1/Kir5.1 in mediating the effect of HK intake on NCC expression in male and female mice.
The finding that the inhibition of AT2R and BK2R hyperpolarizes the DCT membrane and increases NCC expression/activity has also indicated the role of AT2R and BK2R in the regulation of the basolateral Kir4.1/Kir5.1 in the DCT and NCC. 1,2 Furthermore, the inhibition of AT2R and BK2Rinduced stimulation of NCC activity was completely absent in the Ks-Kir4.1 KO mice, suggesting that the basolateral Kir4.1/Kir5.1 in the DCT plays a role in mediating the inhibition of AT2R and BK2R-induced stimulation of NCC. The important role of AT2R and BK2R in the regulation of renal K + excretion is strongly suggested by the finding that the steady-state level of renal K + excretion in the mice treated with PD123319 and HOE140 in response to overnight K + loading was significantly lower than those of untreated mice. Consequently, PD123319+HOE140 treated mice on HK for overnight were hyperkalemic whereas untreated mice on HK diet were normokalemic. We believe that a compromised regulation of NCC during overnight HK loading was responsible for impairing renal K + excretion in the mice treated with AT2R and BK2R antagonists. This notion is supported by 2 lines of evidence: First, HCTZ-induced renal Na + excretion was significantly larger in the mice treated with PD123319+HOE140 than untreated mice on HK for overnight, suggesting a high NCC activity in the treated mice on HK diet. Second, the expression of pNCC was also higher in the mice treated with PD123319+HOE140 than untreated mice on HK for overnight.
Although the inhibitory effect of HK intake on NCC expression/activity was not completely abolished in the mice treated with PD123319 and HOE140, the percentage of the decrease was significantly smaller in the mice treated with AT2R and BK2R antagonists than those of untreated mice. This suggests that HK-induced inhibition of NCC was compromised in the mice treated with AT2R and BK2R antagonists. Moreover, AT2R and BK2R should function in synergistic way in mediating the inhibitory effect of HK intake on NCC because the treatment of the mice with either PD123319 or HOE140 alone had a smaller effect on the HK-induced inhibition of NCC than with combination of 2 antagonists (Wang's unpublished observation). Because NCC plays a key role in the regulation of K + homeostasis and renal K + excretion by controlling the amount of Na + and fluid volume delivery to the late ASDN, 6-9 a high NCC activity means a less Na + and volume delivery to the late part of ASDN. Although the inhibition of BK2R is expected to increase ENaC activity in the collecting duct 29 and ENaC plays an important role in regulating renal K + excretion in the ASDN, 30-33 a diminished Na + and volume delivery should decrease the flow rate in the late ASDN. This should impair the flow-stimulated renal K + excretion through the Ca 2+ -activated big-conductance K + channel (BK) which plays an important role in mediating renal K + excretion during increased dietary K + intake. 34,35 Perspectives A large body of evidence has demonstrated that increased dietary K + intake decreases the expression of NCC, 3,5,16,36-41 an effect is essential for stimulating renal K + excretion. Although it has been established that HK-intake-induced depolarization in the DCT is a key step to initiate the effect of HK intake on NCC, 5 the mechanism by which HK intake depolarizes DCT membrane is not completely understood. Since the plasma K + level in the mice on overnight HK was similar to the mice on NK diet, it was unlikely that high plasma K + was responsible for the DCT depolarization induced by HK, although we could not exclude the possibility that overnight HK loading may transiently increase the plasma K + levels at some points. In this regard, previous study has shown that female rats responding to 3-hour 2% K + loading increased plasma K + levels. 15 However, it is safe to speculate that factors other than plasma K + should also be responsible for the effect of HK diet on Kir4.1/Kir5.1 in the DCT and membrane potential. Because the activation of AT2R or BK2R has been shown to inhibit the basolateral Kir4.1/Kir5.1 in vitro, 1,2 it is most likely that AT2R and BK2R should play an important role in mediating the effect of HK intake on the basolateral K + channels in the DCT.
Thus, our current study has filled the gap by demonstrating that AT2R and BK2R are involved in mediating the effect of HK intake on the basolateral Kir4.1/Kir5.1 in the DCT and NCC. Consequently, the synergistic action of AT2R and BK2R are required for stimulating renal K + excretion during increased dietary K + intake. Considering that both AT2R and BK2R have been reported to increase natriuresis and lowering the blood pressure, 11,42 the activation of AT2R and BK2R may be partially responsible for HK-induced natriuresis and decreasing the blood pressure. The AT2R/BK2R-mediated inhibition of Kir4.1/kir5.1 should be essential for the HK-intake-induced inhibition of NCC. We conclude that AT2R and BK2R play a role in mediating the effect of HK intake on the basolateral Kir4.1/ Kir5.1 of the DCT and NCC.