bicarbonate promotes bk- / 4-mediated k excretion in the renal distal nephron
TRANSCRIPT
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Bicarbonate promotes BK-α/β4-mediated K excretion in the renal distal nephron 5
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Ryan J. Cornelius, Donghai Wen, Lori I. Hatcher, and Steven C. Sansom 8
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Department of Cellular and Integrative Physiology 11
University of Nebraska Medical Center 12
Omaha, Nebraska 13
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Running Head: Bicarbonate promotes BK-mediated K secretion 15
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Corresponding Author: Steven C. Sansom 17
Dept. of Cellular and Integrative Physiology 18
985850 Nebraska Medical Center 19
Omaha, NE 68198-5850 20
(402) 559-2919 21
(402) 559-4438 (Fax) 22
Email: [email protected] 23
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Articles in PresS. Am J Physiol Renal Physiol (September 19, 2012). doi:10.1152/ajprenal.00490.2012
Copyright © 2012 by the American Physiological Society.
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Abstract 28
Ca-activated K channels (BK), which are stimulated by high distal nephron flow, are utilized during high 29
K conditions to remove excess K. Because BK predominantly reside with BK-β4 in acid/base transporting 30
intercalated cells, we determined whether BK-β4 knock-out mice (β4KO) exhibit deficient K excretion when 31
consuming a high K alkaline diet (HK-alk) vs. high K chloride diet (HK-Cl). When WT were placed on HK-alk, 32
but not HK-Cl, renal BK-β4 expression increased (Western blot). When WT and β4KO were placed on HK-Cl, 33
plasma [K] was elevated compared to control K diets; however, K excretion was not different between WT and 34
β4KO. When consuming HK-alk, plasma [K] was lower and K clearance greater in WT compared to β4KO. The 35
urine was alkaline in mice on HK-alk; however, urinary pH was not different between WT and β4KO. 36
Immunohistochemical analysis of pendrin and V-ATPase revealed the same increases in β-IC, comparing WT 37
and β4KO on HK-alk. We found an amiloride-sensitive reduction in Na excretion in β4KO, compared with WT, 38
on HK-alk, indicating enhanced Na reabsorption as a compensatory mechanism to secrete K. Treating mice 39
with an alkaline, Na deficient, high K diet (LNaHK) to minimize Na reabsorption exaggerated the defective K 40
handling of β4KO. When WT on LNaHK were given NH4Cl in the drinking water, K excretion was reduced to 41
the magnitude of β4KO on LNaHK. These results show that WT, but not β4KO, efficiently excretes K on HK-alk 42
but not on HK-Cl and suggest that BK-α/β4 mediated K secretion is promoted by bicarbonaturia. 43
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Key Words: maxi K, collecting duct, intercalated cells, acidosis, alkalosis, potassium secretion, distal 45
nephron 46
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Introduction 48
Modern day humans ingest a diet of high Na, low K and high acid content; however, some remote 49
populations, such as the Yanomami of South America, consume a low Na, high K, alkaline diet, similar to 50
ancient man. The low Na and high alkaline content of the Yanomami diet indicates that the elimination of K 51
may not depend on distal Na reabsorption but rather HCO3 secretion, which could also generate a lumen 52
negative potential that could drive K transport in the distal nephron. 53
Connecting tubule cells and principal cells (PC) of the cortical collecting ducts contain the assembly of 54
epithelial Na channels (ENaC) and renal outer medullary K channels (ROMK) in the apical membrane in series 55
with the Na-K-ATPase in the basolateral membrane; an electrophysiological arrangement that explains Na-56
dependent K secretion. However, in conditions of high K intake distal flow is stimulated (19; 23; 24) primarily 57
because of K recycling and inhibition of Na and Cl reabsorption in the thick ascending limb (50). High flow 58
stimulates large, Ca-activated K channels (BK) to secrete K, as demonstrated ex vivo with isolated tubule 59
perfusion (51; 58) and in vivo by volume expansion (38), genetically eliminating ROMK (2), pharmacologic 60
blockade of vasopressin (V2) receptors (41) or treating with a high K diet (24). Moreover, the arrangement of 61
only ENaC and ROMK in series with the Na-K-ATPase would substantially limit the amount of distal K 62
secretion to a ratio of only two secreted K per three absorbed Na, which is the ratio of the Na-K pump. Isolated 63
perfused tubule studies (rabbit CCDs) using electrophysiological techniques have indicated that the ratio of K 64
secreted to Na absorbed can far exceed the pump ratio in high aldosterone (DOCA-treated) conditions (45); 65
however, the mechanism has never been resolved. 66
ROMK secrete K under the aldosterone-regulated Na for K exchange mechanism in the distal nephron 67
(11; 16; 59). BK secrete K in the distal nephron during conditions of high K intake and may also secrete K 68
during conditions of low Na delivery, in a “Na independent” manner. A recent study showed that blockers of 69
luminal BK or basolateral Na-H exchange inhibited Na-independent K secretion (31). After cell entry, Na would 70
stimulate Na-K-ATPase and drive K into the cell. However, most BK reside in the Na-K-ATPase deficient 71
intercalated cells (IC) of the distal nephron (24; 34). Moreover, if Na independent K secretion involves Na-H 72
exchange, the generated HCO3 would require a cellular exit pathway, such as the Cl/HCO3 exchangers of IC. 73
