Insulin treatment enhances AT1 receptor function in OK cells
http://www.100md.com
《美国生理学杂志》
Heart and Kidney Institute, College of Pharmacy, University of Houston, Houston, Texas
ABSTRACT
Increased renal sodium retention is considered a major risk factor contributing to hypertension associated with chronic hyperinsulinemia and obesity. However, the molecular mechanism involved is not understood. The present study investigates the effect of insulin treatment on AT1 receptor expression and ANG II-induced stimulation of Na/H exchanger (NHE) and Na-K-ATPase (NKA) in opossum kidney (OK) cells, a proximal tubule cell line. The presence of the AT1 receptors in OK cells was confirmed by the specific binding of 125I-sar-ANG II and by detecting 43-kDa protein on Western blot analysis with AT1 receptor antibody and blocking peptide as well as by expression of AT1 receptor mRNA as determined by RT-PCR. Insulin treatment (100 nM for 24 h) caused an increase in 125I-sar-ANG II binding, AT1 receptor protein content, and mRNA levels. The whole cell lysate and membrane showed similar insulin-induced increase in the AT1 receptor protein expression, which was blocked by genistein (100 nM), a tyrosine kinase inhibitor, and cycloheximide (1.5 μg/ml), a protein synthesis inhibitor. Determination of ethyl isopropyl amiloride-sensitive 22Na+ uptake, a measure of the NHE activity, revealed that ANG II (1–100 pM)-induced stimulation of NHE in insulin-treated cells was significantly greater than in the control cells. Similarly, ANG II (1–100 pM)-induced stimulation of ouabain-sensitive 86Rb+ uptake, a measure of NKA activity in insulin-treated cells, was significantly greater than in the control cells. ANG II stimulation of both the transporters was blocked by AT1 receptor antagonist losartan, suggesting the involvement of AT1 receptors. Thus chronic insulin treatment causes upregulation of AT1 receptors, which evoked ANG II-induced stimulation of NHE and NKA. We propose that insulin-induced increase in the renal AT1 receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and thus may contribute to development of hypertension in conditions associated with hyperinsulinemia.
angiotensin II; Na-K-ATPase; Na/H exchanger
ANGIOTENSIN II (ANG II) is an important regulator of renal and cardiovascular functions. ANG II binds mainly to two types of ANG II receptors, namely AT1 and AT2, which on activation initiate a cascade of signaling leading to the cellular response (10). The AT1 receptors promote cell growth and proliferation and produce vasoconstriction and antinatriuresis, (12, 21, 31, 34, 38), whereas AT2 receptors are reported to produce responses opposite to those produced by the AT1 receptors (11). Within the kidney, systemic and locally produced ANG II, via the activation of tubular AT1 receptors, serves as a potent hormone that participates in the reabsorption of the filtered sodium from the lumen and thus helps maintain sodium homeostasis and regulates blood pressure (13, 28). At molecular level, ANG II activates AT1 receptors coupled with Gi proteins and modulates multiple second messenger systems such as lowering of cellular cAMP and activation of phospholipase A2 in proximal tubule epithelial cells (26, 39). The modulation of these second messenger levels by ANG II leads to the stimulation of tubular sodium transporters, namely, Na/H exchanger, Na-K-ATPase, and Na/HCO3 cotransporters, thereby increases tubular sodium reabsorption (7, 8, 15). Numerous studies suggest that an abnormal AT1 receptor function, either caused by excessive availability of ANG II or increased AT1 receptor signaling per se, contributes to the shift in pressure natriuresis and the development of hypertension (9, 17, 25, 28, 36, 37).
It is well established that obesity is a major cause of insulin resistance leading to hyperinsulinemia and essential hypertension (19). Although there is a correlation between hyperinsulinemia and enhanced renal sodium reabsorption, there is evidence suggesting that chronic hyperinsulinemia is not a major factor responsible for the increases in renal sodium reabsorption and blood pressure (20). In vivo studies suggest that the renal AT1 receptor function in obese Zucker rats is increased, which contributes to a greater reabsorption of the filtered sodium and to the development of hypertension in these animals (1, 34). Recently, we provided in vitro evidence that the AT1 receptor numbers are increased in the brush-border membranes (BBM) and ANG II produces a greater stimulation of Na/H exchanger (NHE) in proximal tubules of obese compared with lean Zucker rats (6). We hypothesized that hyperinsulinemia, a characteristic of obese Zucker rats, may be responsible for the increase in the AT1 receptor expression and the enhanced ANG II-mediated stimulation of the sodium transporters. Therefore, to study the effect of insulin on AT1 receptor expression and their influence on the stimulation of sodium transporters, we have utilized proximal tubule cell line derived from the opossum kidney (OK cells), which maintains cellular response to ANG II (24, 36). The OK cells were chronically treated with insulin, followed by measuring AT1 receptor ligand binding and protein and mRNA expression and the ANG II-mediated stimulation of NHE and NKA activities in the treated cell.
MATERIALS AND METHODS
Cell culture media and serum were purchased from GIBCO-BRL. ANG II, cycloheximide, genistein, and insulin were purchased from Sigma (RBI). 86RbCl, 22NaCl, and 125I-sar-angiotensin were purchased from New England Nuclear Life Sciences. Antibodies NHE31-A for NHE3 and AT1 (N-10, SC-1173) for AT1 receptors were purchased from Alpha Diagnostic (San Antonio, TX) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. All other chemicals of highest purity available were purchased from Sigma.
