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Renal glucose reabsorption during hypertonic glucose infusion in female streptozotocin-induced diabetic rats
Life Sciences 68 (2001) 2967–2977
Renal glucose reabsorption during hypertonic glucose infusion in female streptozotocin-induced diabetic rats W.T. Noonan, V.M. Shapiro, R.O. Banks* Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH 45267-0576, USA Received 24 April 2000; accepted 27 October 2000
Abstract Information regarding the renal glucose transport capacity in diabetes mellitus is limited. These data are needed because two weeks following injection of streptozotocin (STZ), mRNA and protein levels of the glucose transporter, GLUT2, are upregulated in the proximal tubule of the rat. Therefore, we measured renal glucose transport and GLUT2 protein levels in female control rats, and in rats one (STZ-1), two (STZ-2), and three weeks (STZ-3) after STZ injection (65 mg kg21, ip). Progressive amounts of glucose were infused into anesthetized rats via the femoral vein and renal clearances collected. The amount of glucose reabsorbed, factored by the glomerular filtration rate (GFR) was significantly greater in STZ-3 rats compared with all other groups. In addition, the amount of glucose reabsorbed factored by the amount of glucose filtered was decreased in STZ-1 and STZ-2 compared with controls but was increased in STZ-3. By contrast, renal GLUT2 levels were elevated in all the STZ-treated rats. These data suggest that other factors, functioning either in conjunction with or independent of GLUT2, are required to support an elevated renal glucose transport capacity. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Type I diabetes; GLUT2; Renal glucose transport capacity
Introduction Two major classes of glucose transporters are responsible for the renal tubular reabsorption of glucose. The transport of glucose across the brush border membrane of the epithelial cells in the proximal tubule is via sodium-glucose transporters (SGLTs [1,2]). This is a secondary active process that requires a sodium gradient between the exterior and interior of the cells. Glucose efflux from the cells occurs through glucose transporters (GLUTs) located in * Corresponding author. Department of Molecular and Cellular Physiology, University of Cincinnati, PO Box 670576, Cincinnati, OH 45267-0576. Tel: (513) 558-3014; fax: (513) 558-5738. E-mail address: [email protected] (R.O. Banks) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 9 0 -6
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the basolateral membrane of cell. To date, five functional GLUTs have been described and they are collectively referred to as GLUT1-GLUT5 [3,4]. In the kidney, mRNA expression has been demonstrated for GLUT1 [5], GLUT2 [5,6], GLUT3 [7], GLUT4 [8], and GLUT5 [9]. However, only the renal expression of GLUT1 [10,11], GLUT2 [12, 10], GLUT4 [13], and GLUT5 [14] protein has been detected. Glucose reabsorption across the basolateral membrane of the S1 segment of the proximal tubule is carried out by the low affinity GLUT2 [10]. A high-affinity GLUT-1 has also been located in the S3 segment of the proximal [10]. The filtered load of glucose is elevated in uncontrolled diabetes mellitus as a result of both hyperglycemia and often hyperfiltration. Since the sodium-dependent reabsorption of glucose in the proximal tubule is increased in diabetes [15], the flux of glucose through the SGLT must be increased. An increased glucose influx into the cell requires that glucose efflux from the cell must be increased if a constant intracellular concentration of glucose is to be maintained. However, Dominguez et al. [16] hypothesized that in uncontrolled diabetes, facilitative efflux of glucose from the cell might be inhibited because of high interstitial glucose concentrations and that adaptations in the expression and function of the basolateral transporters may be necessary. They observed that GLUT2 gene expression and protein levels were, in fact, increased at two, three, and four weeks following streptozotocin-induced diabetes in rats. By contrast, GLUT1 gene expression and protein levels were decreased and SGLT1 protein and mRNA levels were unchanged in diabetic rats [16]. Dominguez et al. [16] also demonstrated that the increased expression of GLUT2 correlated with higher deoxy-D[3H]glucose ([3H]DOG) uptake by isolated proximal tubules. Similarly, in the obese Zucker rat, a model of Type II diabetes, GLUT2 protein levels in renal proximal tubules are increased whereas minor decreases have been observed in GLUT1 protein levels compared to control littermates [17]. The functional correlate of this GLUT2 expression in the whole animal has not been widely studied. Studies in Type I diabetic patients revealed that the transport maximum for glucose (TmG) is significantly increased compared to control subjects [18]. Carney et al. [19] studied glucose reabsorption in rats both during the administration of streptozotocin and sequentially during the following 8 days as hyperglycemia progressed. Although they could not satisfactorily demonstrate evidence of a TmG in either control or diabetic rats, the reabsorptive capacity for glucose was increased in the diabetic animal [19]. The goal of the current study was to determine to what extent increases in renal GLUT2, the only transporter upregulated in the proximal tubule of the diabetic kidney, correlates with changes in the glucose transport capacity. The hypothesis was that an increase in renal GLUT2 is associated with an increase in renal glucose transport capacity. Materials and methods Experiments were performed on a total of 26 female Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 175–250 g; all studies were done in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee. The rats were maintained on standard laboratory chow and water ad libitum until the time of the experiment. Animals were randomly placed in one of four groups: control (n57); one week after streptozotocin injection (STZ-1 week, n57); two weeks after streptozotocin injection (STZ-2
