INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

OUR LABORATORY (36, 37, 40) has shown that along the basolateral membrane of the rat terminal inner medullary collecting duct (tIMCD), NH uptake occurs by the Na,K-ATPase. NH uptake by the Na,K-ATPase provides a source of H⁺ for secretion of net H⁺ equivalents and titration of luminal buffers in this segment (36, 37).

Because NH and K⁺ are competitive substrates for a common extracellular binding site on this transporter (40), interstitial K⁺ concentration should modulate NH uptake by the Na,K-ATPase. Interstitial concentrations of NH and K⁺ have been determined by sampling vasa recta plasma at the same level (9, 11, 14). These studies have demonstrated that K⁺ concentration in the interstitium of rat inner medulla is generally much higher than NH concentration. For example, in untreated rats, vasa recta NH concentration is 8.4 mM (14) whereas K⁺ concentration is 36 mM (11). Moreover, the apparent affinity for the extracellular binding site of the Na,K-ATPase is greater for K⁺ than for NH (40). Therefore, uptake of K⁺ by the Na,K-ATPase should be much greater than uptake of NH under physiological conditions. This raises the possibility that NH uptake by Na,K-ATPase may be of little physiological significance in the tIMCD in vivo. Our laboratory has explored the contribution of NH uptake by Na,K-ATPase to secretion of H⁺ equivalents in tIMCD of normal rats eating a balanced diet. tIMCD tubules from untreated rats were perfused at NH and K⁺ concentrations expected in the interstitium of the inner medulla of these animals. Under physiological conditions (K⁺ concentration = 30 mM; NH concentration = 6 mM), no change in HCO absorption (J_tCO2) was detected with inhibition of Na,K-ATPase by the addition of ouabain to the bath fluid (38).

However, vasa recta K⁺ concentration varies widely with changes in K⁺ homeostasis (9, 11). Dobyan and collaborators (11) showed that after 3 days of a K⁺-restricted diet, vasa recta K⁺ concentration was reduced from 36 to 8.6 mM. These K⁺ concentrations observed in vivo were used in experiments with tIMCD tubules perfused in vitro. tIMCD tubules from rats eating a K⁺-restricted diet were perfused in symmetrical HCO/CO₂-buffered solutions that contained K⁺ and NH at concentrations expected in the interstitium of this treatment group (K⁺ concentration = 10 mM; NH concentration = 6 mM). Under these conditions, inhibition of NH uptake via Na,K-ATPase, by addition of ouabain to the bath, reduced net secretion of H⁺ by 40-50% (38). Thus, in tIMCD tubules from K⁺-depleted rats, when perfused in the presence of physiological concentrations of NH and K⁺, an important role of NH uptake by Na,K-ATPase in the process of secretion of net H⁺ equivalents was demonstrated.

When perfused and bathed under identical conditions (i.e., at a K⁺ concentration of 10 mM and an NH concentration of 6 mM), tubules from K⁺-depleted rats secrete H⁺ equivalents at a higher rate than tubules from K⁺-replete controls (41). These data indicate that in the rat tIMCD a stable adaptation occurs during hypokalemia, which increases net acid secretion. In rat outer medullary collecting duct (OMCD), McDonough and colleagues (23) demonstrated that dietary K⁺ restriction increases Na⁺ pump expression threefold. We reasoned that if Na,K-ATPase expression is upregulated at the level of the plasma membrane in the tIMCD during hypokalemia, greater uptake of NH across the basolateral membrane should occur in tandem with increased secretion of H⁺ equivalents.

The purpose of the present study was therefore to determine whether 1) the Na,K-ATPase is upregulated during hypokalemia and 2) if the increase in NH uptake by Na,K-ATPase observed during hypokalemia occurs because of upregulation of Na,K-ATPase at the level of the plasma membrane or from the reduction in interstitial K⁺ concentration observed in this treatment group.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal conditioning. Tubules from the tIMCD were dissected from pathogen-free male Sprague-Dawley rats weighing 65-120 g (Harlan, Indianapolis, IN). To isolate IMCD cells in suspension, 125- to 150-g rats were used. Animal conditioning was similar to that reported previously by our laboratory (38). Animals were housed in microisolator cages and fed a diet with 7.8 g K⁺/kg food and 0.664 g Na⁺/kg food (Zeigler Brothers, Gardeners, PA) for 2-7 days. Rats were then divided into two treatment groups. Group A rats ate a diet with a normal K⁺ content (diet no. P1868, 2.3 g K⁺/kg food and 1.03 g Na⁺/kg food; ICN Biochemicals, Aurora, OH). Group B rats ate a diet identical to that of group A, but with a low K⁺ content (diet no. 960189, 0.0041 g K⁺/kg food and 1.03 g Na⁺/kg food; ICN Biochemicals). Rats were maintained on diets with either low or normal K⁺ content for 3 or 7 days before death and were pair fed. To induce a rapid diuresis, animals were injected with furosemide (5 mg/100 g body wt ip) 30-45 min before death by decapitation. This furosemide-induced diuresis reduces the inner medullary axial solute concentration gradient (36, 43) and attenuates changes in extracellular osmolality. Serum K⁺ was measured by the Department of Veterinary Medicine, University of Texas at Houston, using ion-sensitive electrodes (Vettest; Idex Laboratories, Westbrook, ME).