Cell culture and drug treatments. OK cells were purchased (American Type Culture Collection, Manassas, VA) and grown in 24-well plastic cell culture plates or 100-mm culture dishes in DMEM culture media supplemented with serum (10%) under constant flow of 5% carbon dioxide mixed with air in a cell culture incubator set at 37°C, as suggested in the manufacturer's manual and reported by us (27). For consistency, cell passages 5–10 were utilized in this study. At 80–90% confluency, the OK cells were washed with serum-free medium and further incubated in serum-free medium with vehicle (control cells) or insulin, 100 nM (treated cells) for 24 h. Also, to investigate the effect of tyrosine kinase in insulin action, OK cells were incubated with insulin in the presence of genistein (100 nM). Furthermore, cells were also chronically coincubated with insulin and cycloheximide to investigate the role of de novo protein synthesis in insulin action. After 24 h of incubation, the cells were washed three times with serum-free media and stabilized for 3 h in same media to ascertain that insulin is absent from the medium and does not have acute effect per se on sodium transporters. Effects of ANG II on the activities of NKA and NHE were measured in the intact cells, while the quantification of AT1 receptor expression was performed in the cell membranes and the cell extracts, as described below.
Quantification of AT1 receptors. OK cell membranes were prepared, as we described earlier (27). Briefly, OK cells were lysed with 10 mM tris[hydroxymethyl]aminomethane (Tris) buffer (pH 7.4) containing a cocktail of protease inhibitors (Complete 1 697 498, Roche Diagnostic) and 1 mM phenylmethylsulfonylfluoride (PMSF) followed by homogenization and sonication. The cell lysates were centrifuged at 200 g for 5 min; resulting supernatant was further centrifuged at 37,000 g for 20 min, to obtain membrane pellets. The pellets were suspended in Tris buffer. In another set of experiments, the cell lysates, without any centrifugation, were used for measuring total contents of AT1 receptor protein in the cell. Protein in the membrane samples and the cell lysates was determined by using a bovine serum albumin (BCA) kit (Pierce) with BSA as standard.
125I-sar-ANG II binding. To the OK cell membranes, 125I-sar-ANG II binding was performed essentially as described earlier (23). The membranes (50 μg protein) were incubated with 30 pM of 125I-sar-ANG II at 30°C for 20 min in a shaking water bath. The assay was terminated by rapid filtration on GF/C filters under vacuum. The radioactivity on the filters was counted in a LKB gamma counter. Nonspecific binding was determined by performing the binding assay in the presence of 1 μM unlabeled ANG II.
Western immunoblotting. The AT1 receptors and NHE3 were detected and quantified by Western blotting, as we have described earlier (23). Basolateral membranes were used a positive control for AT1. Briefly, the sample proteins were solubilized in Laemmeli buffer. Protein samples of the membranes (5–15 μg) and the lysates (10–25 μg) of the control and the treated cells and the proximal tubular membranes (5 μg) were resolved on 10% SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon P membrane (blot). The AT1 receptors and NHE3 were detected by using affinity-purified polyclonal anti-peptide AT1 receptor and anti-NHE3 antibodies, respectively, HRP-linked anti-rabbit IgG and enhanced chemiluminescence. Signals were recorded as bands on Kodak X-ray films. Approximate molecular mass and the density of the protein bands were determined using molecular mass markers and densitometric analysis (Kodak Imaging Station), respectively. The density of the bands was compared between samples from the control and insulin-treated OK cells.
RT-PCR for AT1. Total RNA was isolated from the OK cells and rat renal cortex by the RNeasy mini kit (QIAGEN, Valencia, CA) and 1μg of RNA was used for cDNA synthesis and amplification for AT1 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, used as an internal control) using Advantage cDNA PCR Kit (BD Biosciences, Clonetech, Palo Alto, CA). The PCR for AT1 cDNA was performed utilizing sense (5'-CCA AAG TCA CCT GCA TCA TC-3') and antisense primers (5'-CAC AAT CGC CAT AAT TAT CCT A-3'). The primers used for AT1 cDNA were designed from the cDNA sequences common to rat AT1A and AT1B receptors (3, 33). These primers correspond to regions where no sequence divergence exists between AT1A and AT1B, and amplify a 305-bp cDNA fragment from position 723 to 1028 in the AT1A sequence and from position 630–935 in the AT1B sequence (3, 33). The GAPDH cDNA was amplified using the sense primer (5'- TAC TCC TTG GAG GCC ATG TA-3') and the antisense primer (5'-CGT GGA GTC TAC TGG CGT CT-3') (Ref. 3), which yielded a 723-bp product. The PCR products, were resolved on 1.5% agarose gel, stained with ethidium bromide, and subjected to densitometry of the bands using FluorChem 8800 (Alpha Innotech Imaging System).
