| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Division of Endocrinology (E.C.), University of Alabama at Birmingham, and Veterans Administration Medical Center, Birmingham, Alabama; Developmental Endocrinology Branch (A.M.Z.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; Diabetes Branch (Da.L., De.L.), National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; Department of Nephrology (Da.L.), Children’s Hospital National Medical Center, Washington, DC; Institute of Experimental Clinical Research (H.G.), Aarhus Kommunehospital, DK-8000 Aarhus C., Denmark
Address all correspondence and requests for reprints to: C. A. Bondy, NIH-NICHHD-DEB, Building 10/Room 10N262, 10 Center Drive, MSC-1862, Bethesda, Maryland 20892-1862.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In summary, medullary GLUT1 and GLUT4 mRNA levels are acutely increased in STZ-DM, paralleling the increased renal epithelial metabolic activity accompanying early diabetes. Proximal tubular GLUT2 and 5 mRNA levels were increased in chronic STZ-DM, possibly adapting to the increased need for glucose transport out of these epithelial cells, whereas the concomitant decrease in cortical GLUT1 expression may reflect the decreased requirement for basolateral import of glucose into these same cells. Thus, renal GLUTs demonstrate complex, nephron segment-specific and duration-dependent responses to the effects of STZ-DM.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Diabetes has profound effects on the kidney and is the leading cause of end stage renal disease in the United States (8). Streptozotocin-induced diabetes mellitus (STZ-DM) in rats has been shown to cause renal hypertrophy (9), increase renal gluconeogenesis (10), increase renal glucose use (11), and produce tubular lesions as a result of glycogen accumulation (12). To investigate the role that alterations in facilitative GLUT expression might play in diabetic renal pathology, we used in situ hybridization to quantitate steady-state renal messenger RNA (mRNA) levels for GLUTs 1 2, 4, and 5 in both acute and chronic phases of STZ-DM.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Control rats from the day of injection (day zero, n = 6), diabetes/insulin-treated rats (2 and 7 days, n = 8, respectively), and rats 2, 7, 30, 90, and 180 days after the injections of STZ or placebo (6–8 in each arm of the age groups) were anesthetized with sodium barbital (50 mg/kg ip) and killed. The left kidney was rapidly removed, frozen over dry ice, stored at -70 C, and used for in situ hybridization. Frozen sections were cut at a thickness of 10 µ, thaw-mounted on poly-L-lysine coated slides, and stored at -70 C until use. Sections were cut longitudinally so that all sections included the papilla, outer medulla, and cortex. All sections for a given experiment were prepared, washed, exposed, and analyzed together.
Preparation of complementary RNA (cRNA) probes
The cDNAs used for probe synthesis in the present study were the
rat GLUT1 (14), GLUT2 (15), GLUT4 (16), and GLUT5 (7), which have been
previously described. High specific activity 35S-labeled
cRNA probes were synthesized in 10-µl reactions containing 100 µCi
cytidine 5' (
35S) thiotriphosphate (Amersham SJ 40832),
100 µCi uridine 5' ( 35S) thiotriphosphate (Amersham
SJ 40383), 10 mM NaCl, 6 mM MgCl2,
40 mM Tris (pH 7.5), 2 mM spermidine, 10
mM dithiothreitol, 500 µM each of unlabeled
ATP and GTP, 25 µM each of unlabeled UTP and CTP, 0.5
µg linearized template, 15 U of the appropriate polymerase, and 15 U
RNAsin (Promega, Madison, WI). The reaction was incubated at 42 C for
30 min; then an additional 15 U of the appropriate polymerase and 15 U
RNAsin were added. The reaction was incubated at 42 C for 30 min, after
which the DNA template was removed by digestion with DNase-I at 37 C
for 10 min. Labeled cRNA was column purified (Bio-Spin 6, Bio-Rad) to
separate unincorporated nucleotides. Labeled probe was precipitated
with 5 µl transfer RNA (tRNA,) 10 µl 5 M NaCl, 10 µl
DEPC H20 and 300 µl cold EtOH. Purified probe then
underwent alkaline hydrolysis to produce fragments of an average length
of 150 bases.