Dissection of tubules for immunoblots. Rats were anesthetized with intraperitoneal pentobarbital (5 mg/100 g body wt) before death. An abdominal incision was made, and the aorta was cannulated with polyethylene tubing below the renal arteries. The kidneys were perfused with ice-cold dissection media containing the following (in mM): 144 NaCl, 5 KCl, 6 NH₄Cl, 1 Na₂HPO₄, 2 CaCl₂, 1.2 MgSO₄, 5.5 glucose, and 10 HEPES, pH 7.4 bubbled with 100% O₂. This solution also contained 1 mg/ml collagenase and 1 mg/ml BSA. The kidneys were removed, and the inner medulla was excised. The middle third of the inner medulla was incubated in the same solution for 10 min at 37°C. The incubated tissue was washed once with this solution, but in the absence of collagenase. tIMCD tubules were dissected in the same solution at 11°C, but without albumin and collagenase. On each experiment day, two 20-mm samples of tubules were dissected from a single rat in each treatment group. Tubules were transferred to an Eppendorf tube while being visualized under a microscope. Tubules were centrifuged at 16,000 g for 3 min. The supernatant was discarded, and the pellet was resuspended in the same solution and recentrifuged. The supernatant was discarded and the pellet was resuspended in 20 µl of buffer A, which contained 10 mM triethanolamine, 250 mM sucrose, 1 mg/ml leupeptin (Bachem, Torrance, CA) and 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF; US Biochemical, Toledo, OH) pH 7.6. Membranes were solubilized at 60°C for 15 min in Laemmli sample buffer before being loaded onto SDS-PAGE gels.

Preparation of IMCD cell suspensions for immunoblots and for ATPase assay. IMCD cells were isolated as described previously (4, 30, 40). After the rats were killed by decapitation, the kidneys were removed and placed in chilled HCO-buffered solution (suspension solution) containing (in mM) 118 NaCl, 25 NaHCO, 5 KCl, 4 Na₂HPO₄, 2 CaCl₂, 1.2 MgSO₄, and 5.5 glucose bubbled with 95% air-5% CO₂. On each experiment day, kidney tissue from five rats eating a K⁺-replete diet and five rats eating a K⁺-deficient diet was isolated. Kidney tissue from rats in each treatment group was pooled. The inner medullas were excised, minced, and then incubated in the suspension solution containing 2 mg/ml collagenase B (Roche Molecular Biochemicals, Indianapolis, IN) and 600 U/ml hyaluronidase V (Sigma, St. Louis, MO) for 70 min at 37°C and bubbled continuously with 95% air-5% CO₂. DNase (0.001%, DNase I; Roche Molecular Biochemicals, Indianapolis, IN) was then added to the suspension and incubated another 20 min. The suspension was aspirated through a large-bore pipette every 20 min. At the end of the incubation period, the suspension was centrifuged at 65 g for 37 s at room temperature. The supernatant was removed, and the pellet, which contained the less buoyant structures such as the IMCD, was resuspended in the suspension solution plus 0.1% BSA (without collagenase, hyaluronidase, and DNase). This centrifugation step was repeated twice. The final IMCD suspensions were placed on ice for 10 min to increase the yield of IMCD cells. The supernatant was discarded, and the pellet was resuspended in suspension solution (without albumin, collagenase, hyaluronidase, and DNase) and centrifuged at 813 g for 10 min. When IMCD cells were prepared for immunoblots, the pellet was suspended in 0.5 ml of homogenation buffer A and was homogenized using an Omni tissue homogenizer (Omni/Tech Quest, Warrenton, VA) at 15,000 rpm on ice for 15 s. The homogenization step was repeated twice. The membranes were then centrifuged at 200,000 g for 1 h at 4°C. Expression of N,K-ATPase ₁-subunit (NK-₁) or NK-₁ protein was not detected in the supernatant (data not shown). The supernatant was therefore discarded. Membranes were resuspended in 100 µl of homogenization buffer A. Protein content was measured using the method of Lowry et al. (20). When IMCD cells were prepared for the ATPase assay, the protocol was varied. After the 10-min centrifugation step at 813 g, the pellet was suspended in 0.5 ml of homogenization buffer B, which contained 250 mM sucrose, 10 mM Tris · HCl, 1 mM EDTA-Tris, 1 mM PMSF, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor, pH 7.6. Membranes were homogenized and centrifuged at 200,000 g for 1 h. The pellet was then resuspended in 100 µl of homogenization buffer B.

