Na+-K+-ATPase-mediated basolateral rubidium uptake in the maturing rabbit cortical collecting duct

Alexandru R. Constantinescu1, Jerome C. Lane2, John Mak3, Beth Zavilowitz3, and Lisa M. Satlin3

1 Department of Pediatrics, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey, 2 Northwestern University Medical School, Chicago, Illinois 60614; and 3 Mount Sinai School of Medicine, New York, New York 10029-6574


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Within the renal cortical collecting duct (CCD), transepithelial Na+ absorption and K+ secretion are linked to basolateral Na+-K+-ATPase activity. Our purpose was to examine the developmental changes in basolateral Na+-K+-ATPase-mediated 86rubidium (Rb) uptake, its inhibitor sensitivity and relationship to pump hydrolytic activity and Na+ transport. Multiple CCDs (~6 mm) from maturing rabbits were affixed to coverslips, preincubated at 37°C for 10 min (±1-2.5 mM ouabain or 10 or 100 µM Schering-28080, an inhibitor of H+-K+-ATPase), and then transferred to prewarmed incubation solution containing tracer amounts of 86Rb (±inhibitors). After 1 min at 37°C, tubular samples were rinsed and permeabilized and isotope counts were measured to calculate basolateral Rb uptake. Ouabain-inhibitable Rb uptake, an index of basolateral Na+-K+ pump activity, increased ~3-fold during the 1st 8 wk of postnatal life (P < 0.03). The ~2-fold increase in absolute rate of Rb uptake between 1 and 6 wk (2.64 ± 0.45 to 5.02 ± 0.32 pmol · min-1 · mm-1) did not reach statistical significance. The rate of basolateral Rb uptake increased further after the 6th wk of life to 7.29 ± 0.53 pmol · min-1 · mm-1 in adult animals (P < 0.03 vs. 6 wk). Schering-28080 failed to inhibit Rb uptake, implying that functional H+-K+-ATPase is absent at the basolateral membrane. Na+-K+-ATPase hydrolytic activity, determined by using a microassay that measured inorganic phosphate release from [gamma -32P]ATP under maximum velocity (Vmax) conditions, also increased in the differentiating CCD (from 316.2 ± 44.4 pmol · h-1 · mm-1 at 2 wk to 555.9 ± 105.1 at 4 wk to 789.7 ± 145.0 at 6 wk; r = 1.0 by linear regression analysis; P < 0.005). The parallel ~2.5-fold increases in Na+-K+-ATPase activity and ouabain-sensitive Rb uptake between 2- and 6-wk postnatal age suggest that the developmental increase in basolateral transport capacity is due predominantly to an increase in enzyme abundance. The signals mediating the developmental increase in Na+-K+-ATPase activity in the CCD remain to be defined.

ontogeny; transepithelial sodium transport; ouabain; Schering-28080; microassay


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CORTICAL COLLECTING DUCT (CCD) plays a major role in the final renal regulation of Na+ and K+ homeostasis. Within this segment, principal cells mediate transepithelial Na+ absorption and K+ secretion, processes linked to the activity of basolateral Na+-K+-ATPase. We (23) and others (31) have previously shown that the capacity of the mammalian CCD for net transepithelial Na+ and K+ transport increases during postnatal life, due at least in part to increases in abundance of conducting apical amiloride-sensitive Na+ channels (25) and small-conductance secretory K+ (SK) channels (26), respectively, in differentiating principal cells. It is generally believed that the rate-limiting step in transepithelial Na+ and K+ transport in the CCD resides at the apical channel and not at the basolateral pump, considered to be present in abundance (17). However, the ontogenic profile for activity of Na+-K+-ATPase that maintains the driving force for transepithelial ion transport has not been fully characterized in the CCD.

Ultramicrochemical analysis of Na+-K+-ATPase hydrolytic activity in single microdissected CCDs, determined under maximum velocity (Vmax) conditions in neonatal (2-5 days) and mature rabbits, was shown to double during postnatal life (27); the Michaelis-Menten constant, Km, was identical in the neonatal and mature tubule, suggesting that maturation is associated with an increase in enzyme abundance in this segment. However, the physiological significance of Vmax measurements performed in these permeabilized nephron segments must be interpreted with some caution due to the following observations. First, it is well established that the Na+-K+ pump operates well below the rate of Vmax in the intact cell (3, 4, 7), mainly because of limited intracellular Na+ availability (3). Second, the cell permeabilization necessary to obtain Vmax measurements abolishes normally existing ion gradients, membrane potentials, and conductances (7), all of which influence pump function in the intact tubule.

To measure the functioning rate of basolateral Na+-K+-ATPase activity in individual intact nephron segments, Cheval and Doucet (7) developed a sensitive microassay capable of detecting pump-dependent ouabain-inhibitable basolateral rubidium (Rb) uptake in single nephron segments. Specifically, individual tubule segments were exposed to tracer 86Rb at 37°C for a specific length of time. Tubules were then rinsed free of extracellular Rb, presumably without leakage of intracellular Rb, by sequentially aspirating the segments into a polyethylene tube and rinsing in ice-cold tracer-free medium containing Ba2+ to block the basolateral K+ conductance (7). Although this method allowed for the reliable measurement of 86Rb uptake in single ~1-mm segments isolated from adult animals, we were unable to detect tracer uptake in preliminary studies performed in single small and short segments of CCDs (~80% the tubular width and 45% the maximal length of segments in the adult) characteristic of the neonatal kidney (24). Additionally, the successful transfer of the neonatal tubules between multiple rinse solutions proved to be problematic. We thus modified the latter assay to measure basolateral tracer uptake in a group of microdissected CCDs affixed to a coverslip painted with Cell-Tak, a preparation that allows ready access of adherent membranes to the extracellular medium (8, 29). The coverslip to which the tubules are affixed is easily transferred between sequential solutions and is visually inspected at the conclusion of the assay to confirm retention of all segments throughout the incubation and rinse steps. This assay thus provides a technically simple method sensitive enough to detect basolateral tracer uptake in a small pool of single nephron segments.

