Intracellular pH regulation in cultured renal proximal tubule cells in different stages of maturation H. EKBLAD, Department
A. APERIA,
of Pediatrics,
S-112 81 Stockholm,
AND S. H. LARSSON
St. G6ran’s Sweden
Children’s
Ekblad, H., A. Aperia, and S. H. Larsson. Intracellular pH regulation in cultured renal proximal tubule cells in different stages of maturation. Am. J. Physiol. 263 (Renal Fluid Electrolyte PhysioZ.32): F716-F721, 1992.-This study examinesthe ontogeny of cellular pH regulation in renal proximal tubule cells (RPTC). RPTC from 8 to 40-day-old Sprague-Dawley rats (RPTC-8 to RPTC-40) were studied after 48 h of primary culture. Intracellular pH (pHi) was measuredby quantitative fluorescence microscopy using 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein. Recordingswere madeunder basalconditions and after imposinga cytoplasmic alkalosisand acidosisusing 15 mM NH,+ salt. The net recovery rate (dpHi/dt) from intracellular acidosisincreasessignificantly between 10 and 12 days of age from 0.39 t 0.04 to 0.54 & 0.06 pH units/min (P < 0.05, n = IO vs. 6). This increasecan be completely accountedfor by an increase in the rate of amiloride (100 PM)-inhibitable Na+-H+ exchange (0.29 t 0.04 vs. 0.42 t 0.05 pH units/min, P < 0.05, n = 6 vs. 6). The rate of Na+-H+ exchangeincreases similarly in RPTC-10 and RPTC-40 when the transmembrane Na+ gradient is increasedby Na+ depleting the cells (48 and 49%) respectively). The amiloride-insensitive recovery is Na+ independent and insensitive to 4-acetamido-4’-isothiocyanostilbene-2-2’-disulfonic acid (SITS, 500 PM) (range 0.08-0.14 pH units/min). The net recovery rate from intracellular alkalosisis significantly lower in RPTC-10 than in RPTC-40 (0.16 t 0.02 vs. 0.28 & 0.02 pH units/min, P < 0.01, n = 4 vs. 5). SITS (500PM) inhibits the recovery by 27 t 8 and 26 t 9%, respectively, whereasamiloride has no effect. In RPTC-40 the SITSsensitiverecovery is largely Cl- dependent,and a developmental increasein the activity of Cl--HCO; exchangebetween 12 and 14 days of age is demonstrated. No developmental change is seenin either steady-state pHi (7.27-7.35) or in cytoplasmic buffer capacity (37.6-44.4 mM/pHi unit). In summary, the capacity to protect RPTC from derangementsin pHi is lower in infancy than in adulthood, which might have important clinical implications in the neonate. ontogeny; epithelium; kidney; primary culture; sodium-proton exchange; chloride-bicarbonate exchange; ammonium; buffer capacity; acidosis; alkalosis; 4-acetamido-4’-isothiocyanostilbene-2,2’-disulfonicacid; 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid; amiloride IS COMMON in preterm and in sick full-term infants (2, 7, 10, 11, 22, 28, 30). In general, both renal immaturity and respiratory insufficiency contribute to the development of neonatal acidosis (5, 6, 28). Treatment is based on empirical grounds, since little is known about the regulation of cellular pH (pHi) during postnatal maturation. To learn more about the ontogeny of pHi regulation in epithelial cells, we measured pHi and cellular buffer capacity and compared the responses of immature and mature cultured renal proximal tubule cells (RPTC) to intracellular acidosis and alkalosis. Since RPTC in short-term primary culture maintain much of their in vivo characteristics with regard to the level of differentiation (9, I& 17)) proliferative rate (20)) and morphological characteristics (18)) this was considered
ACIDOSIS
F716
0363-6127/92
Hospital,
Karolinska
Institute,
to be a good model for studying pH regulation. MATERIALS
AND
the ontogeny
of cellular
METHODS
Proximal tubule primary cultures. The preparation of proximal tubule cellsfor primary cultures hasbeendescribedin detail previously (14, 15). Briefly, 8-, lo-, 12-, 14-, 20-, and 40-day-old Sprague-Dawleyrats were anesthetizedwith Inactin (0.08 mg/g body wt ip; Byk-Gulden, Constance, FRG), and the kidneys were perfused with a sterile Hanks’-based0.02-0.05% collagenase solution (type 1; Sigma). The outer 150 pm of the renal cortex wasremovedby the useof a Stadie-Riggsmicrotome and incubated in the collagenaseperfusion solution for a 15min period. In the youngest animals tissue was taken from both kidneys, so that similar amounts of tissue were usedin all age groups.After a seriesof centrifugations, a suspensionconsisting mainly of proximal tubule fragments was obtained (3). This suspensionwas plated onto sterile glasscover slips (Chance Propper) partially replacing the bottoms of plastic Petri dishes (Flow Laboratories). The cellswere cultured in Dulbecco’smodified Eagle’smedium (DMEM, GIBCO Laboratories) with 10% fetal bovine serum (FBS, GIBCO Laboratories) in a waterjacketed incubator (Flow Laboratories) at 37°C with 5% CO,95% air. The composition of our modified DMEM is described as soZution 1 in Table 1. The cellswere serum-deprivedafter 24 h in culture, and the mediumwas carefully rinsed and replaced by DMEM. All experiments were performed after 48 h in culture, when the cellswere nonconfluent and growing in colonies. Coloniesfrom 40-day-old rats tended to be smallerthan from all other agegroups (Fig. 1). Intracellular pH. The method of pHi determination hasbeen describedpreviously (17). pHi was measuredby useof the fluorescent probe 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF, Molecular Probes). The cells were loaded with the acetoxymethyl ester of BCECF for 30-50 min at 37°C (2-6 PM) in DMEM without phenol red (Std-DMEM, solution 1, Table 1). After loading, the medium was replaced by Std-DMEM without BCECF. After an equilibration period of 10 min, pHi was analyzed by use of a Zeiss IM405 inverted microscope equippedwith a computerized spectrophotometersystem (DM3000CM; SPEX, Edison, NJ). The measurementswere always performed in a locally confluent area, i.e., at least three to four cells from the periphery of the colonies, since the two most peripheral layers of cells have been shown to have a lower pHi, a higher growth rate, and a lower expression of the Na+-H+ exchanger than central cells (17). The excitation wavelengths were 485 and 445 nm, with band passesof 5.4 nm, and the emissionwavelengthswere 520-560 nm. The excitation wavelengths were alternated with a frequency of up to 800 Hz. The intensity of emitted light was measuredwith a R928 photon counter (Hammamatsu)and wasaveragedonceper secondover an interval of 0.3 s. Studiesof pHi, calibration, and background measurementswere performed during the samerun of the system and thus under identical instrumental conditions. The emissionat 485-nm excitation is pH sensitive, whereasthat at 445 nm is relatively insensitive to pH changes(23). By calculating the emissionratios after 485- and 445-nm excitation, a pH-dependent, cellular dye content-independent value is gen-
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Society
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POSTNATAL CHANGES IN INTRACELLULAR
Table 1. Experimental
DMEM
Na+ K+
130 5.4 0 0 15 1.8 0.8 130 24 0 0 0.8 20 2.87
baseline following removal of extracellular chloride. The buffer capacity (0) of eachgroup of cellswas determined 130 5.4 0 0 15 5.4 0.8 0 24 126.4 10.8 0.8 20 2.87
All concentrations arein mM unlessotherwisestated.All solutions contained50 IU/ml penicillin,and 50pg/ml streptomycin(Flow Laboratories).pH wassetto 7.40at 37°Candin 5%CO2by titration with NaOHor cholinebase(Sigma)(solution2). Na, K, andosmolalitywere measured regularlyfor all solutions.Osmolalityneverdifferedby more than 10 mosm/lbetweentwo solutionsusedconsecutivelyin an experiment. Spec-DMEM
was without
F717
measuring the SITS-sensitive (250 PM) change in pHi from
solutions
140 0 140 5.4 5.4 5.4 TMA+ 0 116 0 Choline+ 0 24 0 0 0 0 NH: Ca2+ 1.8 1.8 5.4 Mg2+ 0.8 0.8 0.8 Cl125 125 0 24 24 24 HCO, Aspartate0 0 121.4 Lactate0 0 10.8 0.8 0.8 0.8 so;HEPES 20 20 20 2.81 2.87 Spec-DMEMg/l 2.87
PH REGULATION
CaCls, KCl, MgS04, NaCl, NaCOs,
from the alkalinization taking place when the cells were first exposedto NH&I (15 mM) (Fig. 2) (8,17,24). In paired control experiments /3 was determined in the presenceand absenceof both amiloride (100 FM) and 4,4’-diisothiocyanostilbene-2,2’disulfonic acid (DIDS, 250 PM). Thesetransport inhibitors did not change /I (not shown).