Effect of ANG II on NHE and NKA activity in intact OK cells. Activity of the NHE was measured by 22Na+ uptake while the activity of NKA was measured by 86Rb+ uptake as well as by adenosine 5'-triphosphate (ATP) hydrolysis methods, as described earlier (4, 14, 16) with slight modifications. 1) 22Na+ uptake: Briefly, stabilized OK cells were rinsed three times with sodium-free buffer consisting of (in mM) 120 tetramethylammonium chloride (TMA-Cl), 5 KCl, 1 CaCl2, 1 MgCl2 {pH 6.40 ± 0.01 and 295 ± 1 mosmol/KgH2O with 18 Tris base, 28 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), and 18 2-(N-morpholino)ethanesulfonic acid}. The assay was performed in the presence of a 1 mM ouabain and 100 μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) to minimize Na+ exit from the cell. The cells were incubated for 7–10 min in Na+-free buffer containing ANG II (1–100 pM). In AT1 receptor antagonist assays, 1 μM losartan was added 5 min prior to ANG II. The uptake was initiated by addition of 22Na+ (10 μCi/ml) in a buffer containing (in mM) 40 NaCl, 110 TMA-Cl, 5 KCl, 1 CaCl2, 1 MgCl2 (pH 7.40 and 295 ± mosmol/kgH2O with 18 mM Tris base and 28 mM HEPES). The cells were maintained at 37°C for 3 min and the reaction was terminated by washing the wells four times with 1 ml ice-cold isosmotic LiCl-HEPES buffer (pH 7.4). Cells were lysed with 3% sodium dodecyl sulfate (SDS) [0.5 ml/well] and radioactivity was counted directly in cell lysate by gamma-counter. A portion of the lysates was used to determine the protein contents, and the values for each sample were corrected per milligram protein. The uptake was also measured in the presence of ethyl isopropyl ameloride (EIPA, 10 μM), a NHE inhibitor. The NHE activity was determined as the difference of 22Na+ uptake in the absence and presence of EIPA. The effect of ANG II in control and insulin-treated cells was calculated over respective basal 22Na+ uptake. 2)86Rb+ uptake: The control and insulin-treated cells after stabilization for 3 h in the medium free of serum and insulin, as described above, were incubated with vehicle or ANG II (1–100 pM) for 10 min at 37°C. Similar to 22Na+ uptake, AT1 specificty was determined by preincubating the cells with 1μM losartan (AT1 antagonist). The 86Rb+ uptake was initiated by the addition of 10 μl of 86Rb+ in DMEM to obtain final concentration of 3 μCi/ml 86Rb+. After 10 min of incubation at 37°C, the uptake was stopped by aspirating the media and rinsing the wells four times with 1.0 ml/well ice-cold phosphate-buffered saline. Preliminary results show that 86Rb+ uptake is linear for at least 15 min. Similar to the above protocol, the cells were lysed by SDS, radioactivity was counted, and protein contents of the lysates were determined. The NKA activity was determined as the difference between 86Rb+ uptake in the absence (total activity) and presence (ouabain-insensitive) of ouabain (1 mM). Ouabain inhibited 70% of the total uptake. 3) ATP hydrolysis: cells were incubated with or without 10 pM ANG II for 10 min at 37°C. Cell suspension (0.05 mg/ml) was used to assay 1mM ouabain-sensitive NKA activity by end-point phosphate hydrolysis of 4 mM ATP as described earlier (4). The inorganic phosphate (Pi) released was determined calorimetrically.
Statistical analysis. Differences between means were evaluated using the unpaired t-test or analysis of variance with Newman-Keuls multiple test, as appropriate. P < 0.05 was considered statistically significant.
RESULTS
Quantification of AT1 receptors. Western blot analysis with the AT1 receptor antibody revealed the presence of 43-kDa protein band in OK cell membranes and in the rat proximal tubular membranes. The blocking peptide-treated AT1 receptor antibody did not label any band, suggesting the presence of AT1 receptor-specific band detected with the antibody (Fig. 1A). Furthermore, these cells also express a moderate levels of AT1 receptor mRNA. The RT-PCR product in OK cells was of similar size (300 bp), although smaller in intensity compared with rat renal cortex (Fig. 1B), thus suggesting that AT1 message level in OK cells is not markedly lower than proximal tubules. Of the note, the intensity of AT1 protein band in OK cells is smaller than proximal tubules.
As illustrated in (Fig. 1B), the amplification of cDNA for AT1 receptors, synthesized from OK cells and the rat kidney, shows a single band that corresponds to the size of 300 bp on a 100-bp DNA ladder. Since the expected size of our PCR product is 305 bp, we believe that the bands obtained are the ones for the AT1 receptor. Since kidney is known to express AT1 receptors in abundance, we used the mRNA from the rat kidney as a control for the AT1 receptor expression studies. The presence of the bands at the same position in the cDNA of OK cells samples suggesting that OK cells also express the AT1 receptors.
A comparison of the membrane AT1 receptor density revealed that treatment of OK cells with insulin caused more than twofold increase in the AT1 receptor expression compared with the control cells (Fig. 2A). Insulin treatment also caused a significant increase in AT1 receptor message levels (Fig. 2B). 125I-sar-ANG II binding to OK cell membranes revealed specific binding as 30 and 40% of the total binding in control and insulin-treated samples, respectively. The specific binding was approximately twofold higher in the membranes of insulin-treated (1.76 ± 0.03 fmol/mg protein) compared with control cells (0.94 ± 0.09 fmol/mg protein; Fig. 2C). A similar increase in the AT1 receptor expression was observed in the lysates of cells treated with insulin compared with the control cells. When the cells were incubated with insulin in the presence of genistein (10 nM), a tyrosine kinase inhibitor, the upregulatory effect of insulin on AT1 receptor was completely abolished, while genistein alone had no effect on the AT1 receptor protein expression (Fig. 3). This suggested the role of tyrosine kinase, a characteristic of insulin receptors, in the insulin-mediated upregulation of AT1 receptors. Furthermore, when the cells were incubated with insulin in presence of cycloheximide, the insulin-induced increase in the AT1 receptor protein was abolished, suggesting the role of protein synthesis in insulin action on AT1 receptor increase (Fig. 4).
