Kidney International, Vol. 29 (1986), pp. 747—751

TECHNICAL NOTE

23Na NMR spectroscopy of proximal tubule suspensions

ADARSH M. KUMAR, ADRIAN SPITZER, and RAJ K. GUPTA

Departments of Pediatrics, Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA

23Na NMR spectroscopy of proximal tubule suspensions. Intracellularsodium concentrations in proximal tubule suspensions of rat kidneywere measured by NMR spectroscopy. A simple method for thepreparation of proximal tubule suspension is described. Examination bylight microscopy revealed these preparations to contain 93.6 0.6% (N= 5) proximal tubules, and electron microscopy demonstrated that thetubules were open. When incubated with trypan blue for five mm, only2% of tubules picked-up the dye. The basal oxygen consumption ratewas 0.42 0.01 l min mg protein' (N = 6). Addition of succinate(5 mM) resulted in a fivefold increase in the rate of oxygen consumption.The 23Na spectra were obtained in proximal tubules incubated for 30mm in the aqueous shift reagent dysprosium tripolyphosphateDy(PPP1)27. The NMR observable sodium concentration was 34.11.8 mr's at room temperature and 16.3 0.6 mr's (P < 0.001) at 37°C.Addition of ouabain (l0- M) at 37°C resulted in an increase inintracellular sodium to 30.9 2.9 mrs (P < 0.001), while nystatinincreased the concentration of sodium to 72.0 9.1 mr's (P < 0.001),compared to basal concentration. Thus NMR permits the measurementof intracellular concentration of sodium in proximal tubules under basalconditions and to monitor, in the same preparation, the changes thatoccur under various experimental conditions without interfering withthe morphologic integrity of the cells.

Transport of sodium across the proximal tubule cells, as in othertransporting epithelia [1], is regulated by the intracellular Naactivity. This in turn facilitates transport of a variety of sub-stances, including HC03, C1, P1 [2], carbohydrates, lactate andaminoacids [3]. Thus, precise information regarding the intracel-lular concentration of Na is paramount to the understanding oftransport processes across the proximal tubule.

Most of the techniques used for the measurements of intra-cellular concentrations of electrolytes are either invasive orinaccurate. Measurements based on radioisotope exchange ofNa and K in kidney slices [4] or tubule suspensions [5] haveyielded inconsistent values probably because the distribution ofradioactivity between various cell compartments and cytoplasmfollows multiexponential kinetics [6]. Sodium selectivemicroelectrodes involve impalement of the cell membrane andmeasure only local concentration of ions [7]. Radioautographictechniques measure distribution of the element in the cell but donot distinguish between different chemical forms of the electro-lyte. Moreover, this approach can be used only for cells of largesize [8, 9]. Similarly, electron probe x-ray microanalysis [101does not distinguish between various chemical forms and the

Received for publication March 25, 1985,and in revised form July 15, 1985 and October 28, 1985

© 1986 by the International Society of Nephrology

measurements can only be done on freeze-dried tissue. Atomicabsorption spectrophotometry [11] and flame emission photom-etry [12] can be applied only to tissue extracts.

Nuclear magnetic resonance (NMR) is non-invasive andpermits serial measurements of non-bound intracellular NatAmong the limitations of NMR techniques are the requirementfor a dense cell suspension (containing >10% cells) and theinability to ascertain the compartmental localization of NatHowever, its non-invasive property and specificity are signifi-cant advantages over the alternative methods.

Recently Gupta and his collaborators were able to measureintracellular sodium concentration in cell suspensions by nu-clear magnetic resonance (NMR) [13—16] using an anionicparamagnetic shift reagent, dysprosium tripolyphosphateDy(PPP1)27, that permits the separation of the peaks of theintracellular and extracellular sodium ions. Since this reagentdoes not penetrate the cell membrane [13—15, 17], it thus shiftsonly the extracellular 23Na resonance. In the present work,NMR spectroscopy was used to measure intracellular Na in asuspension of proximal tubules of rat kidney subjected tovarious maneuvers known to stimulate or inhibit the transportof sodium.