Results are means -CSE. Group comparisons were made using the Student’s t test, and evaluation of age dependencywas by variance analysis (ANOVA). P < 0.05 was consideredstatistically significant; N is number of colonies studied, and n is
number of animals studied. RESULTS
There was no age-dependent change in steady-state pHi or buffer capacity in RPTC from 8- to 40-day-old rats (mean values, range 7.27-7.35 and 37.6-44.4 mM/pHi unit, respectively; see Table 2). The net rates of recovery from an intracellular acid load for all studied age groups are summarized in Table 3 and typical experiments shown in Fig. 3. The rate increased
andphenolred (GIBCO,customized).TMA, tetramethylammonium. erated (485/445 ratio). Calibration was performed by use of nigericin (25 KM; Sigma) in KC1 buffers of varying pH (6.407.60) with the following composition (in mM): 120 KCl, 20 NaCl, and 20 N-2-hydroxyethylpiperazine-IV’-2-ethanesulfonic acid (HEPES)(29). Calibration wasperformed after eachexperiment on the same population of cells as that used for the experimental measurements,utilizing at least five solutions with different pH. No significant difference was found between age groups for the calibration curves; the slopesof the curves rangedfrom 2.22to 2.34and the intercepts from -11.7 to -12.3. All measurementswereperformed at 37°C and mediumpH 7.40 with 24 mM HCO; and 5% CO,. The Petri dish support allowed complete media exchangeswithin 5 s. Net rate of cytoplasmic pH recovery after cellular alkalosis and acidosis was measuredby exposing the cells to 15 mM NHICl (solution 4, Table 1) (24). Addition of NH&l to the culture medium induced an immediate increasein pH,. After lo-15 min the medium was replaced by Std-DMEM, resulting in a rapid acidification of the cells (Fig. 2). After alkalinization and acidification the cells recovered to baselinepHi. The capacity of cellular pH recovery was determined as initial rate (dPK/dt)In eachagegroup we determinedthe extent to which recovery from the acid load was causedby Na+-H+ exchange or other membranetransporters by repeating the NH&l procedure but allowing the secondrecovery to take place in Na+-free DMEM (solution 2, Table l), in the presence of amiloride (100 PM) (Merck Sharp & Dohme), or in the presenceof both amiloride and 4-acetamido-4’-isothiocyanostilbene-2-2’-disulfonic acid (SITS, 250 PM; Sigma). The rate of Na+-H+ exchange was calculated as the difference in initial rates between measurements in the absenceand presenceof amiloride. In RPTC in primary culture 100 MM amiloride maximally inhibits the Na+-H+ exchanger,but has little effect on Na+-K+-adenosine-
triphosphatase (Na+-K+-ATPase)
(9, 17).
The cellular recovery from an alkaline load was analyzed by measuringthe effect of SITS (500 FM, lo-min preincubation) or amiloride (100PM). The Cl- dependenceof the net recovery was evaluated by Cl- depleting the cells for 10 min (solution 3, Table 1) prior to addition of NH: (solution 5, Table 1). The cellular expressionof Cl--HCO; exchangewas investigated by
Fig. 1. Colonies of renal proximal tubule cells (RPTC) from a lo-day (A) and a 40-day-old (B) Sprague-Dawley rat after 48 h in primary culture. Cells were cultured in absence of fetal bovine serum (FBS) during the last 24 h and then prepared for [3H]thymidine autoradiography. Black silver grains are seen over the few cells which have synthesized DNA (entered S-phase of the cell cycle) during the 6 h [3H]thymidine pulse (17).
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F718
POSTNATAL NH4CI +
NH4Cl +
CHANGES NH4Cl
NH4CI
t
80.
IN INTRACELLULAR Amiloride 1 I
REGULATION
i
Fig. 2. A representative experiment in RPTC from a 20day-old rat, illustrating measurements of cellular pH (pHi) regulation. Addition of NH&l (15 mM) to the culture medium induced a rapid cytoplasmic alkalinization, followed by recovery of pHi* Removal of NH&l after 15 min induced a rapid cellular acidification, followed by recovery of pHi. The experiment was repeated and the second recovery took place in presence of amiloride (100 PM), which caused a marked and reversible decrease in the recovery rate.
7s.
T
z a
PH
70.
65. Table 2. Intracellular pH and buffer capacity in rat RPTC as a function of postnatal age Age, days
0, mM/pHi
PHi
unit
8 7.28t0.03 (6) 40.5t2.1 (6) 10 7.31t0.03 (18) 40.6t4.5 (10) 12 7.3320.04 (6) 43.7t3.2 (6) 14 7.35t0.02 (6) 40.9t3.3 (6) 20 7.27t0.02 (13) 44.4k2.5 (7) 40 7.32t0.03 (8) 37.6t3.8 (7) Values are means t SE; no. of experiments is in parentheses. pHi, intracellular pH; ,f3,buffer capacity. Buffer capacity was determined from the alkalinization following NH&l addition and calculated from the formula A[NH,+]i/ApHi (8, 17,24). [NH,‘], was calculated using a pK of 9.21. RPTC, renal proximal tubule cells.
Table 3. Net and amiloride-insensitive recovery rate from intracellular acidosis in rat RPTC as a function of postnatal age Age, days
Net Rate, pH unit/min
AI Rate, pH unit/min
=L= 0. v)
0
0
Fig. 3. Representative experiments in RPTC illustrating the postnatal increase in net rate of recovery after an acid load. Rate of recovery was 0.42 pH units/min in cells from a lo-day-old (left) and 0.70 pH units/ min in cells from a 40-day-old rat (right).
PHmin
8 0.38t0.04* (6) 0.09t0.02 (6) 6.50t0.03 (6) 10 0.39t0.04* (10) O.OBt0.02 (11) 6.53kO.03 (10) 12 0.5420.06 (6) 0.12t0.02 (6) 6.5720.04 (6) 14 0.59t0.04 (6) O.lOt0.02 (6) 6.55t0.02 (6) 20 0.58t0.06 (6) 0.11t0.03 (6) 6.49t0.04 (6) 40 0.14t0.02 (10) 0.60~10.02 (7) 6.57kO.06 (7) Values are means t SE; no. of experiments is in parentheses. AI, amiloride insensitive; pHmin, intracellular acidosis. * P < 0.05 compared with RPTC from 12-40-day-old rats.
significantly between 10 and 12 days of age (0.39 t 0.04 vs. 0.54 t 0.06 pH units/min, P < 0.05, n = 10 vs. 6). From 12 days of age no significant increase was noted. The developmental increase in the recovery rate from intracellular acidosis could be completely accounted for by an increase in the amiloride-sensitive rate of recovery, most likely the Na+-H+ exchanger, which increased from 0.29 t 0.04 to 0.42 t 0.05 pH units/min between 10 and 12 days of age (P < 0.05, n = 6 vs. 7) (Fig. 4). To further evaluate these differences in Na+-H+ exchange between infant and adolescent cells, we investigated the effect of increasing the transmembrane Na+ gradient in RPTC from lo- and 4O-day-old rats. Cells were incubated in NH&l-DMEM as described above. After lo-15 min the medium was replaced by ONa+-
0,5z g 0,4I e 0) 0,3P
*
*
8
10
l
a
5
w-
x ? x w?a I z 0 12 14 20 40 Age (days) Fig. 4. Age dependence of amiloride-sensitive recovery rate (Na+-H+ exchange) from intracellular acidosis in RPTC. Recovery rate increased significantly between 10 and 12 days of age (n = 6-10, P < 0.05).