Effects of ANG II on NHE and NKA activity. ANG II (1–100 pM) stimulated 22Na uptake over basal in both the control and insulin-treated cells. However, the stimulatory effect of ANG II was significantly greater in the insulin-treated compared with the control cells (Fig. 5A). The basal 22Na+ uptake was similar in the control and insulin-treated cells (basal: control, 4 ± 0.1; insulin-treated, 4.35 ± 0.15 nmol 22Na+·mg protein–1·min–1). To test whether ANG II-induced stimulation of NHE3 was AT1 specific, 22Na+ uptake was carried out in presence of losartan, an AT1 antagonist. As shown in Fig. 4A, 1 μM losartan blocked ANG II-induced stimulation of NHE3 both in control and insulin-treated cells. Losartan per se did not affect the 22Na+ uptake in absence of ANG II. To determine whether ANG II causes any changes in NHE3 protein expression, NHE3 surface antigen was measured in both control and insulin-treated cells. As shown in Fig. 5B, exposure to ANG II alone for 10 min did not affect NHE3 protein contents on the membranes. ANG II (1–100 pM) stimulated 86Rb uptake over basal in both the control and the treated cells, being greater stimulation in the treated compared with the control cells (Fig. 6A). Basal 86Rb uptake in the control and insulin-treated cells was similar (basal: control, 15.2 ± 0.3; insulin-treated, 15.9 ± 0.4 nmol 86Rb+·mg protein–1·min–1). As shown in Fig. 5A, losartan blocked the ANG II effect both in control and insulin- treated cells, suggesting AT1-specific effects. Because OK cells form monolayers by attaching to surface through basolateral membranes, this may impose limitations when pump activity is measured in intact cell. To address this concern, the NKA activity was also measured in whole cell lysate. Cells were scraped from culture dishes to recover maximum basolateral side. As shown Fig. 6B, ANG II (10 pM) stimulated NKA activity both in control and insulin-treated cells with maximum effect being observed in insulin-treated cells. Also, insulin per se did not change the ATPase activity of the pump. These results suggest that attached cells do not impose any limitation on the validity of 86Rb+ uptake as a measure of pump activity.
DISCUSSION
Present study demonstrates that chronic insulin treatment of OK cells causes upregulation of the AT1 receptor numbers with an increase in protein expression of the receptors, which on activation by ANG II produces greater stimulation of NHE and NKA activity.
Obesity is a major cause of hypertension (20). There is evidence suggesting an enhanced function of ANG II receptors in terms of an increase in the renal sodium reabsorption and their contribution to the development of hypertension in obesity (1, 18, 34). Recently, we reported that ANG II binding sites and the stimulation of NHE by ANG II were enhanced in the isolated preparations of proximal tubules of obese Zucker rats compared with the control lean Zucker rats (6). Because obesity is usually associated with insulin resistance and hyperinsulinemia, we speculated that increased level of insulin played a role in the enhanced action of ANG II on sodium transport. We tested this hypothesis by using an in vitro model of OK cells, a cell line of proximal tubule epithelial cells derived from opossum kidney. Several studies have utilized OK cells as an in vitro model to study ANG II-mediated cellular responses (24, 35). However, there are no reports that demonstrate the expression and quantification of AT1 receptors in these cell lines. Therefore, we first determined the expression of AT1 receptor mRNA and protein in OK cells, using RT-PCR and specific AT1 receptor antibody and blocking peptide. We found that OK cell express AT1 receptor mRNA and protein as expressed in the rat proximal tubular membranes and reported earlier (22, 23). A comparison of AT1 receptor expression between control and insulin-treated cells revealed that insulin treatment of OK cells caused an upregulation of the AT1 receptors in the membranes, as well as in the whole cell lysates and was blocked by cycloheximide, a protein synthesis inhibitor. This suggests that the increase in the AT1 receptor expression may be due to an increase in the receptor protein synthesis, which leads to a proportionally higher expression of the receptors on the cell membranes. This is supported by an increase in AT1 mRNA level and ligand binding to the membranes of insulin-treated cells. Insulin-mediated upregulation of the AT1 receptors was blocked by genistein, suggesting the role of tyrosine kinase. Chronic treatment with insulin has been shown to upregulate AT1 receptors in other cells lines, such as vascular smooth cells (30) and rabbit proximal tubule epithelial cells (5). Our studies are consistent with those reported in the smooth muscle cells, in that insulin upregulates the AT1 receptor at the protein synthesis level, which is sensitive to genistein. However, this is the first report showing that OK cells express detectable and quantifiable levels of AT1 receptors and hence these cells may be used as a model of epithelial cells for studying the regulation of endogenously expressed AT1 receptors.
Having established that chronic insulin treatment increases the AT1 receptor expression in OK cell membranes, we tested whether such an increase in the AT1 receptors in response to ANG II produces a greater stimulation of sodium transporters in insulin-treated cells. We found that ANG II indeed produced a greater stimulation of both the NKA and the NHE3 activity in insulin-treated compared with the control cells. It should be noted that after stabilization of cells for 3 h in insulin-free medium, the basal activity of both the transporters was similar in the control and insulin-treated cells. This suggests that the increase in AT1 receptors, and not the transporter per se, might be responsible for the enhanced ANG II-mediated stimulation of the transporters in insulin-treated cells. Although the AT1 receptors expressed in OK cell membranes are very low, the response to picomolar concentrations of ANG II in our study are also supported by other studies (14). Earlier, we have reported an increase in the AT1 receptor number and the enhanced ANG II-mediated stimulation of NHE3 in the isolated proximal tubule of obese Zucker rats (6). Although insulin has been shown to cause mild sodium retention (2, 32), the present study suggests that the upregulation of AT1 receptors and the enhanced ANG II-mediated stimulation of sodium transporters caused by chronic hyperinsulinemia may lead to a powerful antinatriuretic response to ANG II. It is likely that insulin-induced upregulated AT1 receptors may also produce other known cellular effects associated with the AT1 receptors, such as cell growth and cell proliferation (38), and contribute to the pathophysiology associated with hyperinsulinemia and obesity.
In brief, this is the first report, to our knowledge, showing that OK cells express detectable and quantifiable AT1 receptors. Insulin treatment of OK cells upregulates AT1 receptors, which on activation by ANG II produce greater stimulation of sodium transporters. Such an increase in AT1 receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and development of hypertension in conditions associated with hyperinsulinemia.
GRANTS
This study is supported by National Institutes of Health Grant DK-61578.
ACKNOWLEDGMENTS
The authors thank L. Pillai for providing technical assistance in maintaining OK cell cultures and performing Western blot analysis.