In situ hybridization
Tissue sections were prepared in the following manner. Before
hybridization, sections were warmed to 25 C, fixed in 10%
formaldehyde, and soaked for 10 min in 0.25% acetic anhydride/0.1
M triethanolamine hydrochloride/0.9% NaCl. Tissue was then
dehydrated through an ethanol series, delipidated in chloroform,
rehydrated, and air-dried. The 35S-labeled probes
(107 dpm/ml or approximately 50 ng/ml) were added to
hybridization buffer composed of 50% formamide, 0.3 M
NaCl, 20 mM Tris HCl, pH 8, 5 mM EDTA, 500 µg
tRNA, 10% dextran sulfate, 10 mM dithiothreitol, and
0.02% each of BSA, ficoll, and polyvinylpyrolidone. After the
35S-labeled probe in hybridization buffer was added to the
sections, coverslips were placed over the sections, and the slides were
incubated in humidified chambers overnight (14 h) at 55 C.
Slides were washed several times in 4 x SSC to remove cover slips and hybridization buffer, dehydrated and immersed in 0.3 M NaCl, 50% formamide, 20 mM Tris HCl, 1 mM EDTA at 60 C for 10 min. Sections were then treated with RNAse A (20 µg/ml) for 30 min at room temperature, followed by a 15-min wash in 0.1 x SSC at 55 C. Slides were air-dried and apposed to Hyperfilm-ßMax (Amersham) along with autoradiographic standards (ARC Inc., St. Louis, MO) for 1–4 days and then dipped in Kodak NTB2 nuclear emulsion, stored with desiccant at 4 C for 3–30 days, developed, and counterstained with Mayer’s hematoxylin and eosin for microscopic evaluation.
Quantitative densitometry
Quantification of mRNA level was done as follows. Film
autoradiographic images, registered with a solid-state video camera
(Sony XC-77, Sony Corp.) and a 55-mm MicroNikkor lens over a light box
of variable intensity (Northern Light Precision 890, Imaging Research
Inc., Toronto, Canada), were digitized to a 640 x 480 matrix with
light transmittance coded in 256 equal grey levels (LG-3 frame grabber
card, Scion Corp., Frederick, MD). The images were quantified using a
Macintosh II-based image analysis program (Image 1.49, developed by
W.S. Rasband, Research Services Branch, NIMH, Bethesda, MD), in which
the gray scale values from the autoradiographic standard curve were
used in a third-degree polynomial function curve fit. Anatomically
matched, standardized areas of the tubulo-interstitial cortex, outer
stripe of the outer medulla (OS/OM) and inner stripe of the outer
medulla (IS/OM), inner medulla, and papilla were defined by cursor
control and their transmittance measured. Two to three measurements
were taken for each structure in each kidney, and an average value was
determined. Data was normalized as a percentage of the age-matched
control and expressed as mean ± SEM for each group.
One-way ANOVA, followed by unpaired two tailed t tests, was
used to evaluate significant differences between diabetic and control
groups at the different time points.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Blood glucose levels
in the STZ-DM groups are increased 5-fold compared with controls and
are normalized by insulin treatment (Table 1). All diabetic animals
developed glycosuria but not ketonuria (data not shown). Urinary
albumin excretion was markedly elevated in the STZ-DM rats by 30 days,
compared with controls (864 ± 149 vs. 133 ± 34
µg/24 h; P < 0.001), and continued to increase
through 180 days (1560 ± 202 vs. 418 ± 115;
P < 0.001).
|
). GLUT1 mRNA is also detected in the OS/OM, in
medullary rays and in the cortex where we have previously shown that it
is localized in epithelial cells of the proximal straight tubule,
Henle’s loop, distal tubule and collecting duct (7). In the acute
phase of STZ-DM, GLUT1 mRNA levels are significantly increased
throughout the medulla, particularly in the papilla and the IS/OM; the
increases in medullary GLUT1 mRNA levels are progressively normalized
by 30 days and remain normal through the chronic period (Fig. 1).