DMEM (solution 2, Table 1). After 2-3 min the cells were returned to Std-DMEM (solution 1, Table 1). Na+ depletion of the cells before recovery from acidosis resulted in the same relative increase in the rate of recovery in cells
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POSTNATAL
CHANGES
IN INTRACELLULAR
from lo- and 4O-day-old rats (48 and 49%, respectively) (Fig. 5). The amiloride-insensitive rate of recovery was equal in all age groups and mean values ranged from 0.08 to 0.14 pH units/min (Table 3). In a subgroup of the experiments included in Table 3 (5 RPTC-10 and 4 RPTC-40), the amiloride-insensitive rate of recovery was further characterized. In these paired experiments the rate of recovery was determined first in the absence of Na+ (solution 2, Table l), then in the presence of amiloride (100 PM), and finally with both amiloride and SITS (500 PM). In neither age group were any differences found between these three protocols [ 10 day: 0.08 t 0.05,0.05 t 0.01, and 0.04 t 0.01 pH units/min (n = 5), not significant (NS); 40 day: 0.17 t 0.03, 0.18 t 0.02, and 0.17 t 0.03 pH units/ min (n = 4), NS]. For the two latter protocols the rates were significantly lower in RPTC-10 than in RPTC-40 (P < 0.001).
The net capacity of the cells to recover from an intracellular alkaline load was compared in cells from lo- and 40-day-old rats. The recovery was significantly faster in cells from 40-day-old rats (0.16 t 0.02 vs. 0.28 t 0.02 pH units/min, P < 0.01) (Table 4). The maximal pHi from which the recovery started was the same in both age groups (7.82 t 0.09 and 7.81 t 0.06, respectively). Amiloride had no effect on the recovery rate in either age group. In pilot experiments with ado1.escent cells amiloride was corn .bin . .ed with N ‘a+ depletion, result ing in a slight reduction in recovery rate (not sh.own). Thus Na+-H+ exchange does not seem to contribute to the recovery from alkaline load. The SITS-inhibitable component was the same in lo- and 40-day-old RPTC (Table 4; 27 t 8 vs. 26 t 9%, NS). SITS or DIDS at 250 PM had the same effect as SITS at 500 PM. To distinguish the Na+-3HCO; cotransporter from Cl--HCO; exchange we measured the recovery in Cl--depleted cells. Removal of Cl- from the medium resulted in a significant inhibition of recovery in adolescent cells (18 t 1%, N = 7, P < 0.05), whereas in infant cells no significant inhibition was seen 1
F --
E I‘
0,9
W3
PH
F719
REGULATION
Table 4. Recovery from intracellular alkalosis in RPTC from IO-day and 40-day-old rats Age, days
P
10
40
Net rate of recovery, 0.16t0.02 (11) 0.28t0.02 (11) CO.01 pH unit/min Rate with 500 PM, 0.11t0.03 (5) 0.16t0.01 (4) NS SITS pH unit/min Values are means ~frSE; no. of experiments is in parentheses. NS, not significan t.
(-10 t 23%, N = 8, NS). These results suggested that recovery in adolescent cells was to some extent mediated by Cl--HCO, exchange, which was not seen to the same extent in young cells. The low rates of recovery in young cells led to large variations and therefore necessitated another approach to further compare the age groups with respect to Cl--HCO; exchange. Removal of extracellular Cl- resulted in a rapid increase in pHi. The rate of pH change was markedly age dependent (Fig. 6) with the most marked increase after the age of 12 days. The alkalinization was reversib1.e and completely blocked by 250 PM SITS (not shown). DISCUSSION
We show here that a developmental increase in the rate of recovery from an intracellular acid load occurs in the infant rat kidney between 10 and 12 days of age (Table 3) and that it can be completely accounted for by an increase in the capacity of Na+-H+ exchange (Fig. 4). In a previous study of cultured rat RPTC, it was demonstrated that steady-state net Na+ influx increased during early infancy (lo-12 days) (19). The timing of the increase in Na+ influx was exactly the same as for the present increase in the capacity of Na+-H+ exchange. An increased transmembrane Na+ gradient resulted in an identical relative increase in the rate of Na+-H+ exchange in RPTC from lo- and 40-day-old rats (Fig. 5). The acidotic pHi at which the recoveries started were the same in all age groups (Table 3). Previous studies using electron probe analysis have shown that the intracellular
aor7
0’
Na+i-depletion
Normal
Na+i
Fig. 5. Effect of a change in the transmembrane Na+ gradient on the rate of Na+-H+ exchange in RPTC from lo- and 40-day-old rats. Cells with normal Na+ content were acidified with Std-DMEM (solution 1, Table l), whereas Na+-depleted cells were acidified with ONa+-DMEM (solution. 2, Table 1) for 2-3 min. Intracellular Na+ (Na+) depletion caused an identical relative increase in the rate of Na+-H+ exchange in the 2 age groups (48 and 49% for 10 day and 40 day, respectively). Na+-H+ exchange rate in cells from lo-day-old rats was 59% of that in cells from 40-day-old rats both in cells with normal Na+ content and in Na+-depleted cells (** P < 0.01). pHi values from which the initial rates were determined were not different between ages [Na+ depletion: 6.65 t 0.07 vs. 6.67 k 0.04 for 10 vs. 40 day (n = 5 vs. 4), not significant (NS); normal Na+: 6.53 t 0.03 vs. 6.57 t 0.06 for 10 vs. 40 day (n = 10 vs. 7), NS].
.-.. c ? IQ&15-. 2 8 .-i .- I %0,05
O,l-h
'
10
'
20
'
40
'
60
Time in 0 mM Cl-medium
-
300
-
(set)
Fig. 6. Change in pHi in RPTC from lo-day to 40-day-old rats after removal of extracellular Cl- (solution 1 changed to 3, Table 1). After removal of Cl-, pHi increased in all age groups, reaching the same final value within 5 min. However, a developmental increase in the rate of change was noted between 12 and 14 days of age (N = 3-10). The increase in pHi following Cl- removal was completely inhibited by 250 PM SITS (not shown).