Methods

Proximal tubule suspensions were prepared from kidneys ofSprague-Dawley rats weighing 250 to 300 g, fed a commercialdiet and given free access to water. mactin anaesthesia, 100mg/kg body wt, was given to two to three rats for eachexperiment. Kidneys were excised after being perfused throughthe aorta with a normal saline solution, and were placed inchilled Ringer's solution consisting of (in mM): NaC1, 115.0;KC1, 5.0; MgSO4, 0.1; NaH2PO4, 4.0; NaHCO3, 25.0; CaC12,2.3; glucose, 5.0; DL-lactate, 1.0; alanine, 1.0; adjusted to pH7.4 and gassed at 23°C with 95% 02 and 5% CO2 for 20 mm.

Each kidney was cut into two halves along the longitudinalaxis and the superficial cortex was carefully dissected with size#11 blades. The cortical slices were pooled and rinsed in coldRinger's solution, then suspended in 20 ml Ringer's solutioncontaining 1.5 mg/ml collagenase (Type 1, 254 U per mg,Worthington, Freehold, New Jersey, USA) and incubated atroom temperature (22 1°C) in an atmosphere of 95% 02/5%CO2 for 1 hr while gentle mixing was maintained with amagnetic stirrer. At the end of this period, the slices were rinsedtwice and suspended in 15 ml fresh Ringer's solution. In orderto disperse the renal tubule, the cortical slices were passedtwice through six inches of PE-l90 tubing and twice throughPE-90 tubing attached to a 20 ml syringe. The pooled suspen-

747

748 Kumar et a!

sion was filtered through a 150 /.LM stainless steel sieve (USAstandard testing sieve number 100, W. S. Tyler Inc., Mentor,Ohio, USA) and the filtrate was centrifuged at SOxg for i mm.The supernate was discarded and the pellet was suspended inlsx its volume and allowed to settle on ice for seven to ten mm.The top layer, which contained single cells, small segments ofproximal tubules, some distal and collecting duct segments anda few glomeruli, was discarded, The loose pellet obtained afterthe third such wash was rinsed twice in 15 times its volume,suspended in two ml Ringer's solution containing 6% defattedbovine serum albumin (BSA) previously gassed with 95%02/5% C02, and was stored on ice and used within one hour.

Measurements of suspension purity and tubule viabilityThe quality of the preparations was tested by morphologic

and functional methods. Freshly prepared unstained samples oftubule suspensions were examined and photographed with aNikon Diaphot inverted microscope at 100 x magnification. Theproximal tubule segments in suspensions were identified bytheir yellowish color and broad structure. Distal tubules andcollecting duct segments were narrower and transparent. Ali-quots of tubule suspensions were fixed by immersion in 1.25%glutaraldehyde in 0.1M cacodylate buffer and post fixed in 1%osmium tetroxide. After dehydration in graded alcohol, thepellet was embedded in epoxy resin. Thin sections were poststained with uranyl acetate and lead citrate, viewed and photo-graphed with an electron microscope (model JEOL 1200 EX,Japan Electronic Optical Laboratory, Tokyo, Japan).

To evaluate the integrity of the tubules, 0.1 ml of a 0.4%solution of trypan blue was added to 0.5 ml of tubule suspen-sions in Ringer's solution and an aliquot was checked forstained cells after five mm. For measurement of 02 consump-tion, the final suspension of tubules was made in Ringer'ssolution containing 3% dextran instead of 6% BSA [18]. A Clarktype oxygen electrode with a polarographic system mounted ina thermostatically controlled glass chamber at 37°C was usedfor this purpose. Constant mixing in the chamber was accom-plished with a magnetic stirring bar. The tubule suspension waspreincubated at 37°C for 25 to 30 mm in an atmosphere of 95%02/5% CO2 to maintain the pH at 7.4. Oxygen consumption wasmeasured in a 3.0 ml buffer medium containing an aliquot ofpreincubated tubule suspension equivalent to 3 to 5 mg proteinand 4 mivi DL-lactate, and recorded continuously on a chartrecorder. Succinate (5 mM final concentration) was addedthrough the access slot after recording the basal rate for fourmm. The protein content of tubule suspension was measured bythe method of Lowry [19]. Bovine serum albumin was used asa standard.

Measurements of intracellular concentrations of 23Na +

Dysprosium tripolyphosphate Dy(PPP1)27 was prepared bytitrating DyCI3 (Ventron, Alfa Division, Danvers, Massachu-setts, USA) with Na5(PPP) (Sigma, St. Louis, Missouri, USA)to obtain the complex Dy(PPP327, the formation of which wasindicated by the disappearance of a white precipitate. Com-plexes containing less than 2 mole equivalents of PPP1 areinsoluble and give rise to the white precipitate.