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Although the β1 subunit (BK-β1) is localized in PC (17; 37; 39), IC contain BK-α with the BK-β4 subunit 74
as the BK-α/β4 (17). When placed on a high K diet, mice with a knock-out of the BK-β4 (β4KO) exhibit 75
attenuated K excretion (23). Moreover, the BK-α/β4 of IC secrete K as a counter cation to negative ATP ions 76
extruded from IC (22). BK-α/β4 also reside in MDCK-C11 cells, which have many properties consistent with IC 77
of the distal nephron (22). However, in vivo evidence has been elusive for a role for BK-α/β4 in the steady 78
state secretion of K under conditions of high K intake. 79
We have shown previously that the pore-forming BK-α is expressed in the IC of the distal nephron and 80
its expression is enhanced by a high K alkaline diet (24). However, it wasn’t determined whether the 81
associated BK-β4 subunit, also found predominantly in the IC, was enhanced by a high K alkaline vs high K 82
acidic diet. The present studies were performed to determine whether the associated alkaline anionic content 83
of the Yanomami diet played a role in eliminating a high K load with nominally free Na content and whether the 84
BK-α/β4 have a role in eliminating the high K, alkaline diet, as opposed to the high K, chloride diet. To achieve 85
this objective, we determined the effects of high K alkaline vs a high K acidic diet on expression of BK-β4, and 86
we studied the K and Na balance of WT and β4KO mice on diets with varied Na, K and alkaline contents. 87
These results contribute to our understanding between the interaction between K secretion and acid/base 88
transport in the distal nephron. 89
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Methods 90
Animal Studies 91
We maintained the mice in accordance with the Institutional Animal Care and Use Committee of the 92
University of Nebraska Medical Center. Mice had full access to water at all times. For all experiments we fed 93
12-20 week-old wild type (WT; C57Bl/6, Charles River, Wilmington, MA) and β4KO (generously provided by R. 94
Brenner) mice either regular mouse chow (control; 0.6% K+, 0.32% Na+), or one of several special diets 95
(Harlan Teklad, Madison, WI) for 7-10 days before sacrifice. Special diets were either high K with alkaline 96
anions (#TD.07278; HK-alk; 5.0% K+ with 5% of equal carbonate/citrate/Cl and 0.32% Na+), high K with Cl as 97
the counter anion (#TD.09075; HK-Cl; 5.0% K with 5% Cl, 0.32% Na), or a low Na, high K diet with alkaline 98
anions (#TD.08240; LNaHK; 5% K with 5% equal molar carbonate/citrate/Cl and 0.01% Na). A subset of mice 99
on LNaHK was supplied with NH4Cl (280 mM) plus sucrose (2%) in their drinking water to induce an acid load. 100
We collected urine samples using metabolic cages (Nalgene), as previously described (23). After treatment, 101
we collected fresh urine samples for analysis of Na and K with a flame photometer (Jenway Clinical PFP7) as 102
previously described (19) and for pH and osmolality measurements using a Model 215 pH meter (Denver 103
Instruments) and Model 3250 osmometer (Advanced Instruments). At sacrificing, we extracted blood from the 104
carotid artery, measured hematocrit and centrifuged for measurement of plasma K, Na, and osmolality. 105
The transtubular K gradient (TTKG) was calculated as: (U[K]/P[K])(Posm/Uosm), where UK and PK are the 106
urinary [K] and plasma [K], respectively, and Uosm and Posm are the urinary and plasma osmolalities, 107
respectively. 108
Some WT and β4KO were treated with HK-alk + amiloride (5 mg/Kg/day) or HK-alk + vehicle dissolved 109
in water via osmotic pumps (ALZET, DURECT Corp.), implanted subcutaneously in the back, for 48 hrs. until 110
sacrifice. The vehicle values (n >3) were combined with control values when they were not significantly 111
different from controls. 112
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Western blotting 114
Western blotting was performed as described previously (22; 23) following manufacturer’s protocol (Bio-115
Rad Laboratories, Hercules, CA) except RIPA buffer was replaced with PBS containing 0.5% SDS. Primary 116
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antibodies included anti-BK-β4, 24 kDa, (rabbit polyclonal, diluted 1:500, Alomone Labs, Israel), and anti-β-117
actin, 43 kDa (mouse monoclonal, diluted 1:5000, Santa Cruz) with either goat anti-rabbit IgG or donkey anti-118
mouse IgG conjugated horseradish peroxidase (HRP) secondary antibody (diluted 1:20,000 – 1:40,000, Santa 119
Cruz). Expression of primary antibodies was quantified by densitometry using Quantity One (Biorad). 120
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Immunohistochemical staining and quantification 122
For fluorescent immunohistochemical staining (IHC) of kidney sections, the kidneys were harvested, 123
immediately fixed in Histochoice MB (Electron Microscopy Sciences, Hatfield, PA), embedded in paraffin and 124
sectioned onto slides for IHC as previously performed in our laboratory (18). Antibodies were used as follows: 125
anti-V-ATPase (goat polyclonal, diluted 1:100, Santa Cruz), anti-aquaporin 3 (goat polyclonal, diluted 1:100, 126
Santa Cruz), and anti-pendrin (mouse monoclonal, diluted 1:200, MBL). After washing, we incubated the 127
tissue for one hour (23°C) in the dark with the secondary antibody (donkey anti-rabbit IgG conjugated Alexa 128
Fluor 488 and donkey anti-goat IgG conjugated Alexa Fluor 594, diluted 1:200). The coverslips were mounted 129
onto slides overnight with Prolong Gold (Invitrogen), sealed with nail polish. These were viewed on a Leica HC 130