The purpose of the present study was twofold. Our first purpose was to validate our modified microassay for basolateral 86Rb uptake, a method that should be applicable to the study of other basolateral uptake/transport processes in single short nephron segments, such as those isolated from mice or newborn animals. Our second purpose was to examine the developmental changes in basolateral Na+-K+-ATPase-mediated 86Rb uptake, its inhibitor sensitivity, and its relationship to pump hydrolytic activity and Na+ transport.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Adult New Zealand White rabbits were obtained from Charles River (Quebec, Canada) or Covance (Denver, PA). Newborn rabbits were bred in our animal facility and were raised with and fed by their mothers until week 5 of life, when weaning was complete. Thereafter, rabbits were fed a standard laboratory rabbit chow (Ralston, Purina). Animals were studied at each week of postnatal life, as indicated. Adult rabbits were defined as animals >= 8 wk of age. At least four litters of rabbit pups were used for each group of studies. Rabbits were killed by intraperitoneal (neonates) or intravenous injection of pentobarbital sodium (100 mg/kg). All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Isolation of nephron segments. The left kidney was removed via a midline incision, and single tubules were dissected freehand in cold (4°C) incubation solution containing (in mM) 120 NaCl, 5 RbCl, 1 MgSO4, 0.15 Na2HPO4, 0.2 NaH2PO4, 4 NaHCO3, 1 CaCl2, 5 glucose, 2 lactate, ~4 essential and nonessential amino acids, ~3 × 10-2 vitamins, and 20 HEPES; 3% dextran (Mr = 40,000; wt/vol) and 0.1% BSA (fraction V) were also added. Because the hydrolytic activity of Na+-K+-ATPase displays the same apparent affinity for K+ and Rb ions, whereas the Vmax measured in the presence of K+ exceeds that observed in the presence of Rb (7, 33), all 86Rb uptake experiments were performed in the presence of unlabeled Rb rather than K+. The osmotic pressure of the incubation solution was adjusted with mannitol to 300 mosmol/kgH2O, and the pH was adjusted to 7.4. The length of each segment was measured by using an ocular micrometer. Given that these CCDs were nonperfused, we assume that their lumens were collapsed.

Unless specifically indicated, ~6-8 mm total length of CCD were transferred to a 0.5 × 0.2-cm plastic coverslip to which 0.5 µl Cell-Tak (Collaborative Biomedical Products; Bedford, MA) had been previously applied. Once the tubules were well affixed to the Cell-Tak-coated slide, the entire coverslip was easily transferred between different solutions in 1-ml Eppendorf tubes without loss of individual segments. Note that we have previously shown that the basolateral membranes of CCDs attached to a coverslip with Cell-Tak have ready access to the extracellular medium (8, 29). For each animal, at least four coverslips with affixed CCDs were prepared for subsequent assay of Rb uptake in the absence or presence of ouabain or Schering-28080 (see below).

Measurement of basolateral 86Rb influx. Rb influx was measured by using a modification of the method developed and validated by Cheval and Doucet (7). To restore normal ionic gradients, the nephron segments adherent to each coverslip were preincubated at 37°C for 10 min in 1-ml incubation solution. The coverslip was then immersed in 0.5-ml prewarmed incubation solution containing tracer amounts (10-20 nCi/µl; 20,000 cpm/sample) of 86RbCl (10 nCi/µl; New England Nuclear Life Science Products, Boston, MA) where cpm is counts/min. After a 1-min incubation at 37°C, the samples were rapidly cooled by immersing the coverslip for 3 min in 1-ml ice-cold rinse solution containing (in mM) 140 choline chloride, 1.2 MgCl2, 3 BaCl2, and 10 HEPES as well as 0.1% BSA, adjusted to 300 mosmol/kgH2O with mannitol and pH 7.4. Ba2+ was used in all rinse solutions to inhibit Rb leak from the cells into the bathing medium via basolateral K+ channels (7).

To ensure maximal removal of extracellular 86Rb contamination, the latter rinse was repeated twice. The coverslip was then visually inspected under 10× magnification to ensure that all CCDs remained attached to the slide; if tubular segments were lost during the assay, the coverslip was discarded. Each coverslip was finally transferred into a counting vial containing 0.5 ml of 1% (wt/vol) deoxycholate, and the radioactivity determined after addition of 10-ml scintillation solution.

To account for coverslip contamination with 86Rb, a control coverslip pretreated with Cell-Tak was subject simultaneously to the same sequence of incubation steps described above for each tubular sample. The 86Rb uptake measured for the pooled CCD sample was then corrected for its parallel coverslip uptake. The number of counts measured per sample of tubules exceeded the coverslip contamination 7-fold in tubules from 1-2-wk-old animals, 12-fold in tubules from 3-4-wk-old animals, and 15-fold in CCDs isolated from animals >4 wk of age.

