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Am J Physiol Renal Physiol 286: F634-F642, 2004. First published November 25, 2003; doi:10.1152/ajprenal.00217.2003
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Signaling cascade of ANG II-induced inhibition of {alpha}-MG uptake in renal proximal tubule cells

Ho Jae Han, Soo Hyun Park, and Yun Jung Lee

Department of Veterinary Physiology, College of Veterinary Medicine, Hormone Research Center, Chonnam National University, Gwangju 500-757, Korea

Submitted 10 June 2003 ; accepted in final form 10 November 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANG II and Na+-glucose cotransporter have been reported to be associated with the onset of diverse renal diseases. However, the effect of ANG II on Na+-glucose cotransporter activity was not elucidated. The effects of ANG II on {alpha}-methyl-D-[14C]glucopyranoside ({alpha}-MG) uptake and its related signal pathways were examined in the primary cultured rabbit renal proximal tubule cells (PTCs). ANG II (>2 h; >10-9 M) inhibited {alpha}-MG uptake in a time- and concentration-dependent manner and decreased the protein level of Na+-glucose cotransporters, the expression of which was abrogated by both actinomycin D and cycloheximide exposure. ANG II-induced inhibition of {alpha}-MG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD-123319, an ANG II type 2 receptor blocker. ANG II-induced inhibition of {alpha}-MG uptake was blocked by genistein, herbimycin A [tyrosine kinase (TK) inhibitors], mepacrine, and AACOCF3 (phospholipase A2 inhibitors), suggesting the role of TK phosphorylation and arachidonic acid (AA). Indeed, ANG II increased AA release, which was blocked by losartan or TK inhibitors. The effects of ANG II on AA release and {alpha}-MG uptake also were abolished by staurosporine and bisindolylmaleimide I (protein kinase C inhibitors) or PD-98059 (p44/42 MAPK inhibitor), but not SB-203580 (p38 MAPK inhibitor), respectively. Indeed, ANG II increased p44/42 MAPK activity. ANG II-induced activation of p44/42 MAPK was blocked by staurosporine. In conclusion, ANG II inhibited {alpha}-MG uptake via PKC-MAPK-cPLA2 signal cascade through the AT1 receptor in the PTCs.

angiotensin II; kidney; mitogen activated protein kinase; phospholipase A2; protein kinase C; sodium ion-glucose cotransporter; {alpha}-methyl-D-[14C]glucopyranoside

A GROWING BODY OF EVIDENCE indicates that ANG II plays an important role in the pathophysiology of renal diseases in humans (31, 50). Accumulating in vivo evidence also supports the possibility that ANG II may directly cause renal diseases, independent of its blood pressure-elevating effect (7, 31). The renal proximal tubule can synthesize and secrete ANG II in the lumen, and luminal ANG II levels are 100- to 1,000-fold higher than that found in plasma (47). The action of ANG II is mediated by two pharmacologically and biochemically distinct classes of receptors, ANG II type 1 (AT1) and ANG II type 2 (AT2). The signaling events elicited in renal cells by ANG II were considered to be divided into separate linear pathways, where intracellular targets of phospholipase A2 (PLA2) were distinct from those of the phospholipase C (PLC)/protein kinase C (PKC) and tyrosine kinase (TK)/mitogen-activated protein kinase (MAPK)-dependent pathways (21, 44). Although there is a possibility that these major intracellular signaling cascades do not function independently and are actively engaged in cross-talk, there are still unresolved questions and controversies on the active mechanisms of ANG II.

Na+-glucose cotransporters (SGLTs) are expressed in the proximal tubule, where they play a central role in the reabsorption of glucose from the glomerular filtrate. SGLT1 and SGLT2 are expressed in the rabbit renal proximal tubule cells (PTCs; see Ref. 33). Recent reports including our works suggest that the activity of SGLT is related to the development of diabetic nephropathy (15, 16, 29). ANG II also has been reported to be involved in the onset of diabetic nephropathy (5). Our previous studies have demonstrated that ANG II in the PTCs is associated with the pathogenesis of the diabetic nephropathy, since high glucose downregulated ANG II binding because of the increase of ANG II synthesis (35, 36). However, little is known about the effect of ANG II on {alpha}-methyl-D-[14C]glucopyranoside ({alpha}-MG) uptake and its related signal pathways in PTCs, although several lines of evidence suggest that ANG II stimulates glucose transport in rat proximal straight tubules (14) or in cultured vascular smooth muscle cells (37).