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week, n56); and three weeks after streptozotocin injection (STZ-3 week, n56). Following a fast for 24 hours, the animals were rendered diabetic by an intraperitoneal injection of 65 mg/ kg streptozotocin (Sigma, St. Louis, MO). All animals were diabetic at the time of the experiments; this was confirmed by urinary glucose measurements of $ 2,000 mg/dl as measured with Uristix reagent strips (Bayer, Elkhart, IN). On the day of the experiment, rats were anesthetized with sodium pentobarbital (55 mg/ kg) and rectal temperatures maintained at 37 8C with a radiant heat lamp connected to a thermostat. To facilitate respiration, a tracheotomy was performed using PE-200 tubing. The left femoral artery was cannulated with heat-stretched polyethylene tubing and was used for monitoring mean arterial pressure and for collection of blood samples. The left femoral vein was cannulated with heat-stretched polyethylene tubing. The cathether was attached to an infusion pump for infusing 3% creatinine in saline at 0.5 ml kg21 min21 and 3% creatinine plus increasing concentrations of glucose during later portion of the study; the clearance of exogenously administered creatinine is equivalent to the GFR in female rats [20,21]. Saline was infused at 0.5 ml kg21 min21 for two reasons: a) glucose could be rapidly infused during the experimental periods, and b) the infusion rate resulted in baseline urine flow rates in control rats that approximated those in STZ-treated rats. The bladder was cannulated with PE-100 tubing for collection of urine. Upon completion of the surgical procedure, animals were placed on their side above the level of the table to facilitate collection of urine. Following a 60 minute stabilization period, two 20 minute baseline clearances were collected (since these clearances were virtually identical, i.e., the animals were in a steady-state, the values were averaged). Infusion of 3% creatinine plus 10% glucose was then initiated for 30 minutes. Following this period, two additional 20 minute clearances were collected. This procedure was then continued with solution containing 20 and 30% glucose. Blood samples (100 ml) were drawn following baseline urine collections and after the last urine collection during each glucose infusion period. Animals were killed by a lethal dose of sodium pentobarbital. The kidneys were then removed, weighed, snap frozen, and stored at –80 8C until the Western blot was performed (see below). Urine volumes were determined gravimetrically. Creatinine concentrations in the blood and urine were measured by the method of Folin and Wu [22]. Sodium concentrations in the urine and plasma were measured using a flame photometer (Corning 480 Flame Photometer, Medfield, MA). Blood and urine glucose concentrations were measured with Trinder’s reagent (Sigma), a method previously shown to provide values that are in good agreement with the hexokinase and Beckman Glucose Analyzer methods [23]. Western blots were performed on kidneys that were homogenized on ice with a glass/teflon homogenizer in a lysis buffer containing 10mM Tris (pH 7.4), 1% triton X-100, 0.2 mM sodium vanadate, 10mM sodium fluoride, 1mM EDTA, 1mM phenylmethanesulfonyl fluoride, 2 mg/ml leupeptin, and 2 mg/ml aprotinin. Lysates were sonicated for 1 second with a microultrasonic cell disrupter (Kontes, Vineland, NJ) and then centrifuged at 14,000 3 g at 4 8C for 10 minutes to remove the Triton-insoluble fraction. The protein concentration of the lysate was determined using the Bradford method (Bio-Rad), and gel samples were prepared by adding sample buffer containing final concentrations of 50mM Tris (pH 6.7), 2% SDS, 2% b-mercaptoethanol, and bromphenol blue as a marker. Gel samples were boiled for 2 minutes, and then 400 mg of protein was loaded on 9% SDS-polyacrylamide gels. Proteins
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were transferred to nitrocellulose membranes (Schleicher and Schuell) using standard electroblotting techniques. Nitrocellulose membranes were blocked with 5% nonfat dry milk dissolved in buffer containing 10mM sodium phosphate (pH 7.2), 140 mM NaCl, and 0.1% Tween 20 (PBST). Blots were immunolabeled overnight at 4 8C with an antibody (1:400) that recognizes GLUT2 (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were washed in several changes of PBST at room temperature and then incubated with anti-goat Ig linked to horseradish peroxidase (Santa Cruz). Immunoreactivity was detected with enhanced chemiluminescence (Amersham Pharmacia Boitech) according to the manufacturer’s recommended conditions and quantified using densitometric analysis with an ImagePro digital analysis system (Media Cybernetics, Silver Springs, MD). Immunoreactivity for the protein was linear over at least a 3-fold range of protein concentrations. Linear regression analysis was used to evaluate the relationships between two physiological variables. Statistical differences between groups were evaluated using one way analysis of variance (ANOVA) and within groups by a repeated measures ANOVA; the Bonferroni post-hoc test was used when appropriate. Values were accepted as significantly different when the probability (P) of difference was less than 5%. Means 6 SEM are reported. Results The average body weight, kidney weight, and plasma glucose concentration in each group are summarized in Table 1. There were no significant differences in body weights among the groups. By contrast, the kidney weight was significantly greater in diabetic rats compared to controls (P , 0.05). Since differences in kidney weight existed between the control group and the groups treated with STZ, all the renal data were normalized per gram kidney weight (kw). Baseline plasma glucose concentrations were significantly higher in the diabetic rats compared to controls. Figure 1 contains data for the glomerular filtration rate, expressed in ml/min (Figure 1A) and normalized to ml min21 g kw21 (Figure 1B), for each group during baseline clearance periods and for clearance periods during infusion of hypertonic glucose. Although there was some variation from group to group, there was clearly a trend for the GFR to increase during infusion of the hypertonic glucose solutions. In addition, the STZ-1 week group had normalized GFR values that were significantly lower than the corresponding values in the control (untreated) group of rats.