Extracellular contamination with 86Rb was assessed by sampling 10 µl of the final wash solution used to rinse each coverslip. In addition, some coverslips (mean tubular length/coverslip = 6.2 ± 0.1 mm; n = 3) were processed as described above except that the incubation medium contained [14C]inulin (30,000 cpm/µl; New England Nuclear) and not 86Rb. The mean cpm of these latter coverslips (24.3 ± 2.6) was not significantly different from the mean cpm of coverslips to which no tubules were attached (30.3 ± 1.2), consistent with the absence of significant extracellular contamination with tracer.

Because measurements of ion transport are normalized to tubule length (23, 31), Rb influx was expressed as pmol Rb accumulated per minute per millimeter CCD (pmol · min · -1mm-1). The ouabain-sensitive Rb influx for each animal was calculated as the difference between the means of the groups incubated in the absence and presence of 1 or 2.5 mM ouabain added to preincubation, uptake, and rinse solutions. Also measured was the difference in Rb influx between the means of groups of CCDs incubated in the absence and presence of 10 and 100 µM Schering-28080, a relatively specific inhibitor of the H+-K+-ATPase (8, 29, 30) added to preincubation, uptake, and rinse solutions.

In a few experiments, CCDs were dissected from adult kidneys left at room temperature for up to 1 h after the death of the animal to assess the influence of temperature on Na+-K+-ATPase-dependent Rb influx. Basolateral Rb uptake was measured in these CCDs as described above in the absence and presence of 1 mM ouabain.

Measurement of Na+-K+-ATPase hydrolytic activity. Na+-K+-ATPase activity was measured by using a modification of the method reported by Doucet et al. (11), under Vmax conditions defined for neonatal (27) and mature (11, 27) tubules. Single tubules (5-7 mm total length) were affixed to a coverslip to which a 0.5-µl drop of Cell-Tak had been initially applied, as described above. The isotonic solution in which the tubules were dissected was aspirated and replaced by 5 µl of cold distilled water. Fifteen minutes later, the coverslip was placed on a block of dry ice to rapidly freeze the nephron segments. After thawing, a 1-µl droplet of assay medium was deposited on top of the CCDs. After a 15-min incubation at 37°C, the reaction was stopped by inserting the coverslip into an Eppendorf microcentrifuge tube containing 2 ml of a cold suspension of 10% (wt/vol) activated charcoal in 5% (wt/vol) trichloroacetic acid. Thirty minutes later, the contents of the microcentrifuge tube were centrifuged and the radioactivity of 500 µl of the supernatant determined by scintillation counting.

The assay medium for measurement of total ATPase activity contained (in mM) 50 NaCl, 5 RbCl, 10 MgCl2, 1 EGTA, 100 Tris · HCl, and 10 Tris-ATP as well as a tracer amount (5 nCi/µl) of [gamma -32P]ATP (10 Ci/mmol). To determine the ouabain-insensitive Mg-dependent ATPase activity, NaCl and RbCl were omitted from the assay medium, Tris · HCl was increased to 150 mM, and ouabain was added to a final concentration of 1 mM. The pH of each solution was adjusted to 7.4.

Na+-K+-ATPase activity was calculated as the difference between the total and Mg-dependent ATPase, each measured on 2-3 samples per animal. Assays of tubule samples were bracketed by assays of blanks (Cell-Tak coated coverslips to which no tubules were affixed) to determine the nonenzymatic hydrolysis of ATP. ATPase activity was expressed as pmol inorganic phosphate liberated per h/mm tubule length (pmol · h · -1mm-1) by using the formula (11)
ATPase activity<IT>=1/</IT>(<IT>l×t</IT>)<IT>×</IT>(R<IT>−</IT>B)<IT>/</IT>SRA
where l is the total tubular length (mm), t is the incubation time (h), R is the measured radioactivity (cpm) of the sample, B is the measured radioactivity of the companion blank (cpm), and SRA is the specific radioactivity of the [gamma -32P]ATP in counts per minute/picomole, a value derived from the measured radioactivity of the assay medium.

Measurement of net Na+ fluxes in isolated perfused CCDs. Single CCDs were isolated and microperfused in vitro as previously described (23). Tubules were perfused and bathed in Burg's solution containing (in mM) 120 NaCl, 25 NaHCO3, 2.5 K2HPO4, 2.0 CaCl2, 1.2 MgSO4, 4.0 Na lactate, 1.0 Na3 citrate, 6.0 L-alanine, and 5.5 glucose, pH 7.4, 290 ± 2 mosmol/kgH2O; the specimen chamber was continuously suffused with a 95% O2-5% CO2 to maintain pH at 7.4 at 37°C. Because water transport in the neonatal and mature CCD is negligible in the absence of vasopressin (5), and experiments were performed in the absence of transepithelial osmotic gradients, transepithelial water transport was assumed to be zero.

Samples of tubular fluid (4-5/tubule) were collected under water-saturated light mineral oil by timed filling of a precalibrated volumetric constriction pipette. The Na+ concentrations of perfusate and collected tubular fluid were determined by helium glow photometry and used to calculate the rate of net Na+ transport (pmol · min · -1mm-1 tubular length), as previously described (23). The calculated fluxes were then averaged to obtain a single mean rate of ion transport for that CCD.

Statistics. Results are expressed as means ± SE; (n) represents the number of animals. Significant differences between paired data were determined by paired t test. Comparisons of unpaired data were performed by t test or ANOVA and multiple range test, as appropriate. As indicated, maturational trends were identified by linear regression analysis. The software program SigmaStat (Jandel Scientific, CA) was used for all statistical analyses. Significance was asserted if P < 0.05.

