High glucose induces the activity and expression of
Na+/H+
exchange in glomerular mesangial cells
Michael B.
Ganz1,
Karen
Hawkins1, and
Robert F.
Reilly2
1 Section of Nephrology,
Department of Medicine, Case Western Reserve University, Veteran
Affairs Medical Center, Cleveland, Ohio 44106; and
2 Section of Nephrology,
Department of Medicine, University of Colorado, Veteran Affairs
Medical Center, Denver, Colorado 80262
 |
ABSTRACT |
.
Changes in activity or expression of
transporters may account for alterations in cell behavior in diabetes.
We sought to ascertain if mesangial cells (MC) grown in different
glucose concentrations exhibit changes in activity and expression of
acid-extruding transporters, the
Na+/H+
and Na+-dependent
Cl
/HCO
3
exchanger. pHi was determined by
the use of the fluorescent pH-sensitive dye BCECF. In MCs grown in 5 mM glucose (control), the
Na+/H+
exchanger was responsible for 31.8 ± 5.1% of steady-state
pHi, whereas
Na+-dependent
Cl
/HCO
3
contributed 62.9 ± 4.0% (n = 11).
In MCs grown in high glucose for 2 wk,
Na+/H+
exchange contribution to acid-extrusion increased as follows: 42.3 ± 4.6% [n = 8, 10 mM, not
significant (NS)], 51.1 ± 5.1% (n = 8, 20 mM,
P < 0.01), and 64.8 ± 5.5%
(n = 7, 30 mM,
P < 0.001). The
Na+-dependent
Cl
/HCO
3
exchanger contributed less [47.0 ± 4.6, 38.6 ± 5.8, and
21.1 ± 3.8%, for 10, 20, and 30 mM glucose, respectively (n > 7)]. We sought to
ascertain if the magnitude of the acute stimulated response to ANG II
by the
Na+/H+
and Na+-dependent
Cl
/HCO
3
exchanger is changed.
Na+/H+
exchanger (1.89-fold increase in 30 vs. 5 mM,
P < 0.002), but not
Na+-dependent
Cl
/HCO
3
exchange (0.17-fold, NS), exhibited an enhanced response to ANG II (1 µM).
Na+/H+
exchange (NHE1) expression was significantly different (1.72-fold) after prolonged exposure to high glucose. These results suggest that
the
Na+/H+
exchanger, but not Na+-dependent
Cl
/HCO
3
exchanger, may play an early role in the response to hyperglycemia in
the diabetic state.
diabetes; kidney; bicarbonate transport
 |
INTRODUCTION |
IN HUMAN AND EXPERIMENTAL diabetes mellitus, the renal
glomerular lesion is characterized by glomerular hypertrophy followed by the deposition of extracellular matrix (1). This progressive encroachment on capillaries results in the loss of filtration surface
area and is presumed to be responsible for the progressive decline in
glomerular function in diabetic patients (9, 28). There is growing
evidence that the accumulation of matrix in diabetes is related to
changes in mesangial cell (MC) phenotypic behavior (2, 17, 39). These
cells synthesize and release laminin, thrombospondin, collagens
(primarily types I and IV), and proteoglycans (1, 43, 45). Therefore,
changes in MC metabolic properties are responsible, in part, for
glomerular expansion and hence eventual glomerulosclerosis.
How and why MCs have their biological behavior changed in diabetes
still remains unclear. Recent studies in both animals and humans have
demonstrated that strict metabolic (glucose) control appears to delay
the onset of glomerular disease in the diabetic patient and slows
the development of the characteristic glomerular lesion (6,
8, 10). In vitro work has demonstrated that MCs incubated in a
high-glucose milieu have an increase in matrix synthesis,
alteration in protein kinase C (PKC) activity and
expression, and an increase in auto-coid production (1, 5,
17). Moreover, others have found that changes in receptor density and
changes in intracellular Ca2+
release and glucose transport (GLUT-1) activity are evident in the
diabetic model (3, 36).