Table 1 Groups Controls STZ-1 week STZ-2 weeks STZ-3 weeks
Body weight, g
Kidney weight, g
Plasma glucose, mg%
218 6 5 212 610 214 6 5 224 6 15
1.94 6 0.03 2.42 6 0.08* 2.47 6 0.10* 2.43 6 0.10*
124 6 3 444 6 15* 463 6 15* 539 6 30*
Body weights, kidney weights, and plasma glucose concentrations in control rats and in rats injected with (65 mg/kg) streptozotocin.
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Fig. 1. Glomerular filtration rate data in the four groups of rats. In Panel A, the GFR is in ml min21, whereas in panel B the GFR is normalized per gram kidney weight (ml min21 g kw21). * 5 P,0.05 compared with baseline, and † 5 significantly different from corresponding control value.
During infusion of the 30% glucose solution, the plasma glucose concentration increased to 783 6 22, 1856 6 202, 1540 6 65, and 1896 6 191 mg% in controls, STZ-1, STZ-2 and STZ-3, respectively. A number of analyses can be used to evaluate the renal dynamics of glucose reabsorption. Since the both the GFR and the plasma glucose concentration varied considerably both within and among the groups, the amount of glucose reabsorbed (TG) was normalized per unit GFR (TG/GFR). The latter variable was then evaluated as a function of the amount of glu-
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Fig. 2 (A–D). Amounts of glucose reabsorbed (TG) /GFR (mg/ml) as a function of the amount of glucose filtered (mg min21 g kidney21) in the four groups of rats.
cose filtered per min per g kidney weight (Figures 2 A–D). Except for the STZ-3 week group, a quasi plateau value for TG/GFR was observed. TG/GFR values at or above a filtered glucose load of 5 mg min21 g kidney21 averaged 3.27 6 0.16 (n516), 4.50 6 0.5 (n522), 4.63 6 0.23 (n523) and 6.10 6 0.59 (n523) mg/ml in controls, STZ-1, STZ-2 and STZ-3, respectively. Only the TG/GFR value of the STZ-3 group was significantly different from controls. The amount of glucose reabsorbed (mg min21 g kidney21) was also evaluated as a function of the amount of glucose filtered (mg min21 g kidney21); these data are summarized in Figure 3 (A–D). As may be seen, no apparent plateau for the amount of glucose reabsorbed was observed in any of the groups of rats. Indeed, linear regression analysis revealed a significant correlation between these 2 variables in all groups. However, the slope of the regression line was significantly greater in STZ-3 rats (0.423 6 .045) compared with the other 3 groups. Moreover, the slope of the regression line was significantly lower in STZ-1 (0.25 6 0.05) and STD-2 (0.238 6 0.037) compared with the control group (0.363 6 0.044).
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Fig. 3 (A–D). Amount of glucose reabsorbed (mg min21 g kidney weight21) as a function of the amount of glucose filtered (mg min21 g kidney weight21) in the four groups of rats. Linear regression equations and regression lines are illustrated (since no glucose was excreted during baseline periods in control rats, the regression line only reflects data obtained during glucose infusion).
The digital scan of a representative Western blot illustrating the expression of GLUT2 protein in the kidney in control and STZ-treated rats is reproduced in Figure 4A. The GLUT2 protein from the kidney migrated as a band with an apparent molecular mass of 58 kDa. As may seen in this figure, only trace amounts of GLUT2 were measured in the extracts of whole kidneys from control rats. Nonetheless, following treatment with streptozotocin, densitometer analysis of GLUT2 amounts relative to that present in the control lanes of the Western blots revealed increased protein concentrations (expressed as a percent of control) at one, two, and three weeks following STZ (Figure 4B). Of the groups examined, the greatest increase from control was observed in the STZ-1 week group. This group had a percent change from control of 541 6 139%. Finally, the amount of glucose reabsorbed per min correlated, significantly, with the amount of sodium reabsorbed per min in all groups (both variables normalized by kidney
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Fig. 4. Panel A contains a representative Western blot for GLUT2 in extracts of whole kidneys. Panel B illustrates the ratio of GLUT2 (expressed as % of control) in the four groups of STZ-treated rats (n56 kidneys).