Preparation of whole inner medulla and colon plasma membranes for immunoblots. Plasma membranes from rat distal colon and whole inner medulla were isolated as described previously (7). Rats were fed the K⁺-restricted diet for 7 days. Rat distal colon and rat whole inner medulla were excised, minced, and suspended in 10 ml of homogenization buffer C containing (in mM) 10 Tris · HCl, 1 EDTA-Tris, 1 PMSF, and 3 benzamide with 1 ug/ml soybean trypsin inhibitor, pH 7.4. This solution also contained 27% sucrose (wt/vol). Tissue was homogenized on ice using a Polytron (model PT 10/35, Brinkman, Westbury, NY) for 15 s. The homogenization step was repeated twice. Tissue was then homogenized with four or five strokes in a Dounce homogenizer with pestle A (Kontes Glass, Vineland, NJ) and centrifuged at 2,000 g for 4 min. The pellet, which contained nuclei, was discarded. The supernatant was applied to the top of 45% sucrose solution containing homogenization buffer C. The suspension was centrifuged at 200,000 g for 45 min at 4°C. Membranes in the 27/45% sucrose interphase were diluted in buffer C and centrifuged at 25,000 g for 30 min. The pellet was resuspended in buffer C and stored at 70°C. Protein was measured using the method of Lowry et al. (20).

Preparation of immunoblots. Membranes were solubilized at 60°C for 15 min in Laemmli sample buffer. To confirm equal protein loading of IMCD homogenates in each lane, electrophoresis was run on each pair of samples for a given experiment in a single 12% polyacrylamide-SDS gel stained with Coomassie blue. These gels were analyzed by densitometry to provide a quantitative assessment of loading. SDS-PAGE was performed on minigels of 10% polyacrylamide [NK-₁ and H,K-ATPase ₂-subunit (HK-₂)] or 12% polyacrylamide [NK-₁ and aquaporin (AQP)1 and 2]. The proteins were transferred from the gels electrophoretically onto nitrocellulose membranes. After being blocked with 5 g/dl nonfat dry milk, membranes were probed with a mouse monoclonal antibody that recognizes the NK-₁ subunit of rat Na,K-ATPase (Upstate Biotechnology, Lake Placid, NY). This antibody was used at a 1:5,000 titer with individual IMCD tubules or at a 1:10,000 titer with IMCD suspensions. To detect expression of NK-₁ protein, a rabbit polyclonal antibody raised against a rat NK-₁ fusion protein (Upstate Biotechnology) was used at a 1:20,000 titer. Affinity-purified, peptide-derived polyclonal rabbit anti-rat AQP1 and -2 (LL266 and LL752) antibodies were used at titers of 1:2,000 and 1:5,000, respectively (gift of M. A. Knepper, National Heart, Lung, and Blood Institute; Refs. 10, 33). Polyclonal antiserum raised in rabbits against a synthetic peptide that corresponds to amino acids 686-698 of the rat HK-₂ sequence was also used (Ref. 5; a gift of J. Codina and T. D. DuBose, Jr., University of Kansas, Kansas City, KS) at a titer of 1:1,000. Antibodies were diluted in an antibody dilution buffer solution containing 150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dl Tween 20 and 0.1 g/dl BSA (pH 7.5). For NK-₁ subunit immunoblots, sheep anti-mouse IgG conjugated with horseradish peroxidase was used as a secondary antibody (Amersham-Pharmacia Biotech, Piscataway, NJ) at a 1:5,000 dilution. For all other immunoblots, donkey anti-rabbit IgG conjugated to horseradish peroxidase (no. 31458, Pierce, Rockford, IL) was used as a secondary antibody at a 1:5,000 or a 1: 7,500 dilution. Sites of antibody-antigen reaction were visualized using luminol-based enhanced chemiluminescence (KPL; Kirkegaard and Perry, Gaithersburg, MD) before exposure of X-ray film (X-OMAT AR; Eastman Kodak, Rochester, NY). Relative quantification of the resulting band densities was performed using densitometry software running on a SPARC station2 (Sun Microsystems, Mountain View, CA) equipped with an image analysis system (Bio Image, Ann Arbor, MI). Multiple exposures of autoradiograms were made to ensure that densitometry gave a linear relationship between the amount of protein loaded onto the gel and band density, as described previously (22).