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F720
POSTNATAL
CHANGES
IN INTRACELLULAR
Na+ content is equal in serum-deprived RPTC from loand 4O-day-old rats (Na/P = 0.15 t 0.02 and 0.15 t 0.01, respectively) (15, 19; S. Rane, S. H. Larsson, A. Aperia, and C. Lechene, unpublished observations). Thus the difference in recovery rates between RPTC from lo- and 4O-day-old rats cannot be explained by differences in H+ gradients or Na+ gradients over the cell membranes. No developmental change in buffer capacity was seen (Table 2). Furthermore, the low rate of recovery in lo-day-old cells is not a consequence of serum starvation, since these cells cultured 48 h in 10% FBS, if anything, have a lower net rate of recovery (0.30 t 0.06 pH units/min, n = 5, H. Nyctelius and S. H. Larsson, unpublished observations). Consequently, the demonstrated increase in transport rate can only be explained by a developmental change in the number of Na+-H+ exchangers and/or a change in their regulation (19). The rate of recovery after an acid load in RPTC from mature rats (Table 3) is very similar to that previously demonstrated in isolated perfused rabbit S2 segments, using a protocol resembling ours (0.55 pH units/min) (12). Baum (4) studied microperfused juxtamedullary proximal convoluted tubules and measured pHi by fluorescence microscopy. The rate of Na+-H+ exchange was studied in the presence of basolateral SITS and in reverse direction by removing extracellular Na+. The rate of change in pHi was faster than in our study (2.5-3.0 pH units/min). This may be explained entirely by methodological differences, since the rate of pHi change has been documented to be faster if the Na+-H+ exchanger is studied in reverse direction or in the presence of basolateral SITS (21). It may be argued that such a protocol is unphysiological; however, the data are qualitatively very similar to ours, indicating a developmental increase in the capacity of Na+-H+ exchange. The amiloride-insensitive rate of recovery after an acid load appears to be age independent (Table 3) and is similar to that described in the rat proximal convoluted tubule in vivo (21) and in isolated perfused S2 segments from rabbit (12). Because it is SITS insensitive and Na+ independent, it is likely mediated by H+-ATPase (12,25) or passive NH,+ efflux (24). It should be noted that in the subgroup of experiments in which the Na dependence and SITS sensitivity were studied, RPTC-40 showed a significantly faster amiloride-insensitive rate of recovery than RPTC-10. Although no significance was found when all experiments were analyzed together (Table 3), the data may indicate a postnatal increase in H+-ATPase activity. The rate of recovery from an NH,+-induced alkaline load is significantly higher in RPTC from 40-day-old than from lo-day-old rats. The recovery is amiloride insensitive and SITS inhibitable to -25% in both age groups (Table 4). In adolescent, but not in infant cells, the SITS-inhibitable transport is largely Cl- dependent, suggesting a role of Cl--HCO; exchange. An age-dependent increase in the capacity of Cl--HCO; exchange is also supported by the results presented in Fig. 6. The SITS/DIDS-sensitive recovery from intracellular alkalosis is remarkably slow. The principal cytoplasmic acidifier in the proximal tubule, the basolateral Na+-3 HCO; cotransporter (1), does not appear to contribute
PH REGULATION
much to the net recovery. However, it has recently been shown that this transporter is regulated by an intracellular pH-sensitive site and that its activity is completely blocked when the cell interior is made alkaline (pHi above 7.6) (27). Since the recoveries in our studies started at pHi 7.8, the transporter may well have been inactivated. The major pH-regulating membrane transporters thus have a very low capacity to protect the cytoplasm from intracellular alkalosis, and the most important mechanism of recovery in the protocol used in the present experiments may instead be influx of NH,+ (24). We show here that the Na+-H+ exchanger is of crucial importance for recovery from intracellular acidosis in rat RPTC. The transporter is also known to be highly active under steady-state conditions as it mediates up to 87% of Na+ influx (9). In contrast, the Na+-3HCO; cotransporter, which is regarded as the most important regulator of basal pHi in RPTC (1), does not protect the cell from an intracellular alkalosis. During maturation of RPTC the capacities of Na+-H+ and Cl--HCO, exchange increase. The subcellular localization of the participating transporters in cultured RPTC remains to be elucidated. The purpose of the present study was to study cellular net capacity to recover from derangements in pHi. It is noteworthy that the two different transport processes mature at different ages, i.e., the HCO, transport matures -2 days after the Na+-H+ exchanger. The role of Cl--HCO; exchange in proximal tubule pH regulation has been debated (1); our results, however, suggest that this transport may play a role in recovery from alkaline load. A qualitatively similar developmental pattern has previously been demonstrated for the Na+-H+ exchanger and the Na+-3HCO; cotransporter in juxtamedullary proximal tubule cells from rabbit (4) It is unlikely that the stepwise increases in pHi regulatory capacity demonstrated in this study are caused by a change in the ratio of surface area to volume (A/V) (13, 18). Our measurements of pHi were performed on cells in the center of colonies. Single central cells from lo- and 4O-day-old rats cover the same area of the culture support (799 t IO5 vs. 775 t I53 pm’, N = 14 vs.5, NS; cell layer no. 5 from periphery of colonies, S. H. Larsson and J. Luthman, unpublished observations) (Fig. 1). Cellular P content, measured by electron probe analysis, is a good index of cellular mass. The P content is higher in adult than in infant cells, suggesting a difference in cellular height (15). These data suggest that infant rat RPTC, if anything, are smaller than adolescent in primary culture, and consequently A/V is larger in infant than in adolescent cells. Because buffer capacity does not change with age, the described developmental changes in cellular capacity to alter cytoplasmic pH therefore may underestimate developmental changes in the capacity of the cell membrane to transport H+ equivalents. The results of this study indicate that immature RPTC have a lower capacity to recover from derangements in pHi than do mature RPTC. The results support the concept that RPTC H+ and HCO, transport pathways undergo postnatal maturation (4, 26). A maintenance of constant pHi is crucial for normal cellular function. The
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POSTNATAL
CHANGES
IN INTRACELLULAR
low capacity of young cells to recover from intracellular acidosis might have important clinical implications, since dramatic changes in pH homeostasis are particularly common in sick neonates. We wish to thank Anna Johansson, Ingrid Muldin, and Eivor Zettergren for expert technical assistance. This work was supported by grants from the Swedish Medical Research Council, The State Medical Board of Finland, The Emil Aaltonen Foundation, Finland, and the Royal Swedish Academy of Sciences. Address for reprint requests: S. H. Larsson, Department of Pediatrics, St. Goran’s Children’s Hospital, Box 12500, S-112 81 Stockholm, Sweden. Received 16 April 1991; accepted in final form 26 May 1992.
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Am. J. Physiol. F360, 1981.
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and R. Zetterstrom. Hormonal inin developing proximal tubular cells.
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Baum, M. Neonatal rabbit juxtamedullary proximal convoluted tubule acidification. J. Clin. Invest. 85: 499-506, 1990. 5. Campbell, A. G. M. Metabolic changes in the respiratory distress syndrome. Clin. Endocrinol. Metab. 5: 55-87, 1976. 6. Edelmann, C. M., Jr., J. R. Soriano, H. Boichis, A. B. Gruskin, and M. I. Acosta. Renal bicarbonate reabsorption and hydrogen ion excretion in normal infants. J. CZin. Invest. 46: 4.
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Kerpel-Fronius, E., T. Heim, and E. Sulyok. The development of the renal acidifying processes and their relation to acidosis in low-birth-weight infants. Biol. Neonate 15: 156-168,
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Kildeberg, P. Disturbances of hydrogen ion balance occurring in premature infants. II. Late metabolic acidosis. Acta Paediatr. 53:
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Kurtz, I. Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal
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18. Larsson, S. H., L. Larsson, C. Lechene, and A. Aperia. Studies of terminal differentiation of electrolyte transport in the renal proximal tubule using Nephrol. 3: 363-368, 1989.
short-term
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19. Larsson, S. H., S. Rane, Y. Fukuda, A. Aperia, and C. Lechene. Changes in Na influx precede postnatal increase in Na,K-ATPase activity in rat renal proximal tubular cells. Acta Stand.
138: 99-100,
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20 .
Larsson, S. H., S. Yun, S. Kolare, and A. Aperia. Postnatal changes in growth of rat proximal tubule cells. A study of cells in short primary culture. Acta Physiol. Stand. 138: 243-244, 1990. 2l Preisig, P. A., H. E. Ives, E. J. Cragoe, Jr., R. J. Alpern, and F. C. Rector, Jr. Role of the Na+/H+ antiporter in rat proximal tubule bicarbonate absorption. J. Clin. Invest. 80: 970l
978, 1987.
22* Reimold, failure
E. W., T. Dinh
during
the first
year
Don, and H. G. Worthen. of life.
Pediatrics
59: 987-994,
Renal 197%
23. Rink, T. J., R. Y. Tsien, and T. Pozzan. Cytoplasmic pH and free Mg2+ in lymphocytes. J. Cell BioZ. 95: 189-196, 1982. pH. Physiol. Rev. 61: 24. ROOS, A., and W. F. Boron. Intracellular 1981.
