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Third Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto, Japan.
Correspondence to Dr. Mika Ikebe, Third Department of Internal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto, Kumamoto 860, Japan. Phone: 81-96-373-5164; Fax: 81-96-366-8458; E-mail: nono{at}kaiju.medic.kumamoto-u.ac.jp (Hiroshi Nonoguchi)
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NKCC1 has been cloned from mouse terminal inner medullary collecting duct (IMCD) cells, as well as the shark rectal gland and human colon (2,3,4). Northern blot analyses have demonstrated the presence of NKCC1 mRNA not only in the kidney but also in many secretory epithelia, in sites such as the salivary gland, stomach, jejunum, colon, lung, and heart. Immunohistochemical studies in the kidney have demonstrated the presence of NKCC1 proteins in terminal IMCD in mice but not in terminal IMCD in rats (16,17); the NKCC1 protein has been found to be present in the basolateral membrane of intercalated cells in the outer medullary collecting duct (OMCD) in rats. The OMCD is important not only in acid excretion but also in sodium absorption and secretion (18,19). One role of NKCC1 in rat OMCD has been suggested to be ammonium excretion (17). One of the splice variants of NKCC2, i.e., NKCC2F, has been reported to be located also in the OMCD (14). The direction of sodium movement is different for NKCC1 and -2. Recent reports have revealed that mice lacking NKCC1 exhibit signs of deafness and imbalance (20,21). The functional role of NKCC1 in rat OMCD is still unclear, and it is not known whether NKCC1 expression is regulated by pathophysiologic changes such as dehydration and metabolic acidosis. Therefore, we investigated, using competitive PCR and Western blot analysis, whether NKCC1 expression is regulated by pathophysiologic changes in body fluid homeostasis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of Rats
The appropriate conditions for inducing metabolic acidosis were first
examined. Because conventional 0.28 M NH4Cl solution is hypertonic,
rats not only experience acidosis but also become dehydrated. Therefore,
isotonic 0.14 M NH4Cl solution was administered as drinking water.
NH4Cl was then provided in the food. Several doses of
NH4Cl in the food (rat chow, 8 g; NH4Cl, X g;
Bacto agar, 0.25 g; water, 10 ml/100 g body wt per d; X = 0.1, 0.2,
0.25,or 0.5) were administered for 4 d to the acidosis group, for
determination of the appropriate doses of NH4Cl. Arterial blood gas
values, plasma osmolality, and plasma vasopressin concentrations were
measured.
Using the results from the aforementioned experiments, dehydration and chronic metabolic acidosis were induced in male Sprague-Dawley rats. Dehydration was induced by water deprivation for 2 d. Metabolic acidosis was induced by adding NH4Cl to standard rat chow for 6 to 7 d (rat chow, 8 g; NH4Cl, 0.2 g; Bacto agar, 0.25 g; water, 10 ml/100 g body wt per d). Control rats for acidosis received the same diet as did the acidosis group except for the addition of NH4Cl. Control rats and rats with acidosis were given free access to tap water. After anesthesia with sodium pentobarbital, arterial blood was drawn from the abdominal aorta. Blood pH, Pco2, Po2, and HCO3- concentrations were measured using an automatic blood gas analyzer (ABL 510; Radiometer, Copenhagen, Denmark). Plasma levels of vasopressin were measured by RIA (SRL Laboratories, Tokyo, Japan). Plasma osmolality was measured using an osmometer (Fiske, Norwood, MA).
Microdissection of Glomeruli and Nephron Segments
After perfusion of the left kidney with solution A containing 0.1%
collagenase and 0.1% bovine serum albumin, microdissection of glomeruli and
nephron segments was performed in the presence of 10 mM VRC, as described
previously
(22,23).
The microdissected nephron segments were as follows: glomeruli, proximal
convoluted tubules, proximal straight tubules, MAL, CAL, cortical collecting
ducts (CCD), OMCD, and IMCD. After the VRC was washed out of the tubules with
solution A, five glomeruli and 2-mm-long nephron segments were transferred
into Eppendorf tubes, to which 10 µl of solution A2 was added. Solution A
had the following composition: 130 mM NaCl, 5 mM KCl, 1 mM
Na2H2PO4, 1 mM MgSO4, 1 mM calcium
lactate, 2 mM sodium acetate, 5.5 mM glucose, and 10 mM Hepes (pH 7.4 with
NaOH). Solution A2 had the same composition as solution A except that >1
U/µl RNase inhibitor (Boehringer Mannheim) and 1 M DL-dithiothreitol (DTT;
Sigma) were added.