fluorescence microscope with a 40X/0.75NAHCXPL Fluotar objective. Images were captured with an QImaging 131
Retiga EXi CCD camera (Surrey, British Columbia, Canada) and analyzed with ImageJ software (version 1.42, 132
National Institutes of Health, Bethesda, MD). Quantification of V-ATPase signal intensity in IC apical and 133
basolateral membranes was determined following online instructions in single-channel, gray scale images after 134
background correction as performed previously in this lab (23). 135
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Results 138
BK expression with high K alkaline vs high K chloride diets 139
It was shown previously that the pore-forming BK-α subunit, predominantly expressed in the IC of the 140
distal nephron, was intensified in mice on a high K-alk diet by Western blot (24). We performed experiments to 141
determine whether the BK-β4, expressed with the BK-α in IC, (17) was enhanced in the renal cortex of mice on 142
a high K-alk as opposed to a high K-Cl diet. As shown in the representative blot of figure 1A, The BK-β4 143
antibody did not detect protein at the size of BK-β4 in β4KO mice. As shown in the summary bar graph of 144
figure 1B the expression of BK-β4/β-actin is 0.91 ±0.07 (n=5) in HK-alk mice, which was significantly greater 145
(by 82%) than mice treated with control diet (0.50 ±0.06; n=5) and mice treated with HK-Cl (0.49 ± 0.05; n=5). 146
These results indicate that the BK-α/β4 channel has a role to excrete K when the high K diet is alkaline but not 147
when the diet is acidic. 148
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Potassium excretion with high K alkaline vs high K chloride diets 150
We determined the ability of WT and β4KO to excrete K when consuming a high K diet with HCO3 (HK-151
alk) as the major counter anion as opposed to Cl (HK-Cl) for 7-10 days. Figure 2 shows the K clearance (A), 152
plasma K concentration (B) and transtubular K gradient (TTKG; C) for WT and β4KO on control, HK-Cl and 153
HK-alk diets. As shown, the K clearances (figure 2A) of 176 ±18 ml/day/gmKW (n=7) for WT was not 154
significantly different from that of β4KO (167 ±19 ml/day/gmKW; n=10) on a control diet. When placed on HK-155
Cl for 7-10 days, the K clearances for WT and β4KO were significantly elevated, compared to control, to similar 156
values of 1045 ±60 (n=5) and 1188 ±208 ml/day/gmKW (n=5), respectively. However, when placed on HK-alk, 157
the K clearance of WT was 2187 ±116 ml/day/gmKW (n=34), which was significantly greater than the K 158
clearance of WT on HK-Cl and β4KO on HK-alk (1530 ±97 ml/day/gmKW; n=22). 159
The plasma K concentrations of WT and β4KO on HK-Cl and HK-alk are shown in figure 2B. As shown, 160
the plasma K concentrations of both WT and β4KO on were similar, with values of 7.17±0.26 mM (n=6) and 161
7.38 ±0.27 mM (n=5), respectively, which were significantly greater than the plasma K concentrations of mice 162
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on control diets. When placed on HK-alk. The plasma K concentration of β4KO was 5.06 ±0.12 mM (n=22), a 163
value significantly greater than 4.64 ±0.07 mM (n=33); both values were significantly less than the respective 164
values on HK-Cl. 165
The TTKG is a calculation of the luminal to plasma K concentration ratio after secretion/reabsorption of 166
K and before water extraction in the collecting ducts. As shown is figure 2C, there was no significant difference 167
in TTKG between WT (8.5 ±1.1; n= 9) and β4KO (6.4 ±0.7; n=9) on a control diet. When placed on HK-Cl, the 168
TTKGs of WT and β4KO were significantly increased to 12.9 ±0.7 (n=6) and13.2 ±0.7 (n=5), respectively. 169
However, when placed on HK-alk, the TTKG of WT increased to a significantly greater value of 21.0 ±0.7 170
(n=24), compared with WT on HK-Cl, and β4KO on HK-alk, which was 16.4 ±1.0 (n=9). 171
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Effects of high K alkaline and high K chloride diets on IC profile of WT and β4KO 173
Previous studies revealed that the number of cells identified as α-IC increase in acidosis (46). 174
Moreover, the Cl/HCO3 exchanger (pendrin), identified on the apical membrane of β-IC, exhibits increased 175
expression in the distal nephron when mice consume HCO3 orally (21; 55). We first determined the ability of 176
the HK-alk diet to alkalinize the urine. As shown in figure 3, the urinary pH on a control diet was similar in WT 177
and β4KO with values of 6.19 ±0.14 (n=6) and 6.04 ±0.07 (n=7), respectively. The urinary pH values for WT 178
and β4KO on HK-Cl were 5.97 ±0.14 (n=9) and 5.98 ±0.09 (n=6), respectively, which were not different from 179
the respective pH values for WT and β4KO on the control diet. When placed on HK-alk, the urinary pH values 180
of WT and β4KO were significantly elevated, compared to HK-Cl; however, the value for WT (9.00 ±0.16; n=7) 181
was not different than that of β4KO (8.71 ±0.20; n=13). 182
We used double immunohistochemical staining (IHC) with anti-pendrin and anti-V-ATPase to determine 183
whether mice on HK-alk, compared with HK-Cl, elicited an increase in the number of pendrin-marked cells. 184
Pendrin-identified cells mark β-IC plus non A-non B cells (26). We used kidneys from four different mice in 185
each group (control, HK-Cl, HK-alk), with five sections of each kidney for an N of 20 in each group. The means 186
of each group represent the percentage of pendrin-positive cells per V-ATPase positive cells, which mark all IC 187