Deglycosylation of ₁ subunit. The ₁ subunit of Na,K-ATPase was deglycosylated using the protocol of Codina and collaborators (5). Membranes (50 µg) in homogenization buffer A were added to 20 µl of buffer containing 10 mM Tris · HCl, pH 8, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, and 1% CHAPS, pH 7.6, and incubated at 4°C for 1 h. After this incubation period, sodium phosphate (50 mM final concentration, pH 8) and PNGase (1,000 U, no. 7045, New England Biolabs, Beverly, MA) were added and the mixture was incubated for 1 h at 37°C. The reaction was stopped by the addition of Laemmli sample buffer.

ATPase assay. Ouabain-sensitive ATP hydrolysis was measured as reported previously (7, 25). IMCD membranes were incubated for 30 min in 0.75 mg/ml sodium deoxycholate at room temperature. Total and ouabain-sensitive ATP hydrolysis is maximal when membranes are incubated for 30 min at this detergent concentration (data not shown). The purpose of this incubation step is to expose unsealed vesicles (25). The reaction was started by the addition of 2.5 µg of sodium deoxycholate-treated rat IMCD membranes to the reaction mixture, such that the final mixture contained (in mM) 100 NaCl, 10 KCl, 50 Tris · HCl, 1 EDTA-Tris, 0.1 EGTA-Tris, 5 MgCl₂, 3 ATP-Tris containing 1-10 × 10⁶ cpm/200 µl of [-³²P]ATP (Amersham Pharmacia Biotech), 1 PMSF, and 3 benzamidine with 1 µg/ml soybean trypsin inhibitor, pH 7.32. The final volume of the reaction was 200 µl. The mixture was incubated for 30 min at 37°C. The reaction was terminated by the addition of 1 ml of a 50% slurry of activated charcoal in 10 mM Na₂HPO₄, pH 7.5. Samples were vortexed twice, cooled on ice, and centrifuged at 18,544 g for 5 min. The supernatant (450 µl) was used to quantify ³²P released during the incubation. The ATP assay was performed in the presence and absence of 5 mM ouabain in the reaction mixture. Total and ouabain-sensitive [-³²P]ATP hydrolysis was linear with time over the range of 0-60 min and was linear with protein content over the range of 0-10 µg of protein (not shown).

Measurement of protein-to-DNA ratio. IMCD cells were isolated as described in Preparation of IMCD cell suspensions for immunoblots and for ATPase assay. The pellet was resuspended in 0.1 N NaOH plus 0.025% saponin and incubated at 60°C for 1 h. Samples were then split into aliquots for protein and DNA measurements. Protein was measured using the method of Lowry et al. (20) with albumin as a standard. DNA was measured using the Hoesch reagent (Hoesch 33258; Hoefer Scientific Instruments, San Francisco, CA) with calf thymus DNA as a standard (Hoefer Scientific Instruments) as described previously (13). This assay detects DNA but not RNA (13). IMCD suspensions (or DNA standards) were added to 2 ml of solution containing (in mM) 10 mM Tris · HCl, 1 EDTA, and 200 NaCl, pH 7.4 with 10⁴ mg/ml Hoesch 33258, pH 7.4 at 22°C. Fluorescence was measured at excitation and emission wavelengths of 365 and 460 nm, respectively, with a DNA fluorimeter (TKO 100, Hoefer Scientific Instruments). Saponin at the concentrations used did not affect the DNA standard curve (not shown). Nevertheless, the standard curve was performed in the presence of NaOH and saponin at final concentrations found in each of the unknown samples. With this assay, DNA concentration is linear over the range of 0-150 ng of DNA (13).

Dissection of tIMCD tubules for perfusion experiments. The composition of the solutions used in the perfusion experiments is given in Table 1. Coronal slices were cut from the kidneys and placed into a dissection dish containing the chilled experimental solution (11°C). Tubules were dissected in solution 5 for buffering capacity measurements and in solution 8 for experiments that measured changes in pH_i after ouabain addition. tIMCD tubules were dissected, mounted on concentric glass pipettes, and perfused in vitro at 37°C as reported previously by our laboratory (36, 43).

