Vol. 274, Issue 3, F550-F555, March 1998
Development of pH regulatory transport in glomerular
mesangial cells
Michael B.
Ganz and
Brett A.
Saksa
Department of Medicine, Section of Nephrology, Case Western Reserve
University, and Department of Veterans Affairs Medical Center,
Cleveland, Ohio 44106
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ABSTRACT |
Developmental changes in activity or expression of transporters
may account for alterations in cell behavior as the
nonpolarized cell matures. We sought to ascertain whether there is a
maturational change in each of the major acid-base transporters in the
developing mesangial cell (MC), the Na/H exchanger, Na-dependent
Cl/HCO3 exchanger, and the
Cl/HCO3 exchanger. Intracellular
pH (pHi) was determined by the
use of the fluorescent pH-sensitive dye,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).
We assessed transporter activity by studying recovery from an acid load
(NH4/NH3)
in
CO2/HCO3.
In adult MCs, Na/H exchanger was responsible for 35.2 ± 4.3% of
steady-state pHi, whereas the Na-dependent Cl/HCO3 exchanger
contributed 58.7 ± 6.1 (n = 14). In term MCs (tMCs), from days
1-3 after birth, the Na/H exchanger contributes
62.9 ± 7.8% (n = 11, P < 0.001 vs. adult), whereas the
Na-dependent Cl/HCO3 exchanger
contributes 34.0 ± 5.7% (n = 12, P < 0.001 vs. adult), to the rate of
recovery from an acid load in these cells. However, in tMCs
(days 4-6), the Na/H
contributes 47.2 ± 5.9% (n = 8, P < 0.05 vs. adult),
whereas the Na-dependent Cl/HCO3
exchanger contributes 48.7 ± 7.3%
(n = 13, P < 0.05 vs. adult), to the rate of
recovery. tMCs (days 6-12)
yielded transporter activity that was not statistically different than
adult MCs (37.8 ± 4.9 and 54.3 ± 10.2% for Na/H and
Na-dependent Cl/HCO3,
respectively). The magnitude of the stimulated response to angiotensin
II by Na/H and Na-dependent
Cl/HCO3 exchanger in adult and
tMCs is unchanged throughout development. The Na/H exchanger appears to
play a greater role in pHi
homeostasis earlier on in development, and this may reflect
developmental needs of the maturing cell.
angiotensin II; development; sodium/proton exchanger; bicarbonate
transport
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INTRODUCTION |
CONTINUED DEVELOPMENT of the kidney in the pre- and
postnatal period, in most animals, does not involve the formation of
new nephrons but involves, instead, cell proliferation within the existing framework and functional cell maturation (12, 20, 22, 23,
37). The maturation processes are important for functionally active nephric units and appear to involve many
fundamental changes, of which one is the alteration of integral
membrane proteins that function as channels, carriers, or pumps (1,
12). There is growing evidence that the abundance, function, and
sensitivity to circulating hormones of these proteins change during
ontogeny and that these changes may relate to specific needs of the
maturing cell. It is also quite likely that short-term regulation of
membrane proteins such as ion transport by activated membrane receptors and intracellular signal systems such as protein kinase C also undergo
important maturational changes during terminal differentiation. Therefore, the sequential development of ion transporters and hence an
alteration in the homeostatic control of intracellular pH
(pHi) may be critical for
allowing developing cells to alter their biological
behavior.
Unlike epithelia, the development of transport in nonpolarized cells
may reflect a change in the signaling in these cells, such as mesangial
cells (MC), that accounts for alterations in growth and
biosynthesis as the organ undergoes morphogenesis. If this is the case,
then the ontogenic development of ion transport may have important
consequences for the eventual maturation of the cells in the
glomerulus. We therefore sought to ascertain whether there is a
maturational change in each of the major acid-base transporters that
regulate pHi in glomerular MCs,
i.e., the Na/H exchanger, Na-dependent
Cl/HCO3 exchanger, and the
Cl/HCO3 exchanger and in the
response to angiotensin.