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cells. As shown by the representative kidney sections (figure 4A; WT only shown) and the summary bar plots 188
(figure 4B) the proportion of pendrin positive cells for control diet WT and β4KO mice was 34.0 ±7.3 % (n=18) 189
and 21.0 ±9.3 %, respectively, of the total cells with positive staining for V-ATPase. When mice were placed on 190
HK-Cl, the proportion of β-IC was 27.4 ±5.9 % for WT and 30.7 ±3.3 % for β4KO, values not different from 191
those of the control diet groups. The proportion of pendrin-positive cells in the HK-alk treated mice increased to 192
57.8 ±10.4 % for WT and 64.6 ±5.1 % for β4KO, values significantly greater than the respective values for HK-193
Cl. 194
Figure 4C shows results of quantitative fluorescence analysis of the anti-V-ATPase staining of renal 195
sections from WT mice on control and HK-alk diets. As shown, double IHC staining with anti-aquaporin 3 196
(AQP3) and anti-V-ATPase reveals distinct PC and IC, respectively, of cortical collecting ducts. Increased 197
basolateral V-ATPase staining is observed in the section from WT on HK-alk. Figure 4D is a bar plot 198
summarizing the percent β-IC in sections from WT and β4KO on control and HK-alk. On a control diet β-IC WT 199
and β4KO comprised 21.0 ±4.0 % (n=17) and 19.5 ±3.1 % (n=10), respectively. On HK-alk, the proportion of β-200
IC increased similarly and significantly in both WT and β4KO to values of 39.8 ±6.0 % (n=9) and 44.8 ±4.3 % 201
(n=12), respectively. 202
These results showed that HK-alk changed the phenotypic profile of the distal nephron to a high 203
proportion of cells that secrete HCO3 and alkalinize the urine. However, the increase in β-IC and the urine 204
alkalinization with the HK-alk diet is the same for WT and β4KO, suggesting that the BK-α/β4 is not required for 205
secreting HCO3 via pendrin and that the reduced K excretion of β4KO on HK-alk cannot be the result of 206
reduced quantities of β-IC or urine alkalinization. 207
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Compensatory ENaC-mediated Na reabsorption in β4KO 209
It was a concern that β4KO have deficient K secretion on HK-alk because of defective Na 210
reabsorption, not enabling Na-K exchange. However, as shown in figure 5, the data indicate that β4KO on the 211
HK-alk diet is compensating for reduced K secretion by enhancing the ENaC-mediated Na reabsorption in the 212
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distal nephron. As shown by the Na clearance values in figure 5A, the Na excretion is decreased in β4KO on 213
HK-alk. The Na clearance of 4.1 ±0.3 ml/day/gmKW (n=12) for β4KO was significantly less than the WT value 214
of 6.3 ±0.6 ml/day/gmKW (n=15). However, when treated with amiloride to block ENaC-mediated Na 215
reabsorption, the Na clearance of β4KO increased to 7.8 ±0.7 ml/day/gmKW (n=10), which was not 216
significantly greater than the Na clearance value for WT (7.3 ±0.4 ml/day/gmKW; n=7). 217
The Na clearance for WT on HK-alk increased slightly, but insignificantly, when given amiloride, 218
indicating minimal ENaC-mediated Na reabsorption. We used the hematocrit readings as evidence for Na 219
retention and volume expansion in β4KO on HK-alk. As shown in figure 5B, the hematocrit of β4KO was 40.3 220
±0.5 % (n=14), a value significantly less than WT (45.7 ±0.4 %; n=27). When treated with amiloride, the 221
hematocrit of β4KO was 47.9 ±1.3 % (n=9), a value not different from the WT value of 48.6 ±1.3 % (n=9). 222
These data indicate that β4KO are retaining fluid as well as Na when placed on HK-alk. The effect of amiloride, 223
to normalize the hematocrit, indicates that the fluid retention was the result of ENaC-mediated Na reabsorption. 224
Weight gain or loss is also an indicator of ENaC-mediated Na retention. As shown in figure 5C, the 225
weight of WT on HK-alk did not change significantly throughout the course of the diet (Δ wt. = -0.33 ±0.15 gms; 226
n=10). However, weight of β4KO on HK-alk increased by 3.3 ±0.8 gms (n=10). When placed on HK-alk plus 227
amiloride for two days, the weights of WT and β4KO were not significantly changed. 228
The enhanced fluid retention of β4KO and increased Na excretion when placed on amiloride shows that 229
ENaC is over-active in β4KO. β4KO may be attempting to compensate for the decreased K secretion by 230
increasing Na reabsorptive stimulated K secretion. 231
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K handling with low Na, high K diet 233
The results from figure 5 suggest that the β4KO are attempting to compensate for the lack of HCO3 234
promoted K secretion by enhancing the Na-dependent K secretion. If so, then the ability to excrete a high K 235
load should be further compromised when the mice are placed on a low Na, high K, alkaline diet (LNaHK). 236
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As shown in figure 6A, the K clearance of WT on LNaHK was not different from WT on HK-alk. 237
However, β4KO on LNaHK exhibited significantly less K clearance, with a value of 1221 ±129 ml/day/gmKW 238
(n=8), compared to the WT value of 2239 ±153 ml/day/gmKW (n=9). As shown in figure 6B, the plasma [K] of 239
WT on LNaHK was 4.71 ±0.24 mM (n=9), a value not different from WT on HK-alk. However, the plasma K 240
concentration of β4KO on LNaHK was a significantly greater value of 7.53 ±0.23 mM (n=7). The TTKG for WT 241
on LNaHK was 22.9 ±1.5 (n=10), a value not different from WT on HK-alk; however the TTKG for β4KO on 242
LNaHK was only 13.8 ±1.0 (n=8), a value significantly less than WT on LNaHK (figure 6C). 243