Measurement of pH_i in tIMCD tubules perfused in vitro. pH_i was measured using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM. Tubules were perfused and bathed for 15 min at 37°C in either solution 5 (buffering capacity experiments) or solution 8 (ouabain-sensitive alkalinization experiments). The bath was then changed to the same solution but also containing 5 µM BCECF-AM. After 20 min of exposure to BCECF, the bath was changed to the original solution without BCECF. pH_i was determined by measuring the ratio of emitted light at >530 nm when BCECF was excited alternatively at 440 and 495 nm. Readings were calibrated by measuring the ratio of fluorescence at excitations of 495 relative to 440 nm when the tubule was perfused and bathed in either HEPES- or PO-buffered solutions containing 120 mM K⁺ to approximate intracellular concentration of this ion (solutions 1 or 3). All calibration solutions contained 14 µM nigericin in the bath solution. The pH of this solution was varied between 6.8 and 7.8. The other details of pH_i measurement in tubules perfused in vitro were described previously by our laboratory (36, 37). A typical calibration curve performed at K⁺ concentrations of 90 and 120 mM is given in Fig. 1.¹

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Effect of changes in extracellular K+ concentration on intracellular pH (pHi) calibration. Tubules were perfused and bathed in solution 1, which contained 120 mM K+ and 14 µM nigericin (n = 7 tubules). pHi was measured when extracellular pH was varied between 6.6 and 7.8. The bath and perfusate were then switched to solution 2, which contained 90 mM K+, and pHi was measured over the same range in extracellular pH. In some tubules the order of these measurements was reversed. The rate of change in the 495- to 440-nm fluorescence ratio with changes in pHi was similar at K+ concentrations of 90 and 120 mM.

Calculation of H+ fluxes. To quantitate NH uptake through Na,K-ATPase, proton efflux rates (J_H, pmol · mm¹ · min¹) were calculated as J_H = dpH_i/dt × _T × V/L, where dpH_i/dt is the initial rate of change in pH_i (pH units/min) after ouabain addition, _T is the intrinsic intracellular buffering power [mmol/(l · pH unit)], and V/L is the cell volume/tubule length (nl/mm). Thus _T, V/L, and dpH_i/dt were each measured.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

What gene product mediates NH uptake by Na⁺ pump? The relative abundance of the various Na,K-ATPase isoforms in kidney has been studied previously (Ref. 23; see DISCUSSION). We asked whether Na⁺ pumps other than Na,K-ATPase, such as HK-₂, might mediate NH-dependent, ouabain-induced alkalinization reported previously by our laboratory (36, 37). Thus immunoreactivity of NK-₁ was compared with that of HK-₂ in inner medulla from rats that ate the K⁺-deficient diet for 7 days. As shown in Fig. 2, NK-₁ immunoreactivity is readily detectable in the inner medulla of the kidney and in the colon. As shown, NK-₁ protein abundance is at least as great in kidney as in colon. However, HK-₂ immunoreactivity is lower in inner medulla than in colon. Therefore, if protein expression of HK-₂ and NK-₁ are equivalent in colon, then expression of NK-₁ in kidney is much greater than HK-₂. Low HK-₂ immunoreactivity may result from low affinity of the rabbit anti-rat HK-₂ antibody. However, the most likely interpretation of these data is that HK-₂ expression in the tIMCD is low. Thus Na pump-mediated NH uptake in tIMCD was attributed to NK-₁ rather than HK-₂. These results are consistent with previous studies (1, 6), which detected relatively low levels of expression of HK-₂ in the tIMCD. Further experiments therefore studied the effect of hypokalemia on expression of the ₁- and ₁-subunits of the Na,K-ATPase.

View larger version (62K): [in this window] [in a new window]   Fig. 2.   Expression of Na,K-ATPase 1-subunit (NK-1) and H,K-ATPase 2-subunit (HK-2) in K+-restricted rats. Rats received a K+-replete or a K+-restricted diet for 7 days. HK-2 protein expression was readily detected in colon, but less expression was detected in the inner medulla (IM). NK-1 was readily detected in both colon and inner medulla.

Effect of hypokalemia on expression of Na,K-ATPase. The effect of 3 days of dietary K⁺ restriction on NK-₁ subunit expression was examined. As shown in Fig. 3, the anti-rat NK-₁ antibody detected a protein that migrated at 100 kDa, the expected molecular mass of the ₁-subunit of Na,K-ATPase (27). No difference in NK-₁ subunit protein abundance was detected in individual tIMCD tubules from rats that ate either the normal or the low-K⁺ diet for 3 days.²