weight; data not shown). The correlation coefficients from linear regression analysis of the data were 0.64, 0.37, 0.59 and 0.54 in controls, STZ-1, STZ-2 and STZ-3, respectively. Discussion Results of the current study in female rats demonstrate that the renal concentration of the predominant glucose transporter in the kidney, GLUT2, increases within the first week following induction of Type II diabetes mellitus with streptozotocin. On the other hand, an increased transport capacity, defined either by the amount of glucose reabsorbed per unit GFR or by the amount of glucose reabsorbed factored by the amount of glucose filtered, was only observed three weeks following STZ treatment. Results of the current study in female rats differ from those previously reported by Carney et al. [19] in male rats. Although they were also unable to define an apparent TmG in either control or STZ-treated rats (based on an analysis of the amount of glucose reabsorbed as a function of the amount of glucose filtered), they did report an increase in the glucose reabsorptive capacity one week following STZ treatment. In the current study, an increase in the reabsorptive capacity was only observed in rats three weeks following STZ. Whether differences in the results between the current study and those of Carney et al. [19] reflect gender related differences or are due to other factors remains to be determined. As noted above, the renal expression of GLUT2 was elevated at one, two, and three weeks following STZ. By contrast, the glucose transport capacity, defined either by the transport rate of glucose factored by the GFR or by the slope of the linear regression equation relating the amount of glucose reabsorbed vs. the amount of glucose filtered, was only elevated three weeks following STZ treatment. The dissociation between renal GLUT2 expression and re-
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nal glucose transport capacity suggests that other factors, functioning either in conjunction with or independent of GLUT2, are required to support an elevated renal glucose transport capacity. The current study extends the previous results of Dominguez et al. [16] and Chin et al. [6] who demonstrated of an increase in renal GLUT2 protein levels in male rats with STZinduced diabetes mellitus. As early as one week after STZ injection, we observed an increased renal GLUT2 protein level in female rats. Although Chin et al. [6] did not measure GLUT2 protein levels, they did observe an increase in GLUT2 mRNA within this time interval. We also found that whole kidney GLUT2 protein levels were increased two and three weeks after STZ treatment, a finding that was qualitatively similar to the Dominguez group [16]. Since glucose reabsorption across the apical membrane is a sodium dependent process, it was of interest to evaluate the relationship between sodium reabsorption and glucose reabsorption. Thus, there was a significant correlation between these two variables in both control and diabetic animals. Previous studies in isolated perfused rat kidneys have demonstrated that the addition of glucose to the perfusion fluid results in an increase in both sodium and water reabsorption [24,25]. Micropuncture studies in the rat have revealed that the presence of glucose in the lumen increases water reabsorption by the proximal tubule [26]. Bank and Aynedjian [15] found that progressive increases in intraluminal glucose concentrations markedly stimulate net sodium reabsorption in proximal tubules from both normal and diabetic rats. Overall, the data in the current study are consistent with previous reports relating to the importance of sodium in glucose reabsorption in diabetes mellitus. Glomerular hyperfiltration is often observed in early stages of diabetes [e.g., 27]. However, in the current study none of the STZ-treated groups of rats had an elevated GFR relative to controls. A plausible explanation for the lack of hyperfiltration in STZ-treated rats is related to the fact that control rats in the current study were saline-volume expanded during the baseline and experimental periods. Saline expansion, which was employed to rapidly administer glucose and to produce the same baseline urine flow rates in control rats as in STZtreated rats, is a procedure typically associated with hyperfiltration [e.g. 28]. Finally, the GFR tended to increase in all groups during infusion of the hypertonic glucose solutions. Although this increase was significant only in the control and STZ-1 week groups, administration of hypertonic sugar solutions is generally associated with an increase in the GFR [e.g., 29]. In summary, GLUT2 levels in the kidney are elevated at one, two, and three weeks following injection of streptozotocin. By contrast, diabetic rats only have an increased reabsorptive capacity for glucose at three weeks following injection of STZ. The dissociation between renal GLUT2 expression and renal glucose transport capacity suggests that other factors, functioning either in conjunction with or independent of GLUT2, are required to support an elevated renal glucose transport capacity.
Acknowledgments The authors are grateful to P. William Conrad, Ph.D. for expert technical assistance with the Western blotting procedures.
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This work was supported by a grant (96-03-S) from the American Heart Association (to R.O.B.) and the National Institutes of Health Grant HL07571 (to W.T.N). References 1. Kanai Y, Lee WS, You G, Brown D, Hediger MA. The human kidney low affinity Na1/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose. Journal of Clinical Investigation 1994;93:397–404. 2. Lee WS, Kanai Y, Wells RG,. Hediger MA. The high affinity Na1/glucose cotransporter. Re-evaluation of function and distribution of expression. Journal of Biological Chemistry 1994;269:12032–12039. 3. Thorens B. Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes. American Journal of Physiology 1996;270:G541–G553. 4. Thorens B. Facilitated glucose transporters in epithelial cells. Annual Review of Physiology 1193;55:591– 608. 5. Bell GI, Kayano T, Buse JB, Burant CF, TAKEDA J, Lin D, Fukumoto H, Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care 1990;13:198–208 6. Chin E, Zamah AM, Landau D, Gronbeck H, Flyvbjerg A, Leroith D, Bondy CA. Changes in facilitative glucose transporter messenger ribonucleic acid levels in the diabetic rat kidney. Endocrinology 1997;138:1267–1275. 