25. Schwartz,
G. J., and Q. Al-Awqati.
exocytosis
of vesicles
proximal
containing
and collecting tubules.
Carbon dioxide causes H+ pumps in isolated perfused J. Clin. Invest. 75: 1638-1644,
1985. 26.
27
Schwartz, G. J., and A. P. Evan. Development of solute transport in rabbit proximal tubule. I. HCO, and glucose absorption. Am. J. Physiol. F390, 1983. .
Soleimani, McKinney.
245 (Renal
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M., G. A. Lesione,
Electrolyte
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J. A. Bergman,
14): F382-
and T. D.
modifier site regulates activity of the Na+: HCO;-cotransporter in basolateral membranes of kidney proximal tubules. J. CZin. Invest. 88: 1135-1140, 1991. 28. Svenningsen, N. W. Renal acid-base titration studies in infants
A pH
with and without Pediatr.
metabolic acidosis in the postneonatal
Res. 8: 659-672,
period.
1974.
29.
Thomas, J. A., R. N. Buchsbaum, A. Zimniak, and E. Racker. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry
30.
Ylppo, A. Neugeborenen-, Hunger- und Intoxikationsacidosis in ihren Beziehungen zueinander. Studien uber Acidosis bei Sauglingen, inbesondere im Lichte des Wasserstoffionen-“Stoffwechsels.”
18: 2210-2218, tubule
Physiol.
17* Larsson, S. H., Y. Fukuda, S. Kolare, and A. Aperia. Proliferation and intracellular pH in cultured proximal tubular cells.
Cytoplasmic pH
by an amiloride-sensitive 83: 341-369, 1984. and C. Lechene. Coupling
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Larsson, S. H., A. Aperia, and C. Lechene. Studies on terminal differentiation of rat renal proximal tubular cells in culture: ouabain-sensitive K and Na transport. Acta Physiol. Stand. 132:
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S., S. Cohen, and A. Rothstein.
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Ekblad, H., P. Kero, and J. Takala. Slow sodium acetate infusion in the correction of metabolic acidosis in premature infants.
8.
Stand.
F721
S., A. Aperia, and C. Lechene. Studies on final differentiation of rat renal proximal tubular cells in culture. Am. J.
Physiol.
S. K. Acute renal failure in the neonate. Pediatr.
REGULATION
15. Larsson,
1990.
2.
North
Physiol.
PH
2. Kinderheikd.
1979.
14: l-184,
1916.
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A. APERIA,
of Pediatrics,
S-112 81 Stockholm,
AND S. H. LARSSON
St. G6ran’s Sweden
Children’s
Ekblad, H., A. Aperia, and S. H. Larsson. Intracellular pH regulation in cultured renal proximal tubule cells in different stages of maturation. Am. J. Physiol. 263 (Renal Fluid Electrolyte PhysioZ.32): F716-F721, 1992.-This study examinesthe ontogeny of cellular pH regulation in renal proximal tubule cells (RPTC). RPTC from 8 to 40-day-old Sprague-Dawley rats (RPTC-8 to RPTC-40) were studied after 48 h of primary culture. Intracellular pH (pHi) was measuredby quantitative fluorescence microscopy using 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein. Recordingswere madeunder basalconditions and after imposinga cytoplasmic alkalosisand acidosisusing 15 mM NH,+ salt. The net recovery rate (dpHi/dt) from intracellular acidosisincreasessignificantly between 10 and 12 days of age from 0.39 t 0.04 to 0.54 & 0.06 pH units/min (P < 0.05, n = IO vs. 6). This increasecan be completely accountedfor by an increase in the rate of amiloride (100 PM)-inhibitable Na+-H+ exchange (0.29 t 0.04 vs. 0.42 t 0.05 pH units/min, P < 0.05, n = 6 vs. 6). The rate of Na+-H+ exchangeincreases similarly in RPTC-10 and RPTC-40 when the transmembrane Na+ gradient is increasedby Na+ depleting the cells (48 and 49%) respectively). The amiloride-insensitive recovery is Na+ independent and insensitive to 4-acetamido-4’-isothiocyanostilbene-2-2’-disulfonic acid (SITS, 500 PM) (range 0.08-0.14 pH units/min). The net recovery rate from intracellular alkalosisis significantly lower in RPTC-10 than in RPTC-40 (0.16 t 0.02 vs. 0.28 & 0.02 pH units/min, P < 0.01, n = 4 vs. 5). SITS (500PM) inhibits the recovery by 27 t 8 and 26 t 9%, respectively, whereasamiloride has no effect. In RPTC-40 the SITSsensitiverecovery is largely Cl- dependent,and a developmental increasein the activity of Cl--HCO; exchangebetween 12 and 14 days of age is demonstrated. No developmental change is seenin either steady-state pHi (7.27-7.35) or in cytoplasmic buffer capacity (37.6-44.4 mM/pHi unit). In summary, the capacity to protect RPTC from derangementsin pHi is lower in infancy than in adulthood, which might have important clinical implications in the neonate. ontogeny; epithelium; kidney; primary culture; sodium-proton exchange; chloride-bicarbonate exchange; ammonium; buffer capacity; acidosis; alkalosis; 4-acetamido-4’-isothiocyanostilbene-2,2’-disulfonicacid; 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid; amiloride IS COMMON in preterm and in sick full-term infants (2, 7, 10, 11, 22, 28, 30). In general, both renal immaturity and respiratory insufficiency contribute to the development of neonatal acidosis (5, 6, 28). Treatment is based on empirical grounds, since little is known about the regulation of cellular pH (pHi) during postnatal maturation. To learn more about the ontogeny of pHi regulation in epithelial cells, we measured pHi and cellular buffer capacity and compared the responses of immature and mature cultured renal proximal tubule cells (RPTC) to intracellular acidosis and alkalosis. Since RPTC in short-term primary culture maintain much of their in vivo characteristics with regard to the level of differentiation (9, I& 17)) proliferative rate (20)) and morphological characteristics (18)) this was considered
ACIDOSIS
F716
0363-6127/92
Hospital,
Karolinska
Institute,
to be a good model for studying pH regulation. MATERIALS
AND
the ontogeny
of cellular
METHODS
Proximal tubule primary cultures. The preparation of proximal tubule cellsfor primary cultures hasbeendescribedin detail previously (14, 15). Briefly, 8-, lo-, 12-, 14-, 20-, and 40-day-old Sprague-Dawleyrats were anesthetizedwith Inactin (0.08 mg/g body wt ip; Byk-Gulden, Constance, FRG), and the kidneys were perfused with a sterile Hanks’-based0.02-0.05% collagenase solution (type 1; Sigma). The outer 150 pm of the renal cortex wasremovedby the useof a Stadie-Riggsmicrotome and incubated in the collagenaseperfusion solution for a 15min period. In the youngest animals tissue was taken from both kidneys, so that similar amounts of tissue were usedin all age groups.After a seriesof centrifugations, a suspensionconsisting mainly of proximal tubule fragments was obtained (3). This suspensionwas plated onto sterile glasscover slips (Chance Propper) partially replacing the bottoms of plastic Petri dishes (Flow Laboratories). The cellswere cultured in Dulbecco’smodified Eagle’smedium (DMEM, GIBCO Laboratories) with 10% fetal bovine serum (FBS, GIBCO Laboratories) in a waterjacketed incubator (Flow Laboratories) at 37°C with 5% CO,95% air. The composition of our modified DMEM is described as soZution 1 in Table 1. The cellswere serum-deprivedafter 24 h in culture, and the mediumwas carefully rinsed and replaced by DMEM. All experiments were performed after 48 h in culture, when the cellswere nonconfluent and growing in colonies. Coloniesfrom 40-day-old rats tended to be smallerthan from all other