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Effect of 3 days of dietary K+ restriction on NK-1 protein expression in terminal inner medullary collecting duct (tIMCD) tubules. A: rats ate either a normal (N) or a K+-restricted diet (L) for 3 days. Two sets of 20-mm IMCD tubules were dissected from a single rat in each treatment group on a single experimental day. Homogenates from each of these 2 samples taken from a rat in each treatment group were loaded in separate lanes on a single gel. Thus each lane is loaded with equal lengths of IMCD tubules. B: band density of immunoblots given in A. Band density data were normalized to the means observed in tubules from K+-replete control rats isolated on the same day. No change in band density was detected with 3 days of dietary K+ restriction. NS, not significant.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Purity of IMCD suspensions. One-tenth of the inner medulla suspension was collected and homogenized (whole IM). The remaining nine-tenths of the inner medullary suspension was centrifuged 3 times (65 × g for 37 s) to separate the final pellet (IMCD) from the lighter non-IMCD structures found in the pooled supernatants (non-IMCD). Samples from each fraction were homogenized and dissolved in Laemmli sample buffer and loaded on SDS-PAGE gels (12%; Ref. 4). A: aquaporin-1 (AQP1) immunoreactivity was low in IMCD suspensions relative to either whole IM or non-IMCD structures, which indicated little contamination by loops of Henle. B: AQP2 expression was high in IMCD suspensions relative to either whole IM or non-IMCD structures, which indicates enrichment of IMCD cells in these suspensions.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   NK-11 protein expression in IMCD suspensions from rats eating a normal (N) or a K+-restricted (L) diet for 3 days. Immunoblots of NK-11 subunits are shown. Each lane was loaded with 6 (1), 20 (native 1), or 6 (core 1) µg protein.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   NK-11 protein expression in IMCD suspensions from rats eating a normal or a K+-restricted diet for 3 days. A: band density of immunoblots given in Fig. 5. Band densities obtained from homogenates of K+-restricted rats were normalized to the band density of homogenates from K+-replete control animals prepared the same day. B: Na,K-ATPase activity in tIMCD suspensions from rats eating a normal or a K+-restricted diet for 3 days. In this figure, ouabain-sensitive ATPase activity was 6.42 ± 0.70 µmol Pi · mg protein1 · h1 (n = 5) in IMCD cell homogenates from K+-restricted rats and 5.03 ± 0.35 µmol Pi · mg protein1 · h1 (n = 5) in K+-replete control rats. Ouabain-sensitive ATPase activity obtained from homogenates of K+-restricted rats was normalized to the ATPase activity observed in homogenates of K+-replete control animals prepared the same day. *P < 0.05. Values represent means ± SE.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Relative cell volume of tIMCD tubules from rats eating a normal or a K+-restricted diet for 3 days. Relative cell volume was measured in IMCD suspensions (protein-to-DNA ratio, A) and in tIMCD tubules perfused in vitro (cross-sectional area, B). No effect of dietary restriction on relative cell volume was noted. #P = NS.

View larger version (41K): [in this window] [in a new window]   Fig. 8.   NK-11 protein expression in IMCD suspensions from rats eating a normal (N) or a K+-restricted (L) diet for 7 days. Immunoblots of NK-11 subunit protein are shown. Each lane was loaded with 6 (1), 20 (native 1), or 6 (core 1) µg protein.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   NK-11 protein expression in IMCD suspensions from rats eating a normal (N) or a K+-restricted (L) diet for 7 days. A: band density of immunoblots given in Fig. 8. Band densities obtained from homogenates of K+-restricted rats were normalized to the band density of homogenates from K+-replete control animals prepared the same day. B: Na,K-ATPase activity in tIMCD suspensions from rats eating a normal or a K+-restricted diet for 7 days. In this figure, ouabain-sensitive ATPase activity was 11.55 ± 2.50 µmol Pi · mg protein1 · h1 (n = 5) in K+-restricted rats and 5.47 ± 1.09 µmol Pi · mg protein1 · h1 (n = 5) in K+-replete control rats. Ouabain-sensitive ATPase activity in homogenates of K+-restricted rats was normalized to the ATPase activity observed in homogenates of K+-replete control animals prepared the same day. Values represent means ± SE. *P < 0.05.

View larger version (41K): [in this window] [in a new window]   Fig. 10.   Effect of 7 days of dietary K+ restriction on NK-1 protein expression in tIMCD tubules. A: rats ate either a normal (N) or a K+-restricted (L) diet for 7 days. Two sets of 20-mm IMCD tubules were dissected from a single rat in each treatment group on a single experimental day. Homogenates from each of these 2 samples obtained from a rat in each treatment group were loaded in separate lanes on a single gel. Thus each lane is loaded with equal lengths of IMCD tubules. B: densitometry of 1-subunit protein expression obtained from normal and K+-restricted rats. Band density data were normalized to the means observed in tubules from K+-replete control rats isolated on the same day and were expressed as means ± SE. Band density was increased with 7 days of dietary K+ restriction. *P < 0.05.

