1Department of Molecular Biomedicine, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Mexico City 07360; 2Department of Pharmacology, Facultad de Medicina, Universidad Autónoma de San Luis Potosi, San Luis Potosí 78000; and 3Department of Pathology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico
Submitted 21 March 2003 ; accepted in final form 11 September 2003
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ABSTRACT |
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renovascular hypertension; sodium reabsorption; ANG II type 1 receptor; glucose uptake
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MATERIALS AND METHODS |
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Aortic coartation. Aortic coarctation was performed according to published procedures (16). We used male Wistar rats (weighing 300-350 g). The animals were divided into the following groups: normotensive (sham operated), hypertensive (aortic coarctation), and hypertensive treated with either ramipril (an angiotensin-converting enzyme inhibitor; 2.5 mg·kg-1·day-1 for 21 days) or losartan (an AT1-receptor antagonist; 10 mg·kg-1·day-1 for 21 days). Systolic blood pressure measurements were made 21 days after surgery (16). The kidneys were quickly perfused, and the right kidney was removed to isolate brush-border membrane vesicles (BBMV) and perform immunohistochemical studies, Western blot analysis, and RT-PCR analysis.
BBMV preparation. The preparation of BBMV was carried out by two-step MgCl2 precipitation, as previously described (17). Each BBMV preparation required the use of 10 animals to obtain enough BBMV for one uptake experiment. Briefly, the renal cortices from 10 normotensive, 10 hypertensive, 10 ramipril-treated, or 10 losartantreated rats were minced and suspended in a hypotonic buffer (10 mM mannitol, 2 mM Tris-H2SO4, pH 7.4) and homogenized for 2 min with a polytron (Ultra-Turrax T-25, Janke& Kunkel, IKA Labortechnik) at 20,500 rpm. Samples of the initial homogenate and the final suspension were obtained for protein and enzyme determination. The initial homogenate was subjected to a series of MgCl2 (10 mM) precipitations and centrifugation (Sorval RC-5B, DuPont Instruments) steps. Between steps, the pellet was resuspended and homogenized with a Dounce glass pestle homogenizer (10 strikes). At the end of the procedure, the vesicles were suspended in the intravesicular buffer (100 mM mannitol, 100 mM KCl, 20 mM HEPES/Tris), frozen, and stored in liquid nitrogen until required. Protein content was determined according to the Bradford method (3). The purity of the BBMV was monitored by measuring the specific activities of leucine aminopeptidase and alkaline phosphatase (a typical brush-border marker enzyme) and Na+-K+-ATPase (a basolateral marker enzyme) according to Haase et al. (8) and Berner and Kinne (1), respectively. Enrichment factors were calculated by the relationship between enzymatic activity in the vesicles and the initial homogenate.
Transport experiments. The transport of D-glucose was measured at room temperature by a rapid filtration technique (29). The protocols were as follows.
Na+-glucose cotransporter immunoblotting. Immunoblotting analysis was used to identify the Na+-glucose cotransporter in BBMVs. Blots were then incubated overnight at 4°C with SGLT2 antibody (Alpha Diagnostic International) diluted in blocking solution (1:1,000). Blots were stained for horseradish peroxidase activity using the enhanced chemiluminescence detection system (ECL kit, Amersham Pharmacia Biotech, Piscataway, NJ) (26). After detection, samples were measured by densitometry with a Kodak electrophoresis documentation and analysis system (EDAS 290).
Immunohistochemistry. Kidneys (n = 6) from normotensive, hypertensive, and hypertensive rats treated with either ramipril or losartan were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Kidney sections (3 µm) were incubated with SGLT2 antibody (Alpha Diagnostic International) diluted 1:10. The immunoreactive signal was detected with an streptavidin-biotin-immunoperoxidase reaction (LSAB+ kit, Dako) and visualized by exposure to diaminobenzidine. Expression of SGLT2 was evaluated by computer image analysis according to a previously published method (27). We analyzed 20 noncrossed fields (770 x 58 µm, enlarged x40) per biopsy, using light microscopy with a Olympus B x51 microscope (Olympus American, Melville, NY) covering at least 80% of the cortical part of the core captured with a digital video camera. Each picture was processed on a computer and analyzed using Image-Pro and Photoshop 7, an image-processing software (Adobe Systems, San Jose, CA). Using the capabilities of color recognition by this software, we selected a specific brownish color for positive areas. After selection, these areas were quantified (pixel unit) using the histogram function of the software. For each field, the number of positive areas was expressed as a fraction of the tubule-interstisium area (positive areas divided by the overall field area). Finally, for each biopsy, the fractional amount of expression of antibody was obtained by averaging the values obtained from 20 fields examined.