agegroups (Fig. 1). Intracellular pH. The method of pHi determination hasbeen describedpreviously (17). pHi was measuredby useof the fluorescent probe 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF, Molecular Probes). The cells were loaded with the acetoxymethyl ester of BCECF for 30-50 min at 37°C (2-6 PM) in DMEM without phenol red (Std-DMEM, solution 1, Table 1). After loading, the medium was replaced by Std-DMEM without BCECF. After an equilibration period of 10 min, pHi was analyzed by use of a Zeiss IM405 inverted microscope equippedwith a computerized spectrophotometersystem (DM3000CM; SPEX, Edison, NJ). The measurementswere always performed in a locally confluent area, i.e., at least three to four cells from the periphery of the colonies, since the two most peripheral layers of cells have been shown to have a lower pHi, a higher growth rate, and a lower expression of the Na+-H+ exchanger than central cells (17). The excitation wavelengths were 485 and 445 nm, with band passesof 5.4 nm, and the emissionwavelengthswere 520-560 nm. The excitation wavelengths were alternated with a frequency of up to 800 Hz. The intensity of emitted light was measuredwith a R928 photon counter (Hammamatsu)and wasaveragedonceper secondover an interval of 0.3 s. Studiesof pHi, calibration, and background measurementswere performed during the samerun of the system and thus under identical instrumental conditions. The emissionat 485-nm excitation is pH sensitive, whereasthat at 445 nm is relatively insensitive to pH changes(23). By calculating the emissionratios after 485- and 445-nm excitation, a pH-dependent, cellular dye content-independent value is gen-
$2.00 Copyright 0 1992 the American Physiological
Society
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POSTNATAL CHANGES IN INTRACELLULAR
Table 1. Experimental
DMEM
Na+ K+
130 5.4 0 0 15 1.8 0.8 130 24 0 0 0.8 20 2.87
baseline following removal of extracellular chloride. The buffer capacity (0) of eachgroup of cellswas determined 130 5.4 0 0 15 5.4 0.8 0 24 126.4 10.8 0.8 20 2.87
All concentrations arein mM unlessotherwisestated.All solutions contained50 IU/ml penicillin,and 50pg/ml streptomycin(Flow Laboratories).pH wassetto 7.40at 37°Candin 5%CO2by titration with NaOHor cholinebase(Sigma)(solution2). Na, K, andosmolalitywere measured regularlyfor all solutions.Osmolalityneverdifferedby more than 10 mosm/lbetweentwo solutionsusedconsecutivelyin an experiment. Spec-DMEM
was without
F717
measuring the SITS-sensitive (250 PM) change in pHi from
solutions
140 0 140 5.4 5.4 5.4 TMA+ 0 116 0 Choline+ 0 24 0 0 0 0 NH: Ca2+ 1.8 1.8 5.4 Mg2+ 0.8 0.8 0.8 Cl125 125 0 24 24 24 HCO, Aspartate0 0 121.4 Lactate0 0 10.8 0.8 0.8 0.8 so;HEPES 20 20 20 2.81 2.87 Spec-DMEMg/l 2.87
PH REGULATION
CaCls, KCl, MgS04, NaCl, NaCOs,
from the alkalinization taking place when the cells were first exposedto NH&I (15 mM) (Fig. 2) (8,17,24). In paired control experiments /3 was determined in the presenceand absenceof both amiloride (100 FM) and 4,4’-diisothiocyanostilbene-2,2’disulfonic acid (DIDS, 250 PM). Thesetransport inhibitors did not change /I (not shown).
Results are means -CSE. Group comparisons were made using the Student’s t test, and evaluation of age dependencywas by variance analysis (ANOVA). P < 0.05 was consideredstatistically significant; N is number of colonies studied, and n is
number of animals studied. RESULTS
There was no age-dependent change in steady-state pHi or buffer capacity in RPTC from 8- to 40-day-old rats (mean values, range 7.27-7.35 and 37.6-44.4 mM/pHi unit, respectively; see Table 2). The net rates of recovery from an intracellular acid load for all studied age groups are summarized in Table 3 and typical experiments shown in Fig. 3. The rate increased
andphenolred (GIBCO,customized).TMA, tetramethylammonium. erated (485/445 ratio). Calibration was performed by use of nigericin (25 KM; Sigma) in KC1 buffers of varying pH (6.407.60) with the following composition (in mM): 120 KCl, 20 NaCl, and 20 N-2-hydroxyethylpiperazine-IV’-2-ethanesulfonic acid (HEPES)(29). Calibration wasperformed after eachexperiment on the same population of cells as that used for the experimental measurements,utilizing at least five solutions with different pH. No significant difference was found between age groups for the calibration curves; the slopesof the curves rangedfrom 2.22to 2.34and the intercepts from -11.7 to -12.3. All measurementswereperformed at 37°C and mediumpH 7.40 with 24 mM HCO; and 5% CO,. The Petri dish support allowed complete media exchangeswithin 5 s. Net rate of cytoplasmic pH recovery after cellular alkalosis and acidosis was measuredby exposing the cells to 15 mM NHICl (solution 4, Table 1) (24). Addition of NH&l to the culture medium induced an immediate increasein pH,. After lo-15 min the medium was replaced by Std-DMEM, resulting in a rapid acidification of the cells (Fig. 2). After alkalinization and acidification the cells recovered to baselinepHi. The capacity of cellular pH recovery was determined as initial rate (dPK/dt)In eachagegroup we determinedthe extent to which recovery from the acid load was causedby Na+-H+ exchange or other membranetransporters by repeating the NH&l procedure but allowing the secondrecovery to take place in Na+-free DMEM (solution 2, Table l), in the presence of amiloride (100 PM) (Merck Sharp & Dohme), or in the presenceof both amiloride and 4-acetamido-4’-isothiocyanostilbene-2-2’-disulfonic acid (SITS, 250 PM; Sigma). The rate of Na+-H+ exchange was calculated as the difference in initial rates between measurements in the absenceand presenceof amiloride. In RPTC in primary culture 100 MM amiloride maximally inhibits the Na+-H+ exchanger,but has little effect on Na+-K+-adenosine-
triphosphatase (Na+-K+-ATPase)
(9, 17).
The cellular recovery from an alkaline load was analyzed by measuringthe effect of SITS (500 FM, lo-min preincubation) or amiloride (100PM). The Cl- dependenceof the net recovery was evaluated by Cl- depleting the cells for 10 min (solution 3, Table 1) prior to addition of NH: (solution 5, Table 1). The cellular expressionof Cl--HCO; exchangewas investigated by
Fig. 1. Colonies of renal proximal tubule cells (RPTC) from a lo-day (A) and a 40-day-old (B) Sprague-Dawley rat after 48 h in primary culture. Cells were cultured in absence of fetal bovine serum (FBS) during the last 24 h and then prepared for [3H]thymidine autoradiography. Black silver grains are seen over the few cells which have synthesized DNA (entered S-phase of the cell cycle) during the 6 h [3H]thymidine pulse (17).
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F718
POSTNATAL NH4CI +
NH4Cl +
CHANGES NH4Cl
NH4CI
t
80.
IN INTRACELLULAR Amiloride 1 I
REGULATION
i
Fig. 2. A representative experiment in RPTC from a 20day-old rat, illustrating measurements of cellular pH (pHi) regulation. Addition of NH&l (15 mM) to the culture medium induced a rapid cytoplasmic alkalinization, followed by recovery of pHi* Removal of NH&l after 15 min induced a rapid cellular acidification, followed by recovery of pHi. The experiment was repeated and the second recovery took place in presence of amiloride (100 PM), which caused a marked and reversible decrease in the recovery rate.
7s.
T
z a
PH
70.