Effect of K+ restriction on NH uptake by Na,K-ATPase. As an index of NH uptake by Na,K-ATPase at the level of the plasma membrane during steady-state conditions, NH-dependent, ouabain-induced alkalinization was measured in tIMCD tubules. With this value, as well as _T and V/L, ouabain-induced net H⁺ efflux (J; dpH_i/dt · _T · V/L) was determined. tIMCD tubules from rats eating the K⁺-restricted or the K⁺-replete diet for 3 days were studied (36, 37). Values reported for V/L (Fig. 7) and _T (Fig. 11) were similar in tIMCDs from rats in both treatment groups.

View larger version (13K): [in this window] [in a new window]   Fig. 11.   Buffering capacity in tIMCD tubules perfused in vitro from rats eating a normal or a K+-deficient diet for 3 days.

View larger version (24K): [in this window] [in a new window]   Fig. 12.   Effect of K+ restriction on NH uptake by the Na,K-ATPase in tIMCD tubules perfused in vitro. A: the effect of ouabain on pHi was studied in IMCD tubules from rats on a normal or K+-restricted for 3 days. Tubules were studied at K+ concentrations of 10 and 30 mM (solutions 8 and 9). Nine tubules were taken from previously published data (38). B: NH uptake by Na,K-ATPase (J) was taken to be equal to dpHi/dt · T · V/L, where dpHi/dt is the initial rate of change in pHi after the addition of ouabain to the bath (A), T is buffering capacity (Fig. 11), and V/L is the tubule volume per tubule length. Initial (baseline) pHi measured in tIMCD tubules from K+-restricted rats was 7.24 ± 0.03 (n = 6) at a K+ concentration of 10 mM and 7.18 ± 0.02 (n = 5) at a K+ concentration of 30 mM. In tubules from K+-replete rats, initial pHi was 7.13 ± 0.03 (n = 8) at a K+ concentration of 10 mM and 7.10 ± 0.04 (n = 6) at a K+ concentration of 30 mM.

View larger version (14K): [in this window] [in a new window]   Fig. 13.   Effect of extracellular K+ concentration on resting pHi. Rats ate a K+-restricted diet for 3 days. Tubules were perfused and bathed in solution 10 (K+ = 10 mM, NH = 6 mM). Resting pHi was 7.16 ± 0.04 (n = 6). Bath and perfusate K+ concentration was increased to 30 mM (solution 11), and pHi was measured 4 min later (7.21 ± .04, P < 0.05). Bath and perfusate were returned to the initial solution (solution 10), and pHi was measured 4 min later (7.11 ± 0.03, P < 0.05). Thus pHi increased when extracellular K+ concentration was increased, consistent with inhibition of net H+ uptake at higher K+ concentrations (n = 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study demonstrates that expression of both the ₁- and ₁-subunits of Na,K-ATPase is upregulated during chronic hypokalemia. However, the dominant mechanism for the increase in Na⁺ pump-mediated NH uptake that occurs after 3 days of dietary K⁺ restriction (38) is the change in interstitial K⁺ concentration, which occurs during hypokalemia in vivo, rather than upregulation of Na⁺ pump expression at the level of the plasma membrane.

Rates of NH uptake by Na,K-ATPase after changes in extracellular K⁺ concentration measured in the present study were compared with rates predicted by the model of Kurtz and Balaban (19). In rat IMCD, the Michaelis constant (K_m) and transport rate when all binding sites of a carrier are saturated (V_max) for the Na,K-ATPase with NH and K⁺ as substrates were reported previously by our laboratory (40).⁴ NH concentration was taken to be 6 mM. Incorporating these values, the model predicts that NH uptake by the Na⁺ pump is reduced by 61% when interstitial K⁺ concentration is increased from 10 to 30 mM, a prediction consistent with the experimental observations of this study. In the present study, NH-dependent, ouabain-sensitive J was used as an index of the rate of NH uptake by Na,K-ATPase at steady state. We observed that J was reduced by 84% when extracellular K⁺ concentration was increased from 10 to 30 mM in either normal or K⁺-restricted rats. Thus changes in extracellular K⁺ concentration over the physiological range observed in the interstitium of the inner medulla in vivo produce a dramatic change in Na pump-mediated NH uptake. However, we cannot exclude the possibility that trafficking of the Na⁺ pump between the plasma membrane and the cytosol occurs after changes in K⁺ concentration.

Our laboratory reported previously that in IMCD tubules from K⁺-restricted rats, J_tCO2 is upregulated relative to K⁺-replete controls (41). Because tubules were perfused and bathed in identical solutions (K⁺ = 10 mM, NH = 6 mM), a stable adaptation must occur during hypokalemia that upregulates net acid secretion. Most likely, uptake of H⁺ equivalents across the basolateral membrane is increased in series with increased secretion of H⁺ equivalents into the luminal fluid. However, after 3 days of dietary K⁺ restriction, increased net H⁺ uptake across the basolateral membrane does not occur through increased expression of Na,K-ATPase. Expression of another transporter that localizes to the basolateral membrane, such as anion exchange, may be upregulated during hypokalemia, which augments secretion of H⁺ equivalents.