When grown in a hormonally defined medium, primary cultured renal PTCs form confluent monolayers of polarized cells, which retain a number of differentiated transport functions typical of the renal PTCs (9). Included among these transport functions are a probenecid-sensitive p-aminohippurate transport system, an Na+-dependent sugar transport system, and an Na+-dependent Pi transport system (9, 17, 52). The results of studies concerning these membrane transport systems in PTCs are directly comparable to results obtained with original renal tissue (49). The PTCs respond to a number of hormones known to affect renal PTCs in vivo, including insulin (which inhibits phosphoenolpyruvate carboxykinase activity at physiological concentrations; see Ref. 48) and parathyroid hormone (which is stimulatory to adenylate cyclase; see Ref. 43). The PTCs lack a similar responsiveness to arginine vasopressin and calcitonin, indicating the PTC culture preparation is highly purified (9). More recently, we have reported a dose-dependent, biphasic effect of ANG II on Na+ uptake by the PTCs, consistent with results obtained with intact renal tissue (18). Therefore, PTCs in hormonally defined, serum-free culture conditions would be a powerful tool for studying the effect of ANG II on {alpha}-MG uptake of renal PTCs. Thus we investigated the effect of ANG II on {alpha}-MG uptake and its related signal cascades in the PTCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. New Zealand White male rabbits (1.5–2.0 kg) were purchased from Dae Han Experimental Animals (Chungju, Korea). Class IV collagenase and soybean trypsin inhibitor were purchased from Life Technologies (Grand Island, NY). ANG II, actinomycin D, cycloheximide, ouabain, mepacrine, AACOCF3, staurosporine, bisindolylmaleimide I, arachidonic acid (AA), genistein, herbimycin A, indomethacin, econazole, nordihydroguaiaretic acid (NDGA), 12-O-tetradecanoylphorbol-13-acetate (TPA), PD-98059, and SB-203580 were obtained from Sigma Chemical (St. Louis, MO). SQ-22536, the Rp diastereomer of cAMP (Rp-cAMP), and protein kinase inhibitor amide-(14–22) (PKI) were purchased from Calbiochem (San Diego, CA). {alpha}-MG, AA, 3-O-methyl-D-[14C]glucose (3-O-[14C]MG), [3H]fructose, L-[3H]arginine, and [3H]alanine were purchased from Dupont/NEN (Boston, MA). The [3H]cAMP assay system (code TRK 432) was purchased from Amersham International (Buckinghamshire, UK). Rabbit anti-SGLT1 was purchased from Chemicon International (Temecula, CA), and rabbit anti-SGLT2 was from Alpha Diagnostic International (San Antonio, TX). Phospho-p44/42 MAPK or p44/42 MAPK antibody was purchased from New England Biolabs (Herts, UK). Goat anti-rabbit-IgG was purchased from Jackson ImmunoResearch (West Grove, PA). All other reagents were of the highest purity commercially available. Liquiscint was obtained from National Diagnostics (Parsippany, NY).

Cell preparation and culture condition. Primary rabbit renal PTC cultures were prepared by the method of Chung et al. (9). The PTCs were grown in DMEM-Ham's nutrient mixture F-12 (F-12) medium (GIBCO-BRL, Gaithersburg, MD) with 15 mM HEPES and 20 mM sodium bicarbonate (pH 7.4). Immediately before the use of the medium, three growth supplements (5 µg/ml insulin, 5 µg/ml transferrin, and 5 x 10-8 M hydrocortisone) were added. The kidneys of a rabbit were perfused via the renal artery, first with PBS and then with medium containing 0.5% iron oxide. Renal cortical slices were prepared and homogenized. The homogenate was poured first through a 253-µm and then an 83-µm mesh filter. Tubules and glomeruli on top of the 83-µm filter were transferred to sterile medium. Glomeruli (containing iron oxide) were removed with the stirring bar. The remaining tubules were incubated briefly in medium. The tubules were then washed by centrifugation, resuspended in medium containing the three supplements, and transferred to culture dishes. Medium was changed 1 day after plating and every 2 days thereafter. Primary cultured rabbit kidney PTCs were maintained in a 37°C, 5% CO2 humidified environment in a serum-free basal medium supplemented with three growth supplements. PTCs were confluent and quiescent for 48 h, and then experiments were conducted.