WT should maintain K balance when placed on a low Na, high K diet as well as a normal Na, high K 244
diet as long as the diet is also alkaline. Our LNaHK diet was designed to mimic that of the Yanomami, based 245
on their urinary outputs (33). However, WT may have an exaggerated defect with low Na if the animal is made 246
acidic. The results of acidifying the mice on LNaHK, by giving NH4Cl in the drinking water, are shown in figure 247
7. Figure 7A shows that the urinary pH of WT on LNaHK was 8.59 ±0.04 (n=3) with regular water, but 248
significantly lower, at 6.63 ±0.04 (n=3) when drinking acid water. For β4KO on LNaHK, the urinary pH was 8.70 249
±0.07 (n=2) when drinking regular water and 6.77 ±0.22 (n=5) when drinking acid water. As shown in figure 7B, 250
WT exhibited a significant decrease in K clearance to a value of 1083 ±132 ml/day/gmKW (n=5) when 251
consuming LNaHK with acid water. This value was approximately 50% the value for WT consuming LNaHK 252
with regular drinking water. However, the value of 1215 ±208 ml/day/gmKW (n=5) for β4KO on LNaHK plus 253
acid water was not different from the value for β4KO on LNaHK with regular drinking water. When mice on 254
LNaHK were treated with acid water the plasma concentrations of WT and β4KO were significantly elevated, 255
compared with LNaHK, to values of 6.8 ±0.4 mM (n=6) and 6.6 ±0.7 mM (n=5), respectively (figure 7C). The 256
TTKGs for WT and β4KO on LNaHK plus acid water were significantly decreased, compared with LNaHK, to 257
similar values of 15.1 ±1.1 (n=5) and 14.4 ±1.5 (n=5), respectively (figure 7D). These results show that WT 258
maintained K balance when placed on a low Na, high K diet, as long as it is also alkaline; however, if made 259
acidic, the ability of WT to maintain K balance on LNaHK was diminished to the capacity of β4KO. 260
The amounts of food consumption and urine output for WT and β4KO on each diet are shown in table 261
1. All groups of mice on high K diets consumed nearly twice as much chow as mice on the control diets. There 262
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was no significant difference in the quantity of chow consumed by WT and β4KO on any diet. Urinary excretion 263
rates of WT on HK-alk, HK-Cl and LNaHK were significantly greater than control. Significance was not 264
detected in urinary excretion rates among all groups other than groups on a control diet, which excreted urine 265
at about 20% the rate of mice on high K diets. 266
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268
Discussion 269
Our results show that wild type C57Bl/6 mice handle a high K alkaline diet, regardless of the Na 270
content, with a minimal increase in plasma [K]. The expression of BK-β4 subunit, found in IC of the distal 271
nephron, is enhanced in kidneys of mice on HK-alk, but not HK-Cl. When given the HK-Cl diet, or when NH4Cl 272
is added to the drinking water of mice on LNaHK, the plasma [K] increases significantly in WT, demonstrating 273
that mice consuming high K maintain K balance more easily when the diet is alkaline. However, the stimulatory 274
effect of renal HCO3 does not enhance K secretion in β4KO. We found that the defective K secretion of β4KO 275
is not a result of less β-IC, or deficient Na reabsorption-K secretion exchange. Rather, Na reabsorption-K 276
secretion exchange is enhanced in β4KO on HK-alk, probably as a compensatory mechanism. 277
278
BK-β4 expression with high K diets 279
A previous study showed that mRNA for BK-α and BK-β4 was increased in isolated cortical collecting 280
ducts of rabbit on a high K diet (32). The anionic content of this diet was not revealed but it is presumed 281
alkaline since rabbits are vegetarians. We have found by western blot that HK-alk enhances the BK-α in the 282
renal cortex of mice (24). We show here that the BK-β4, the subunit identified in IC, is up-regulated by HK-alk 283
but not by HK-Cl. This is consistent with the notion that the BK-α/β4 of intercalated cells, which are endowed 284
with acid/base transporters, is involved in HCO3-promoted K secretion. 285
286
Potassium excretion with alkaline vs acidic high K diets 287
Our results confirm a previous study showing an impairment of K excretion in β4KO (23). The high K 288
diet of that study was the same as the HK-alk diet of the present study. The anionic content of HK-alk is a 289
mixture of carbonate, citrate and Cl. The alkaline anions are ultimately converted to HCO3, where they are 290
secreted by the β-IC of the distal nephron to produce alkaline urine. 291
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We have not determined the role of alkalinity on K handling in WT vs β4KO on a normal K diet. 292
However, the BK-α/β4 should be only relevant with a high K load, which induces a flow rate of more than four 293
fold. Therefore, we would not expect that the alkalinity of the diet would make a difference between WT and 294
β4KO in handling a normal K load. In support of this notion, studies showed no change in K handling when rats 295
on a normal K diet were treated with NaHCO3 in their drinking water (12; 40). 296
The TTKG, which estimates the K gradient across the final CCD, before the K is concentrated in the 297
medullary collecting ducts with water reabsorption, is an indicator of distal K secretion in response to an 298
electro-negative lumen potential generated by an active driving force (54; 57). The TTKGs of mice on a 299
control diet were close to the value of 6.3 found for humans (4). The TTKG was greater when mice consumed 300
HK-alk, in comparison to HK-Cl (20.9 vs 13.6, figure 2). This finding supports an earlier study showing that 301