A variety of techniques have been used to study transporter expression at the cell surface. At the level of the plasma membrane, protein expression has been studied extensively with immunocytochemical techniques. However, this technique is technically challenging when studying changes in Na,K-ATPase expression along the collecting duct because of the extensive infolding of the basolateral membrane (31). Cellular fractionation has also been used (47), but it requires considerable tissue, limiting its use in IMCD suspensions. Ouabain binding and ouabain-sensitive Rb⁺ uptake have been used in other cell types for study of changes in Na⁺ pump expression along the basolateral membrane. However, because the tIMCD suspensions represent tubule fragments, functional measurements such as Rb⁺ uptake are not useful because of the large variability in measurements (Wall SM and Trinh HN, unpublished observations). Moreover, ouabain binding studies in rats are difficult because of the low affinity of Na,K-ATPase for ouabain in rat (16). Our laboratory therefore used tubules perfused in vitro as a means by which to study expression of functional Na⁺ pumps along the basolateral membrane. We showed previously (36) that in the presence of NH, pH_i is increased after ouabain addition to the bath. Through a series of ion substitution experiments we showed (36, 37) that this effect of ouabain on pH_i occurs through inhibition of NH uptake by Na,K-ATPase. Using this approach, we observed that cell surface expression of functional Na,K-ATPase is not changed with 3 days of dietary K⁺ restriction.

After a dietary K⁺ load, both Na,K-ATPase (12, 35) and ROMK channels (35) are upregulated in the cortical collecting duct (CCD). Upregulation of these transporters occurs in series and results in increased secretion of K⁺ into the luminal fluid of the CCD (21, 26). Conversely, with dietary K⁺ depletion these transporters are downregulated, which promotes K⁺ conservation in this segment (18, 35). K⁺ absorption is observed in the OMCD and IMCD after dietary K⁺ restriction (17), which also promotes K⁺ conservation. However, in the medullary collecting duct the mechanism of this adaptive response probably differs from that of the CCD. The Na⁺ pump is upregulated in the CCD during hyperkalemia (12, 35), whereas in the medullary collecting duct it is upregulated during hypokalemia (23).⁵ Why Na,K-ATPase protein expression is upregulated in the medullary collecting duct after 7 days of K⁺ restriction is unknown. This increased expression observed during hypokalemia may generate a latent pool of Na⁺ pumps, which are available for translocation into the plasma membrane. Such a mechanism may serve to maintain intracellular volume or intracellular K⁺ concentration after rapid changes in extracellular K⁺ concentration. Whether Na,K-ATPase-mediated transport across the basolateral membrane is increased in the medullary collecting duct after >1 wk of K⁺ restriction has not been explored. Therefore, it remains to be determined whether increased Na⁺ pump expression augments NH-stimulated H⁺ secretion after prolonged intake of a low-K⁺ diet.

The Na,K-ATPase is composed of - and -subunits. In some renal segments, such as in rat inner medulla, NK-₁₁ also assembles with a -subunit (2, 24). In kidney, although the ₁-and ₁-isoforms of Na,K-ATPase are very abundant, protein expression of the other isoforms of the - and -subunits (_2-4 and _2-3) has not been demonstrated (15, 23, 28, 29, 34). In the IMCD Na,K-ATPase activity correlates more closely with ₁- than with ₁-subunit expression, similar to previous observations in rat OMCD (23).

Although changes in expression of NK-₁₁ during hypokalemia were studied in detail, changes in expression of the -subunit in this treatment group remain to be determined. The physiological role of the -subunit is not understood. However, it is known to alter the affinity of NK-₁₁ for K⁺ and Na⁺ (2). The present study did not discern the effect of hypokalemia on -subunit expression, nor did it determine the effect of changes in -subunit expression on Na⁺ pump-mediated NH uptake. However, the measurements of NH uptake by the Na⁺ pump in IMCD tubules perfused in vitro reported in the present study reflect changes in expression of each subunit, including , when assembled as a heterotrimer in native tissue and studied under physiological conditions.

In conclusion, Na⁺ pump expression is upregulated in the rat IMCD during hypokalemia. However, the primary mechanism for the increase in NH uptake mediated by Na,K-ATPase that follows 3 days of dietary K⁺ restriction results from changes in interstitial K⁺ concentration observed in vivo rather than changes in protein expression.