7. Kayano T, Fukumoto H, Eddy RL, Fan YS, Byers MG, Shows TB, Bell GI. Evidence for a family of human glucose transporter-like proteins. Sequence and gene localization of a protein expressed in fetal skeletal muscle and other tissues. Journal of Biological Chemistry 1988;263:15245–15248. 8. Birnbaum M. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell 1989;57:305–315. 9. Kayano T, Burant CF, Fukumoto H, Gould GW, Fan YS, Eddy RL, Byers MG, Shows TB, Seino S,. Bell GI. Human facilitative glucose transporters. Isolation, functional characterization, and gene localization of cDNAs encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle, and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6). Journal of Biological Chemistry 1990;265:13276–13282. 10. Dominguez JH, Camp K, Maiani L, Garvey WT. Glucose transporters of rat proximal tubule: differential expression and subcellular distribution. American Journal of Physiology 1992;262:F807–F812. 11. Thorens B, Lodish HF, Brown D. Differential localization of two glucose transporter isoforms in rat kidney. American Journal of Physiology 1990;259:C286–C294. 12. Thorens B, Cheng ZQ, Brown D, Lodish HF. Liver glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells. American Journal of Physiology 1990;259:F279–F285. 13. Brosius FC, Briggs JP, Marcus RG, Barac-Nieto M, Charron MJ. Insulin-responsive glucose transporter expression in renal microvessels and glomeruli. Kidney International 1992;42:1086–1092. 14. Shepherd PR, Gibbs EM, Wesslau C, Gould GW, Kahn BB. Human small intestine facilitative fructose/ glucose transporter (GLUT5) is also present in insulin-responsive tissues and brain. Investigation of biochemical characteristics and translocation. Diabetes 1992;41:1360–1365. 15. Bank N, Aynedjian HS. Progressive increases in luminal glucose stimulate proximal sodium absorption in normal and diabetic rats. Journal of Clinical Investigation 1990;86:309–316. 16. Dominguez JH, Camp K, Maianu L, Feister H, Garvey WT. Molecular adaptations of GLUT1 and GLUT2 in renal proximal tubules of diabetic rats. American Journal of Physiology 1994;266:F283–F290. 17. Kamran M, Peterson RG, Dominguez JH. Overexpression of GLUT2 gene in renal proximal tubules of diabetic Zucker rats. Journal of the American Society of Nephrology 1997;8:943–948. 18. Mogensen CE. Maximum tubular reabsorption capacity for glucose and renal hemodynamcis during rapid hypertonic glucose infusion in normal and diabetic subjects. Scandinavian Journal of Clinical Laboratory Investigation 1971;28:101–109. 19. Carney SL, Wong NL, Dirks JH. Acute effects of streptozotocin diabetes on rat renal function. Journal of Laboratory and Clinical Medicine 1979;93:950–961. 20. Banks RO, Cesci AJ, Ooi BS, Pollak VE. Effect of aminonucleoside on transport maximum of para-amino-
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hippurate and intrarenal blood flow distribution in rats. Journal of Laboratory and Clinical Medicine 1977;90:341–346. Harvey AM, Malvin RL. Comparison of creatinine and inulin clearances in male and female rats. American Journal of Physiology 1965;209:849–852. Folin O, Wu H. Journal of Biological Chemistry 1919;38:81–110. Lott JA, Turner K. Evaluation of Trinder’s glucose oxidase method for measuring glucose in serum and urine. Clinical Chemistry 1975;21:1754–1760. Frega NS, Weinberg JM, Ross BD, Leaf A. Stimulation of sodium transport by glucose in the perfused rat kidney. American Journal of Physiology 1977;233:F235–F240. Trimble ME. Effects of L-glucose on sodium reabsorption in the isolated perfused rat kidney. Life Sciences 1975;17:1799–1806. Weinman EJ, Suki N, Eknoyang. D-Glucose enhancement of water reabsorption in proximal tubule of the rat kidney. American Journal of Physiology 1976;231:777–780. Mathis KM, Banks RO. Role of nitric oxide and angiotensin II in diabetes mellitus-induced glomerular hyperfiltration. Journal of the American Society of Nephrology 1996;7:105–112. Noonan WT, Banks RO. The role of nitric oxide in saline-induced natriuresis and diuresis in rats. Proceedings of the Society for Experimental Biology and Medicine 1999;221:376–381. Capasso G, Saviano C, Ciani F, Lang F, Russo F, De Santo NG. A decrease in renal medullary tonicity stimulates anion transport in Henle’s loop of rat kidneys. American Journal of Physiology 1998;274:F693–F699.
Renal glucose reabsorption during hypertonic glucose infusion in female streptozotocin-induced diabetic rats W.T. Noonan, V.M. Shapiro, R.O. Banks* Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH 45267-0576, USA Received 24 April 2000; accepted 27 October 2000
Abstract Information regarding the renal glucose transport capacity in diabetes mellitus is limited. These data are needed because two weeks following injection of streptozotocin (STZ), mRNA and protein levels of the glucose transporter, GLUT2, are upregulated in the proximal tubule of the rat. Therefore, we measured renal glucose transport and GLUT2 protein levels in female control rats, and in rats one (STZ-1), two (STZ-2), and three weeks (STZ-3) after STZ injection (65 mg kg21, ip). Progressive amounts of glucose were infused into anesthetized rats via the femoral vein and renal clearances collected. The amount of glucose reabsorbed, factored by the glomerular filtration rate (GFR) was significantly greater in STZ-3 rats compared with all other groups. In addition, the amount of glucose reabsorbed factored by the amount of glucose filtered was decreased in STZ-1 and STZ-2 compared with controls but was increased in STZ-3. By contrast, renal GLUT2 levels were elevated in all the STZ-treated rats. These data suggest that other factors, functioning either in conjunction with or independent of GLUT2, are required to support an elevated renal glucose transport capacity. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Type I diabetes; GLUT2; Renal glucose transport capacity
Introduction Two major classes of glucose transporters are responsible for the renal tubular reabsorption of glucose. The transport of glucose across the brush border membrane of the epithelial cells in the proximal tubule is via sodium-glucose transporters (SGLTs [1,2]). This is a secondary active process that requires a sodium gradient between the exterior and interior of the cells. Glucose efflux from the cells occurs through glucose transporters (GLUTs) located in * Corresponding author. Department of Molecular and Cellular Physiology, University of Cincinnati, PO Box 670576, Cincinnati, OH 45267-0576. Tel: (513) 558-3014; fax: (513) 558-5738. E-mail address: [email protected] (R.O. Banks) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 9 0 -6