Incubation Study
To elucidate whether low pH stimulates NKCC1 mRNA expression,
microdissected OMCD (2 mm) were incubated in 100 µl of isotonic medium at
37°C. The time course of the effect of low pH on NKCC1 mRNA expression was
investigated first. Microdissected OMCD were incubated for 0, 5, 15, 30, 60,
or 90 min at 37°C in medium with normal or low pH (pH 7.4 and 6.7,
respectively). OMCD were then incubated at pH 7.4, 7.1, or 6.7 for 90 min at
37°C. The pH of the medium was adjusted by adding HCl to solution A.
Reverse Transcription-Competitive PCR
Reverse transcription (RT)-competitive PCR was performed using a cDNA
synthesis kit and PCR master kit, as described previously
(22,23).
Each sample was centrifuged at 15,000 rpm for 5 min at 4°C. After the
supernatant was discarded, 3.5 µl of 2% Triton X-100 solution with RNase
inhibitor and DTT was added. Then, 4.4 µl of a RT mixture containing random
primers was added. RT was performed by incubating the samples at 42°C for
60 min. After RT was stopped by heating the sample at 90°C for 5 min, the
samples were subjected to PCR.
Specific primers for NKCC1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed. NKCC1 sense and antisense primers were defined by bases 2494 to 2511 (5'-GGTTCTCCAAACTCACGG-3') and bases 3040 to 3059 (5'-GTCTTGCCATCCTCTTCCTC-3'), respectively, of the mouse NKCC1 cDNA sequence (3). These correspond to bases 2486 to 2505 and 3032 to 3051, respectively, of the rat sequence (24). The amplified cDNA was predicted to be 566 bp in length. The PCR product was sequenced. GAPDH sense and antisense primers were defined by bases 506 to 525 (5'-TCCCTCAAGATTGTCAGCAA-3') and bases 794 to 813 (5'-AGATCCACAACGGATACATT-3'), respectively (25). The predicted length of the amplified cDNA was 308 bp.
NKCC1 mRNA expression was quantified using competitive PCR. The DNA competitor for the competitive PCR was synthesized using overlap-extension PCR, as described previously (22,23,26,27). The inner antisense and inner sense primers were defined by bases 2682 to 2701 (5'CCACATCCCTCATATCTGCTCCCGCAAGTCATCTGCGTGA-3') and bases 2790 to 2809 (5'-TCACGCAGATGACTTGCGGGAGCAGATATGAGGGATGTGG-3'), respectively. The inner sense and inner antisense primers were made to be complementary by adding complementary sequences to each 5'-end (the complementary sequences added to each 5'-end are underlined). The first PCR was performed with the combinations of the outer sense and inner antisense primers and the outer antisense and inner sense primers. Two PCR products (208 and 270 bp) were combined in the second PCR. The final PCR product, the DNA competitor, was electrophoresed and purified using a nucleic acids extraction kit, Nucleotrap (Macherey-Nagel, Duren, Germany). The size of the DNA competitor was 478 bp. A series of dilutions of the competitor and NKCC1 cDNA were used to establish the standard curve.
Another DNA competitor was produced using a competitive DNA construction kit (Takara, Shiga, Japan), according to the instructions provided by the manufacturer. Sense and antisense primers for NKCC1 mRNA were used to produce the competitor. The size of the DNA competitor was 456 bp.
GAPDH mRNA was amplified using the usual PCR method, and NKCC1 mRNA was amplified using competitive PCR. The DNA competitor (1 to 20 fg) was added to the NKCC1 samples. The annealing temperatures for NKCC1 and GAPDH were 60°C and 62°C, respectively. The PCR cycle numbers for NKCC1 and GAPDH were 30 and 28 cycles, respectively.
Ethidium Bromide Staining and Southern Blotting
After PCR, the PCR product was precipitated with ethanol and
electrophoresed in a 2% agarose gel with Tris-ethylenediaminetetra-acetate
(EDTA) buffer. The PCR products were observed by ethidium bromide staining.
The intensity of the bands in the NKCC1 samples was quantitated using a
densitometer (Atto, Tokyo, Japan) for competitive PCR assays. The NKCC1
cDNA/competitor ratio was corrected for the differences in molecular weight.
The Tris-EDTA buffer had the following composition: 40 mM Tris, 1 mM EDTA (pH
8.3 with acetic acid).