Tubule suspensions in 6% BSA Ringer's solution mixed with10 times its volume of medium containing 4 mivi Dy(PPP)27were incubated for 30 mm at either 22° 1° or 37°C in

siliconized flasks to allow equilibration of the shift reagentthroughout the extracellular space. Equilibration over longerintervals did not change the levels of intracellular 23Na mea-sured. Dy(PPP)27 at this concentration shifted the extracellu-lar 23Na resonance by approximately 8 ppm. No toxic effects ofDy(PPP1)77 have previously been demonstrated in epithelialcells in concentrations of up to 5 mivi [20]. Oxygenation andgentle stirring were maintained throughout the incubation pe-riod. Tubules were then centrifuged for 30 sec to obtain adensity of approximately 20% in the final suspension.

23Na spectra were obtained at 53 MHz on a Varian XL-200 FTNMR spectrometer using a spinning sample. The spectra wereaccumulated with an acquisition time of 0.2 sec (T1 < 40 msec)following 90° pulses, over an interval of three mm. The intensitiesof the NMR signals were measured by integration of the areaunder the resonance peaks. In estimating levels of intracellular23Na, the ratio of the size of the extracellular 23Na signalrecorded from the tubule suspension (A0J to that of the mediumalone (A0) was used as an estimate of the extracellular space (S0)within the NMR window. As previously described [14]:

= A0/A0 and[Nath] = (Ajn/Aout) (S01/l-S0) [Na0j/W

where, [Naj] is in mM or zmole/ml cell H20, A1 is the measuredintensity of the intracellular 23Na signal and W is the fractionalwater content of the tissue, expressed in units of volumewater/total tissue volume.

The volume water/total tissue volume was calculated bymultiplying the fractional water content of 0.78 g H2O/g tissue[21] with the specific gravity of 1.03 for the rat kidney cortexdetermined in our laboratory. This yielded a value of 0.8 for Win equation 2.

In order to alter the sodium content of the cells, tubulesuspensions were incubated with or without a freshly preparedsolution of ouabain (l04M) or nystatin (10'M) for 1 hr at 37°Cin an oxygen atmosphere. Tubules were subsequently incu-bated with the medium containing shift reagent for 30 mm at37°C, and 23Na signals were obtained as described above.

ResultsThe tubule suspensions (Fig. 1) contained 93.6 0.6% (N =

5) proximal tubule segments. A representative electron micro-graph (Fig. 2) of a tubule segment revealed the open lumen andthe intact brush border microvilli of the luminal membrane.Large numbers of mitochondria were seen in the cytoplasm.The basal membrane with its infoldings also appeared intact.

Following incubation with trypan blue for five mm, 98% oftubules excluded the dye. The basal oxygen consumption ratewas 0.42 0.01 pJ min mg protein' (Table I). The valueincreased by fivefold (1.95 .08 itl/min per mg protein) afteraddition of succinate.

The NMR measureable intracellular 23Na concentration was34.1 1.8 m (Table 2, Figs. 3 and 4) at 22 1°C. Raising thetemperature to 37°C resulted in a fall in the intracellular 23Naconcentration to 16.3 0.6 mvi (Pc .001). Addition of ouabain(104M) resulted in a twofold increase to 30.9 2.9 mM (P C.001), while in the presence of nystatin (105M) there was afourfold increase in 23Na to a value of 72.0 9.1 mri (P C0.0005). No significant differences in values were observedwhether the measurements were performed sequentially on the

(1)(2)

23Na NMR spectroscopy of proximal tubule suspensions 749

Table 1. Rate of 02 consumption (Qo2) by rat proximal tubules,u.l min' mg protein '

Basal Rate Succinate

0.42 .011 1.95 .08N=6 N=5

Proximal tubule suspension was preincubated for 25 mm at 37°C in95% 02/5% CO2. Basal rate was recorded for four mm and succinate (5m final concentration) was then added. Results are given as meansSE; N represents number of tubule suspensions.