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the basolateral membrane of cell. To date, five functional GLUTs have been described and they are collectively referred to as GLUT1-GLUT5 [3,4]. In the kidney, mRNA expression has been demonstrated for GLUT1 [5], GLUT2 [5,6], GLUT3 [7], GLUT4 [8], and GLUT5 [9]. However, only the renal expression of GLUT1 [10,11], GLUT2 [12, 10], GLUT4 [13], and GLUT5 [14] protein has been detected. Glucose reabsorption across the basolateral membrane of the S1 segment of the proximal tubule is carried out by the low affinity GLUT2 [10]. A high-affinity GLUT-1 has also been located in the S3 segment of the proximal [10]. The filtered load of glucose is elevated in uncontrolled diabetes mellitus as a result of both hyperglycemia and often hyperfiltration. Since the sodium-dependent reabsorption of glucose in the proximal tubule is increased in diabetes [15], the flux of glucose through the SGLT must be increased. An increased glucose influx into the cell requires that glucose efflux from the cell must be increased if a constant intracellular concentration of glucose is to be maintained. However, Dominguez et al. [16] hypothesized that in uncontrolled diabetes, facilitative efflux of glucose from the cell might be inhibited because of high interstitial glucose concentrations and that adaptations in the expression and function of the basolateral transporters may be necessary. They observed that GLUT2 gene expression and protein levels were, in fact, increased at two, three, and four weeks following streptozotocin-induced diabetes in rats. By contrast, GLUT1 gene expression and protein levels were decreased and SGLT1 protein and mRNA levels were unchanged in diabetic rats [16]. Dominguez et al. [16] also demonstrated that the increased expression of GLUT2 correlated with higher deoxy-D[3H]glucose ([3H]DOG) uptake by isolated proximal tubules. Similarly, in the obese Zucker rat, a model of Type II diabetes, GLUT2 protein levels in renal proximal tubules are increased whereas minor decreases have been observed in GLUT1 protein levels compared to control littermates [17]. The functional correlate of this GLUT2 expression in the whole animal has not been widely studied. Studies in Type I diabetic patients revealed that the transport maximum for glucose (TmG) is significantly increased compared to control subjects [18]. Carney et al. [19] studied glucose reabsorption in rats both during the administration of streptozotocin and sequentially during the following 8 days as hyperglycemia progressed. Although they could not satisfactorily demonstrate evidence of a TmG in either control or diabetic rats, the reabsorptive capacity for glucose was increased in the diabetic animal [19]. The goal of the current study was to determine to what extent increases in renal GLUT2, the only transporter upregulated in the proximal tubule of the diabetic kidney, correlates with changes in the glucose transport capacity. The hypothesis was that an increase in renal GLUT2 is associated with an increase in renal glucose transport capacity. Materials and methods Experiments were performed on a total of 26 female Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 175–250 g; all studies were done in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee. The rats were maintained on standard laboratory chow and water ad libitum until the time of the experiment. Animals were randomly placed in one of four groups: control (n57); one week after streptozotocin injection (STZ-1 week, n57); two weeks after streptozotocin injection (STZ-2
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week, n56); and three weeks after streptozotocin injection (STZ-3 week, n56). Following a fast for 24 hours, the animals were rendered diabetic by an intraperitoneal injection of 65 mg/ kg streptozotocin (Sigma, St. Louis, MO). All animals were diabetic at the time of the experiments; this was confirmed by urinary glucose measurements of $ 2,000 mg/dl as measured with Uristix reagent strips (Bayer, Elkhart, IN). On the day of the experiment, rats were anesthetized with sodium pentobarbital (55 mg/ kg) and rectal temperatures maintained at 37 8C with a radiant heat lamp connected to a thermostat. To facilitate respiration, a tracheotomy was performed using PE-200 tubing. The left femoral artery was cannulated with heat-stretched polyethylene tubing and was used for monitoring mean arterial pressure and for collection of blood samples. The left femoral vein was cannulated with heat-stretched polyethylene tubing. The cathether was attached to an infusion pump for infusing 3% creatinine in saline at 0.5 ml kg21 min21 and 3% creatinine plus increasing concentrations of glucose during later portion of the study; the clearance of exogenously administered creatinine is equivalent to the GFR in female rats [20,21]. Saline was infused at 0.5 ml kg21 min21 for two reasons: a) glucose could be rapidly infused during the experimental periods, and b) the infusion rate resulted in baseline urine flow rates in control rats that approximated those in STZ-treated rats. The bladder was cannulated with PE-100 tubing for collection of urine. Upon completion of the surgical procedure, animals were placed on their side above the level of the table to facilitate collection of urine. Following a 60 minute stabilization period, two 20 minute baseline clearances were collected (since these clearances were virtually identical, i.e., the animals were in a steady-state, the values were averaged). Infusion of 3% creatinine plus 10% glucose was then initiated for 30 minutes. Following this period, two additional 20 minute clearances were collected. This procedure was then continued with solution containing 20 and 30% glucose. Blood samples (100 ml) were drawn following baseline urine collections and after the last urine collection during each glucose infusion period. Animals were killed by a lethal dose of sodium pentobarbital. The kidneys were then removed, weighed, snap frozen, and stored at –80 8C until the Western blot was performed (see below). Urine volumes were determined gravimetrically. Creatinine concentrations in the blood and urine were