{alpha}-MG uptake. {alpha}-MG uptake experiments were conducted as described by the method of Sakhrani et al. (38). To study {alpha}-MG uptake, the culture medium was removed by aspiration, and monolayers were washed gently two times with the uptake buffer (136 mM NaCl, 5.4 mM KCl, 0.41 mM MgSO4, 1.3 mM CaCl2, 0.44 mM Na2HPO4, 0.44 mM KH2PO4, 5 mM HEPES, 2 mM glutamine, and 0.5 µg/ml BSA, pH 7.4). After the washing procedure, the monolayers were incubated at 37°C for 30 min in an uptake buffer that contained 0.5 mM {alpha}-MG and {alpha}-[14C]MG (0.5 µCi/ml). At the end of the incubation period, the monolayers were again washed three times with ice-cold uptake buffer, and the cells were solubilized in 1 ml of 0.1% SDS. To determine the {alpha}-[14C]MG incorporated intracellulary, 900 µl of each sample were removed and counted in a liquid scintillation counter (LS 6500; Beckman Instruments, Fullerton, CA). The remainder of each sample was used for protein determination (4). 3-O-[14C]MG uptake was conducted using 3-O-[14C]MG instead of {alpha}-[14C]MG. The next steps were conducted as described in {alpha}-MG uptake. Fructose, L-arginine, and alanine uptake was conducted according to the method of Yudilevich and Sweiry (53), Crouzoulon and Korieh (10), and Mounfield and Robson (24), respectively. The radioactivity counts in each sample were then normalized with respect to protein and were corrected for time 0 uptake per milligram protein. All uptake measurements were made in triplicate.

AA release. [3H]AA release experiments were performed by a modification of the method of Xing et al. (51). Confluent monolayers of PTC cultures were incubated for 24 h in DMEM-F-12 medium containing 0.5 µCi [3H]AA/ml and the three growth supplements. The monolayers were then washed three times with DMEM-F-12 (pH 7.4) and incubated (at 37°C) for 1 h in uptake buffer containing the specified agents at appropriate concentrations. At the end of the incubation period, the incubation medium was removed by aspiration and transferred to ice-cold tubes containing 100 µl of 55 mM EGTA (final concentration, 5 mM each). The uptake buffer was then centrifuged at 12,000 g to eliminate cell debris. To determine the level of radioactivity in the supernatant, the samples were placed in scintillation vials containing scintillation fluid, and the radioactivity was counted using a liquid scintillation counter. The cells that remained attached to the plate were scraped into 1 ml of 0.1% SDS. The 900 µl of the resulting cell lysate were used for scintillation counting. The remaining 100 µl of the cell lysate were used for protein determinations. For each condition, the quantity of [3H]AA that had been released (determined as described above) was first standardized with respect to protein. Subsequently, this standardized level of released [3H]AA was compared by the percentage to the total level of [3H]AA that had been incorporated in the cells at the beginning of the incubation period (the total released radioactivity plus the total cell-associated radioactivity at the end of the stimulation period).

Membrane preparation for SGLTs blotting. Medium of confluent PTCs was exchanged 1 day before the experiment. The medium was then removed, and the cells were washed two times with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in buffer A [137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF, and 10 µg/ml leupeptin (pH 7.5)]. The resuspended cells were then mechanically lysed on ice by trituration with a 21.1-gauge needle. The lysates were first centrifuged at 1,000 g for 10 min at 4°C. The supernatants were centrifuged at 100,000 g for 1 h at 4°C to prepare cytosolic and total particulate fractions. The particulate fractions, which contained the membrane fraction, were washed two times and resuspended in buffer A containing 1% (vol/vol) Triton X-100. The protein in each fraction was quantified with a Bradford (4) procedure.

Western blot analysis. Cell homogenates (20 µg protein) were separated on 10% SDS-PAGE and transferred to nitrocellulose paper. Blots were then washed with H2O, blocked with 5% skimmed milk powder in 10 mM Tris·HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween 20 for 1 h, and incubated with the appropriate primary antibody at dilutions recommended by the supplier. Next, the membrane was washed, and primary antibodies were detected with goat anti-rabbit-IgG conjugated to horseradish peroxidase, and the bands were visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech)

125I-labeled ANG II binding. The ANG II binding assays were performed as described by Becker and Harris (3). To summarize, after the incubation of PTCs with losartan or PD-123319 at different dosages (10-9, 10-7, and 10-5 M) for 30 min, confluent monolayers of PTCs were washed two times with ice-cold PBS containing 0.1% albumin (PBS-A), and cells were incubated in PBS-A, supplemented with 125I-labeled [Sar1,Ile8]ANG II (0.1 nM) at 4°C for 4 h, followed by three washes with the same ice-cold PBS-A. After solubilization in 0.5 N NaOH (1 ml), 900 µl of each sample were transferred to a scintillation tube and counted in a gamma counter (Wizard 1470; Wallac, Turku, Finland). The remainder of each sample was used for protein determination by the Bradford (6) method.

cAMP assay. The confluent PTCs were then incubated with a different dosage of ANG II (0 to 10-7 M) for 8 h at 37°C in a humidified, 5% CO2-95% air environment. Subsequently, samples were prepared for intracellular cAMP determinations by homogenization in serum-free media containing 4 mM EDTA using a Polytron PT 1200, followed by a 5-min incubation at 100°C. After centrifugation at 890 g for 5 min, the supernatants were transferred to new tubes and stored at 4°. These samples were used for cAMP assays, using a [3H]cAMP assay system. Values were expressed as picomoles cAMP per milligram protein.