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METHODS |
Isolation and culture of MCs. MCs were
obtained from the isolated glomeruli of young adult male Sprague-Dawley
rat kidneys according to previously described protocols (13). Glomeruli were washed twice with Hanks' balanced salt solution and were plated
onto 75-cm2 tissue culture flasks
in culture media containing DMEM, 5 µg/ml insulin, 5 µg/ml
transferrin, 5 ng/ml selenous acid, 25 mM glucose, 400 ng/ml
penicillin, 500 ng/ml streptomycin, and 25 mM
HCO3, with 20% FBS. Routine
identification of MCs was performed by indirect immunofluorescence
microscopy using rabbit IgG directed against vascular smooth muscle
myosin and mouse anti-rabbit FITC-conjugated IgG. Cells showed
uniformly strong positive staining of longitudinal filaments, a pattern
that is characteristic of MCs (15). In addition, MCs were stained
uniformly with anti-Thy 1.1, which has also been considered to be
indicative of rat MCs (24, 25).
The functionally immature rat kidney at term, prior to weaning (up to
15 days), allows us to study maturational processes that parallel human
fetal and term kidney development (10, 21). Our procedure to isolate
term MCs (tMCs, 1-12 days after birth) has been similar to that of
isolating adult MCs from kidneys of neonatal Sprague-Dawley rats as
previously reported (34). tMC populations where characterized in
identical fashion to adult MCs and where grown in the same media.
For pHi experiments, third to
eighth passages, adult MCs were grown on glass coverslips (9 × 50 mm) in the culture media containing 10% FBS. MCs outgrowths were
suitable for experiments in 7-9 days after plating, when they were
then 70-90% confluent. For tMC experiments, primary cells were
from term days 1-3,
4-6, and
7-12 days subcultured on glass
coverslips, but, unlike in adult MCs, they were not passaged. Twenty-four hours prior to all pHi
studies, the medium was changed from 10% FBS to 0.5% FBS to halt cell
growth.
Determination of
pHi.
pHi was determined by the use of
the fluorescent pH-sensitive dye,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Both adult and tMCs on coverslips were loaded with BCECF that had been
initially dissolved in DMSO to a concentration of 5 mM and then diluted
to a final concentration of 5 µM in our standard saline solution.
Fluorescent measurements were made with a Perkin-Elmer LS-5B
spectrofluorometer (Norwalk, CT), with the coverslip mounted in a
temperature-controlled flow-through cuvette at an angle of 60° to
the incident beam, as we have previously described (13). Intracellular
dye was alternately excited at wavelengths of 500 and 440 nm (3-nm
bandwidth), and the emission was monitored at 530 nm (5-nm bandwidth).
We continuously monitored the emission while exciting at 500 nm and
periodically (every 2 min) obtained measurements at 440-nm excitation.
The excitation ratio (500/440) was calculated from these measurements.
The problem of dye leakage was minimized by continuously perfusing the
cuvette. The nigericin/high-K technique described by Thomas et al. (16)
was used to clamp pHi to
predetermined values and thereby obtain an intracellular calibration of
the excitation ratio. We used extracellular pH values from
6.20 to 7.80 to calibrate pHi by
generating a calibration curve as reported previously (14).
Rate of recovery and
HCO3 transport statistics.
Potential mechanisms regulating acid extrusion can be studied as
pHi returns to near basal level
after an acid load in the presence of
HCO3 (Fig.
1A).
The application and subsequent removal of 20 mM
NH3/NH4
causes pHi to decrease rapidly.
The pHi recovers from this acid
load because of the activity of the ethylisopropylamiloride (EIPA)-sensitive Na/H exchanger and the stilbene derivative,
SITS-sensitive Na-dependent
Cl/HCO3 exchanger. To determine
the proportion of the change in acid extrusion
(Jext) that is
due to the Na/H exchanger, we acid loaded MCs that have been
preincubated with SITS (5, 6). Since SITS fluoresces, we
preincubated MCs with SITS (1 h) and then washed them; SITS
irreversibly blocks all HCO3
transport, and therefore Na/H exchange is the only means by which MCs
can recover from an acid load. To calculate what percentage of recovery is the result of Na/H and/or Na-dependent
Cl/HCO3 exchanger, one then
measures the mean rate of pHi
recovery, from one pHi point to
another pHi (6.8 to 6.9).
Recovery, therefore, is a change of
pHi over time
(dpHi/dt).
We present
dpHi/dt
as 10
4 pH units/s as
reported by others (14).
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Fig. 1.
Representative tracings of recovery from an acid load of 20 mM
NH4/NH3
for 5 min of term mesangial cells (tMC), day
7 in bicarbonate. A:
control, with starting intracellular pH
(pHi) ~7.30.
B: MCs preexposed to SITS.
Starting pHi is ~6.90. Recovery
was then assessed at pHi 6.80. C: MCs exposed (arrow) to
ethylisopropylamiloride (EIPA) (50 µM). Resultant
pHi is ~7.25. Recovery was then
assessed at pHi 6.80.
