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
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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.---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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table 1.   Recovery of pHi


                              
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Table 2.   Stimulation at pHi 6.80 


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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; triangle , recovery of MCs in 20 mM glucose; open circle , 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; triangle , recovery of MCs in 20 mM glucose; open circle , 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; triangle , recovery of MCs in 20 mM glucose; open circle , 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; triangle , recovery of MCs in 20 mM glucose; open circle , recovery of MCs in 30 mM glucose.

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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