For calculated values derived from two different mean measurements (i.e., the ratio of the mean rate of Rb uptake to the mean rate of maximum Rb pumping, the latter calculated from the ATPase activity of the pump; see RESULTS), the SD (Sz) of the computed results was determined as previously described (24), according to Baird (2)
S<SUB><IT>z</IT></SUB><IT>=z</IT>(S<SUB><IT>x</IT></SUB><IT>/x</IT>)<SUP><IT>2</IT></SUP><IT>+</IT>(S<SUB><IT>y</IT></SUB><IT>/y</IT>)<SUP><IT>2</IT></SUP>
where z is the calculated mean derived from the two mean measurements, x and y, and Sx, Sy, and Sz are the standard deviations of x, y, and z, respectively. The average n, no, rounded to the nearest whole number, was calculated as (20)
n<SUB>o</SUB><IT>=</IT>(<IT>n<SUB>x</SUB>+n<SUB>y</SUB></IT>)<IT>−</IT>(<IT>n</IT><SUP><IT>2</IT></SUP><SUB><IT>x</IT></SUB><IT>+n</IT><SUP><IT>2</IT></SUP><SUB><IT>y</IT></SUB>)<IT>/</IT>(<IT>n<SUB>x</SUB>+n<SUB>y</SUB></IT>)


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Linearity of Rb uptake. To determine the optimal length of tubule necessary to reliably detect Rb uptake, we examined the relationship between total tubular length and the initial (1 min) rate of basolateral Rb uptake in CCDs isolated from animals >= 5 wk of age. As shown in Fig. 1, a linear correlation (P < 0.01 by linear regression analysis) was obtained between tubular length and Rb uptake for CCD samples ranging in total tubular length from ~1 to 7.5 mm. Based on this analysis and the low rates of tracer uptake anticipated for neonatal segments, we chose to use a total tubular length of at least 6 mm for all assays.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Linearity of basolateral Rb uptake as a function of cortical collecting duct (CCD) length. The relationship between the 1 min rate of basolateral Rb uptake (pmol · min-1 · mm-1) and tubular length (mm) was linear for CCD samples comprised of 1 to 7.5 mm total length (r = 0.99 by linear regression analysis; P < 0.01). Paired data from 3 animals (>= 5 wk of age) were used to generate each data point; the rate of uptake for 1 mm reflects a single measurement. Values are means ± SE.

Effect of dissection temperature on basolateral Rb uptake. Prolonged exposure of human proximal tubules (32) and cultured A6 cells (1) to cold temperature leads to disruption of the cytoskeleton and redistribution of membrane-associated proteins. To examine whether exposure of tubules to 4°C during the ~1-h time period required for microdissection systematically altered the polarized expression and activity of the Na+-K+ pump, we compared the initial rates of basolateral Rb uptake in two groups of CCDs isolated from the same animal; one group of CCDs was dissected from kidney sections kept at room temperature after the death of the animal whereas the other group was isolated from sections maintained in incubation solution chilled to 4°C. All CCD samples were subject to the same 86Rb uptake assay, as described above. As shown in Fig. 2, there was no significant difference in total or ouabain-sensitive Rb uptake between CCDs isolated at room temperature and those dissected at 4°C. These data support the immunocytochemical evidence reported by Breton and Brown (6) that basolateral distribution of Na+-K+-ATPase remains unchanged during cold exposure (to 4 h) followed by rewarming.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of dissection temperature on the rate of basolateral Rb uptake. Tubules dissected at room temperature (21°C) within 60 min of the death of the animal exhibited similar rates of baseline, ouabain (O)-insensitive, and ouabain-sensitive (O-sensitive) basolateral Rb uptake (pmol · min-1 · mm-1) compared with those dissected at 4°C. Values are means ± SE; n, number of animals studied in each age group.

Ouabain-sensitive basolateral Rb uptake. The initial rate of ouabain-sensitive Rb uptake, an index of the rate of basolateral Na+-K+-ATPase activity, increased gradually during the first 6 wk of postnatal life (r = 0.93 by linear regression analysis; P < 0.03)(Fig. 3). Although the absolute rate of basolateral Rb uptake doubled during this interval, from 2.64 ± 0.45 at 1 wk to 5.02 ± 0.32 pmol · min-1 · mm-1 at 6 wk, the difference in rates between these two time points did not achieve statistical significance based on analysis of variance.1 The rate of basolateral Rb uptake increased further after the 6th wk of life to 7.29 ± 0.53 pmol · min-1 · mm-1 in adult animals (P < 0.03 vs. 6 wk).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Postnatal maturation of basolateral Na+-K+-ATPase-dependent Rb influx in single rabbit CCDs. The rate of ouabain-sensitive Rb uptake (pmol · min-1 · mm-1), an index of basolateral Na+-K+ pump activity in the intact CCD, increased gradually between 1 and 6 wk of age (r = 0.93 by linear regression analysis; P < 0.03). Although the absolute rate of basolateral Rb uptake doubled during this interval, the difference between these 2 time points did not achieve statistical significance based on analysis of variance. The rate of basolateral Rb uptake increased significantly after the 6th wk of life. Data from 6-11 CCD samples from (n) number of animals were studied in each age group. Values are means ± SE. *, P < 0.05 compared with an 8-wk-old by ANOVA.