RNA isolation and RT-PCR. Renal cortex was obtained from all four groups, and total RNA was isolated by the TRIzol method (GIBCO BRL, Life Technologies). Total RNA (2 µg) was converted to cDNA using a Superscript II RNAse H-Reverse Transcriptase Kit (GIBCO BRL, Life Technologies). PCR was performed with the PerkinElmer Gene Amp 2400 PCR system for 35 cycles at 94°C for 1 min, 64°C for 1 min, and 72°C for 1 min followed by a 10-min extension at 72°C. The primers utilized were 5'-ccaatagaggcacagttggtgg-3' (sense) and 5'-cgtaaatgttccacaacgg-3' (antisense) for SGLT2 and 5'-ggatttggccgtattggcc-3' (sense) and 5'-catgtcagatccacaacgg-3' (antisense) for GAPDH (28, 31). The final PCR products were 388 and 715 bp in size, respectively. The bands were analyzed with a Kodak electrophoresis documentation and analysis system (EDAS 290).
Statistics. All results are expressed as means ± SE. Significance was determined by Student's t-test or by ANOVA for cases with multiple comparisons. Significance is considered as P < 0.05.
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RESULTS |
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Purity of membrane vesicles. Table 1 shows the specific activities of enzymes in homogenate and BBMV from normotensive, hypertensive, ramipril-treated hypertensive, or losartan-treated hypertensive rats. The brush-border markers alkaline phosphatase and leucine aminopeptidase were enriched 11-fold in the final BBMV in all four experimental groups compared with the initial homogenate. Because enrichment of the brush-border markers was not different among preparations of BBMV obtained from all four groups, these preparations are suitable for comparison in glucose transport. In contrast, there was a decrease in Na+-K+-ATPase specific activity in the BBMV for all four groups compared with the initial homogenate. Enrichment of this marker was very low (0.13), indicating very little basolateral contamination in the BBMV preparations.
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Glucose uptake in kidney cortex BBMV. Figure 1 shows the time course of glucose uptake into BBMV prepared from normotensive and hypertensive rat kidney cortex. In the presence of a Na+ gradient across the vesicle membrane, there was a transient increase in the intravesicular concentration of glucose (15 s). The magnitude of the accumulation ratio and the initial rate of glucose uptake were significantly higher in the BBMV from hypertensive rats compared with the BBMV from normotensive rats. Moreover, treatment of the hypertensive rats with either ramipril or losartan decreased the maximal value (15 s) of glucose accumulation in the BBMV (Fig. 2). Elimination of the Na+ gradient by the imposition of a K+ gradient resulted in elimination of the transient increase in glucose (Fig. 1). Furthermore, inhibition of the Na+-glucose cotransporter with phlorizin decreased maximal intravesicular glucose accumulation by 84, 93, 89, and 90% in BBMV from normotensive, hypertensive, ramipril-treated hypertensive, and losartan-treated hypertensive rats, respectively. Uptake of glucose at equilibrium (5 min) was identical in the presence and absence of a Na+ gradient in all four experimental groups, indicating a similar size of membrane vesicles in all four groups of animals (14, 10, 13, and 12 pmol/mg protein) for BBMV from normotensive, hypertensive, ramipril-treated hypertensive, and losartan-treated hypertensive rats, respectively. Furthermore, in the absence of an Na+ gradient (50 mM of Na+ inside and outside the vesicle), the glucose uptake at 15 s was 0.5 ± 0.01 and 0.6 ± 0.03 pmol/mg protein for BBMV from normotensive and hypertensive rats, respectively.
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To evaluate whether alterations in the glucose transport observed in the BBMV from hypertensive rats were related to changes in maximal transport capacity (Vmax) and/or Na+-glucose transporter affinity (Km), glucose kinetics were determined. As shown in Fig. 3, Na+-dependent glucose uptake was saturable and conformed to Michaelis-Menten kinetics in BBMV from normotensive and hypertensive rats. Furthermore, glucose uptake was higher in BBMV from hypertensive rats. Kinetic parameters for all four experimental groups are shown in Table 2. Vmax values were higher in the BBMV from hypertensive rats compared with BBMV from normotensive rats. Moreover, treatment with either ramipril or losartan restored Vmax values similar to that for the BBMV from normotensive rats. In contrast, similar Km values for glucose were found in the BBMV from all experimental groups.