acetazolamide-induced bicarbonaturia enhanced the TTKG of humans (4). 302
The competition between H and K secretion in the distal nephron is well recognized. Metabolic acidosis 303
with excess H secretion tends to cause hyperkalemia (1; 52) and metabolic alkalosis with excess HCO3 304
secretion tends to cause hypokalemia when consuming normal K diets (36). Decreased intracellular pH can 305
inhibit ROMK (30; 53); however, it is unclear how an alkaline diet affects the intracellular pH of ROMK-residing 306
PC, which are not specialized to transport acid-base. An increase in K secretion may occur with merely a 307
neutralization of the active H transport by the distal nephron, rather than by HCO3-driven K secretion. 308
However, the urinary pH of the LNaHK mice given acid water was 6.6-6.8, which was consistent with no 309
acidification by the α-IC because the pH of the urine entering the distal nephron is approximately 6.7 (7). 310
Therefore, the high pH of 8.3 in the final urine of LNaHK could only be derived from HCO3 secreted by the β-311
IC, which we found equally enhanced, with respect to total IC, in WT and β4KO on HK-alk. 312
How does an alkaline diet promote K excretion independent of Na reabsorption? One scenario is that 313
HCO3 is secreted from β-IC via pendrin, a Cl/HCO3 exchanger shown in this study and others to be up-314
regulated in animals given an alkaline diet (42; 47; 56). In other cells known to secrete high amounts of HCO3, 315
such as the pancreas (35) and airway serous cells (14), the Cl entering the cell in exchange for HCO3 is 316
recycled back across the apical membrane via a Cl channel such as CFTR, thereby generating an electro-317
15
negative lumen potential. However, the presence of CFTR or another Cl channel in the apical membrane of β-318
IC has not been established. 319
320
Defective β4KO secretion is disassociated from IC phenotypic change 321
Using either apical pendrin or basolateral V-ATPase staining, we found an approximate increase in the 322
proportion of β-IC, from comprising 20 to 35% of total IC in WT and β4KO on a control diet, to 40 to 60% of 323
total IC when mice were on HK-alk diet. That IC convert from β-IC to α-IC according to acid/base status was 324
shown previously (13; 46), with a recent study showing that preventing the conversion of β-IC to α-IC causes 325
acidosis (13). That pendrin estimated a higher percentage of β-IC with HK-alk than basolateral V-ATPase is 326
consistent with the view that pendrin also labels non A-non B cells (26), whereas basolateral V-ATPase should 327
only label β-IC. Both antibodies show that β-IC are increased equally in WT and β4KO, indicating that the 328
phenotypic switch is not dependent on BK-α/β4 function and decreased proportion of β-IC cannot be 329
responsible for the diminished TTKG and K excretion of β4KO on HK-alk. 330
331
Compensatory role of Na reabsorption in β4KO 332
Two recent studies supported the notion that there are two types of K secretion: the well-studied 333
exchange of Na reabsorption for K secretion in the distal nephron and another type of K stimulated transport 334
that is independent of Na reabsorption. Rats treated a high K diet for several days exhibited a mild increase in 335
plasma [K] with amiloride treatment; however, when treated with high K overnight, amiloride increased plasma 336
[K] to an extremely high value of 8.9 mM (10). Because these overnight-treated rats were not K adapted, they 337
did not develop a Na-independent mechanism for secreting K. In isolated perfused rat collecting ducts, 338
iberiotoxin, a specific BK channel blocker, inhibited K secretion in the absence of luminal Na (31) 339
demonstrating that BK channels mediate Na-independent K secretion. 340
16
It might be expected that the Na-dependent component of K secretion would be enhanced if the Na 341
independent component is compromised. This was evident for β4KO as there was less Na excretion in the 342
β4KO than the WT on HK-alk (figure 4). We showed previously that β4KO exhibited reduced Na excretion on 343
HK-alk, along with a reduced hematocrit and increased blood pressure, which was evidence for Na retention 344
and volume expansion.(24). However, we have shown here that amiloride treatment returned the Na excretion 345
to WT levels. Thus, in the absence of HCO3-stimulated K secretion, β4KO compensate by increasing ENaC-346
mediated Na reabsorption to stimulate K secretion. Further evidence of compensatory Na reabsorption was 347
shown in the β4KO on LNaHK. In the absence of substantial dietary Na, the Na reabsorptive compensatory 348
mechanism was also compromised in β4KO as indicated by a further reduction in TTKG and an increase in 349
plasma [K] to near lethal levels. 350
The finding that plasma [K] increased to extremely high levels in β4KO on LNaHK indicates that BK-351
α/β4 is used for Na independent K secretion. The LNaHK diet, which is alkaline and has a K/Na ratio of near 352
500, might mimic the diet of the Yanomami. Similar to previous studies by our laboratory (19; 23), the plasma 353
[K] of WT increased only slightly, by approximately 300 μM, in mice consuming HK-alk, compared with a 354
normal diet (figure 1) (19). Moreover, the plasma [K] was not greater in WT mice consuming LNaHK compared 355
with HK-alk. We did not determine the ability to excrete K with a low Na, high K chloride diet; however, 356
consuming acid water considerably reduced their ability to handle a Na-deficient high K load. This result shows 357