The role, however, of ion transporters remains ill defined. It has been
readily demonstrated that hyperactivity of the
Na+/H+
exchanger has been implicated in the vascular injury associated with
diabetes (3, 14, 40, 41, 46). Changes in exchanger activity have been
reported late in diabetic disease (rabbit model). Chronic
hyperosmolarity in proximal tubule cells was responsible for an
increase in NHE3 exchanger expression (3). Moreover, in vascular smooth
muscle cells, an increase in PKC mass was associated with an increase
in
Na+/H+
exchange activity in the diabetic and hypertensive patient (7, 16, 18,
24, 31, 33) that may or may not be related to ANG II. Therefore, we
sought to ascertain if there is a change in activity and expression of
the
Na+/H+
and Na+-dependent
Cl
/HCO
3
exchanger in different glucose concentrations and in their response to angiotensin.
 |
METHODS |
Isolation and culture of MCs. MCs were
obtained from the isolated glomeruli of young adult male Sprague-Dawley
rat kidneys according to a previously described protocol (19, 20).
Glomeruli were washed two times with Hanks' balanced salt solution and
were plated on 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
HCO
3 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 (21). In addition, MCs were stained with
anti-Thy 1.1, which has also been considered to be indicative of rat
MCs (21, 35).
For pHi experiments, third- to
eighth-passage adult MCs were grown on glass coverslips (9 × 50 mm) in the culture media containing 10% FBS. MC outgrowths were
suitable for experiments 7 days after plating as they were then
70-90% confluent. The cells were then placed in the respective
glucose concentration for 7 and/or 14 days. Twenty-four hours before
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 BCECF. MCs 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 (20). 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
nigericin/high-K+ technique was
used to clamp pHi to predetermined
values and thereby obtain an intracellular calibration of the
excitation ratio. We used pHo
values from 6.20 to 7.80 to calibrate
pHi by generating a calibration
curve as reported previously (20, 22).
Rate of recovery and
HCO
3 transport
statistics. Potential mechanisms regulating acid
extrusion can be studied, as pHi
returns to near-basal levels after an acid load in the presence of
HCO
3 as we have previously described
(12). The pHi recovers from this acid load because of the activity of the ethylisopropyl amiloride (EIPA)-sensitive
Na+/H+
exchanger and the stilbene derivative SITS-sensitive
Na+-dependent
Cl
/HCO
3
exchanger. To determine the proportion of the change in acid extrusion
that is due to the
Na+/H+
exchanger, we acid loaded MCs that were preincubated with SITS (13).
Because SITS fluoresces, we preincubated MCs with SITS (1 h)
and then washed them. SITS irreversibly blocks all
HCO
3 transport; therefore,
Na+/H+
exchange is the only means by which MCs can recover from an acid load
(20). To calculate what percentage of recovery is the result of
Na+/H+
and/or Na+-dependent
Cl
/HCO
3
exchange, one measures the mean rate of pHi recovery starting at one
pHi point to another
pHi (6.8-6.9). Recovery,
therefore, is a change of pHi over
time
(dpHi/dt).
Expression of the
Na+/H+
exchanger.
Northern Blot experiments were performed. In brief, MCs were lysed
using guanidium thiocyanate and were purified by ultracentrifugation through cesium chloride. For Northern analysis, RNA was fractionated on
a 1.2% denaturing agarose gel and transferred to a nylon membrane. Labeling of the cDNA probe of NHE1 was accomplished by using a random
prime labeling kit. Hybridization was carried out in 2× sodium
chloride-sodium phosphate-EDTA, 0.1% SDS, and 5× Denhardt's solution. The blots were developed using alkaline phosphatase conjugate
and lumigen phosphodiesterase chemiluminescent substrate. Each
individual blot was hybridized with a radiolabeled probe to p-RGAPDH-1
mRNA. Hybridization signals were quantified by scan analysis and were
normalized for changes in p-RGAPDH-1 mRNA expression, and the results
of the experiments were averaged and tested for statistical significance.