The percentage of basolateral Rb uptake inhibited by ouabain remained constant after birth, averaging 78% under the conditions employed in this study. There was no significant difference in the % inhibition detected in experiments using 1 vs. 2.5 mM ouabain in 1 to 2-wk-old animals (80.3 ± 4.1, n = 7 vs. 80.8 ± 4.0, n = 12, respectively) or rabbits >= 6 wk of age (74.8 ± 2.7, n = 7 vs. 77.2 ± 1.5, n = 9). An increase in ouabain preincubation time from 10 to 20 min did not significantly increase the % inhibition of basolateral Rb uptake (incremental change of 1.7 ± 6.6%; n = 3 3-wk-old animals).

Effect of Schering-28080 on basolateral Rb uptake. The effect of 10 or 100 µM Schering-28080 on basolateral Rb uptake was sought to assess the contribution of H+-K+-ATPase towards ouabain-insensitive basolateral Rb uptake. As shown in Fig. 4, neither concentration of Schering-28080 significantly altered basolateral Rb uptake in CCDs isolated from 1- to 2-, 4- to 6-, and >= 8-wk-old animals. These results are consistent with the absence of functional H+-K+-ATPase activity at the basolateral membrane of the rabbit CCD under baseline conditions.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of SCH (Schering)-28080 on basolateral Rb uptake. Neither 10 nor 100 µM Sch-28080, an inhibitor of H+-K+-ATPase had a significant effect on the initial rate of basolateral Rb uptake (pmol · min-1 · mm-1) in single CCDs. Values are means ± SE of 7 animals studied in each age group.

Na+-K+-ATPase activity. The hydrolytic activity of Na+-K+-ATPase measured under Vmax conditions increased significantly from 316.2 ± 44.4 pmol · h-1 · mm-1 at 2 wk (n = 5), to 555.9 ± 105.1 at 4 wk (n = 3) to 789.7 ± 145.0 at 6 wk of age (n = 5; r = 1.0 by linear regression analysis; P < 0.005). The percentage of total ATPase activity that was ouabain-insensitive (Mg- ATPase activity) did not differ among the three age groups, averaging 49.0 ± 2.5% (P = NS). The initial rate of Na+-K+-ATPase-mediated Rb uptake, expressed as a % of the maximum pumping rate, the latter value calculated from the ATPase hydrolytic activity of the pump, assuming a stoichiometry of two Rb ions transported per molecule of ATP hydrolyzed, did not differ among the three age groups studied (19.5 ± 0.1% at 2 wk, 19.1 ± 0.2% at 4 wk, and 19.1 ± 0.1% at 6 wk; P = NS). These percentages are identical to those reported by Cheval and Doucet (7) by using a similar assay in mature rabbit CCDs.

Net tubular Na+ absorption at slow flow rates. To correlate rates of Na+-K+-ATPase-mediated Rb uptake measured in nonperfused tubules (i.e., stationary flow) with net Na+ absorption, measurements of net Na+ transport were performed in isolated CCDs perfused at a tubular flow rate of 0.6 nl · min-1 · mm-1, the slowest rate we could consistently reproduce. The observed rates of net Na+ absorption at this slow flow rate in CCDs isolated from 1- to 2-, 4-, 6-, and 8-wk-old animals are shown as open bars in Fig. 5. At this tubular flow rate, net Na+ absorption increased significantly from 3.7 ± 1.9 pmol · min-1 · mm-1 in the first 2 wk of postnatal life, a value not significantly different from zero (P = 0.100), to 16.7 ± 2.1 pmol · min-1 · mm-1 at 8 wk (P < 0.05).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   Postnatal maturation of net Na+ absorption in CCDs perfused at slow tubular flow rates. The predicted active Na+ absorptive fluxes calculated from the initial rates of basolateral ouabain-sensitive Rb uptake in nonperfused CCDs (i.e., stationary flow) are shown as solid black bars; the assumptions made in performing this calculation are presented in the DISCUSSION. The rates of net Na+ absorption actually measured in single CCDs isolated from maturing rabbits and perfused at 0.6 nl · min-1 · mm-1 are identified by open bars. Note the similarity between predicted and observed rates of Na+ absorption (in pmol · min-1 · mm-1). Values are means ± SE; n, number of animals studied in each age group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The microassay described above, modified from that originally described by Cheval and Doucet (7), provides a sensitive and reliable measurement of the functioning rate of basolateral uptake processes in single tubular segments. The assay is particularly well suited to measuring basolateral uptake in small and short tubules, such as those in neonatal animals or mice, whose low individual uptake rates may necessitate the pooling of multiple segments into each sample to obtain meaningful results. The use of Cell-Tax to affix the group of tubules to a coverslip facilitates the sequential transfer of the segments between the various incubation, uptake, and rinse solutions, and allows for visual inspection of the sample at the conclusion of the experiment to ensure that all tubules are retained throughout the assay. As is evident from Fig. 1, Rb uptake was linear within a wide range of tubule segment lengths. This basolateral uptake assay should be widely applicable to the measurement of activity of other basolateral transporters in single defined nephron segments.

Our values for the initial rate of basolateral ouabain-sensitive Rb uptake in intact CCDs isolated from adult rabbits are within the range published by others for rat (7) and rabbit (4). The results of the present developmental study indicate that basolateral Na+-K+ pump activity in rabbit CCDs increases gradually during the first 6 wk of postnatal life, a time interval during which the rates of net transepithelial Na+ and K+ transport in the CCD attain mature values (23, 31) and surges thereafter. Consistent with the low rates of Na+-K+-ATPase-dependent Rb influx detected in CCDs early in life is our previous observation of simple basolateral surfaces, devoid of elaborate infoldings, in neonatal compared with mature principal cells (12).