Statistical analysis. Results were expressed as means ± SE. The difference between two mean values was analyzed by the nonparametric Wilcoxon sign test or ANOVA. The difference was considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of ANG II on {alpha}-MG uptake. To determine whether ANG II affects {alpha}-MG uptake in a time-dependent manner, PTCs were exposed to 10-7 M ANG II for various time intervals (Fig. 1A). ANG II significantly inhibited {alpha}-MG uptake over 2 h and exhibited a maximal inhibitory effect between 8 and 12 h. To determine the concentration dependency of ANG II on {alpha}-MG uptake, PTCs were exposed to various concentrations of ANG II for 8 h. ANG II above 10-9 M inhibited {alpha}-MG uptake, and maximum inhibition of {alpha}-MG uptake was observed at 10-7 M ANG II (Fig. 1B). To validate the above-mentioned effect of ANG II on {alpha}-MG uptake in the PTCs, we additionally examined the effect of ANG II on Na+ transport. As shown in Fig. 1C, ANG II has biphasic effects on Na+ uptake; a low concentration of ANG II (10-11 M) stimulates Na+ uptake, whereas a high concentration of ANG II (>=10-9 M) inhibits it. On the other hand, in the experiments to examine specificity of the ANG II on {alpha}-MG uptake, ANG II did not affect 3-O-MG (control: 470 ± 34 vs. ANG II: 447 ± 34 pmol·mg protein-1·min-1), fructose (83 ± 6 vs. 89 ± 4 pmol·mg protein-1·min-1), L-arginine (122 ± 5 vs. 118 ± 8 pmol·mg protein-1·min-1), and alanine (75 ± 3 vs. 73 ± 7 pmol·mg protein-1·min-1) uptakes. On the other hand, ANG II-induced inhibition of {alpha}-MG uptake may be, in part, mediated by the inhibition of Na+-K+-ATPase, since ouabain in the absence of ANG II inhibited {alpha}-MG uptake (Fig. 2).



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  		Fig. 1. Time and dose response of ANG II on {alpha}-methyl-D-[14C]glucopyranoside ({alpha}-MG) uptake and Na+ uptake. Proximal tubule cells (PTCs) were treated with ANG II (10-7 M) for different times (A; 0–12 h) or dosages of ANG II (B and C; 0–10-6 M) before the uptake experiment for 8 h. Values are means ± SE of 4 independent experiments with triplicate dishes. *P < 0.05 vs. control.

 


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  		Fig. 2. Effect of ouabain on ANG II-induced inhibition of {alpha}-MG uptake. PTCs were treated with ouabain (5 x 10-7 M) for 30 min before the treatment of ANG II (10-7 M) for 8 h. Values are means ± SE of 5 independent experiments with triplicate dishes. P < 0.05 vs. control (*) or ANG II alone (**).

 

To determine whether RNA and protein synthesis are involved in the ANG II-induced inhibition of {alpha}-MG uptake, PTCs were treated with actinomycin D (a transcription inhibitor, 10-7 M) or cycloheximide (a translation inhibitor, 4 x 10-5 M) for 30 min before the treatment of ANG II. Actinomysin D and cycloheximide blocked ANG II-induced inhibition of {alpha}-MG uptake (Fig. 3). In experiments to determine effects of ANG II on the protein expression level of SGLTs, ANG II (10-7 M, 8 h) significantly decreased the SGLT1 and SGLT2 protein levels. Western blots probed with anti-{beta}-actin showed no differences in the loading protein levels. Consistent with the {alpha}-MG uptake result, actinomysin D and cycloheximide blocked the ANG II-induced decrease of both SGLT1 and SGLT2 protein expression (Fig. 4). Densitometric analysis demonstrated that 8 h of exposure to 10-7 M ANG II significantly decreased SGLT1 or SGLT2 band density to 73 ± 8 and 49 ± 25% of control, respectively.



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  		Fig. 3. Effects of actinomycin D and cycloheximide on ANG II-induced inhibition of {alpha}-MG uptake. PTCs were treated with actinomycin D (10-7 M) and cycloheximide (4 x 10-5 M) for 30 min before the treatment of ANG II (10-7 M) for 8 h. Values are the means ± SE of 3 independent experiments with triplicate dishes. P < 0.05 vs. control (*) and ANG II alone (**).