The gel of the GAPDH sample was then denatured and neutralized. The PCR products were transferred onto a nylon membrane with 20x SSC buffer (3 M NaCl, 0.3 M sodium citrate) and left overnight. The membrane was baked in an ultraviolet baking system and stored until Southern hybridization. The nonisotopic digitoxigenin (DIG)-nucleotide detection system was used for Southern hybridization and DNA detection. A specific probe for GAPDH was produced with a PCR DIG probe synthesis kit, using each cDNA and specific primers, as described in the instruction manual. Observation of DNA was performed using a DIG luminescence detection kit.
Western Blotting
Western blotting was performed using membrane fractions from the cortex,
outer medulla, and inner medulla and microdissected OMCD and MAL, as described
previously
(22,23,28).
In brief, the kidney was removed and cut into blocks of the cortex, outer
medulla, and inner medulla. Each part was cut into small pieces with a razor
blade and homogenized with glass homogenizer. The homogenate was centrifuged
at 7600 rpm for 15 min at 4°C. The supernatant was then centrifuged at
15,000 rpm for 30 min at 4°C. The pellet was dissolved in lysis buffer,
and the protein content was measured using the bicinchoninic acid protein
assay reagent (Pierce, Rockford, IL). Microdissected OMCD (20-mm long) and MAL
(20-mm long) were transferred to Eppendorf tubes, to which lysis buffer was
added. Samples (30 µg of the membrane fractions and microdissected OMCD and
MAL) were mixed with 2x sample buffer and then subjected to sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After
SDS-polyacrylamide gel electrophoresis, the proteins were transferred to a
nylon membrane (Immobilon-P; Millipore, Bedford, MA). After blocking with
Tris-buffered saline with Tween 20 and 5% milk, the membrane was incubated
overnight with the primary antibody against NKCC1 (diluted 1:2000 for the
membrane fractions and 1:500 for the microdissected nephron segments). The
specific polyclonal antibody against NKCC1 protein was a kind gift from Dr. R.
James Turner, National Institutes of Health (Bethesda, MD)
(24). The antibody was raised
against a 6 x His fusion protein corresponding to amino acids 750 to
1203 of rat NKCC1. The membrane was then incubated with horseradish
peroxidase-linked antirabbit IgG F(ab')2 for 1 h at room
temperature. Protein expression was detected with enhanced chemiluminescence
Western blotting detection reagents (Amersham). Protein expression was
measured with a densitometer (Atto). The composition of the lysis buffer was
0.5 mM Na3VO4, 1 mM EDTA, 5 mM ethyleneglycol
bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetate, 2 mM
DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leucine, 5 mM ß-glycerophosphate, and 20 mM Hepes (pH 7.4). The
composition of the 2x sample buffer was 20% glycerol, 4.6% SDS, 10%
ß-mercaptoethanol, 130 mM DTT, 0.01% bromphenol blue, and 130 mM Tris-HCl
(pH 6.8). Tris-buffered saline contained 20 mM Tris and 137 mM NaCl.
Statistical Analyses
Results are expressed as mean ± SEM. Statistical analyses were
performed using the t test for two groups and ANOVA or Kruskal-Wallis
analysis followed by Dunnett's multiple-comparison test for more than three
groups, as appropriate. P < 0.05 was considered significant.
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NKCC1 mRNA Distribution along the Nephron in Mice and Rats
NKCC1 mRNA distribution along the nephron was compared between mice and
rats (Figure 1). NKCC1 mRNA
expression was most abundant in the glomeruli and IMCD in mice. Low expression
was observed in other nephron segments, such as the proximal convoluted
tubules, proximal straight tubules, MAL, CAL, CCD, OMCD, and IMCD. In
contrast, NKCC1 mRNA expression was abundant in the collecting ducts,
especially the OMCD, in rats. Low expression was observed in the glomeruli,
MAL, and CAL. Competitive PCR revealed that NKCC1 mRNA expression in the OMCD
was 5 times higher than that in the CCD and 2 times higher than that in the
IMCD (Figures 2 and
3).
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Effects of Dehydration and Metabolic Acidosis on Rat NKCC1 mRNA
Expression in the Collecting Duct
NKCC1 mRNA expression was compared in control rats, dehydrated rats, and
rats with acidosis. Competitive PCR was used in these experiments. Dehydration
significantly increased NKCC1 mRNA expression in the CCD, OMCD, and IMCD
(Figure 2). Chronic metabolic
acidosis also caused increases in NKCC1 mRNA expression in the CCD, OMCD, and
IMCD (Figure 3). Expression was
most abundant in the OMCD in both dehydrated rats and rats with metabolic
acidosis. There was no difference in GAPDH mRNA expression in the three
segments in dehydrated rats, rats with acidosis, and control rats (data not
shown). These data show that NKCC1 mRNA expression is regulated under such
pathophysiologic conditions.