To correlate our measurements of pump-mediated basolateral Rb uptake in nonperfused CCDs with rates of transepithelial Na+ transport, we measured the rate of net Na+ absorption in CCDs microperfused at slow flow rates. Figure 5 presents a comparison of the observed rates (open bars) of net Na+ absorption with the predicted rates (filled bars) of pump-mediated active Na+ absorption at stationary flow. The latter calculations were performed assuming a 3Na:2Rb stoichiometry for the pump and a ratio of pump activity measured in 5 mM Rb to that in 5 mM K+ of ~70% in the rabbit CCD (7, 33). Thus the initial rate of ouabain-sensitive Rb influx of 7.3 pmol · min-1 · mm-1 in the >= 8-wk-old rabbit CCD (Fig. 3) predicts a unidirectional lumen-to-bath active Na+ flux of ~15 pmol · min-1 · mm-1 in the absence of tubular flow, a value similar to that (16.7 ± 2.1 pmol · min-1 · mm-1) actually observed in adult CCDs microperfused at slow flow rates. Indeed, the observed rates of net Na+ absorption at the slow tubular fluid flow rate closely paralleled the rates predicted in the absence of luminal flow in all CCDs. To the extent that our measurements of net Na+ absorption reflect active transport,2 this analysis suggests that pump activity is sufficient to account for the active component of Na+ absorption in the maturing CCD.

We (23) and others (31) have previously identified a surge in the capacity of the rabbit CCD for net Na+ absorption early in postnatal life such that mature rates of transport are attained by 4 wk of age. The results of the present study indicate that the rate of basolateral Rb uptake does not reach adult levels until after 6-wk postnatal life, and thus follows the postnatal maturation of net Na+ absorption. Schwartz and Evan (28) also reported a lag between the early appearance of HCO3- absorption and later increase in Na+-K+-ATPase activity in the maturing rabbit juxtamedullary proximal tubule. These investigators suggested that the developmental induction of pump activity over the first 6 wk of postnatal life was a consequence of an increase in apical Na+ entry into the cell due to insertion of newly synthesized Na+ transporters into the luminal membrane.

Cumulative evidence identifies a pivotal role of intracellular Na+ in short- and long-term regulation of Na+-K+-ATPase activity. Within the rabbit CCD, an increase in the intracellular Na+ concentration acutely (within minutes) stimulates Na+-K+-ATPase activity, presumably due to the recruitment and/or activation of pumps in the basolateral membrane (3, 4, 9, 14). The characteristic aldosterone-induced stimulation of Na+-K+-ATPase activity is not observed in CCDs isolated from adrenalectomized rabbits pretreated with amiloride, an agent that inhibits passive luminal entry of Na+ (16, 21). More recently, studies performed in hepatocyte (18), vascular smooth muscle (34), and aortic (22) cell lines provide evidence that enhanced Na+ influx into cells increases steady state levels of Na+-K+-ATPase alpha 1- and beta 1-subunit mRNA. In the principal cell in the CCD, the number of apical conducting amiloride-sensitive Na+ channels increases significantly after the first week of postnatal life, reaching a value similar to that detected in 5-wk-old animals (25). Whether this postnatal increase in channel activity results in an increase in cell Na+ concentration, which in turn, contributes to short or long term regulation of basolateral pump turnover rate remains to be tested.

To begin to explore whether the developmental increase in ouabain-sensitive Rb uptake is mediated by a maturational increase in substrate availability or increase in number of active Na+-K+-ATPase proteins, we measured Na+-K+-ATPase hydrolytic activity in single microdissected permeabilized rabbit CCDs under substrate-saturating conditions. The ~2.5-fold increase in Na+-K+-ATPase activity detected between 2 and 6 wk postnatal age (P < 0.3 by linear regression analysis), similar to that reported previously by Schmidt and Horster (27) in the maturing rabbit CCD, is consistent with an increase in abundance of active enzyme. Comparison of the rates of basolateral Rb uptake and Na+-K+-ATPase activity indicates that the basolateral pump operates far below Vmax at all ages, likely reflecting the low intracellular Na+ concentration (rate-limiting condition) in intact tubules.

Rb uptake in the CCD was not completely inhibited by either 1 or 2.5 mM ouabain, a finding similar to that reported by others (7, 27). Given that the Na+-K+ pump in the rabbit CCD is exquisitely sensitive to ouabain (10), the ouabain-insensitive Rb influx may reflect passive basolateral Rb influx or the presence of a ouabain-insensitive basolateral uptake Rb pathway. Rb influx through a basolateral K+ channel has been proposed as a possibility (7, 19). However, movement along this conductive pathway in rabbit CCD would be expected to be partially inhibited by Rb (33). An alternate Rb uptake route that we sought to explore was that mediated by a basolateral Schering-28080-sensitive H+-K+-ATPase.