 


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  		Fig. 4. Effect of ANG II on Na+/glucose cotransporter (SGLT) protein expression. SGLT protein expression was determined by Western blotting with membrane protein obtained from PTCs that were treated with ANG II (10-7 M) for 8 h. Blots were probed with SGLT1, SGLT2, or {beta}-actin specific antibody. Bands represent 70–77 kDa of SGLT1 and SGLT2 and 41 kDa of {beta}-actin. Bars denote means ± SE of 3 experiments for each condition determined from densitometry relative to {beta}-actin. AD, Actinomycin D; CHX, cycloheximide D. *P < 0.05 vs. each control.

 

To determine whether the inhibition of {alpha}-MG uptake induced by ANG II is mediated by AT receptors, PTCs were treated with losartan or PD-123319 in a dose-dependent manner before [125I]ANG II binding assay. Losartan inhibits [125I]ANG II binding in a dose-dependent manner (Fig. 5A). However, PD-123319 even at high dosage (10-5 M) has a mild inhibition effect of [125I]ANG II binding. To investigate the specific receptor on ANG II-induced inhibition of {alpha}-MG uptake, losartan (10-6 M), an AT1 receptor antagonist, or PD-123319 (10-6 M), an AT2 receptor antagonist, was added to the PTCs for 30 min before the treatment of ANG II. Losartan, not PD-123319, blocked ANG II-induced inhibition of {alpha}-MG uptake (Fig. 5B).



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  		Fig. 5. Distribution of ANG II receptor subtype. A: effect of losartan and PD-123319 on specific binding of 125I-ANG II. PTCs were treated with losartan or PD-123319 (0 to 10-5 M) for 30 min before the treatment of 125I-ANG II (0.1 nmol/ml). B: effect of losartan and PD-123319 on ANG II-induced inhibition of {alpha}-MG uptake. PTCs were treated with losartan or PD-123319 (10-6 M, respectively) for 30 min before treatment of ANG II (10-7 M) for 8 h. Values are means ± SE of 3 independent experiments with triplicate dishes. P < 0.05 vs. control (*) and ANG II alone (**).

 

Role of AA in ANG II-induced inhibition of {alpha}-MG uptake. To determine whether phosphorylation of TK is involved in the effect of ANG II on {alpha}-MG uptake, PTCs were treated with genistein or herbimycin A (10-6 M, TK inhibitors) before the treatment of ANG II (10-7 M). Genistein and herbimycin A completely blocked ANG II-induced inhibition of {alpha}-MG uptake (Fig. 6). We also investigated the relation of the PLA2 signal pathway in ANG II-induced inhibition of {alpha}-MG uptake. Thus PTCs were treated with mepacrine or AACOCF3 (10-6 M), PLA2 inhibitors, for 30 min before the treatment of ANG II (10-7 M). Mepacrine and AACOCF3 blocked ANG II-induced inhibition of {alpha}-MG uptake, suggesting that AA is involved in ANG II-induced inhibition of {alpha}-MG uptake (Fig. 7A). Indeed AA inhibits {alpha}-MG uptake. Furthermore, indomethacin (a cyclooxygenase inhibitor) and econazole (a cytochrome P-450 epoxygenase inhibitor), but not NDGA (a lipoxygenase inhibitor, 10-6 M), abolished ANG II-induced inhibition of {alpha}-MG uptake (Fig. 7B). PGE2 (>10-9 M), one of cyclooxygenase metabolites, and 5,6-epoxy-eicosatrienoic acid (EET, >10-8 M), one of the cytochrome P-450 metabolites, inhibited {alpha}-MG uptake (Fig. 7B, inset). ANG II (10-7 M, 1 h) increased AA release (Fig. 8). Mepacrine and AACOCF3 prevented the ANG II-induced increase of AA release (data not shown). When PTCs were treated with losartan (an AT1 receptor antagonist), genistein, or herbimycin A (TK inhibitors; 10-6 M) before the treatment of ANG II, AA release induced by ANG II was also blocked. AA, PGE2, or 5,6-EET inhibits {alpha}-MG uptake in the presence of TK inhibitors (Fig. 8, inset) or PLA2 inhibitors (data not shown).



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  		Fig. 6. Effect of genistein or herbimycin A on ANG II-induced inhibition of {alpha}-MG uptake. PTCs were treated with genistein or herbimycin A (10-6 M, respectively) for 30 min before treatment of ANG II (10-7 M) for 8 h. Values are means ± SE of 3 independent experiments with triplicate dishes. P < 0.05 vs. control (*) and ANG II alone (**).