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To assess HCO3 transport, we
calculated fluxes of HCO3 as
induced by Cl removal then readdition. Fluxes were calculated as the
product of
dpHi/dt
and 
total (where
total is total buffer capacity and where
total = HCO3 + non-HCO3). Values of
HCO3 at
pHi 7.5 were 72.5 mM, as we have
reported previously (14). The units of flux for
Cl/HCO3 exchange as presented in
Table 1 were micromolar per second,
as we have used in the past (14). Data were judged by
ANOVA by the Bonferroni method in Tables 1 and 2.
Solutions. The standard
HCO3 solution contained (in
mM) 145 Na+, 5 K+, 1 Mg2+, 1.8 Ca2+, 122 Cl, 25 
, 1.0 
, 1.0 
, and 10 glucose and was
buffered to pH 7.40 with
CO2/HCO3.
The nigericin solution contained (in mM) 105 K+, 105 Cl, 0 Na+, 1 MgCl2, 30 of buffer (either HEPES
for pH 6.8-7.4, PIPES for pH 6.0-6.8, or MOPS for pH
7.4-8.4) to achieve the desired extracellular pH, 40 N-methyl-D-glucamine (NMDG) and 10 µM of
nigericin. Solutions containing
NH4 were
prepared by replacing 20 mM NaCl with 20 mM NH4/NH3.
Angiotensin II was dissolved in a saline solution on the day of the
experiment to the appropriate concentration. EIPA was dissolved in the
standard buffer solution with 145 mM of Na.
Materials. BCECF was obtained from
Molecular Probes (Eugene, OR). DMEM, FBS, penicillin, streptomycin, and
PBS solution were purchased from Life Technologies ( Grand Island,
NY). Fibroblast growth factor, insulin, transferrin, and
selenium were obtained from Collaborative Research (Bedford, MA).
Anti-Thy 1.1 was purchased from Chemicon (El Segundo, CA). Horseradish
peroxidase-conjugated anti-rabbit antibodies, nigericin, NMDG, plastic
cuvettes, and other laboratory chemicals were purchased from Sigma (St.
Louis, MO). Tissue culture flasks and petri dishes were obtained from Falcon (Lincoln Park, NJ).
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RESULTS |
Acid extruders. We examined the
ability of MCs to recovery from an acute intracellular acid load
imposed by pulsing a solution containing 20 mM
NH4/NH3
as previously described (13, 14). When MCs are studied in the presence
of HCO3, both the Na/H exchange and the Na-dependent Cl/HCO3
exchange contribute to the recovery of
pHi from acid load (5, 6). By
comparing the rates of recovery from an acid load as the cell raises
its pHi from MCs pretreated with
SITS, we determined whether there is a change in Na/H exchanger maximal
activity altering net acid extrusion (Fig.
1B). To ascertain whether maximal
activity of the Na-dependent Cl/HCO3 exchanger is altered, we
acid loaded MCs preincubated with EIPA; therefore, the Na-dependent
Cl/HCO3 exchanger is
the only means by which MCs can recover from an acid load
(Fig. 1C). The data represent
percent recovery at pHi 6.80 for
Na/H and Na-dependent Cl/HCO3
exchange.
As shown in Table 1, by executing these experiments, we were able
to ascertain that the Na-dependent
Cl/HCO3 exchanger is responsible
for approximately two-thirds of recovery from an acid load and that the
Na/H exchange is responsible for ~30% in adult MCs, as has been
reported previously (14). The remaining 5-8% has been attributed
to other proton transporters (i.e., H-K-ATPase) not yet defined in
cultured MCs. However, in tMCs, the rate of recovery is initially more
dependent on Na/H exchange activity (Table 1). There is a steady
increase in the percentage of recovery from an acid load that is
dependent on the Na-dependent
Cl/HCO3 exchanger until after
day 6. Term MCs assume the same
transport activity as adult MCs with the expected decrease in
Na/H exchange activity after day 6.
To ascertain whether there is a difference in acid-loading activity, we
examined the Cl/HCO3 exchanger.