The mammalian CCD is comprised not only of principal cells, the majority cell population, but also of intercalated cells, which transport H+/HCO3- and participate in K+ absorption in response to hypokalemia and metabolic acidosis (29, 30). Although functional studies (8, 29, 30) identify a predominant apical polarization of H+-K+-ATPase in intercalated cells in the CCD, evidence does exist to support the presence of basolateral H+-K+-ATPase that may contribute to HCO3- secretion under some conditions. By using digital ratio fluorometry and a pH-sensitive dye, we identified a basolateral K+-dependent H+ extrusion pathway in acutely acid-loaded intercalated cells in neonatal and adult microperfused rabbit CCDs (8). Gifford et al. demonstrated that basolateral Schering-28080 reduced HCO3- secretion in microperfused CCDs isolated from chronically alkalotic rats, consistent with a basolateral localization of H+-K+-ATPase (15). Additional support for basolateral H+-K+-ATPase derives from the observation that steady state expression of mRNA encoding the colonic isoform of this protein is upregulated in intercalated cells immunodissected from alkalotic adult rabbits (13). The results of our present study, however, provide no evidence for a functional Schering-28080-sensitive basolateral H+-K+-ATPase in the rabbit CCD under steady state conditions.

In conclusion, postnatal maturation of the rabbit CCD is associated with an increase in Na+-K+-ATPase hydrolytic activity and ouabain-sensitive basolateral Rb uptake, an index of basolateral Na+-K+ pump activity in the intact CCD. The absence of effect of Schering-28080 on basolateral Rb uptake implies that H+-K+-ATPase is not present and functional at the basolateral membrane of the CCD at any age. The signals mediating the developmental increase in Na+-K+-ATPase activity remain to be defined.


    ACKNOWLEDGEMENTS

The authors thank Beth Zavilowitz for expert technical assistance.


    FOOTNOTES

Funding for the study was provided by the National Institute of Diabetes and Digestive and Kidney Diseases Research Grant DK-38470 and an American Heart Association Grant-in-Aid. Parts of this work were presented at the 1996 and 1999 (November) Annual Meetings of the American Society of Nephrology in New Orleans, LA, and Miami Beach, FL, respectively.

Address for reprint requests and other correspondence: L. M. Satlin, Box 1664, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574 (E-mail: lisa.satlin{at}mssm.edu).

1 Our failure to demonstrate that the developmental increase in basolateral Rb uptake is statistically significant during the first 6 wk of postnatal life is similar to that reported by Schwartz and Evan (28) in their analysis of the development of Na-K-ATPase activity in the proximal tubule. In the latter paper, the authors were also unable to demonstrate a significant increase in pump activity during the first 6 wk of life, despite a 70% rise in activity, an observation they attributed to the low power of the analysis of variance, as discussed in detail in that manuscript.

2 Vehaskari (31) showed that the ouabain-sensitive lumen-to-bath 22Na flux in maturing rabbit CCDs increased ~2.5-fold between 2 and 4 wk of age, identical to the ~2.5-fold increase in net Na+ absorption observed between birth and 4 wk in the present study (Fig. 5). These data suggest that, at very slow flow rates, measurements of net Na+ absorption predominantly reflect active transport. Although the rate of basolateral Rb uptake increased 1.5-fold between 2 and 4 wk (Fig. 3), this change did not achieve statistical significance (P = 0.10).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 January 2000; accepted in final form 11 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alejandro, VS, Nelson WJ, Huie P, Sibley RK, Dafoe D, Kuo P, Scandling Jr JD, and Myers BD. Postischemic injury, delayed function and Na+/K+-ATPase distribution in the transplanted kidney. Kidney Int 48: 1308-1315, 1995[ISI][Medline].

2.   Baird, DC An Introduction to Measurement, Theory and Experimental Design. Englewood Cliffs: Prentice-Hall, 1962, p. 61-62.

3.   Barlet-Bas, C, Cheval L, Khadouri C, Marsy S S, and Doucet A. Difference in the Na affinity of Na+-K+-ATPase along the rabbit nephron: modulation by K. Am J Physiol Renal Fluid Electrolyte Physiol 259: F246-F250, 1990[Abstract/Free Full Text].

4.   Blot-Chabaud, M, Jaisser F, Gingold M, Bonvalet JP, and Farman N. Na+-K+-ATPase-dependent sodium flux in cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 255: F605-F613, 1988[Abstract/Free Full Text].

5.   Bonilla-Felix, M, Vehaskari VM, and Hamm LL. Water transport in the immature rabbit collecting duct. Pediatr Nephrol 13: 103-107, 1999[ISI][Medline].

6.   Breton, S, and Brown D. Cold-induced microtubule disruption and relocalization of membrane proteins in kidney epithelial cells. J Am Soc Nephrol 9: 155-166, 1998[Abstract].

7.   Cheval, L, and Doucet A. Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron. Am J Physiol Renal Fluid Electrolyte Physiol 259: F111-F121, 1990[Abstract/Free Full Text].

8.   Constantinescu, A, Silver RB, and Satlin LM. H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct. Am J Physiol Renal Physiol 272: F167-F177, 1997[Abstract/Free Full Text].

9.   Coutry, N, Blot-Chabaud M, Mateo P, Bonvalet JP, and Farman N. Time course of sodium-induced Na+-K+-ATPase recruitment in rabbit cortical collecting tubule. Am J Physiol Cell Physiol 263: C61-C68, 1992[Abstract/Free Full Text].

10.   Doucet, A, and Barlet C. Evidence for differences in the sensitivity to ouabain of Na,K-ATPase along the nephrons of rabbit kidney. J Biol Chem 261: 993-995, 1986[Abstract/Free Full Text].

11.   Doucet, A, Katz AI, and Morel F. Determination of Na-K-ATPase activity in single segments of the mammalian nephron. Am J Physiol Renal Fluid Electrolyte Physiol 237: F105-F113, 1979[ISI][Medline].