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. Section 1734 solely to indicate this fact.
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ABSTRACT
Increased renal sodium retention is considered a major risk factor contributing to hypertension associated with chronic hyperinsulinemia and obesity. However, the molecular mechanism involved is not understood. The present study investigates the effect of insulin treatment on AT1 receptor expression and ANG II-induced stimulation of Na/H exchanger (NHE) and Na-K-ATPase (NKA) in opossum kidney (OK) cells, a proximal tubule cell line. The presence of the AT1 receptors in OK cells was confirmed by the specific binding of 125I-sar-ANG II and by detecting 43-kDa protein on Western blot analysis with AT1 receptor antibody and blocking peptide as well as by expression of AT1 receptor mRNA as determined by RT-PCR. Insulin treatment (100 nM for 24 h) caused an increase in 125I-sar-ANG II binding, AT1 receptor protein content, and mRNA levels. The whole cell lysate and membrane showed similar insulin-induced increase in the AT1 receptor protein expression, which was blocked by genistein (100 nM), a tyrosine kinase inhibitor, and cycloheximide (1.5 μg/ml), a protein synthesis inhibitor. Determination of ethyl isopropyl amiloride-sensitive 22Na+ uptake, a measure of the NHE activity, revealed that ANG II (1–100 pM)-induced stimulation of NHE in insulin-treated cells was significantly greater than in the control cells. Similarly, ANG II (1–100 pM)-induced stimulation of ouabain-sensitive 86Rb+ uptake, a measure of NKA activity in insulin-treated cells, was significantly greater than in the control cells. ANG II stimulation of both the transporters was blocked by AT1 receptor antagonist losartan, suggesting the involvement of AT1 receptors. Thus chronic insulin treatment causes upregulation of AT1 receptors, which evoked ANG II-induced stimulation of NHE and NKA. We propose that insulin-induced increase in the renal AT1 receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and thus may contribute to development of hypertension in conditions associated with hyperinsulinemia.
angiotensin II; Na-K-ATPase; Na/H exchanger
ANGIOTENSIN II (ANG II) is an important regulator of renal and cardiovascular functions. ANG II binds mainly to two types of ANG II receptors, namely AT1 and AT2, which on activation initiate a cascade of signaling leading to the cellular response (10). The AT1 receptors promote cell growth and proliferation and produce vasoconstriction and antinatriuresis, (12, 21, 31, 34, 38), whereas AT2 receptors are reported to produce responses opposite to those produced by the AT1 receptors (11). Within the kidney, systemic and locally produced ANG II, via the activation of tubular AT1 receptors, serves as a potent hormone that participates in the reabsorption of the filtered sodium from the lumen and thus helps maintain sodium homeostasis and regulates blood pressure (13, 28). At molecular level, ANG II activates AT1 receptors coupled with Gi proteins and modulates multiple second messenger systems such as lowering of cellular cAMP and activation of phospholipase A2 in proximal tubule epithelial cells (26, 39). The modulation of these second messenger levels by ANG II leads to the stimulation of tubular sodium transporters, namely, Na/H exchanger, Na-K-ATPase, and Na/HCO3 cotransporters, thereby increases tubular sodium reabsorption (7, 8, 15). Numerous studies suggest that an abnormal AT1 receptor function, either caused by excessive availability of ANG II or increased AT1 receptor signaling per se, contributes to the shift in pressure natriuresis and the development of hypertension (9, 17, 25, 28, 36, 37).
It is well established that obesity is a major cause of insulin resistance leading to hyperinsulinemia and essential hypertension (19). Although there is a correlation between hyperinsulinemia and enhanced renal sodium reabsorption, there is evidence suggesting that chronic hyperinsulinemia is not a major factor responsible for the increases in renal sodium reabsorption and blood pressure (20). In vivo studies suggest that the renal AT1 receptor function in obese Zucker rats is increased, which contributes to a greater reabsorption of the filtered sodium and to the development of hypertension in these animals (1, 34). Recently, we provided in vitro evidence that the AT1 receptor numbers are increased in the brush-border membranes (BBM) and ANG II produces a greater stimulation of Na/H exchanger (NHE) in proximal tubules of obese compared with lean Zucker rats (6). We hypothesized that hyperinsulinemia, a characteristic of obese Zucker rats, may be responsible for the increase in the AT1 receptor expression and the enhanced ANG II-mediated stimulation of the sodium transporters. Therefore, to study the effect of insulin on AT1 receptor expression and their influence on the stimulation of sodium transporters, we have utilized proximal tubule cell line derived from the opossum kidney (OK cells), which maintains cellular response to ANG II (24, 36). The OK cells were chronically treated with insulin, followed by measuring AT1 receptor ligand binding and protein and mRNA expression and the ANG II-mediated stimulation of NHE and NKA activities in the treated cell.
MATERIALS AND METHODS
Cell culture media and serum were purchased from GIBCO-BRL. ANG II, cycloheximide, genistein, and insulin were purchased from Sigma (RBI). 86RbCl, 22NaCl, and 125I-sar-angiotensin were purchased from New England Nuclear Life Sciences. Antibodies NHE31-A for NHE3 and AT1 (N-10, SC-1173) for AT1 receptors were purchased from Alpha Diagnostic (San Antonio, TX) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. All other chemicals of highest purity available were purchased from Sigma.