Effects of Low Medium pH on Rat NKCC1 mRNA Expression In Vitro
Time-course experiments revealed that NKCC1 mRNA expression was stimulated
by 30- to 90-min incubations in low-pH medium
(Figure 4). Although incubation
of OMCD at low pH for 5 min decreased NKCC1 mRNA expression, the initial
decrease in NKCC1 mRNA expression produced by low pH could be attributable to
mRNA degradation. It is not known whether this degradation is caused by the
activation of RNase. On the basis of these findings, OMCD were incubated at pH
7.4, 7.1, and 6.7 for 90 min. Incubation of OMCD in pH 6.7 medium
significantly stimulated NKCC1 mRNA expression in vitro by 62%,
compared with incubation at pH 7.4. The increase in expression at pH 7.1 was
not statistically significant (6.8 ± 0.6, 7.9 ± 1.0, and 11.0
± 1.4 fg/mm at pH 7.4, 7.1, and 6.7, respectively; P < 0.05
for pH 6.7 versus pH 7.4) (Figure
5).
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Rat NKCC1 Protein Expression in Dehydrated Rats, Rats with Metabolic
Acidosis, and Control Rats
The antibody recognized a single broad band at 170 kD, which was compatible
with the predicted size of the NKCC1 protein. The membrane fraction from the
inner medulla exhibited higher levels of expression of NKCC1 protein than did
the cortex or the outer medulla (Figure
6). Dehydration and acidosis significantly increased NKCC1 protein
expression in the membrane fraction from the outer medulla, by 122 ± 35
and 95 ± 29%, respectively. This increase was not observed in other
parts of the kidney.
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NKCC1 protein expression was observed in microdissected OMCD but not in MAL, confirming that this antibody is specific for NKCC1 (Figure 7). Dehydration and chronic metabolic acidosis significantly increased expression, by 113 ± 23 and 86 ± 30%, respectively.
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NKCC2 has been considered to be more important than NKCC1 for sodium absorption and secretion. The distribution of NKCC2 is only in the kidney. Immunohistochemical studies have demonstrated the presence of NKCC2 in the luminal membrane of MAL and CAL, where the capacity for NaCl absorption is very high and is sensitive to arginine vasopressin. NKCC2 protein levels in MAL are not upregulated with water restriction or vasopressin treatment, although vasopressin increases NKCC2-mediated sodium reabsorption (9,29). Therefore, vasopressin seems to regulate NKCC2 through another mechanism. Furosemide and bumetanide are known to cause potent diuresis by inhibiting NKCC2. In contrast, NKCC1 is located in various secretory epithelia, including the OMCD of rat kidney. Although OMCD have a lower sodium-absorbing capacity than do MAL and CAL (19), the epithelial sodium channel is known to be activated by vasopressin and aldosterone (30). OMCD also have a high capacity for acid excretion (18). In this study, chronic metabolic acidosis increased NKCC1 mRNA expression in OMCD. Furthermore, incubation of OMCD in low-pH medium for >30 min stimulated NKCC1 mRNA expression in vitro, suggesting that low pH is one of the stimulators of NKCC1 mRNA expression. The initial decrease in NKCC1 mRNA expression could be attributable to the increased degradation of mRNA. RNase activation may play some role. Wall et al. (31) reported that increased extracellular pH stimulated bumetanide-sensitive rubidium uptake in mouse IMCD cells, which is a change in the opposite direction, compared with our results. Their study examined very acute effects of the changes in extracellular pH on NKCC1 activity. The stimulation of NKCC1 activity was probably caused without changes in NKCC1 mRNA or protein expression. Our study focused on chronic effects of extracellular pH. Initial decreases in NKCC1 mRNA expression with low pH may reflect acute inhibitory effects of low pH on NKCC1 mRNA expression. Therefore, it might be reasonable to assume that low extracellular pH inhibits NKCC1 mRNA expression in the acute phase but stimulates it in the chronic phase.
