Upregulation of the Secretory-Type Na⁺/K⁺/2Cl^--Cotransporter in the Kidney by Metabolic Acidosis and Dehydration in Rats

Materials

The cDNA synthesis kit and PCR master kit were obtained from Boehringer Mannheim (Mannheim, Germany). Enhanced chemiluminescence Western blotting detection reagents were purchased from Amersham Pharmacia Biotec (Buckinghamshire, UK). Vanadyl ribonucleoside complex (VRC) was supplied by Life Technologies BRL (Gaithersburg, MD). Agarose was obtained from SeaKem (Rocklane, ME). Bacto agar was purchased from Difco Laboratories (Detroit, MI).

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 20× 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 2× 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 × 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(β-aminoethyl ether)-N,N,N′,N′-tetraacetate, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leucine, 5 mM β-glycerophosphate, and 20 mM Hepes (pH 7.4). The composition of the 2× sample buffer was 20% glycerol, 4.6% SDS, 10% β-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.

Physiologic Parameters for Rats

The effect of isotonic 0.14 M NH₄Cl on plasma bicarbonate concentrations was first examined. However, the reduction in plasma bicarbonate concentrations was not enough (24.5 ± 0.6 and 22.1 ± 1.1 mEq/liter HCO₃^- in control and NH₄Cl-treated rats, respectively; n = 5). The effect of NH₄Cl in the food was then examined. Table 1 summarizes the effects of several doses of NH₄Cl in the food on several parameters in control rats, dehydrated rats, and rats with metabolic acidosis. Dehydration caused increases in plasma osmolality and arginine vasopressin levels. In contrast, NH₄Cl in the food did not increase plasma osmolality or arginine vasopressin levels. Decreases in plasma pH and bicarbonate concentrations were observed at all tested doses. NH₄Cl in the food dose-dependently decreased plasma bicarbonate concentrations. However, 0.5 g of NH₄Cl/8 g of food caused severe metabolic acidosis and a loss of body weight. On the basis of these results from a variety of in vivo models, 0.2 g of NH₄Cl/8 g of standard chow was used to induce metabolic acidosis in the following experiments. These data demonstrated that metabolic acidosis could be induced by our new method without dehydration.

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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Figure 1.

NKCC1 mRNA expression along the nephron in mice and rats. NKCC1 mRNA expression was measured using reverse transcription (RT)-competitive PCR. (Top) Typical examples of ethidium bromide-stained gels. The upper bands for mice and rats represent the PCR products derived from NKCC1 mRNA, and the lower bands represent those derived from the competitor. (Bottom) Summary of the results of five experiments ([UNK], mice; ▪, 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.

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Figure 2.

Rat NKCC1 mRNA expression in the collecting ducts in control and dehydrated rats. (Top) Typical example of an ethidium bromide-stained gel. (Bottom) Summary of NKCC1 mRNA expression in collecting ducts in control and dehydrated rats, from nine experiments. The amounts of competitor used in the gel were 5, 15, and 10 fg/sample for CCD, OMCD, and IMCD, respectively. The 456-bp DNA competitor was used in this study. Two days of dehydration significantly increased NKCC1 mRNA expression in the CCD, OMCD, and IMCD. C, control; D, dehydration. ^*P < 0.05 versus the control value for each segment.

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Figure 3.

Rat NKCC1 mRNA expression in the collecting ducts in control rats and rats with metabolic acidosis. (Top) Typical example of an ethidium bromide-stained gel. (Bottom) Summary of NKCC1 mRNA expression in collecting ducts in control rats and rats with acidosis, from 10 experiments. The amounts of competitor used in the gel were 4, 15, and 12 fg/sample for CCD, OMCD, and IMCD, respectively. The 456-bp DNA competitor was used in this study. Chronic metabolic acidosis significantly increased NKCC1 mRNA expression in the CCD, OMCD, and IMCD. C, control; A, metabolic acidosis. ^*P < 0.05 versus the control value for each segment.

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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Figure 4.

Time course of the effect of low pH on NKCC1 mRNA expression in OMCD in vitro. Microdissected OMCD were incubated in normal medium (pH 7.4) (C and ▪) or acidic medium (pH 6.7) (A and [UNK]) for 0, 5, 15, 30, 60, and 90 min at 37°C. (Top) Typical example of an ethidium bromide-stained gel. (Bottom) Summary of six experiments. NKCC1 mRNA expression in OMCD was decreased at 5 min of incubation at pH 6.7. However, expression was stimulated at 30 to 90 min of incubation at low pH. ^*P < 0.05 versus pH 7.4.

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Figure 5.

Effects of low medium pH on rat NKCC1 mRNA expression in OMCD in vitro. Microdissected OMCD were incubated for 90 min in medium of pH 7.4, 7.1, or 6.7. Incubation at pH 6.7 stimulated NKCC1 mRNA expression 62% more than did incubation at pH 7.4. Horizontal bar, mean value of five experiments. ^*P < 0.05 versus pH 7.4 (ANOVA and Dunnett's multiple-comparison test).

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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Figure 6.

Rat NKCC1 protein expression in the kidney in control rats, rats with metabolic acidosis, and dehydrated rats. Membrane fractions from the cortex (CX), the outer medulla (OM), and the inner medulla (IM) were used for Western blotting. (Top) Typical example of a Western blot. (Bottom) Summary of NKCC1 protein expression in the three parts of the kidney, from nine experiments. NKCC1 protein abundance in the membrane fraction from the outer medulla was significantly increased by metabolic acidosis and dehydration. C, control; A, metabolic acidosis; D, dehydration. ^*P < 0.05 versus the control value for each part.

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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Figure 7.

Rat NKCC1 protein expression in the OMCD and MAL in control rats, dehydrated rats, and rats with metabolic acidosis. Microdissected OMCD (20-mm long) and MAL (20-mm long) were used for Western blotting. (Top) Typical example of a Western blot. (Bottom) Summary of NKCC1 protein expression in OMCD and MAL, from seven experiments. NKCC1 protein bands at 170 kD were observed only in the OMCD and not in the MAL. NKCC1 protein expression in the OMCD was significantly stimulated by 2 d of dehydration and chronic metabolic acidosis. C, control; A, metabolic acidosis; D, dehydration. ^*P < 0.05 versus control.