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Na+-glucose cotransporter immunoblotting. Western blot analysis performed to detect expression of the Na+-glucose transporter (SGLT2) in the renal homogenate showed a small detection of the protein (16 ± 5 and 20 ± 9 arbitrary units for normotensive and hypertensive rats, respectively). However, when the immunoblotting was performed in the same batch of BBMV used for transport studies, the expression of the Na+-glucose transporter in the BBMV isolated from the hypertensive rats was increased when compared with the normotensive rats, as shown in Fig. 4. In contrast in the BBMV isolated from the rats treated with either ramipril or losartan, Na+-glucose cotransporter expression was not different to the levels found in the BBMV from normotensive rats.
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Na+-glucose cotransporter immunohistochemistry. Figure 5 shows paraffin-embedded sections with periodic acid-Schiff staining. Light microscopy revealed glomerullar hypoperfusion, glomerulatis, and mesangial expansion. However, the renal tissue from neither ramiprilnor losartan-treated hypertensive rats demonstrated any of these morphological abnormalities. Intensive, positive Na+-glucose cotransporter was observed in the renal tissue of hypertensive rats (Fig. 5B). Na+-glucose cotransporters were localized in the membrane of epithelial proximal tubular cells. Less immunostaining was observed in the renal tissue of normotensive and ramipril- or losartan-treated hypertensive rats (Fig. 5, A, C, and D). Moreover, expression of the transporter evaluated by computerassisted analysis of 20 different fields for 5 rats for each group showed that the the Na+-glucose cotransporter was expressed as 1,029 ± 71, 5,003 ± 292, 2,662 ± 136, 1,830 ± 125 arbitrary units in the normotensive, hypertensive, ramipril-, and losartan-treated hypertensive rats, respectively.
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Na+-glucose mRNA expression. To obtain information as to whether the increased protein levels of the Na+-glucose cotransporter were associated with changes in transporter mRNA expression, we performed RT-PCR analysis. PCR amplification using primers specific for the Na+-glucose cotransporter (SGLT2) was normalized with the data of PCR for GAPDH in the tissue from normotensive, hypertensive, ramipril-treated hypertensive, or losartan-treated hypertensive rats. Figure 6A shows how mRNA of Na+-glucose transporter expression in renal tissue from hypertensive rats was increased fivefold compared with the expression of the mRNA of the transporter in renal tissue from normotensive rats. Furthermore, mRNA expression of the transporter in renal tissue from ramipril- or losartan-treated hypertensive rats was not different from that in normotensive rats. Moreover, to establish whether ANG II levels were responsible for induction of SGLT2 mRNA expression, we chronically (7 days) infused rats with a dose of 200 ng·kg.-1·min-1 of ANG II. Chronic ANG II infusion increased expression of renal SGLT2 mRNA to levels similar to those in rats with aortic coarctation (Fig. 6B). When we prevented the hypertensive effect of ANG II by treating ANG II-infused rats with the antihypertensive drug nifedipine (10 mg·kg-1·day-1), although an increase in blood pressure was prevented (from 160 ± 2 to 100 ± 3 mmHg for ANG II and ANG II-nifedipine-treated rats, respectively), the effect of ANG II on SGLT2 mRNA expression was not affected (Fig. 6B). Finally, to explore prostaglandins as the possible mediators of the effect of ANG II on SGLT2 mRNA expression, we treated the ANG II-infused rats with indomethacin (6 mg·kg-1·day-1). This did not affect blood pressure (153 ± 3 mmHg), and as can be seen in Fig. 6B inhibition of prostaglandin synthesis did not affect ANG II-dependent induction of SGLT2 mRNA expression.
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DISCUSSION |
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Our data agree and disagree with previous reports. Morduchowicz et al. described a decrease in Na+-dependent D-glucose uptake in renal BBMV prepared from SHR compared with control WKY rats; the reduction in glucose uptake was suggested to be related to a decrease in the density of the Na+-glucose cotransporter (18). However, ANG II levels are also decreased in SHR (4). Thus differences between our data and these previous reports may be related to the presence of ANG II. Furthermore, Parenti et al. (21) reported that under specific experimental conditions, Na+ uptake by BBMV from Milan hypertensive rats was faster than in the normotensive rats. Moreover, recently it has been reported that Na+-coupled glucose transport through SGLT1 activity is increased in renal BBMV from high-salt-loaded DS rats (14). Kinetic studies of Na+-dependent glucose uptake in jejunal BBMV from SHR and WKY suggested that enhancement in glucose transport in SHR could be due to an increase in the number of transport molecules (30).