measured by the method of Folin and Wu [22]. Sodium concentrations in the urine and plasma were measured using a flame photometer (Corning 480 Flame Photometer, Medfield, MA). Blood and urine glucose concentrations were measured with Trinder’s reagent (Sigma), a method previously shown to provide values that are in good agreement with the hexokinase and Beckman Glucose Analyzer methods [23]. Western blots were performed on kidneys that were homogenized on ice with a glass/teflon homogenizer in a lysis buffer containing 10mM Tris (pH 7.4), 1% triton X-100, 0.2 mM sodium vanadate, 10mM sodium fluoride, 1mM EDTA, 1mM phenylmethanesulfonyl fluoride, 2 mg/ml leupeptin, and 2 mg/ml aprotinin. Lysates were sonicated for 1 second with a microultrasonic cell disrupter (Kontes, Vineland, NJ) and then centrifuged at 14,000 3 g at 4 8C for 10 minutes to remove the Triton-insoluble fraction. The protein concentration of the lysate was determined using the Bradford method (Bio-Rad), and gel samples were prepared by adding sample buffer containing final concentrations of 50mM Tris (pH 6.7), 2% SDS, 2% b-mercaptoethanol, and bromphenol blue as a marker. Gel samples were boiled for 2 minutes, and then 400 mg of protein was loaded on 9% SDS-polyacrylamide gels. Proteins
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were transferred to nitrocellulose membranes (Schleicher and Schuell) using standard electroblotting techniques. Nitrocellulose membranes were blocked with 5% nonfat dry milk dissolved in buffer containing 10mM sodium phosphate (pH 7.2), 140 mM NaCl, and 0.1% Tween 20 (PBST). Blots were immunolabeled overnight at 4 8C with an antibody (1:400) that recognizes GLUT2 (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were washed in several changes of PBST at room temperature and then incubated with anti-goat Ig linked to horseradish peroxidase (Santa Cruz). Immunoreactivity was detected with enhanced chemiluminescence (Amersham Pharmacia Boitech) according to the manufacturer’s recommended conditions and quantified using densitometric analysis with an ImagePro digital analysis system (Media Cybernetics, Silver Springs, MD). Immunoreactivity for the protein was linear over at least a 3-fold range of protein concentrations. Linear regression analysis was used to evaluate the relationships between two physiological variables. Statistical differences between groups were evaluated using one way analysis of variance (ANOVA) and within groups by a repeated measures ANOVA; the Bonferroni post-hoc test was used when appropriate. Values were accepted as significantly different when the probability (P) of difference was less than 5%. Means 6 SEM are reported. Results The average body weight, kidney weight, and plasma glucose concentration in each group are summarized in Table 1. There were no significant differences in body weights among the groups. By contrast, the kidney weight was significantly greater in diabetic rats compared to controls (P , 0.05). Since differences in kidney weight existed between the control group and the groups treated with STZ, all the renal data were normalized per gram kidney weight (kw). Baseline plasma glucose concentrations were significantly higher in the diabetic rats compared to controls. Figure 1 contains data for the glomerular filtration rate, expressed in ml/min (Figure 1A) and normalized to ml min21 g kw21 (Figure 1B), for each group during baseline clearance periods and for clearance periods during infusion of hypertonic glucose. Although there was some variation from group to group, there was clearly a trend for the GFR to increase during infusion of the hypertonic glucose solutions. In addition, the STZ-1 week group had normalized GFR values that were significantly lower than the corresponding values in the control (untreated) group of rats.
Table 1 Groups Controls STZ-1 week STZ-2 weeks STZ-3 weeks
Body weight, g
Kidney weight, g
Plasma glucose, mg%
218 6 5 212 610 214 6 5 224 6 15
1.94 6 0.03 2.42 6 0.08* 2.47 6 0.10* 2.43 6 0.10*
124 6 3 444 6 15* 463 6 15* 539 6 30*
Body weights, kidney weights, and plasma glucose concentrations in control rats and in rats injected with (65 mg/kg) streptozotocin.
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Fig. 1. Glomerular filtration rate data in the four groups of rats. In Panel A, the GFR is in ml min21, whereas in panel B the GFR is normalized per gram kidney weight (ml min21 g kw21). * 5 P,0.05 compared with baseline, and † 5 significantly different from corresponding control value.
During infusion of the 30% glucose solution, the plasma glucose concentration increased to 783 6 22, 1856 6 202, 1540 6 65, and 1896 6 191 mg% in controls, STZ-1, STZ-2 and STZ-3, respectively. A number of analyses can be used to evaluate the renal dynamics of glucose reabsorption. Since the both the GFR and the plasma glucose concentration varied considerably both within and among the groups, the amount of glucose reabsorbed (TG) was normalized per unit GFR (TG/GFR). The latter variable was then evaluated as a function of the amount of glu-
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Fig. 2 (A–D). Amounts of glucose reabsorbed (TG) /GFR (mg/ml) as a function of the amount of glucose filtered (mg min21 g kidney21) in the four groups of rats.
cose filtered per min per g kidney weight (Figures 2 A–D). Except for the STZ-3 week group, a quasi plateau value for TG/GFR was observed. TG/GFR values at or above a filtered glucose load of 5 mg min21 g kidney21 averaged 3.27 6 0.16 (n516), 4.50 6 0.5 (n522), 4.63 6 0.23 (n523) and 6.10 6 0.59 (n523) mg/ml in controls, STZ-1, STZ-2 and STZ-3, respectively. Only the TG/GFR value of the STZ-3 group was significantly different from controls. The amount of glucose reabsorbed (mg min21 g kidney21) was also evaluated as a function of the amount of glucose filtered (mg min21 g kidney21); these data are summarized in Figure 3 (A–D). As may be seen, no apparent plateau for the amount of glucose reabsorbed was observed in any of the groups of rats. Indeed, linear regression analysis revealed a significant correlation between these 2 variables in all groups. However, the slope of the regression line was significantly greater in STZ-3 rats (0.423 6 .045) compared with the other 3 groups. Moreover, the slope of the regression line was significantly lower in STZ-1 (0.25 6 0.05) and STD-2 (0.238 6 0.037) compared with the control group (0.363 6 0.044).