Solutions. The standard
HCO
3 solution contained (in mM) 145 Na+, 5 K+, 1 Mg2+, 1.8 Ca2+, 122 Cl
, 25 HCO
3, 1.0 SO2
3, 1.0 PO3
4, and 5 glucose and was
buffered to a pH of 7.40 with
CO2/HCO
3. To maintain the same tonicity, we reduced the
Na+ by reducing NaCl by equal
amounts (mM) when increasing glucose. The nigericin solution contained
(in mM) 105 K+, 105 Cl
, 0 Na+, 1 MgCl2, 30 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 pHo, 40 N-methyl-D-glutamine
(NMDG), and 10 µM nigericin. NH+4 solutions
were prepared by replacing 20 mM NaCl with 20 mM
NH+4/NH3. ANG 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 (145 mM
Na+). Experiments performed
using mannitol were executed in identical fashion to those for glucose.
Materials. BCECF was obtained from
Molecular Probes (Eugene, OR). DMEM, FBS, penicillin, streptomycin, and
PBS were purchased from GIBCO Laboratories (Grand Island, NY).
Fibroblast growth factor, insulin, 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).
Statistics. Data are reported as means ± SE in Tables 1 and
2. Statistical significance was judged by
the unpaired Student's t-test. In
experiments wherein glucose or mannitol concentrations were used
(Tables 1 and 2), the data were compared with control (normal glucose).
 |
RESULTS |
Acid extruders. We examined the
ability of MCs to recover from an acute intracellular acid load imposed
by pulsing with a solution containing 20 mM
NH+4/NH3.
By comparing the rates of recovery from an acid load in the presence of
SITS, we determined if there is a change in
Na+/H+
exchanger maximal activity that would alter net acid extrusion. To
determine whether maximal activity of the
Na+-dependent
Cl
/HCO
3
exchanger exchange is altered, we acid load MCs preincubated with EIPA.
The data represent percent recovery at
pHi of 6.80 for
Na+/H+
and Na+-dependent
Cl
/HCO
3 exchange.
As shown in Fig. 1,
Na+/H+
exchange increases in activity in increasing concentrations of glucose.
After 2 wk in high-glucose media, the
Na+/H+
exchange activity increased by 33.0% (10 mM), 60.7% (20 mM), and
103.7% (30 mM). The increase in activity occurs after 7 days (data not
shown) and is maximal by 14 days. However, the percentage of the
activity of the Na+-dependent
Cl
/HCO
3
transporter that is responsible for acid extrusion is reduced by 22.1%
[10 mM, not significant (NS)], 38.5% (20 mM), and 65.1%
(30 mM, P < 0.01; Fig.
2). Interestingly, the decrease of the
Na+-dependent
Cl
/HCO
3
exchanger contribution is not equal to the increase in
Na+/H+
exchange activity. There is an increase from 6.1% to almost 15.1% of other acid extruders (i.e.,
H+-ATPase) in the regulation
of acid extrusion. These results are summarized in Table 1.

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Fig. 1.
Representative tracings of recovery from an acid load of 20 mM
NH+4/NH3
and exposure to SITS for 1 h in
CO2/HCO 3
examining
Na+/H+
exchange activity. , Control (5 mM glucose); x, recovery of MCs in
10 mM glucose; , recovery of MCs in 20 mM glucose; , recovery of
MCs in 30 mM glucose.
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Fig. 2.
Representative tracings of recovery from an acid load of 20 mM
NH+4/NH3
and exposure to ethylisopropyl amiloride for 5 min in
HCO 3 examining
Na+-dependent
Cl /HCO 3.