Fig. 1. Micrograph of a tubule suspension examined and photographedby a Nikon Diapho: inverted microscope. Large and small segments ofproximal tubules are predominant. (x60)

Table 2. NMR measured intracellular 23Na concentration (mM) in ratproximal tubule suspensions

Temperature ControlOuabain1O- M

Nystatin1O M

22 1°C 34.06 1.75N= 6

37°C 16.30 0.58*N=7

30.92 2.93**N=4

72.00 9.07**N=3

Tubules were incubated with ouabain and nystatin for 60 mm andthen exposed to the shift reagent for 30 mm. Oxygenation was main-tained throughout the incubation period. The incubation and the NMRmeasurements were done at the same temperature. Values are meanSE, N represents number of tubule suspensions.

* Different (P < 0.001) from 22 1°C.** Different (P < 0.001) from control at 37°C.

Table 3. The effect of temperature on intracellular 23Naconcentrations in rat proximal tubule suspensions

TemperatureSame

preparationsDifferent

preparations

22 1°C 37.4 1.90N=3

30.7 0.82N=3

37°C 16.5 0.77N=3

16.2 0.94N=4

A change in the temperature of the media was associated with achange in opposite direction in intracellular sodium concentration. Themagnitude of the changes were similar whether measurements weredone in succession on the same preparation or on different tubulesuspensions.

Discussion

Fig. 2. Electron micrograph of a proximal tubule obtained from asuspension prepared from rat kidney cortex. The tubule shows intactbrush border (BB); open lumen, and well preserved basolateral mem-brane (BM) with infoldings. Large numbers of mitochondria (M) areseen scattered in the cytoplasm. (x3300)

same preparation or on different preparations incubated ateither 22° or 37°C (Table 3).

To our knowledge, this represents the first report of intracel-lular Na measurements by NMR spectroscopy at 22° and at37°C in rat kidney proximal tubule suspensions. Preparation ofthe suspensions by the procedure described gave consistentlygood yield of viable tubules. Use of PE-190 and PE-90 tubingwas helpful in the dispersion of tubules from their adheringtissue in the slices. One of the salient features of our techniqueis the use of a stainless sieve of 150 M size that allowed onlythe tubule segments to pass through. The suspension thusobtained was free of connective tissue. The centrifugation stepsduring the washing procedure used by other investigators [22,23] were eliminated. Instead, suspensions were allowed tosediment at 1 xg at 4°C for seven to ten mm, a much moregentle treatment. The longer and thicker proximal tubule seg-ments, being heavier due to their high protein content per mmtubule length [24], sedimented earlier than the distal segments,

.tf

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4

-

750 Kumar et al

23NO

1000 Hz

Fig. 3. Sample 23Na NMR spectra from the medium containing 4 mMDy(PPF1)27 and 140 mM of Na (upper trace) and from the renalproximal tubule suspension (lower trace). Notice the spectral separa-tion of intra-(23Naj) from the extracellular (23Na0,) resonances, Thefractional extracellular space (8,,,,,) of the tissue sample was 0.85 andthe intracellular 23Na was 35 tmole/ml cell H20, or 35 mist

resulting in a nearly pure preparation of proximal tubule seg-ments (Fig. 1). Electron micrographs revealed the preservationof cellular morphology, the presence of open lumens and intactbrush border and basolateral membranes (Fig. 2), Exclusion ofvital dye trypan blue and the rate of oxygen consumptiondemonstrated that the viability and integrity of tubule cells werewell preserved. Indeed, the rate of oxygen consumption (Qos)was comparable to that reported for whole kidney [25], kidneyslices [26] and other preparations of renal cortical tubules[27—29]. Moreover, a fivefold stimulation of oxygen consump-tion was induced by addition of succinate, indicating that theproximal tubule cells possessed intact transport systems forcarboxylic acids [301 and were able to oxidize a substraterequired to meet the energy demands for gluconeogenesis andmonovalent cation transport [31].