65. Table 2. Intracellular pH and buffer capacity in rat RPTC as a function of postnatal age Age, days
0, mM/pHi
PHi
unit
8 7.28t0.03 (6) 40.5t2.1 (6) 10 7.31t0.03 (18) 40.6t4.5 (10) 12 7.3320.04 (6) 43.7t3.2 (6) 14 7.35t0.02 (6) 40.9t3.3 (6) 20 7.27t0.02 (13) 44.4k2.5 (7) 40 7.32t0.03 (8) 37.6t3.8 (7) Values are means t SE; no. of experiments is in parentheses. pHi, intracellular pH; ,f3,buffer capacity. Buffer capacity was determined from the alkalinization following NH&l addition and calculated from the formula A[NH,+]i/ApHi (8, 17,24). [NH,‘], was calculated using a pK of 9.21. RPTC, renal proximal tubule cells.
Table 3. Net and amiloride-insensitive recovery rate from intracellular acidosis in rat RPTC as a function of postnatal age Age, days
Net Rate, pH unit/min
AI Rate, pH unit/min
=L= 0. v)
0
0
Fig. 3. Representative experiments in RPTC illustrating the postnatal increase in net rate of recovery after an acid load. Rate of recovery was 0.42 pH units/min in cells from a lo-day-old (left) and 0.70 pH units/ min in cells from a 40-day-old rat (right).
PHmin
8 0.38t0.04* (6) 0.09t0.02 (6) 6.50t0.03 (6) 10 0.39t0.04* (10) O.OBt0.02 (11) 6.53kO.03 (10) 12 0.5420.06 (6) 0.12t0.02 (6) 6.5720.04 (6) 14 0.59t0.04 (6) O.lOt0.02 (6) 6.55t0.02 (6) 20 0.58t0.06 (6) 0.11t0.03 (6) 6.49t0.04 (6) 40 0.14t0.02 (10) 0.60~10.02 (7) 6.57kO.06 (7) Values are means t SE; no. of experiments is in parentheses. AI, amiloride insensitive; pHmin, intracellular acidosis. * P < 0.05 compared with RPTC from 12-40-day-old rats.
significantly between 10 and 12 days of age (0.39 t 0.04 vs. 0.54 t 0.06 pH units/min, P < 0.05, n = 10 vs. 6). From 12 days of age no significant increase was noted. The developmental increase in the recovery rate from intracellular acidosis could be completely accounted for by an increase in the amiloride-sensitive rate of recovery, most likely the Na+-H+ exchanger, which increased from 0.29 t 0.04 to 0.42 t 0.05 pH units/min between 10 and 12 days of age (P < 0.05, n = 6 vs. 7) (Fig. 4). To further evaluate these differences in Na+-H+ exchange between infant and adolescent cells, we investigated the effect of increasing the transmembrane Na+ gradient in RPTC from lo- and 4O-day-old rats. Cells were incubated in NH&l-DMEM as described above. After lo-15 min the medium was replaced by ONa+-
0,5z g 0,4I e 0) 0,3P
*
*
8
10
l
a
5
w-
x ? x w?a I z 0 12 14 20 40 Age (days) Fig. 4. Age dependence of amiloride-sensitive recovery rate (Na+-H+ exchange) from intracellular acidosis in RPTC. Recovery rate increased significantly between 10 and 12 days of age (n = 6-10, P < 0.05).
DMEM (solution 2, Table 1). After 2-3 min the cells were returned to Std-DMEM (solution 1, Table 1). Na+ depletion of the cells before recovery from acidosis resulted in the same relative increase in the rate of recovery in cells
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POSTNATAL
CHANGES
IN INTRACELLULAR
from lo- and 4O-day-old rats (48 and 49%, respectively) (Fig. 5). The amiloride-insensitive rate of recovery was equal in all age groups and mean values ranged from 0.08 to 0.14 pH units/min (Table 3). In a subgroup of the experiments included in Table 3 (5 RPTC-10 and 4 RPTC-40), the amiloride-insensitive rate of recovery was further characterized. In these paired experiments the rate of recovery was determined first in the absence of Na+ (solution 2, Table l), then in the presence of amiloride (100 PM), and finally with both amiloride and SITS (500 PM). In neither age group were any differences found between these three protocols [ 10 day: 0.08 t 0.05,0.05 t 0.01, and 0.04 t 0.01 pH units/min (n = 5), not significant (NS); 40 day: 0.17 t 0.03, 0.18 t 0.02, and 0.17 t 0.03 pH units/ min (n = 4), NS]. For the two latter protocols the rates were significantly lower in RPTC-10 than in RPTC-40 (P < 0.001).
The net capacity of the cells to recover from an intracellular alkaline load was compared in cells from lo- and 40-day-old rats. The recovery was significantly faster in cells from 40-day-old rats (0.16 t 0.02 vs. 0.28 t 0.02 pH units/min, P < 0.01) (Table 4). The maximal pHi from which the recovery started was the same in both age groups (7.82 t 0.09 and 7.81 t 0.06, respectively). Amiloride had no effect on the recovery rate in either age group. In pilot experiments with ado1.escent cells amiloride was corn .bin . .ed with N ‘a+ depletion, result ing in a slight reduction in recovery rate (not sh.own). Thus Na+-H+ exchange does not seem to contribute to the recovery from alkaline load. The SITS-inhibitable component was the same in lo- and 40-day-old RPTC (Table 4; 27 t 8 vs. 26 t 9%, NS). SITS or DIDS at 250 PM had the same effect as SITS at 500 PM. To distinguish the Na+-3HCO; cotransporter from Cl--HCO; exchange we measured the recovery in Cl--depleted cells. Removal of Cl- from the medium resulted in a significant inhibition of recovery in adolescent cells (18 t 1%, N = 7, P < 0.05), whereas in infant cells no significant inhibition was seen 1
F --
E I‘
0,9
W3
PH
F719
REGULATION
Table 4. Recovery from intracellular alkalosis in RPTC from IO-day and 40-day-old rats Age, days
P
10
40
Net rate of recovery, 0.16t0.02 (11) 0.28t0.02 (11) CO.01 pH unit/min Rate with 500 PM, 0.11t0.03 (5) 0.16t0.01 (4) NS SITS pH unit/min Values are means ~frSE; no. of experiments is in parentheses. NS, not significan t.
(-10 t 23%, N = 8, NS). These results suggested that recovery in adolescent cells was to some extent mediated by Cl--HCO, exchange, which was not seen to the same extent in young cells. The low rates of recovery in young cells led to large variations and therefore necessitated another approach to further compare the age groups with respect to Cl--HCO; exchange. Removal of extracellular Cl- resulted in a rapid increase in pHi. The rate of pH change was markedly age dependent (Fig. 6) with the most marked increase after the age of 12 days. The alkalinization was reversib1.e and completely blocked by 250 PM SITS (not shown). DISCUSSION
We show here that a developmental increase in the rate of recovery from an intracellular acid load occurs in the infant rat kidney between 10 and 12 days of age (Table 3) and that it can be completely accounted for by an increase in the capacity of Na+-H+ exchange (Fig. 4). In a previous study of cultured rat RPTC, it was demonstrated that steady-state net Na+ influx increased during early infancy (lo-12 days) (19). The timing of the increase in Na+ influx was exactly the same as for the present increase in the capacity of Na+-H+ exchange. An increased transmembrane Na+ gradient resulted in an identical relative increase in the rate of Na+-H+ exchange in RPTC from lo- and 40-day-old rats (Fig. 5). The acidotic pHi at which the recoveries started were the same in all age groups (Table 3). Previous studies using electron probe analysis have shown that the intracellular
aor7
0’
Na+i-depletion
Normal
Na+i
Fig. 5. Effect of a change in the transmembrane Na+ gradient on the rate of Na+-H+ exchange in RPTC from lo- and 40-day-old rats. Cells with normal Na+ content were acidified with Std-DMEM (solution 1, Table l), whereas Na+-depleted cells were acidified with ONa+-DMEM (solution. 2, Table 1) for 2-3 min. Intracellular Na+ (Na+) depletion caused an identical relative increase in the rate of Na+-H+ exchange in the 2 age groups (48 and 49% for 10 day and 40 day, respectively). Na+-H+ exchange rate in cells from lo-day-old rats was 59% of that in cells from 40-day-old rats both in cells with normal Na+ content and in Na+-depleted cells (** P < 0.01). pHi values from which the initial rates were determined were not different between ages [Na+ depletion: 6.65 t 0.07 vs. 6.67 k 0.04 for 10 vs. 40 day (n = 5 vs. 4), not significant (NS); normal Na+: 6.53 t 0.03 vs. 6.57 t 0.06 for 10 vs. 40 day (n = 10 vs. 7), NS].