 


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  		Fig. 7. Effect of mepacrine and AACOCF3 on ANG II-induced inhibition of {alpha}-MG uptake. A: PTCs were treated with mepacrine or AACOCF3 for 30 min before the treatment of ANG II (10-7 M) for 8 h or were incubated with ANG II alone or together with arachidonic acid (AA, 10-7 M) for 8 h. B: effect of nordihydroguiareic acid (NDGA), indomethacin, and econazole on ANG II-induced inhibition of {alpha}-MG uptake. PTCs were treated with NDGA, indomethacin, or econazole for 30 min at a concentration of 10-7 M before the treatment of ANG II for 8 h. Inset represents the effect of PGE2 and 5,6 epoxy-eicosatrienoic acid (EET) on {alpha}-MG uptake. PTCs were treated with PGE2 (10-9 or 10-7 M) or 5,6-EET (10-8 or 10-6 M) for 8 h. Values are means ± SE of 4 independent experiments with triplicate dishes. P < 0.05 vs. control (*) and ANG II alone (**).

 


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  		Fig. 8. Effect of losartan, genistein, and herbimycin A on ANG II-induced increase of [3H]AA release. PTCs were treated with losartan, genistein, or herbimycin (10-6 M) for 30 min before treatment of ANG II (10-7 M) for 1 h. Inset: effect of genistein and herbimycin A on AA-induced inhibition of {alpha}-MG uptake. PTCs were treated with genistein or herbimycin A for 30 min before treatment of AA (10-7 M) for 8 h. Values are means ± SE of 3 independent experiments with triplicate dishes. P < 0.05 vs. control (*) or ANG II alone (**).

 

Involvement of cAMP/PKC in ANG II-induced increase of AA release and inhibition of {alpha}-MG uptake. To identify the role of the cAMP signal pathway in the ANG II-induced inhibition of {alpha}-MG uptake, PTCs were treated with SQ-22536 (10-6 M, an adenylate cyclase inhibitor), Rp-cAMP (10-5 M, cAMP analog), and myristoylated PKI amide-(14–22) (10-5 M, a protein kinase A inhibitor) before the treatment of ANG II. SQ-22536, Rp-cAMP, or PKI did not block the ANG II-induced inhibition of {alpha}-MG uptake (Fig. 9A). Indeed, ANG II did not affect cAMP content (Fig. 9B). This result suggests that cAMP pathways are not involved in the effect of ANG II on {alpha}-MG uptake. To examine the involvement of PKC on the ANG II-induced increase of AA, PTCs were treated with TPA (100 ng/ml, a PKC activator) alone or in combination with ANG II. TPA increased [3H]AA release by 68 ± 9% compared with control, suggesting the possibility that PKC is involved in the ANG II-induced increase of [3H]AA release (Fig. 10). TPA also inhibits {alpha}-MG uptake by 72 ± 11% compared with control. Thus, to investigate the involvement of PKC in the effect of ANG II, PTCs were treated with staurosporine (10-7 M) or bisindolylmaleimide I (10-6 M) (PKC inhibitors) for 30 min before the treatment of ANG II. Staurosporine and bisindolylmaleimide I completely blocked the ANG II-induced increase of [3H]AA release and inhibition of {alpha}-MG uptake (Fig. 10). These results indicate that PKC is a more upstream regulator than PLA2 in the ANG II-induced increase of [3H]AA release and inhibition of {alpha}-MG uptake. In addition, we examined whether TPA could inhibit {alpha}-MG uptake in the presence of TK inhibitors. TPA-induced inhibition of {alpha}-MG uptake was not blocked by genistein and herbimycin A (Fig. 10, inset). These results indicate that the inhibitory effect of ANG II and {alpha}-MG uptake via the PKC signaling pathway is downstream of TK phosphorylation.



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  		Fig. 9. Effect of cAMP on ANG II-induced inhibition of {alpha}-MG uptake. A: PTCs were treated with SQ-22536 (10-5 M), the Rp diastereomer of cAMP (Rp-cAMP, 10-5 M), or protein kinase inhibitor (PKI, 10-6 M) for 30 min before treatment of ANG II (10-7 M) for 8 h. B: PTCs were treated with different dosages of ANG II (0–10-7 M) for 8 h before cAMP assay. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control.

 


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  		Fig. 10. Effect of staurosporine, bisindolylmaleimide I, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on ANG II-induced increase of [3H]AA (A) and inhibition of {alpha}-MG uptake (B). PTCs were treated with staurosporine (10-7 M) or bisindolylmaleimide I (10-7 M) for 30 min before the treatment of ANG II (10-7 M) or were incubated with ANG II (10-7 M) alone or together with TPA. Inset represents the effect of genistein and herbimycin A on TPA-induced inhibition of {alpha}-MG uptake. PTCs were treated with genistein or herbimycin A for 30 min before the treatment of TPA (100 ng/ml) for 8 h. Values are means ± SE of 3 independent experiments with triplicate dishes. P < 0.05 vs. control (*) or ANG II alone (**).