Cell culture and drug treatments. OK cells were purchased (American Type Culture Collection, Manassas, VA) and grown in 24-well plastic cell culture plates or 100-mm culture dishes in DMEM culture media supplemented with serum (10%) under constant flow of 5% carbon dioxide mixed with air in a cell culture incubator set at 37°C, as suggested in the manufacturer's manual and reported by us (27). For consistency, cell passages 5–10 were utilized in this study. At 80–90% confluency, the OK cells were washed with serum-free medium and further incubated in serum-free medium with vehicle (control cells) or insulin, 100 nM (treated cells) for 24 h. Also, to investigate the effect of tyrosine kinase in insulin action, OK cells were incubated with insulin in the presence of genistein (100 nM). Furthermore, cells were also chronically coincubated with insulin and cycloheximide to investigate the role of de novo protein synthesis in insulin action. After 24 h of incubation, the cells were washed three times with serum-free media and stabilized for 3 h in same media to ascertain that insulin is absent from the medium and does not have acute effect per se on sodium transporters. Effects of ANG II on the activities of NKA and NHE were measured in the intact cells, while the quantification of AT1 receptor expression was performed in the cell membranes and the cell extracts, as described below.
Quantification of AT1 receptors. OK cell membranes were prepared, as we described earlier (27). Briefly, OK cells were lysed with 10 mM tris[hydroxymethyl]aminomethane (Tris) buffer (pH 7.4) containing a cocktail of protease inhibitors (Complete 1 697 498, Roche Diagnostic) and 1 mM phenylmethylsulfonylfluoride (PMSF) followed by homogenization and sonication. The cell lysates were centrifuged at 200 g for 5 min; resulting supernatant was further centrifuged at 37,000 g for 20 min, to obtain membrane pellets. The pellets were suspended in Tris buffer. In another set of experiments, the cell lysates, without any centrifugation, were used for measuring total contents of AT1 receptor protein in the cell. Protein in the membrane samples and the cell lysates was determined by using a bovine serum albumin (BCA) kit (Pierce) with BSA as standard.
125I-sar-ANG II binding. To the OK cell membranes, 125I-sar-ANG II binding was performed essentially as described earlier (23). The membranes (50 μg protein) were incubated with 30 pM of 125I-sar-ANG II at 30°C for 20 min in a shaking water bath. The assay was terminated by rapid filtration on GF/C filters under vacuum. The radioactivity on the filters was counted in a LKB gamma counter. Nonspecific binding was determined by performing the binding assay in the presence of 1 μM unlabeled ANG II.
Western immunoblotting. The AT1 receptors and NHE3 were detected and quantified by Western blotting, as we have described earlier (23). Basolateral membranes were used a positive control for AT1. Briefly, the sample proteins were solubilized in Laemmeli buffer. Protein samples of the membranes (5–15 μg) and the lysates (10–25 μg) of the control and the treated cells and the proximal tubular membranes (5 μg) were resolved on 10% SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon P membrane (blot). The AT1 receptors and NHE3 were detected by using affinity-purified polyclonal anti-peptide AT1 receptor and anti-NHE3 antibodies, respectively, HRP-linked anti-rabbit IgG and enhanced chemiluminescence. Signals were recorded as bands on Kodak X-ray films. Approximate molecular mass and the density of the protein bands were determined using molecular mass markers and densitometric analysis (Kodak Imaging Station), respectively. The density of the bands was compared between samples from the control and insulin-treated OK cells.
RT-PCR for AT1. Total RNA was isolated from the OK cells and rat renal cortex by the RNeasy mini kit (QIAGEN, Valencia, CA) and 1μg of RNA was used for cDNA synthesis and amplification for AT1 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, used as an internal control) using Advantage cDNA PCR Kit (BD Biosciences, Clonetech, Palo Alto, CA). The PCR for AT1 cDNA was performed utilizing sense (5'-CCA AAG TCA CCT GCA TCA TC-3') and antisense primers (5'-CAC AAT CGC CAT AAT TAT CCT A-3'). The primers used for AT1 cDNA were designed from the cDNA sequences common to rat AT1A and AT1B receptors (3, 33). These primers correspond to regions where no sequence divergence exists between AT1A and AT1B, and amplify a 305-bp cDNA fragment from position 723 to 1028 in the AT1A sequence and from position 630–935 in the AT1B sequence (3, 33). The GAPDH cDNA was amplified using the sense primer (5'- TAC TCC TTG GAG GCC ATG TA-3') and the antisense primer (5'-CGT GGA GTC TAC TGG CGT CT-3') (Ref. 3), which yielded a 723-bp product. The PCR products, were resolved on 1.5% agarose gel, stained with ethidium bromide, and subjected to densitometry of the bands using FluorChem 8800 (Alpha Innotech Imaging System).