This exchanger normally operates with Cl being pumped into the cell and
HCO3 exiting the cell, which
results in a net acidification as the cell is losing HCO3. Removal of the
outside Cl forms a concentration gradient (whereupon Cl inside is now
greater than the bathing solution), thereby reversing the direction of
the exchanger. With zero Cl in the bathing solution, the
Cl/HCO3 exchanger now pumps
HCO3 in and Cl out. In these
experiments, Cl was replaced by equal molar amounts of aspartate (6,
29). To determine whether the exchanger activity is different in
development, we examined the rate of recovery in the presence of
HCO3 (at the point wherein Cl
readded) from this protocol in adult MC and tMCs
(days 1-3, 4-6 and
7-12; Fig.
2). Since this is the only acid extruder
known in MCs, net efflux of HCO3
(Jefflux) is
used to ascertain Cl/HCO3 activity. Jefflux
of HCO3 for all MCs studied is
measured at pHi 7.50 (14). As
shown in Table 1, there is no difference in recovery rate in tMCs vs.
adult.
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Fig. 2.
Representative tracing of effect of a rapid removal of Cl in tMCs,
day 7. Upon return of Cl, one is then
able to assess the activity of
Cl/HCO3 exchange at
pHi 7.50.
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Agonist stimulation of transporters.
The rate of pHi increase over time
(dpHi/dt)
allows us to ascertain whether the exchanger is stimulated or not. To
ascertain whether maximal transport activity is altered throughout
development, we studied the effect of agonist stimulation on
transporter activity. Term and adult MCs were acid-loaded, and the
effect of ANG II on recovery was assayed. As above, we compared the
rates of recovery
(dpHi/dt)
of MCs pretreated with SITS with and without ANG II (1 µM); the
effect on Na/H exchanger maximal activity therefore was ascertained
(Fig.
3A). To
ascertain whether ANG II altered maximal activity of the Na-dependent
Cl/HCO3 exchanger during
development, we acid loaded MCs preincubated with EIPA with and without
ANG II (Fig. 3B).
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Fig. 3.
Representative tracings of the effect of ANG II on recovery from an
acid load in tMCs, day 7.
A: MCs were preexposed to SITS, and
then recovery is assessed in absence ( ) and presence of ANG II
( ). Rate of recovery is assessed at
pHi 6.70. B: MCs were exposed (arrow) to EIPA
(50 µM), and then recovery was assessed in absence () and presence
of ANG II ().
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As shown in Table 2, ANG II (1 µM)
maximally stimulates the Na/H exchanger in adults (1.97-fold increase),
and this was unchanged throughout development. Moreover,
the effect of ANG II on Na-dependent Cl/HCO3 exchanger (1.95-fold
increase) was also unaffected throughout all stages of MC development.
In addition, using the identical protocol outlined above for assaying
the Cl/HCO3 exchanger, we determined that ANG II effect on maximal return to basal pH was also
unchanged throughout development (Fig. 4;
Table 2).
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Fig. 4.
Representative tracing assessing the effect of application of ANG II on
effect of activity of Cl/HCO3 as
Cl is readded in tMCs, day
7; , control; , addition of ANG II
immediately prior to return of Cl. Activity is assessed at
pHi 7.50.
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DISCUSSION |
The ontogeny of several membrane transporting proteins has been only
recently studied in renal tubular epithelia but not in nonpolarized
cells (17). The main function of tubular epithelium is transport. An
increase in the size of transporting membranes and an increase in the
integral membrane proteins that function as transporters or channels
(7) have characterized late differentiation. It has been proposed that
developmental changes in ion transport in "nonpolarized" cells
may reflect differences in signal transduction properties for the
transporters as the cell grows rather than transport needs. However,
the development of pH regulatory transport in nonpolarized cells has
not been fully examined. It appears that
HCO3 transport maturation follows
Na/H maturation, whereas the maximal activity of these transporters is
unaffected.
These results are supported, in part, by recent studies on
HCO3 transport in the rabbit. In
the fetal rabbit, electroneutral HCO3 transport, which appears to
be linked to the maturation of the Na-K-ATPase and Na/H exchanger, is
30% of the transport rate of adult kidney (2, 26). In addition, the
maturation of the Na-HCO3
cotransporter is dependent on the enzyme, carbonic anhydrase (8, 23).
However, in rats, it appears that carbonic anhydrase increases
postnatally, whereas immunohistochemical and biochemical studies in
humans suggest that the enzyme is so abundant early in
fetal development that it appears unlikely to be a rate-limiting step
in transport development.