 

Involvement of p44/42 MAPK in ANG II-induced increase of AA release and inhibition of {alpha}-MG uptake. Because MAPK signal pathways are involved in the action of ANG II, we examined the role of MAPKs in the ANG II-induced effect on AA release and {alpha}-MG uptake. PTCs were treated with MAPK blockers for 30 min before the treatment of ANG II. PD-98059 (>10-7 M) completely blocked the ANG II-induced increase of AA release and inhibition of {alpha}-MG uptake, whereas SB-203580 did not (Fig. 11). TK inhibitor or p44/42 MAPK inhibitor does not abolish the inhibition of {alpha}-MG uptake induced by AA (data not shown). It suggests that TK or MAPK is upstream of the AA signal pathway. These results suggest that p44/42 MAPK activation is needed to have an inhibitory effect of {alpha}-MG uptake by ANG II. Indeed, ANG II rapidly induced p44/42 MAPK activation over 5 min (Fig. 12A). There was a sustained activation of p44/42 MAPK by 2 h. In addition, ANG II-induced activation of p44/42 MAPK was blocked by PKC inhibitor (Fig. 12B).



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  		Fig. 11. Effect of PD-98059 and SB-203580 on ANG II-induced increase of [3H]AA release (A) and inhibition of {alpha}-MG uptake (B). PTCs were treated with PD-98059 (a p44/42 MAPK inhibitor) and SB-203580 (a p38 MAPK inhibitor, 10-7 and 10-6 M) for 30 min before the treatment of ANG II (10-7 M) for 8 h. Values are means ± SE of 3 independent experiments with triplicate dishes. P < 0.05 vs. control (*) or ANG II alone (**).

 


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  		Fig. 12. Time course of phosphorylated p44/42 MAPK by ANG II (A) and effect of staurosporine or PD-98059 on ANG II-induced phosphorylated p44/42 MAPK (B). PTCs were treated with different time periods (0–480 min) of ANG II (10-7 M) or were treated with staurosporine (10-7 M) or PD-98059 (10-6 M) for 30 min before the treatment of ANG II (10-7 M) for 8 h. A and B: top, phosphorylated p44/42 MAPK; bottom, total p44/42 MAPK. The example shown is representative of 4 experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
First, this study demonstrated that ANG II inhibited {alpha}-MG uptake via TK-PKC-MAPK-cytosolic (c) PLA2 signal cascades in the PTCs. We previously reported that the treatment of ANG II for 4 h has a biphasic effect on Na+ transport in the PTCs; a picomolar range of ANG II stimulates Na+ transport, whereas a micromolar range of ANG II inhibits it (18). However, there is no biphasic effect of ANG II on SGLT, indicating that ANG II differently regulates {alpha}-MG uptake, unlike Na+ transport. Glucose in the lumen is reabsorbed by SGLTs in the brush-border membrane, accumulated within the cells, and transported out of the cell across the basolateral membrane by a facilitated sugar transporter (33). In the present study, ANG II did not affect 3-O-MG, fructose, L-arginine, and alanine transporter, which is expressed in the renal proximal tubule (12, 41). It suggests that ANG II specifically regulates SGLTs. The concentration of ANG II used in this study has an important pathophysiological significance in the regulation of renal proximal tubule functions, since 1) the concentration of ANG II in the kidney is 1,000 times higher than the normal range of plasma (39), 2) plasma ANG II concentrations have been estimated to range from 10-11 to 3 x 10-9 M (under conditions of sodium depletion; see Ref. 32), and 3) the concentration of ANG II is increased in diabetic animals (46). Recently, we have demonstrated that high glucose inhibited {alpha}-MG uptake in PTCs (15, 16). Others have demonstrated that high glucose resulted in a reduction of the expression and activity of SGLTs (23) and that SGLT activity was decreased in brush-border membrane vesicles of diabetic rats (19). In the present study, we also found that ANG II reduces the expression of SGLT1 and SGLT2 and {alpha}-MG uptake. Our results and those of others suggest that ANG II is involved in the high glucose-induced inhibition of {alpha}-MG uptake through increasing of ANG II by high glucose (15, 16). Consistent with our present and previous results (18), Burns and colleague (6) reported that the binding of ANG II to the brush-border membrane of rabbit renal PTCs was inhibited by AT1 antagonist but not with an AT2 antagonist. It suggests that the effects of ANG II are mediated by AT1 receptor in rabbit renal PTCs. However, Dulin et al. (11) reported that Dup-753 did not displace binding of 125I-[Sar1]ANG II from rabbit brush-border membrane.