Intracellular 23Na concentration in the proximal tubules in-cubated at 22°C for 30 mm was 34.1 1.8 m. When tubuleswere incubated at 37°C for 30 mm, the sodium concentration inthe cell decreased to 16.3 0.6 m. This demonstrates that theNMR-visible Na concentration is temperature dependent, pre-sumably due to the temperature dependence of Nat Kt-ATPaseactivity. Similar effects have been recently observed by Sudo andMorel [32] in single medullary collecting ducts (MCT) of ratkidney. In these studies, sodium was measured by flamemicrophotometry in extracts of renal tubules. MCT incubated at4°C for 60 mm had a cell sodium content of 86.3, which decreasedto 17.4 mEqlliter cell volume when tubules were incubated at

xb

Fig. 4. 23Na NMR spectra of renal proximal tubules at 53 MHz and 221°C accumulated over the first three-mm interval (lower trace)

compared with that accumulated during the following three mm on thesame sample (upper trace). Note the steadiness of the intracellular Na"level during NMR measurements, Both spectra yielded essentiallyidentical (< 5% variation) intracellular 23Na levels of 35 j.tmole/ml cellH20, or 35

37°C. Temperature dependence of intracellular sodium concentra-tions had also been noticed in rabbit proximal tubules whenmeasured by radioactive 24Na exchange [5]. A decrease from 155to 65.8 mEq!liter cell water was observed with an increase oftemperature from 4° to 25°C. Flelium glow photometric measure-ments of intracellular sodium in rabbit proximal tubules incubatedin hypotonic medium at 10°C for 15 mm had also given a higherNa's concentration (64.7 mEqlliter cell water) than that observedat 37°C (33 mEqlliter cell water) [12].

The mechanism by which temperature affects the sodiumgradient across the renal cell membrane is not clear. Onepossible explanation is that Na" K-ATPase (Natpump) ac-tivity increases with the temperature. This would increase theextrusion of Na" through the basolateral membrane and thusdecrease the intracellular Na concentration. The temperaturedependence of mitochondrial respiration and of Nat K''-ATPase activity in rabbit proximal tubules have been demon-strated recently by Soltoff and Mandel [33]. These investigatorshave demonstrated that a tight coupling exists between energymetabolism and net ion transport between 15 and 37°C. In ourstudy, the addition of l0- M ouabain, a Na KtATPaseinhibitor, prevented the decrease in intracellular sodium con-centration observed when the ambient temperature was in-creased from 22° to 37°C. Thus, a higher sodium concentrationobserved at lower temperature may be a reflection of a lowerNa"' Kt-ATPase activity. Incubation of the tubules at 37°C withnystatin, an agent that increases the permeability of the luminal

1000 Hz

23Na NMR spectroscopy of proximal tubule suspensions 751

membrane to sodium, resulted in an increase in cell sodiumconcentration from 16.3 0.6 to 72 9.17 m. The lack of fullequilibration between the extra- and intracellular compartmentsin regard to sodium was interpreted to represent evidence thatthe Na pumps located in the basolateral membrane continuedto function properly. Experiments performed on suspensions ofrabbit proximal tubules have demonstrated that the higherconcentrations of intracellular Na induced by the presence ofnystatin results in the stimulation of the Na pump [33].

NMR-measured changes in intracellular 23Na concentrationin the presence of nystatin and different concentrations ofouabain in suspensions of rabbit proximal tubules [34], andamiloride and ouabain in rat outer medullary tubules [15], havealso been recently reported. In these studies, 10 mm incuba-tions with 10 LM ouabain at 37°C increased the intracellularsodium concentration by 50% in rabbit proximal tubules,whereas two hours of incubation with io M ouabain at roomtemperature caused an increase from a value of 32 m to 50 min rat outer medullary tubules [15].

In summary, we have developed a simple and reliable methodfor the preparation of nearly pure suspensions of proximaltubule segments. The tubules in these suspensions are morpho-logically intact and behave predictably from a functional pointof view. The preparations are well suited for determinations ofintracellular concentrations of ions by NMR spectroscopy. The23Na values observed can serve as a basis of reference forfurther inquiries.

AcknowledgmentsA preliminary report of this study was published in abstract form

(Kidney mt 27:314A, 1984). This work was supported in part by NIHGrant AM 32030 awarded to Dr. R. K. Gupta and by the core Grant CA13330 supporting the NMR facility. The authors express their appreci-ation to Dr. Sasha Englard and Sze Fong Ng for allowing them accessto and providing guidance for the use of their oxygen analyzer, Dr. JaneFant and Frank Macaluso for performing the electron microscopy, Drs.David Spray and David Hall for the light microscopy, Ms. BethZavilowitz for technical assistance and to Ms. Frances Andrade fortyping the manuscript.

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