12.   Evan, AP, Satlin LM, Gattone VH, III, Connors B, and Schwartz GJ. Postnatal maturation of the rabbit renal collecting duct. II. Morphologic observations. Am J Physiol Renal Fluid Electrolyte Physiol 261: F91-F107, 1991[Abstract].

13.   Fejes-Toth, G, Rusvai E, Longo KA, and Naray-Fejes-Toth A. Expression of colonic H-K-ATPase mRNA in cortical collecting duct: regulation by acid/base balance. Am J Physiol Renal Fluid Electrolyte Physiol 269: F551-F557, 1995[Abstract/Free Full Text].

14.   Fujii, Y, Takemoto F, and Katz AI. Early effects of aldosterone on Na-K pump in rat cortical collecting tubules. Am J Physiol Renal Fluid Electrolyte Physiol 259: F40-F45, 1990[Abstract/Free Full Text].

15.   Gifford, JD, Rome L, and Galla JH. H+-K+-ATPase activity in rat collecting duct segments. Am J Physiol Renal Fluid Electrolyte Physiol 262: F692-F695, 1992[Abstract/Free Full Text].

16.   Hayhurst, RA, and O'Neil RG. Time-dependent actions of aldosterone and amiloride on Na+-K+-ATPase of cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 254: F689-F696, 1988[Abstract/Free Full Text].

17.   Katz, A. Renal Na-K-ATPase: its role in tubular sodium and potassium transport. Am J Physiol Renal Fluid Electrolyte Physiol 242: F207-F219, 1982[ISI][Medline].

18.   Kirtane, A, Ismail-Beigi N, and Ismail-Beigi F. Role of enhanced Na+ entry in the control of Na-K-ATPase gene expression by serum. J Membr Biol 137: 9-15, 1994[ISI][Medline].

19.   Koeppen, BN, Biagi BA, and Giebisch GH. Intracellular microelectrode characterization of the rabbit cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 244: F35-F47, 1983[ISI][Medline].

20.   Li, JCR Statistical inference. Ann Arbor, MI: Edwards, 1969, p. 193-197.

21.   Petty, KJ, Kokko JP, and Marver D. Secondary effect of aldosterone on Na-K-ATPase activity in the rabbit cortical collecting tubule. J Clin Invest 68: 1514-1521, 1981[ISI][Medline].

22.   Ruiz-Opazo, N, Cloix JF, Melis MG, Xiang XH, and Hererra VLM Characterization of a sodium-response transcriptional mechanism. Hypertension 30: 191-198, 1997[Abstract/Free Full Text].

23.   Satlin, LM. Postnatal maturation of potassium transport in rabbit cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 266: F57-F65, 1994[Abstract/Free Full Text].

24.   Satlin, LM, Evan AP, Gattone VH, III, and Schwartz GJ. Postnatal maturation of the rabbit cortical collecting duct. Pediatr Nephrol 2: 135-145, 1988[ISI][Medline].

25.   Satlin, LM, and Palmer LG. Apical Na+ conductance in maturing rabbit principal cell. Am J Physiol Renal Fluid Electrolyte Physiol 270: F391-F397, 1996[Abstract/Free Full Text].

26.   Satlin, LM, and Palmer LG. Apical K+ conductance in maturing rabbit principal cell. Am J Physiol Renal Physiol 272: F397-F404, 1997[Abstract/Free Full Text].

27.   Schmidt, U, and Horster M. Na-K-activated ATPase: activity maturation in rabbit nephron segments dissected in vitro. Am J Physiol Renal Fluid Electrolyte Physiol 233: F55-F60, 1977[ISI][Medline].

28.   Schwartz, GJ, and Evan AP. Development of solute transport in rabbit proximal tubule. III. Na-K-ATPase activity. Am J Physiol Renal Fluid Electrolyte Physiol 246: F845-F852, 1984[ISI][Medline].

29.   Silver, RB, Mennitt PA, and Satlin LM. Stimulation of H-K-ATPase in intercalated cells of cortical collecting duct with chronic metabolic acidosis. Am J Physiol Renal Fluid Electrolyte Physiol 270: F539-F547, 1996[Abstract/Free Full Text].

30.   Silver, RB, and Soleimani M. H+-K+-ATPases: regulation and role in pathophysiological states. Am J Physiol Renal Physiol 276: F799-F811, 1999[Abstract/Free Full Text].

31.   Vehaskari, VM Ontogeny of cortical collecting duct sodium transport. Am J Physiol Renal Fluid Electrolyte Physiol 267: F49-F54, 1994[Abstract/Free Full Text].

32.   Verrey, F, Groscurth P, and Bolliger U. Cytoskeletal disruption in A6 kidney cells: impact on endo/exocytosis and NaCl transport regulation by antidiuretic hormone. J Membr Biol 145: 193-204, 1995[ISI][Medline].

33.   Warden, DH, Hayashi M, Schuster VL, and Stokes JB. K+ and Rb+ transport by the rabbit CCD: Rb+ reduces K+ conductance and Na+ transport. Am J Physiol Renal Fluid Electrolyte Physiol 257: F43-F52, 1989[Abstract/Free Full Text].

34.   Yamamoto, K, Ikeda U, Okada K, Saito T, Kawakami K, and Shimada K. Sodium ion mediated regulation of Na/K-ATPase gene expression in vascular smooth muscle cells. Cardiovasc Res 28: 957-962, 1994[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 279(6):F1161-F1168
0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Google Scholar
Articles by Constantinescu, A. R.
Articles by Satlin, L. M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Constantinescu, A. R.
Articles by Satlin, L. M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online