, Control (5 mM glucose); x, recovery of MCs in 10 mM glucose; ,
recovery of MCs in 20 mM glucose; , recovery of MCs in 30 mM
glucose.
|
|
To ascertain whether the effect was the result of the change of
Na+ or a specific effect of
glucose, we substituted mannitol for glucose and performed identical
experiments to those described above. After 2 wk in media with
mannitol, the
Na+/H+
exchange was unchanged. The increase in activity was 5.1% (10 mM, NS),
4.1% (20 mM, NS) and 3.7% (30 mM, NS;
n = 3 for each concentration of
mannitol). The percentage of the activity of the
Na+-dependent
Cl
/HCO
3
transporter that is responsible for acid extrusion was also unchanged
[decrease by 3.2% (10 mM, NS), 2.9% (20 mM, NS), and 3.1% (30 mM, NS); n = 3 for each concentration of mannitol]. These experiments suggest that the effect is due to
the exposure to glucose rather than reduction of
Na+.
Agonist stimulation of transporters.
The rate of pHi increase over time
(dpHi/dt)
allows us to ascertain whether or not the exchanger is stimulated. To
ascertain if maximal transport activity is altered in high-glucose
media, we studied the effect of agonist stimulation on transporter
activity. MCs were acid loaded, and the effect of ANG II on
recovery was assayed. As above, we compared the rates of recovery
(dpHi/dt)
in MCs pretreated with SITS with and without ANG II (1 µM); the
effect on
Na+/H+
exchanger maximal activity therefore was ascertained in varying glucose
concentrations. Although ANG II enhanced
Na+/H+
activity (Fig. 3) there
was no further stimulatory effect on the
Na+-dependent
Cl
/HCO
3
exchanger in any glucose concentration (Fig.
4). These results are also summarized in
Table 2.

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Fig. 3.
Representative tracings of effect of ANG II on recovery from an acid
load of 20 mM
NH+4/NH3
and exposure to SITS for 5 min in
CO2/HCO 3
examining
Na+/H+
exchange activity. Arrow represents addition of 1 µM ANG II. ,
Control without ANG II; x, recovery of MCs in 5 mM glucose; *, recovery
of MCs in 10 mM glucose; , recovery of MCs in 20 mM glucose; ,
recovery of MCs in 30 mM glucose.
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Fig. 4.
Representative tracing assessing the effect of application of ANG II on
the activity of the Na+-dependent
Cl /HCO 3
exchanger. , Control without ANG II; x, recovery of MCs in 5 mM
glucose; *, recovery of MCs in 10 mM glucose; , recovery of MCs in
20 mM glucose; , recovery of MCs in 30 mM glucose.
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|
Finally, to ascertain if there is a change in NHE1 expression, as that
is the only
Na+/H+
exchanger expressed in these nonpolarized cells, we performed Northern
blot analysis under different glucose concentrations. As shown in Fig.
5, expression of NHE1 increased after the
cells were placed in a high-glucose media (30 mM) for 2 wk. This
increase in activity was not evident at 10 and 20 mM but was
statistically significant for 30 mM glucose
(n = 3 for each glucose
concentrations). These data demonstrate that, under high-glucose
conditions, activity of the
Na+/H+
exchanger is enhanced, and expression is increased.

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Fig. 5.
Representative Northern blot examining NHE1 expression in mesangial
cells in 5 and 30 mM glucose. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
|
|
 |
DISCUSSION |
There is a growing body of evidence that suggests that
hyperglycemic-induced glomerular injury modulates MC biological
behavior, and this is reflected in early changes in subcellular events, including membrane transport (3, 27, 29, 30). We have demonstrated that
MCs subcultured over time in a culture media that represents the
diabetic milieu (high glucose) enhance the activity of
Na+/H+
exchange and stimulate its expression. This effect is not seen on the
other major acid-base transporter, the
Na+-dependent
Cl
/HCO
3 exchanger.