The Na/H exchange activity has been demonstrated to be present in fetal
tissue and has been found to have enhanced activity as the rabbit
kidney matures; activity also increases in the first 2 wk of life until
it reached the adult rate (~6 wk) (10, 40). Moreover, recent work has
shown that activity of a fetal Na/H transporter in tubular epithelium
is 25% that of adult, and the administration of glucocorticoid
significantly enhanced the
Vmax of the
exchanger, whereas the
Km was unaffected
(3, 21, 22). This was confirmed by demonstrating an age-related
increase in ileal brush border Na/H (presumed
Na-HE2 isotype) exchange activity. However, precise analysis of transport in cortical vesicles (7-day-old rats) revealed that Na/H exchange activity was increased compared with
adult kidney. Furthermore, a decrease in the
Vmax of Na/H exchange in basolateral membrane vesicles was found when the comparison was made to adult (10, 11). Species and perhaps tissue exchanger (isotypes) specificity differences may account for the discrepancy in
the results. Additional investigation into cell development should
further our understanding of transport maturation.
The ontogeny of sodium transport has been studied in greater detail. It
has been demonstrated that the relative abundance of each isoform of
the Na-K-ATPase has been shown to change throughout ontogeny (18, 30,
36). The mRNA for 
1- and
1-subunits of the Na-K-ATPase
increases in the rat renal cortex throughout fetal life and remains
stable for the first 2 wk postnatally, only to increase again during
weaning. Moreover, maximal activity of the ATPase has been shown to be
only 10-50% of the adult rat and rabbit kidney activity levels.
The distribution, however, of the Na-K-ATPase along the nephron was
found to be virtually identical in both neonatal and adult rabbit
kidneys.
The factors influencing the developmental increase in Na-K-ATPase
activity are less clear (4, 38). Although Larsson and co-workers
(20-22) have suggested that there is an increase of Na entry into the proximal tubule cells that precedes by 4 days an
increase in maximal velocity of the Na-K-ATPase, others have demonstrated that developmental changes in Na-K-ATPase activity are
preceded by an increase in HCO3
transport (26, 27, 33). Manipulations in which Na/H exchange activity
was inhibited demonstrated that the development of the Na-K-ATPase was
greatly retarded. These results suggest that the maturational increase
in Na-K-ATPase velocity follows passive Na entry and is in part
mediated via the activity of the Na/H exchanger in rabbit kidney. In
addition to Na availability, maturation of the Na-K-ATPase also appears to be dependent on the increases in serum levels of glucocorticoid seen
throughout development and weaning: adrenalectomy abolishes maturation,
whereas treatment with glucocorticoid restores Na-K-ATPase activity (9,
36). Although these changes appear to be correlated with developmental
changes that occur during weaning, they may relate to the expression
and sensitivity of the glucocorticoid receptor rather than changes in
hormone level (19).
Although its role in hypertension (renin-angiotensin-aldosterone) has
been well defined, the role of ANG II in development of the kidney has
only recently been appreciated (31, 35). It has been demonstrated,
through binding studies, that AT2
ANG II receptors are prominent in developing kidneys, especially in the
mesenchymal and interstitial tissue but not the epithelial tissue (28,
40). However, in development, both types (1 and 2) of receptors are
present and are in higher levels in the developing fetus, with
AT1 receptors in the developing
mesonephros. Autoradiographic analysis has revealed the highest density
in glomeruli found within the cortex in both neonatal and adult kidney
(32). Moreover, fetal kidneys express the angiotensinogen gene early in
gestation, and the protein product can be identified in the proximal
tubules. These results have lead to the proposal that ANG II may play a critical role during the S-shape developmental phase of the glomerulus and hence in the functional maturation of the glomerular mesangium (39). We and others have shown that ANG II stimulates enhanced Na
influx via Na/H exchange and or Na/Ca exchangers in addition to the
Na-Cl/HCO3 exchanger in both adult
and tMCs (40). It has been proposed that ANG II may play a role in
mesangial cell proliferation during development. This however, is
speculation in light of the fact that in mice with a knockout for the
AT2 receptor gene, no structural
abnormalities have been defined. Therefore, the precise
signaling role of ANG II in development remains ill defined.
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ACKNOWLEDGEMENTS |
M. B. Ganz was supported by American Heart Established
Investigatorship 9600485 and by a Veterans Affairs Merit Review.
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FOOTNOTES |
Address for reprint requests: M. B. Ganz 111K(W), Section of
Nephrology, Cleveland VA Medical Center, 10701 East Blvd., Cleveland,
OH 44106.
Received 1 October 1997; accepted in final form 1 December 1997.
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AJP Renal Physiol 274(3):F550-F555
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Abstract
Introduction