Effect of ANG II on NHE and NKA activity in intact OK cells. Activity of the NHE was measured by 22Na+ uptake while the activity of NKA was measured by 86Rb+ uptake as well as by adenosine 5'-triphosphate (ATP) hydrolysis methods, as described earlier (4, 14, 16) with slight modifications. 1) 22Na+ uptake: Briefly, stabilized OK cells were rinsed three times with sodium-free buffer consisting of (in mM) 120 tetramethylammonium chloride (TMA-Cl), 5 KCl, 1 CaCl2, 1 MgCl2 {pH 6.40 ± 0.01 and 295 ± 1 mosmol/KgH2O with 18 Tris base, 28 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), and 18 2-(N-morpholino)ethanesulfonic acid}. The assay was performed in the presence of a 1 mM ouabain and 100 μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) to minimize Na+ exit from the cell. The cells were incubated for 7–10 min in Na+-free buffer containing ANG II (1–100 pM). In AT1 receptor antagonist assays, 1 μM losartan was added 5 min prior to ANG II. The uptake was initiated by addition of 22Na+ (10 μCi/ml) in a buffer containing (in mM) 40 NaCl, 110 TMA-Cl, 5 KCl, 1 CaCl2, 1 MgCl2 (pH 7.40 and 295 ± mosmol/kgH2O with 18 mM Tris base and 28 mM HEPES). The cells were maintained at 37°C for 3 min and the reaction was terminated by washing the wells four times with 1 ml ice-cold isosmotic LiCl-HEPES buffer (pH 7.4). Cells were lysed with 3% sodium dodecyl sulfate (SDS) [0.5 ml/well] and radioactivity was counted directly in cell lysate by gamma-counter. A portion of the lysates was used to determine the protein contents, and the values for each sample were corrected per milligram protein. The uptake was also measured in the presence of ethyl isopropyl ameloride (EIPA, 10 μM), a NHE inhibitor. The NHE activity was determined as the difference of 22Na+ uptake in the absence and presence of EIPA. The effect of ANG II in control and insulin-treated cells was calculated over respective basal 22Na+ uptake. 2)86Rb+ uptake: The control and insulin-treated cells after stabilization for 3 h in the medium free of serum and insulin, as described above, were incubated with vehicle or ANG II (1–100 pM) for 10 min at 37°C. Similar to 22Na+ uptake, AT1 specificty was determined by preincubating the cells with 1μM losartan (AT1 antagonist). The 86Rb+ uptake was initiated by the addition of 10 μl of 86Rb+ in DMEM to obtain final concentration of 3 μCi/ml 86Rb+. After 10 min of incubation at 37°C, the uptake was stopped by aspirating the media and rinsing the wells four times with 1.0 ml/well ice-cold phosphate-buffered saline. Preliminary results show that 86Rb+ uptake is linear for at least 15 min. Similar to the above protocol, the cells were lysed by SDS, radioactivity was counted, and protein contents of the lysates were determined. The NKA activity was determined as the difference between 86Rb+ uptake in the absence (total activity) and presence (ouabain-insensitive) of ouabain (1 mM). Ouabain inhibited 70% of the total uptake. 3) ATP hydrolysis: cells were incubated with or without 10 pM ANG II for 10 min at 37°C. Cell suspension (0.05 mg/ml) was used to assay 1mM ouabain-sensitive NKA activity by end-point phosphate hydrolysis of 4 mM ATP as described earlier (4). The inorganic phosphate (Pi) released was determined calorimetrically.
Statistical analysis. Differences between means were evaluated using the unpaired t-test or analysis of variance with Newman-Keuls multiple test, as appropriate. P < 0.05 was considered statistically significant.
RESULTS
Quantification of AT1 receptors. Western blot analysis with the AT1 receptor antibody revealed the presence of 43-kDa protein band in OK cell membranes and in the rat proximal tubular membranes. The blocking peptide-treated AT1 receptor antibody did not label any band, suggesting the presence of AT1 receptor-specific band detected with the antibody (Fig. 1A). Furthermore, these cells also express a moderate levels of AT1 receptor mRNA. The RT-PCR product in OK cells was of similar size (300 bp), although smaller in intensity compared with rat renal cortex (Fig. 1B), thus suggesting that AT1 message level in OK cells is not markedly lower than proximal tubules. Of the note, the intensity of AT1 protein band in OK cells is smaller than proximal tubules.
As illustrated in (Fig. 1B), the amplification of cDNA for AT1 receptors, synthesized from OK cells and the rat kidney, shows a single band that corresponds to the size of 300 bp on a 100-bp DNA ladder. Since the expected size of our PCR product is 305 bp, we believe that the bands obtained are the ones for the AT1 receptor. Since kidney is known to express AT1 receptors in abundance, we used the mRNA from the rat kidney as a control for the AT1 receptor expression studies. The presence of the bands at the same position in the cDNA of OK cells samples suggesting that OK cells also express the AT1 receptors.
A comparison of the membrane AT1 receptor density revealed that treatment of OK cells with insulin caused more than twofold increase in the AT1 receptor expression compared with the control cells (Fig. 2A). Insulin treatment also caused a significant increase in AT1 receptor message levels (Fig. 2B). 125I-sar-ANG II binding to OK cell membranes revealed specific binding as 30 and 40% of the total binding in control and insulin-treated samples, respectively. The specific binding was approximately twofold higher in the membranes of insulin-treated (1.76 ± 0.03 fmol/mg protein) compared with control cells (0.94 ± 0.09 fmol/mg protein; Fig. 2C). A similar increase in the AT1 receptor expression was observed in the lysates of cells treated with insulin compared with the control cells. When the cells were incubated with insulin in the presence of genistein (10 nM), a tyrosine kinase inhibitor, the upregulatory effect of insulin on AT1 receptor was completely abolished, while genistein alone had no effect on the AT1 receptor protein expression (Fig. 3). This suggested the role of tyrosine kinase, a characteristic of insulin receptors, in the insulin-mediated upregulation of AT1 receptors. Furthermore, when the cells were incubated with insulin in presence of cycloheximide, the insulin-induced increase in the AT1 receptor protein was abolished, suggesting the role of protein synthesis in insulin action on AT1 receptor increase (Fig. 4).