that mice can excrete a high K load absent of Na as long as the diet is alkaline. The naturally high alkaline 358
content associated with the high K diet may be an important component of Na independent K secretion by the 359
Yanomami. 360
361
K balance 362
There is a decreased output of urinary K per K consumed in the WT mice on HK-Cl vs HK-alk. 363
However, the kidneys are not the only avenue for excreting K and the WT on HK-Cl are likely utilizing several 364
extrarenal epithelia to maintain K balance. Several studies have described K adapted (44) and aldosterone 365
regulated K secretion in the colon (6; 8), which handles 10% of K excretion under normal conditions. Even the 366
17
mice on a high K-alk diet excrete 10% less urinary K than they consume. Although water intake is a highly 367
variable measurement, we observe that mice excrete no more than 80% of the volume of water consumed, 368
showing that they are utilizing extrarenal avenues to eliminate the volume as well as the K. WNK4 and WNK1 369
kinases are present in a variety of extrarenal epithelial cells, including colonic crypts, pancreatic ducts, bile 370
ducts, and sweat glands (5; 25), indicating adjustments in Na, Cl and K homeostasis by several extrarenal 371
tissues. We have also noted that the mice on HK-Cl were very wet, indicating profuse sweating as an avenue 372
to eliminate K after achieving a high plasma [K] level. 373
374
Role of IC cells in K secretion 375
Although we have shown that the BK-α/β4 of IC have a role in reducing the size of IC during high flow 376
conditions (23), it has been controversial whether the BK-α/β4 can secrete K because of the paucity of Na-K-377
ATPase to deliver K across the basolateral membrane of IC. However, a recent study with the isolated rat 378
collecting duct has shown that K secretion is reduced by inhibiting NKCC1 (28), localized in the basolateral 379
membrane of IC (15). In the colon, K uptake via basolateral NKCC1 is the putative source of K that is secreted 380
via apical BK (48; 49). The question remains concerning the mechanism for cellular Na extrusion after Na 381
enters via NKCC1. 382
It is not unprecedented that IC transport electrolytes other than acid/base. Renal IC contain a pathway 383
for neutral Na-Cl absorption via a Na-dependent Cl/HCO3 exchanger (NDCBE) (9; 27). It will be interesting to 384
determine how specific transport pathways in IC sustain basolateral K entry and Na extrusion in a cell that is 385
nearly devoid of Na-K-ATPase (43). 386
387
Significance 388
We found that an alkalinizing diet not only enhanced TTKG, a high K diet with Cl as the counter ion is 389
detrimental, causing a large increase in plasma [K] due to a failure to excrete the high K load. Our results 390
explain why sustained-release KCl tablets can elevate plasma [K] to dangerously high levels and why the 391
18
antidote to “KCl poisoning” is NaHCO3 (20). On the other hand, KCl is the best treatment for the condition of 392
hypokalemic alkalosis (3; 29). 393
394
19
Acknowledgments 395
This project was funded by National Institutes of Diabetes and Digestive and Kidney Diseases Grants RO1 396
DK071014 and RO1 DK73070 (to SCS), and a fellowship (#11PRE7530018) from the American Heart 397
Association MWA Affiliate (RJC). 398
399
400
401
402
403
20
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534
535
536
27
Figure Legends 537 538 Figure 1. A. Western blots of BK-β4 (24 kDA) and b-actin (43 kDA) from mice on a control, HK-alk and HK-Cl 539
diet. Negative control shows absence of BK-β4 in β4KO kidneys. B. Summary bar plot of BK-β4/β-actin 540
expression in control, HK-alk, and HK-Cl treated mice. *p<0.05 using one-way ANOVA plus SNK test. 541
542 Figure 2. Bar plots illustrating differences in K clearance (A), plasma [K] (B) and TTKG (C) between WT and 543
β4-KO mice on control, HK-Cl and HK-alk diets. �p<0.001 compared with control diet, *p<0.001 compared with 544
WT and #p<0.001 compared with HK-Cl using one-way ANOVA plus SNK test. . 545
546
Figure 3. Bar plots illustrating the urinary pH values of WT and β4-KO mice on control, HK-Cl and HK-alk 547
diets. Symbols are same as figure 1. 548
549
Figure 4. A. Double IHC with anti-pendrin (arrows; green) and anti-V-ATPase (red) of renal sections from 550
control, HK-alk and HK-Cl diet mice. B. Bar graph summarizing the % pendrin-positive cells/V-ATPase-551
positive cells. (*p<0.01 compared with control diet). C. Double IHC with anti-Aquaporin 3 (AQP3; green) and 552
anti-V-ATPase (red) of renal sections from WT on a control and HK-alk diet. D. Summary bar plots of % 553
basolateral V-ATPase per total V-ATPase marked cells, comparing WT and β4KO on control and HK-alk. 554
555
Figure 5. Bar plots illustrating effects of amiloride on Na clearance (A), hematocrit (B) and weight change (C) 556
of WT and β4-KO mice on HK-alk diets. *p<0.001 compared with WT and #p<0.001 compared with HK-alk, 557
using ANOVA plus SNK test. 558
559
Figure 6. Summary bar plots depicting differences in K clearance (A), plasma [K] (B), and TTKG (C) between 560
WT and β4KO given HK-alk and LNaHK diets. *p<0.001 compared with WT. #p<0.001 compared with HK-alk 561
using one-way ANOVA plus SNK test. 562
563
28
Figure 7. Summary bar plots depicting differences in urinary pH (A), K clearance (B), plasma [K] (C), and 564
TTKG (D) between WT and β4KO mice on LNaHK plus normal or acid (NH4Cl) drinking water. *p<0.001 565
compared with WT and #p<0.001 compared with LNaHK using one-way ANOVA plus SNK test. 566
567
29
Table 1. Food intake and urinary volumes 568
mouse Diet food in
SEM
P vs. WT
PvsCon
UV SEM2
P vs. WT3
PvsCon2
Hct.