Increases in glucose induce numerous changes in various cell phenotypic
behaviors. The pleotypic nature of these changes has been proposed to
be related to glucose and its metabolites. It is presumed that the flux
of glucose leading to an alteration in intracellular metabolic
processes may be responsible for the eventual changes seen in enhanced
cell proliferation and matrix deposition that so characterize the
microvascular disease of diabetes (25, 26). Other subcellular
mechanisms are proposed to be responsible for high-glucose-induced
changes. These include that of nonenzymatic glycation processes,
oxidative-reductive stress, aldose-reductase activation, and/or
diacylglycerol-PKC activation (15, 34, 42, 46). However, these events
are distal to the plasma membrane events that are not only an early
first response to changes in the high-glucose milieu, but one that
persists, allowing the cell to continue to exhibit pathophysiological changes.
Changes in the activity and expression of ion transporters have been
studied in a variety of experimental conditions (46). Intrinsic
properties of proximal tubular transport require adaptation to various
metabolic states. With chronic (>2 wk) metabolic acidosis, enhanced
Na+/H+
exchange activity (along with HCO
3
symport) was evident (4). Significant changes in
pHi were not evident, as enhanced
transport on the both basolateral and apical sides was evident (37),
allowing steady-state pHi to be
maintained. This adaptation of ion transporters will defend pH but
adjust to the change in the tonicity of the extracellular environment. These same investigators also demonstrated that enhanced exchange activity was evident in experimental models of hyperfiltration with
increased cell hypertrophy along with enhanced
HCO
3 transport; again, the maintenance
of the steady-state pHi was achieved (38). Interestingly, they found that transporter stimulation was out of proportion to the cell hypertrophy (increased surface area),
suggesting an increase in expression, although that was not examined.
Changes in extracellular osmolarity via a chronic exposure to
hyperosmolarity have recently been shown to alter
Na+/H+
exchange activity and expression in a variety of cells (3). We and
others have shown that acute exposure to hyperosmolar conditions decreases exchanger activity acutely in a wide variety of cells (32,
44). However, chronic exposure to hyperosmolarity was shown to increase
Na+/H+
exchanger activity for NHE1, -2, -3, and -4 (3, 11, 23). These studies
used hypertonic media by exposing the cells to glucose concentrations.
These investigators showed that chronic exposure to a high-glucose
media stimulated transcriptional and translational mechanisms of
exchanger regulation. In these studies, the effect of stimulatory
agonists that may be important in disease, on transporter function, was
not studied.
Our results extend these findings. We demonstrated that, under normal
tonicity but increased glucose (lowering of
Na+ to maintain tonicity), we were
able to reproduce the effects of increasing tonicity alone,
supporting a role for glucose. However, this change is
transporter specific in the MCs. The
Na+-dependent
Cl
/HCO
3 exchanger
is unaffected, whereas other ill-defined acid extruders (perhaps the
H+-K+-ATPase)
are increased. This is also supported by the findings that, with
angiotensin stimulation, there is a further increase in
Na+/H+
exchange activity but not that of
Na+-dependent
Cl
/HCO
3
transport; increased numbers of transporters lead to a greater role in
pH regulation.
The mechanisms by which these changes occur have not been ascertained.
Moreover, the changes that occur under chronic conditions may also
reflect changes from the exposure to glucose to that of subcellular
signaling mechanisms (i.e., PKC, G proteins). Defining the role by
which glucose alters ion transport activity and the subcellular
signaling mechanisms may further our understanding of the effects of
the diabetic milieu.
 |
ACKNOWLEDGEMENTS |
M. B. Ganz was supported by American Heart Association Established
Investigator Award 9600485 and by a Veteran's Affair Merit Review. R. F. Reilly was supported by a Veterans Affairs Research Associate Award
and a Merit Review.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. B. Ganz,
Section of Nephrology, 111K (W), Cleveland VA Medical Center, 10701 East Blvd., Cleveland, Ohio 44106 (E-mail: mbg4{at}po.cwru.edu).
Received 26 March 1999; accepted in final form 17 August 1999.
 |
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