Effects of ANG II on NHE and NKA activity. ANG II (1–100 pM) stimulated 22Na uptake over basal in both the control and insulin-treated cells. However, the stimulatory effect of ANG II was significantly greater in the insulin-treated compared with the control cells (Fig. 5A). The basal 22Na+ uptake was similar in the control and insulin-treated cells (basal: control, 4 ± 0.1; insulin-treated, 4.35 ± 0.15 nmol 22Na+·mg protein–1·min–1). To test whether ANG II-induced stimulation of NHE3 was AT1 specific, 22Na+ uptake was carried out in presence of losartan, an AT1 antagonist. As shown in Fig. 4A, 1 μM losartan blocked ANG II-induced stimulation of NHE3 both in control and insulin-treated cells. Losartan per se did not affect the 22Na+ uptake in absence of ANG II. To determine whether ANG II causes any changes in NHE3 protein expression, NHE3 surface antigen was measured in both control and insulin-treated cells. As shown in Fig. 5B, exposure to ANG II alone for 10 min did not affect NHE3 protein contents on the membranes. ANG II (1–100 pM) stimulated 86Rb uptake over basal in both the control and the treated cells, being greater stimulation in the treated compared with the control cells (Fig. 6A). Basal 86Rb uptake in the control and insulin-treated cells was similar (basal: control, 15.2 ± 0.3; insulin-treated, 15.9 ± 0.4 nmol 86Rb+·mg protein–1·min–1). As shown in Fig. 5A, losartan blocked the ANG II effect both in control and insulin- treated cells, suggesting AT1-specific effects. Because OK cells form monolayers by attaching to surface through basolateral membranes, this may impose limitations when pump activity is measured in intact cell. To address this concern, the NKA activity was also measured in whole cell lysate. Cells were scraped from culture dishes to recover maximum basolateral side. As shown Fig. 6B, ANG II (10 pM) stimulated NKA activity both in control and insulin-treated cells with maximum effect being observed in insulin-treated cells. Also, insulin per se did not change the ATPase activity of the pump. These results suggest that attached cells do not impose any limitation on the validity of 86Rb+ uptake as a measure of pump activity.
DISCUSSION
Present study demonstrates that chronic insulin treatment of OK cells causes upregulation of the AT1 receptor numbers with an increase in protein expression of the receptors, which on activation by ANG II produces greater stimulation of NHE and NKA activity.
Obesity is a major cause of hypertension (20). There is evidence suggesting an enhanced function of ANG II receptors in terms of an increase in the renal sodium reabsorption and their contribution to the development of hypertension in obesity (1, 18, 34). Recently, we reported that ANG II binding sites and the stimulation of NHE by ANG II were enhanced in the isolated preparations of proximal tubules of obese Zucker rats compared with the control lean Zucker rats (6). Because obesity is usually associated with insulin resistance and hyperinsulinemia, we speculated that increased level of insulin played a role in the enhanced action of ANG II on sodium transport. We tested this hypothesis by using an in vitro model of OK cells, a cell line of proximal tubule epithelial cells derived from opossum kidney. Several studies have utilized OK cells as an in vitro model to study ANG II-mediated cellular responses (24, 35). However, there are no reports that demonstrate the expression and quantification of AT1 receptors in these cell lines. Therefore, we first determined the expression of AT1 receptor mRNA and protein in OK cells, using RT-PCR and specific AT1 receptor antibody and blocking peptide. We found that OK cell express AT1 receptor mRNA and protein as expressed in the rat proximal tubular membranes and reported earlier (22, 23). A comparison of AT1 receptor expression between control and insulin-treated cells revealed that insulin treatment of OK cells caused an upregulation of the AT1 receptors in the membranes, as well as in the whole cell lysates and was blocked by cycloheximide, a protein synthesis inhibitor. This suggests that the increase in the AT1 receptor expression may be due to an increase in the receptor protein synthesis, which leads to a proportionally higher expression of the receptors on the cell membranes. This is supported by an increase in AT1 mRNA level and ligand binding to the membranes of insulin-treated cells. Insulin-mediated upregulation of the AT1 receptors was blocked by genistein, suggesting the role of tyrosine kinase. Chronic treatment with insulin has been shown to upregulate AT1 receptors in other cells lines, such as vascular smooth cells (30) and rabbit proximal tubule epithelial cells (5). Our studies are consistent with those reported in the smooth muscle cells, in that insulin upregulates the AT1 receptor at the protein synthesis level, which is sensitive to genistein. However, this is the first report showing that OK cells express detectable and quantifiable levels of AT1 receptors and hence these cells may be used as a model of epithelial cells for studying the regulation of endogenously expressed AT1 receptors.
Having established that chronic insulin treatment increases the AT1 receptor expression in OK cell membranes, we tested whether such an increase in the AT1 receptors in response to ANG II produces a greater stimulation of sodium transporters in insulin-treated cells. We found that ANG II indeed produced a greater stimulation of both the NKA and the NHE3 activity in insulin-treated compared with the control cells. It should be noted that after stabilization of cells for 3 h in insulin-free medium, the basal activity of both the transporters was similar in the control and insulin-treated cells. This suggests that the increase in AT1 receptors, and not the transporter per se, might be responsible for the enhanced ANG II-mediated stimulation of the transporters in insulin-treated cells. Although the AT1 receptors expressed in OK cell membranes are very low, the response to picomolar concentrations of ANG II in our study are also supported by other studies (14). Earlier, we have reported an increase in the AT1 receptor number and the enhanced ANG II-mediated stimulation of NHE3 in the isolated proximal tubule of obese Zucker rats (6). Although insulin has been shown to cause mild sodium retention (2, 32), the present study suggests that the upregulation of AT1 receptors and the enhanced ANG II-mediated stimulation of sodium transporters caused by chronic hyperinsulinemia may lead to a powerful antinatriuretic response to ANG II. It is likely that insulin-induced upregulated AT1 receptors may also produce other known cellular effects associated with the AT1 receptors, such as cell growth and cell proliferation (38), and contribute to the pathophysiology associated with hyperinsulinemia and obesity.
In brief, this is the first report, to our knowledge, showing that OK cells express detectable and quantifiable AT1 receptors. Insulin treatment of OK cells upregulates AT1 receptors, which on activation by ANG II produce greater stimulation of sodium transporters. Such an increase in AT1 receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and development of hypertension in conditions associated with hyperinsulinemia.
GRANTS
This study is supported by National Institutes of Health Grant DK-61578.
ACKNOWLEDGMENTS
The authors thank L. Pillai for providing technical assistance in maintaining OK cell cultures and performing Western blot analysis.
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. Section 1734 solely to indicate this fact.
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