SEM3
PvsWTPvsCon
P vs. con3
WT control 1.5 0.2 3.5 0.3 45.6
0.4
β4ΚΟ control 1.7 0.3 NS 3.8 0.4 NS 45.9
0.8
WT HK-alk 3.5 0.2 <0.001
19.3 1.1 <0.001
44.3
0.3 0.066
β4ΚΟ HK-alk 3.5 0.3 NS 0.001 19.7 1.4 NS <0.001
40.2
0.5 <0.001 <0.001
WT HK-Cl 3.3 0.2 0.002 16.1 1 <0.001
47 0.7 0.485
β4ΚΟ HK-Cl 2.9 0.4 NS 0.145 14.9 3.3 NS <0.001
40 0.9 <0.001 <0.001
WT HK-alk am
3 0.4 0.004 14.9 1.8 <0.001
48.6
1.3 0.02
β4ΚΟ HK-alk am
2.6 0.4 NS 0.069 14.8 0.6 NS <0.001
47.2
0.7 NS 0.518
WT LNaHK 3.6 0.3 <0.001
18.3 1.3 <0.001
46.2
0.8 0.771
β4ΚΟ LNaHK 3.5 0.5 NS 0.006 16 1.2 NS <0.001
44.1
0.7 NS 0.314
WT LNaHK+A
4 0.1 <0.001
13.4 1.2 <0.001
43.5
0.9 0.212
β4ΚΟ LNaHK+A
2.9 0.2 NS 0.086 9.3 1.1 NS 0.034 42.8
0.6 NS 0.123
569
Table 1. Values represent means for food consumption (food: gms/day) and urinary excretion rate (UV: 570 ml/day/gm kidney weight). SEM is standard error of mean. P vs WT is the probability that β4KO is different 571 from WT on same diet/drug using ANOVA plus SNK test. NS (not significant) is P > 0.05. P vs con is the 572 probability that WT or β4KO is different from respective genotypes on a control diet. The number of samples 573 (N) is >5 in all groups. Differences between groups were determined by the ANOVA plus SNK test. 574
575
BK-β4
control HK-alk HK-Cl
25 kD
WT β4-KO
A. Immunoblots
B BK β4/β actin densitometry
BK-β4
β-actin 37 kD50 kD
25 kD
B. BK-β4/β-actin densitometry
)
1.0
1.2
*
β4/ β
-act
in (A
u)
0.6
0.8
t l HK lk HK Cl
BK
- β
0.0
0.2
0.4
control HK-alk HK-Cl
A. Potassium Clearance)
2500WT #
B. Plasma K concentration10
WT
ml/d
ay/g
mK
W)
1500
2000
WTβ4KO
*#ɟ ɟ
a [K
], m
M
6
8
WT β4KO *
##*
a [K
] (m
M)
K c
lear
ance
(m
500
1000
Plas
ma
2
4
Plas
ma
control HK-Cl HK-alk0
C. Transtubular K gradient
control HK-Cl HK-alk0
20
25 WT β4KO
*#
#
#ɟ ɟ
TTK
G
5
10
15 #ɟ ɟ
control HK-Cl HK-alk0
5
Urinary pH
10WT β4KO
# #pH 6
8
#
4
control HK-Cl HK-alk2
t l
A. WT – pendrin + V-ATPase B. % β-IC (pendrin-identified)
80# #
control
HK Cl / V-A
TPas
e +
40
60
WTβ4KO
#
#
HK-alk
HK-Cl
% p
endr
in +
20
40 #
HK alk
control HK-Cl HK-alk0
C. V-ATPase + AQP3B % β IC (V ATPase on BLM)
control HK-alk
B. % β-IC (V-ATPase on BLM)
40
50
60
WT β4KO
##
% β
-IC20
30
40
0
10
control HK-alk
A. Na clearance
10
B. Hematocrit
55WT
ml/d
ay/g
mK
W)
6
8
WTβ4KO
#
(%) 45
50
WT β4KO
*
# #
Na
clea
ranc
e (m
2
4*
Hct
.
35
40*
HK-alk HK-alk + amil
N
0HK-alk HK-alk+amil
30
C. Weight change
ms)
2
3
4
5
WT β4KO
*
Δ w
iegh
t (gm
-1
0
1
2
HK-alk HK-alk + amil
-2
-1
#
B. Plasma K concentration
8 * #A. K Clearance
] (m
M) 6
8WTβ4KO
*
#
day/
gmK
W)
1500
2000
2500WTβ4KO
*
* #
Plas
ma
[K]
2
4
K c
lear
ance
(ml/d
500
1000
HK-alk LNaHK0
HK-alk LNaHK
K
0
C. TTKG
20
25 WT β4KO
** #
TTK
G
5
10
15 * #
HK-alk LNaHK0
5
A. Urinary pH10 WT
β4KO
B. K Clearance
3000WT
pH
6
8 # #
ml/d
ay/g
mK
W)
1500
2000
2500
WTβ4KO
*p
2
4
K c
lear
ance
(m
500
1000
1500#
LNaHK LNaHK + acid 0
C. Plasma K concentration10
LNaHK LNaHK + acid0
D. TTKG
25
(mM
)
6
8
WTβ4KO
*#
15
20
25WT β4KO
*#
Plas
ma
[K]
2
4 TTK
G
5
10
15
LNaHK LNaHK + acid 0
LNaHK LNaHK + acid 0
5