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Fig. 3 (A–D). Amount of glucose reabsorbed (mg min21 g kidney weight21) as a function of the amount of glucose filtered (mg min21 g kidney weight21) in the four groups of rats. Linear regression equations and regression lines are illustrated (since no glucose was excreted during baseline periods in control rats, the regression line only reflects data obtained during glucose infusion).
The digital scan of a representative Western blot illustrating the expression of GLUT2 protein in the kidney in control and STZ-treated rats is reproduced in Figure 4A. The GLUT2 protein from the kidney migrated as a band with an apparent molecular mass of 58 kDa. As may seen in this figure, only trace amounts of GLUT2 were measured in the extracts of whole kidneys from control rats. Nonetheless, following treatment with streptozotocin, densitometer analysis of GLUT2 amounts relative to that present in the control lanes of the Western blots revealed increased protein concentrations (expressed as a percent of control) at one, two, and three weeks following STZ (Figure 4B). Of the groups examined, the greatest increase from control was observed in the STZ-1 week group. This group had a percent change from control of 541 6 139%. Finally, the amount of glucose reabsorbed per min correlated, significantly, with the amount of sodium reabsorbed per min in all groups (both variables normalized by kidney
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Fig. 4. Panel A contains a representative Western blot for GLUT2 in extracts of whole kidneys. Panel B illustrates the ratio of GLUT2 (expressed as % of control) in the four groups of STZ-treated rats (n56 kidneys).
weight; data not shown). The correlation coefficients from linear regression analysis of the data were 0.64, 0.37, 0.59 and 0.54 in controls, STZ-1, STZ-2 and STZ-3, respectively. Discussion Results of the current study in female rats demonstrate that the renal concentration of the predominant glucose transporter in the kidney, GLUT2, increases within the first week following induction of Type II diabetes mellitus with streptozotocin. On the other hand, an increased transport capacity, defined either by the amount of glucose reabsorbed per unit GFR or by the amount of glucose reabsorbed factored by the amount of glucose filtered, was only observed three weeks following STZ treatment. Results of the current study in female rats differ from those previously reported by Carney et al. [19] in male rats. Although they were also unable to define an apparent TmG in either control or STZ-treated rats (based on an analysis of the amount of glucose reabsorbed as a function of the amount of glucose filtered), they did report an increase in the glucose reabsorptive capacity one week following STZ treatment. In the current study, an increase in the reabsorptive capacity was only observed in rats three weeks following STZ. Whether differences in the results between the current study and those of Carney et al. [19] reflect gender related differences or are due to other factors remains to be determined. As noted above, the renal expression of GLUT2 was elevated at one, two, and three weeks following STZ. By contrast, the glucose transport capacity, defined either by the transport rate of glucose factored by the GFR or by the slope of the linear regression equation relating the amount of glucose reabsorbed vs. the amount of glucose filtered, was only elevated three weeks following STZ treatment. The dissociation between renal GLUT2 expression and re-
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nal glucose transport capacity suggests that other factors, functioning either in conjunction with or independent of GLUT2, are required to support an elevated renal glucose transport capacity. The current study extends the previous results of Dominguez et al. [16] and Chin et al. [6] who demonstrated of an increase in renal GLUT2 protein levels in male rats with STZinduced diabetes mellitus. As early as one week after STZ injection, we observed an increased renal GLUT2 protein level in female rats. Although Chin et al. [6] did not measure GLUT2 protein levels, they did observe an increase in GLUT2 mRNA within this time interval. We also found that whole kidney GLUT2 protein levels were increased two and three weeks after STZ treatment, a finding that was qualitatively similar to the Dominguez group [16]. Since glucose reabsorption across the apical membrane is a sodium dependent process, it was of interest to evaluate the relationship between sodium reabsorption and glucose reabsorption. Thus, there was a significant correlation between these two variables in both control and diabetic animals. Previous studies in isolated perfused rat kidneys have demonstrated that the addition of glucose to the perfusion fluid results in an increase in both sodium and water reabsorption [24,25]. Micropuncture studies in the rat have revealed that the presence of glucose in the lumen increases water reabsorption by the proximal tubule [26]. Bank and Aynedjian [15] found that progressive increases in intraluminal glucose concentrations markedly stimulate net sodium reabsorption in proximal tubules from both normal and diabetic rats. Overall, the data in the current study are consistent with previous reports relating to the importance of sodium in glucose reabsorption in diabetes mellitus. Glomerular hyperfiltration is often observed in early stages of diabetes [e.g., 27]. However, in the current study none of the STZ-treated groups of rats had an elevated GFR relative to controls. A plausible explanation for the lack of hyperfiltration in STZ-treated rats is related to the fact that control rats in the current study were saline-volume expanded during the baseline and experimental periods. Saline expansion, which was employed to rapidly administer glucose and to produce the same baseline urine flow rates in control rats as in STZtreated rats, is a procedure typically associated with hyperfiltration [e.g. 28]. Finally, the GFR tended to increase in all groups during infusion of the hypertonic glucose solutions. Although this increase was significant only in the control and STZ-1 week groups, administration of hypertonic sugar solutions is generally associated with an increase in the GFR [e.g., 29]. In summary, GLUT2 levels in the kidney are elevated at one, two, and three weeks following injection of streptozotocin. By contrast, diabetic rats only have an increased reabsorptive capacity for glucose at three weeks following injection of STZ. The dissociation between renal GLUT2 expression and renal glucose transport capacity suggests that other factors, functioning either in conjunction with or independent of GLUT2, are required to support an elevated renal glucose transport capacity.
Acknowledgments The authors are grateful to P. William Conrad, Ph.D. for expert technical assistance with the Western blotting procedures.
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