Net acid excretion is the sum of ammonium and titratable acid excretion if there is no bicarbonate-wasting. Ammonia is primarily produced in proximal tubules by phosphate-dependent glutaminase and accumulates in the medullary interstitium (11). The ammonium ion (NH4+) is absorbed via NKCC2 in MAL and CAL by substituting for K+ (10,11,12). The luminal membrane of MAL and CAL is impermeable to NH3 (10,12). The nonionic diffusion of NH3 into the lumen of OMCD, which is stimulated by low luminal pH, has been thought to be the primary route of ammonium excretion, at least under control conditions (18). NH4+ has also been shown to substitute for K+ for NKCC1 (31). Wall (32) reported that the participation of NKCC1 in ammonium excretion in the OMCD is only 6% under control conditions. However, the upregulation of NKCC1 mRNA and protein by dehydration and metabolic acidosis suggests that the role of NKCC1 in dehydration and chronic metabolic acidosis should be much larger. When these findings are taken together, it can be speculated that NH4+ is absorbed via NKCC2 in MAL and CAL and is secreted via nonionic diffusion and via NKCC1 in the OMCD. Whether the participation of NH4+ secretion via NKCC1 in metabolic acidosis plays some role in ammonium secretion must be examined.
Our results showing the highest level of NKCC1 protein expression in the inner medulla of control rats differ slightly from the results of Ginns et al. (17). Those authors reported the highest level of expression in the base region of the inner medulla and the inner stripe of the outer medulla. We combined the base and tip regions of the inner medulla and the outer and inner stripes of the outer medulla. This high level of expression in the inner medulla is thought to be caused by the difference in the populations of collecting ducts in the outer and inner medulla; the IMCD occupies approximately 20% of the inner medulla, whereas the OMCD occupies only 10% of the outer medulla (33). It is not known whether NKCC1 exists not only in IMCD but also in other types of cells, such as interstitial cells of the inner medulla. NKCC1 protein expression must be precisely examined by Western blot analysis using microdissected OMCD and IMCD.
It is interesting that dehydration stimulated both NKCC1 mRNA and protein expression in the OMCD in our study. Arterial blood gas analysis revealed that rats experience respiratory alkalosis under dehydration conditions. Plasma HCO3 concentrations were decreased in response to reduced Pco2, and plasma vasopressin levels were significantly increased. Low plasma HCO3- concentrations, high blood vasopressin levels, or high osmolality in the medullary interstitium and urine in dehydrated rats could stimulate NKCC1 expression in the OMCD (34). Hypertonic cell shrinkage is known to stimulate rat parotid NKCC1 activity, suggesting a role for NKCC1 in acute regulatory volume increases (35). Chronic cell volume regulation takes place via the accumulation of osmolytes in the cells. The participation of NKCC1 in chronic cell volume regulation, however, is not known. Protein kinase inhibitors (specifically staurosporine and K252a) are known to blunt the stimulation, suggesting the participation of protein kinase C in NKCC1 activation (35). Volume-sensitive phosphorylation of NKCC1 by c-Jun amino-terminal kinase has also been reported (36). NKCC1 activity in the parotid gland is also increased by ß-adrenergic and muscarinic stimulation (37,38). Recent reports unexpectedly revealed that mice lacking NKCC1 exhibit signs of deafness and imbalance (20,21). Although epithelial chloride secretion has been reported to be impaired, the characteristics of ion transport in the OMCD in such knockout animals are not yet known. The specific mechanisms of NKCC1 regulation in OMCD must be examined further.
In summary, NKCC1 mRNA and protein expression in OMCD is upregulated under conditions of dehydration and chronic metabolic acidosis. These data suggest that NKCC1 may play an important role in renal adaptation to these physiologic conditions. Low pH and possibly hypertonicity seem to stimulate NKCC1 mRNA expression.
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, rats). The amounts of
competitor used in the gels were 50, 10, 10, 8, 2, 2, 15, and 80 fg/sample for
the glomeruli (Glm), proximal convoluted tubules (PCT), proximal straight
tubules (PST), medullary thick ascending limbs (MAL), cortical thick ascending
limbs (CAL), cortical collecting ducts (CCD), outer medullary collecting ducts
(OMCD), and inner medullary collecting ducts (IMCD) for mice and 25, 1, 1, 1,
2, 5, 20, and 15 fg/sample for rats, respectively. Five glomeruli and
2-mm-long nephron segments per sample were used for RT-competitive PCR. The
478-bp DNA competitor was used in this study. NKCC1 mRNA was most abundant in
the IMCD in mice and in the OMCD in rats.