.-.. c ? IQ&15-. 2 8 .-i .- I %0,05
O,l-h
'
10
'
20
'
40
'
60
Time in 0 mM Cl-medium
-
300
-
(set)
Fig. 6. Change in pHi in RPTC from lo-day to 40-day-old rats after removal of extracellular Cl- (solution 1 changed to 3, Table 1). After removal of Cl-, pHi increased in all age groups, reaching the same final value within 5 min. However, a developmental increase in the rate of change was noted between 12 and 14 days of age (N = 3-10). The increase in pHi following Cl- removal was completely inhibited by 250 PM SITS (not shown).
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F720
POSTNATAL
CHANGES
IN INTRACELLULAR
Na+ content is equal in serum-deprived RPTC from loand 4O-day-old rats (Na/P = 0.15 t 0.02 and 0.15 t 0.01, respectively) (15, 19; S. Rane, S. H. Larsson, A. Aperia, and C. Lechene, unpublished observations). Thus the difference in recovery rates between RPTC from lo- and 4O-day-old rats cannot be explained by differences in H+ gradients or Na+ gradients over the cell membranes. No developmental change in buffer capacity was seen (Table 2). Furthermore, the low rate of recovery in lo-day-old cells is not a consequence of serum starvation, since these cells cultured 48 h in 10% FBS, if anything, have a lower net rate of recovery (0.30 t 0.06 pH units/min, n = 5, H. Nyctelius and S. H. Larsson, unpublished observations). Consequently, the demonstrated increase in transport rate can only be explained by a developmental change in the number of Na+-H+ exchangers and/or a change in their regulation (19). The rate of recovery after an acid load in RPTC from mature rats (Table 3) is very similar to that previously demonstrated in isolated perfused rabbit S2 segments, using a protocol resembling ours (0.55 pH units/min) (12). Baum (4) studied microperfused juxtamedullary proximal convoluted tubules and measured pHi by fluorescence microscopy. The rate of Na+-H+ exchange was studied in the presence of basolateral SITS and in reverse direction by removing extracellular Na+. The rate of change in pHi was faster than in our study (2.5-3.0 pH units/min). This may be explained entirely by methodological differences, since the rate of pHi change has been documented to be faster if the Na+-H+ exchanger is studied in reverse direction or in the presence of basolateral SITS (21). It may be argued that such a protocol is unphysiological; however, the data are qualitatively very similar to ours, indicating a developmental increase in the capacity of Na+-H+ exchange. The amiloride-insensitive rate of recovery after an acid load appears to be age independent (Table 3) and is similar to that described in the rat proximal convoluted tubule in vivo (21) and in isolated perfused S2 segments from rabbit (12). Because it is SITS insensitive and Na+ independent, it is likely mediated by H+-ATPase (12,25) or passive NH,+ efflux (24). It should be noted that in the subgroup of experiments in which the Na dependence and SITS sensitivity were studied, RPTC-40 showed a significantly faster amiloride-insensitive rate of recovery than RPTC-10. Although no significance was found when all experiments were analyzed together (Table 3), the data may indicate a postnatal increase in H+-ATPase activity. The rate of recovery from an NH,+-induced alkaline load is significantly higher in RPTC from 40-day-old than from lo-day-old rats. The recovery is amiloride insensitive and SITS inhibitable to -25% in both age groups (Table 4). In adolescent, but not in infant cells, the SITS-inhibitable transport is largely Cl- dependent, suggesting a role of Cl--HCO; exchange. An age-dependent increase in the capacity of Cl--HCO; exchange is also supported by the results presented in Fig. 6. The SITS/DIDS-sensitive recovery from intracellular alkalosis is remarkably slow. The principal cytoplasmic acidifier in the proximal tubule, the basolateral Na+-3 HCO; cotransporter (1), does not appear to contribute
PH REGULATION
much to the net recovery. However, it has recently been shown that this transporter is regulated by an intracellular pH-sensitive site and that its activity is completely blocked when the cell interior is made alkaline (pHi above 7.6) (27). Since the recoveries in our studies started at pHi 7.8, the transporter may well have been inactivated. The major pH-regulating membrane transporters thus have a very low capacity to protect the cytoplasm from intracellular alkalosis, and the most important mechanism of recovery in the protocol used in the present experiments may instead be influx of NH,+ (24). We show here that the Na+-H+ exchanger is of crucial importance for recovery from intracellular acidosis in rat RPTC. The transporter is also known to be highly active under steady-state conditions as it mediates up to 87% of Na+ influx (9). In contrast, the Na+-3HCO; cotransporter, which is regarded as the most important regulator of basal pHi in RPTC (1), does not protect the cell from an intracellular alkalosis. During maturation of RPTC the capacities of Na+-H+ and Cl--HCO, exchange increase. The subcellular localization of the participating transporters in cultured RPTC remains to be elucidated. The purpose of the present study was to study cellular net capacity to recover from derangements in pHi. It is noteworthy that the two different transport processes mature at different ages, i.e., the HCO, transport matures -2 days after the Na+-H+ exchanger. The role of Cl--HCO; exchange in proximal tubule pH regulation has been debated (1); our results, however, suggest that this transport may play a role in recovery from alkaline load. A qualitatively similar developmental pattern has previously been demonstrated for the Na+-H+ exchanger and the Na+-3HCO; cotransporter in juxtamedullary proximal tubule cells from rabbit (4) It is unlikely that the stepwise increases in pHi regulatory capacity demonstrated in this study are caused by a change in the ratio of surface area to volume (A/V) (13, 18). Our measurements of pHi were performed on cells in the center of colonies. Single central cells from lo- and 4O-day-old rats cover the same area of the culture support (799 t IO5 vs. 775 t I53 pm’, N = 14 vs.5, NS; cell layer no. 5 from periphery of colonies, S. H. Larsson and J. Luthman, unpublished observations) (Fig. 1). Cellular P content, measured by electron probe analysis, is a good index of cellular mass. The P content is higher in adult than in infant cells, suggesting a difference in cellular height (15). These data suggest that infant rat RPTC, if anything, are smaller than adolescent in primary culture, and consequently A/V is larger in infant than in adolescent cells. Because buffer capacity does not change with age, the described developmental changes in cellular capacity to alter cytoplasmic pH therefore may underestimate developmental changes in the capacity of the cell membrane to transport H+ equivalents. The results of this study indicate that immature RPTC have a lower capacity to recover from derangements in pHi than do mature RPTC. The results support the concept that RPTC H+ and HCO, transport pathways undergo postnatal maturation (4, 26). A maintenance of constant pHi is crucial for normal cellular function. The
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POSTNATAL
CHANGES
IN INTRACELLULAR
low capacity of young cells to recover from intracellular acidosis might have important clinical implications, since dramatic changes in pH homeostasis are particularly common in sick neonates. We wish to thank Anna Johansson, Ingrid Muldin, and Eivor Zettergren for expert technical assistance. This work was supported by grants from the Swedish Medical Research Council, The State Medical Board of Finland, The Emil Aaltonen Foundation, Finland, and the Royal Swedish Academy of Sciences. Address for reprint requests: S. H. Larsson, Department of Pediatrics, St. Goran’s Children’s Hospital, Box 12500, S-112 81 Stockholm, Sweden. Received 16 April 1991; accepted in final form 26 May 1992.
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