ANG II elicits highly implicated cascades of intracellular signal transduction that lead to renal function, such as regulation of SGLT in the proximal tubules. Binding of ANG II to the AT1 receptor stimulates PLC/PKC, PLA2, TK, and MAPK, and its events are early signaling processes occurring within minutes (21, 44). In the present study, TK activation is required for the action of ANG II. ANG II via AT1 receptor rapidly activates TK in the rabbit PTCs (22). Our present studies have shown that the effects of ANG II on {alpha}-MG uptake are mediated by cPLA2 and the metabolites of cPLA2. This result is supported by the reports that the proximal tubule is rich in cytochrome P-450 enzyme activity (13) and that cyclooxygenase inhibitor blocked the effect of ANG II and ANG II increased PGE2 synthesis in rabbit PTCs although the level of cyclooxygenase in proximal tubules is minimal (1, 28, 18). Of interest, in the present study, the treatment of 5,6-EET and PGE2, metabolites of AA, for 8 h mimicked the effect of ANG II, but they did not affect {alpha}-MG uptake in the short time of incubation (30 min; unpublished data). This result suggests that metabolites of AA may be involved in the reduction of SGLT expression, resulting in the decrease of {alpha}-MG uptake of PTCs exposed to ANG II.

The present results also demonstrated that ANG II-induced AA release is involved in PKC in PTCs. Several investigators have demonstrated that PKC inhibitors block AA or PGE2 release induced by ANG II in rat vascular smooth muscle cells (27) or aortic endothelial cells (30), and rabbit renal SGLT1 has a PKC-binding site and is modulated by PKC (20). In addition, ANG II elicited translocation of PKC-{alpha} in the PTCs (8) and stimulated Na+-K+(NH4+)-2Cl- cotransport via the PKC pathway in rat medullary thick ascending limbs (2). Our present results and those of others suggest that PKC activates the expression or translocation of SGLT, leading to a change of the kinetics of the transporter such as substrate affinity, maximum velocity, or turnover number. In rabbit aortic smooth muscle cells, PD-98059 attenuates the ANG II-induced increase of AA release and cPLA2 activation (27). Parenti et al. (34) demonstrated that ANG II activated p44/42 MAPK phosphorylation in PTCs from Wistar-Kyoto rats. Here, we demonstrated that p44/42 MAPK mediated ANG II-induced inhibition of {alpha}-MG uptake. Indeed, we observed that ANG II activated p44/42 MAPK. Recent reports also have demonstrated that ANG II-induced activation of p44/42 MAPK is regulated by PKC in PTCs and human embryo kidney cell lines (41, 45). However, some studies suggested that ANG II-induced p44/42 MAPK activation was PKC independent (25, 26). Our results show that PKC regulates the activation of p44/42 MAPK induced by ANG II, indicating the flow of ANG II-PKC-p44/42 MAPK. This is the first report, to our knowledge, that PKC, MAPK, and AA are responsible for the regulation of SGLT by ANG II in PTCs. These results suggest that alterations of these highly regulated SGLTs by ANG II may be a pivotal role in pathogenesis of renal disease, such as diabetic nephropathy, which increased the intrarenal ANG II level (40, 54).

Taken together, we illustrated a hypothetical model of the signaling mechanisms involved in mediating the ANG II-induced inhibition of SGLT (Fig. 13). In summary, ANG II activates AT1 receptor and stimulates PKC, which triggers p44/42 MAPK activation. Subsequently, p44/42 MAPK activation induces the release of [3H]AA, which is metabolized to 5,6-EET or PGE2 by cyclooxygenase and cytochrome P-450. These molecules may induce the decrease of SGLTs protein expression, which is involved in inhibition of SGLT activity of renal PTCs.



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  		Fig. 13. Hypothesized model of inhibition of SGLTs ({alpha}-MG uptake) by ANG II in renal PTCs. ANG II binding to AT1 receptor stimulates TK or PKC, which induces p44/42 MAPK activation. Subsequently, p44/42 MAPK activation induces the release of [3H]AA, which may be metabolized to cyclooxygenase and cytochrome P-450 metabolites, such as PGE2 and 5,6-EET. These molecules may induce the decrease of SGLT expression, which is involved in inhibition of the {alpha}-MG uptake. AT1R, ANG II receptor type 1; TK, tyrosine kinase; PKC, protein kinase C; MAPK (p44/42), p44/42 mitogen-activated protein kinase; cPLA2, cytosolic phospholipase A2; AA, arachidonic acid. Solid line, proposed pathway; broken line, suspected pathway.

 


   ACKNOWLEDGMENTS
 
GRANTS

This research was supported by Grant SC14032 from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Han, Dept. of Veterinary Physiology, College of Veterinary Medicine, Chonnam National Univ., Gwangju 500-757, Korea (E-mail: hjhan{at}chonnam.ac.kr).

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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