Increased Na+-dependent glucose transport has been suggested to be due to several cellular mechanisms, such as increased phosphorylation of the cotransporter (30) or induction of trafficking of the Na+-glucose cotransporter from an intracellular pool into the BBMV (5). Thus to investigate the mechanism by which renovascular hypertension might regulate the increase in Na+-dependent glucose uptake, kinetics studies were performed as well as mRNA and protein expression in BBMV from hypertensive and normotensive rats. As can be seen in Table 2, there were no differences in the Km values in all four experimental groups. However, the Vmax value was increased in the BBMV from hypertensive rats compared with normotensive or hypertensive rats treated with the angiotensinconverting inhibitor or the AT1 receptor blocker. Thus kinetics of Na+-dependent glucose uptake suggested that increased glucose transport in the BBMV from hypertensive rats could be related to an increase in the number of transporter molecules, because the apparent Vmax of glucose transport was twofold higher in the BBMV from hypertensive rats than those from normotensive rats. In contrast, the affinity of the cotransporter was similar in BBMV from hypertensive and normotensive rats, because the apparent Km was not different in both groups of rats. An increased number of Na+-glucose cotransporters was further supported by our observations that either immunostaning or immunoblotting of the Na+-glucose cotransporter in BBMV from hypertensive rats was higher compared with the BBMV from normotensive rats. We suggest that this increase in the number of cotransporters is related to the synthesis of the protein rather than a translocation from the intracellular organelle into the membrane, as suggested previously (5). We support this hypothesis based in our observation that increased expression of protein is observed in both homogenate or BBMV, that, in immnohistochemistry, increased expression of protein is observed in cytoplasm and membrane, and that increased expression of Na+-glucose cotransporter mRNA was observed concomitantly with an increase in protein and activity. Therefore, we suggest that during development of renovascular hypertension, increased expression of Na+-glucose transporter mRNA leads to induction of cotransporter protein that expresses a functional increase in Na+-dependent glucose uptake. Thus the present results suggest the existence of an intrarenal or systemic mechanism responsible for induction of the Na+-glucose cotransporter during development of renovascular hypertension. The aortic coarctaction model of renovascular hypertension is associated with increased activity of the renin-angiotensin system.
We (11) and others (15) have reported a direct effect of ANG II on mRNA expression in renal tissue. Therefore, we explored the role of ANG II in the regulation of the expression of Na+-glucose cotransporter and increased Na+-dependent glucose uptake in renovascular hypertension by testing the hypothesis that either inhibition of ANG II synthesis by inhibition of the angiotensin-converting enzyme or blockade of the AT1 receptor could prevent increased expression of the Na+-glucose cotransporter and, thereby, increased Na+-dependent glucose uptake. Indeed, treatment of hypertensive rats with either ramipril or losartan prevented the induction of Na+-glucose transporter protein, mRNA, and activity, suggesting that ANG II could be responsible for the regulation of the Na+-glucose cotransporter. However, blockade of the ANG II system also prevents an increase in blood pressure in rats with aortic coarctation. Thus further experiments were performed to explore the role of altered tension on the regulation of Na+-glucose cotransporter status. Therefore, we used a high-ANG II-concentration model through chronic infusion of ANG II and prevented hypertension with the vasodilator nifedipine. We demonstrated that chronic ANG II infusion was associated with an increase in SGLT2 mRNA and that prevention of hypertension did not affect this effect of ANG II, suggesting that hypertension was not participating in the regulation of SGLT2 mRNA expression. We also discarded prostaglandins as the mediators of the effect of ANG II by showing that ANG II induction of SGLT2 mRNA expression was not modified by prostaglandin inhibition. Thus our data suggest that ANG II was directly responsible for increased expression of the Na+-glucose cotransporter and thereby increased epithelial Na+ and glucose uptake. Therefore, induction of the Na+-glucose cotransporter could represent a mechanism by which ANG II increases Na+ reabsorption and fluid transport across proximal tubular epithelial cells (9).
In conclusion, we have shown that aortic coarctation-induced hypertension is associated with increased Na+-glucose cotransporter activity through induction of SGLT2 protein and mRNA expression, probably by an ANG II-dependent mechanism. Increased Na+ reabsorption through the Na+-glucose cotransporter may be participating in the development of hypertension. Additionally, increased glucose epithelial uptake may play an important role in the adaptation of glucose transport to hypertension.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by Mexican Council of Science and Technology (CONACYT) Grant 31048. R. Bautista was a fellow of CONACYT.
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FOOTNOTES |
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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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REFERENCES |
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