Cortical GLUT1 mRNA levels, however, begin to decrease by day 7 and
remain significantly decreased up to 6 months after the onset of
STZ-DM.
|
),
where it is localized in epithelial cells of the TAL, as previously
described (7). Medullary GLUT4 mRNA levels are increased by almost 90%
in the acute phase of STZ-DM but are similar to control levels by day
30 and thereafter (Fig. 2C). Insulin treatment had little effect on
GLUT4 mRNA levels at the 2-day time point, but with 7 days of insulin
treatment (Fig. 2C), GLUT4 mRNA levels were normalized in diabetic
kidneys.
|
), and increase by 30–50% in PSTs and by 400% in
PCTs 1 month after induction of STZ-DM (Fig. 3, B–D).
|
), where it is localized in PSTs (7). STZ-DM had no
detectable effect on this mRNA in the acute phase, but PST GLUT5 mRNA
levels were increased significantly at 30 and 90 days after onset of
diabetes, returning to normal thereafter.
|
).
Interestingly, insulin demonstrates segment specific effects on GLUT2
mRNA, producing a normalization of elevated levels in the PST of the
OS/OM, but a further increase GLUT2 mRNA in cortical PCTs. After 7 days
of insulin, however, all GLUT mRNA levels were normalized, except for a
persistent slight elevation in GLUT1 in the IS/OM. No groups were
treated with insulin for longer than 7 days; and, thus, there is no
data on effects of insulin on the cortical changes in GLUT1, 2, and 5
mRNAs seen in chronic diabetes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
STZ-DM has been shown to increase glomerular filtration rate, tubular sodium transport, and Na+/K+ ATPase activity (17, 18). The distribution of renal GLUT1 expression is correlated with renal tubular glycolysis in support of active transport mechanisms (5). Thus, the increase in GLUT1 mRNA levels throughout the medulla in acute STZ-DM may be related to the increased metabolic demands of early diabetes. The present, data showing decreases in cortical GLUT1 mRNA levels during chronic diabetes, confirm the results of a recent study that evaluated GLUT1 protein and mRNA levels in proximal tubules fractionated from the renal cortex in STZ-DM (19). The reduction in GLUT1 expression in proximal tubules during prolonged diabetes may be caused by the saturation of these tubular cells with glucose reabsorbed from the filtrate; and thus, expression of the high affinity GLUT1 (which normally facilitates the passage of glucose from the circulation across the basolateral membranes into these cells) would be gratuitous. Interestingly, cortical GLUT1 mRNA levels were stable early in diabetes when medullary levels were increased and were decreased later in diabetes, by which time medullary levels had normalized, suggesting that local tubular factors are more important in regulation of renal GLUT1 expression than are systemic factors.
The present study demonstrates that proximal tubule GLUT2 mRNA levels increase dramatically in chronic diabetes, with maximal elevation of almost 5 times normal from 1–3 months and continued elevation after 6 months of diabetes. A previous study also reported increased renal GLUT2 mRNA and protein in chronic STZ-DM (19). Interestingly, the use of in situ hybridization in our study reveals that the balance of proximal tubule GLUT2 gene expression shifts from the straight portion in normal rats to the convoluted segment in the diabetic rats. The adaptive significance of this increase in proximal tubule GLUT2 expression seems clear; these cells actively reabsorb a large load of filtered glucose in diabetes via a Na+-GLUT. The glucose that accumulates in these epithelial cells must be transferred back to the circulation across the basolateral membranes largely via the low-affinity, high-capacity GLUT2 transporter, which is localized on these membranes (20, 21). Hence, the increase in proximal tubule GLUT2 may serve to accommodate the increased glucose transport load. The significance of the selective increase in PCT GLUT2 mRNA in STZ-DM is not clear, but it is noteworthy that insulin treatment also produced striking segment-specific effects, with a marked increase in PCT and decrease or normalization in PST GLUT2 mRNA levels. The present data suggests that regulation of renal GLUT2 is similar to the small intestine, where GLUT2 mRNA and protein both are increased after 45 days of STZ-DM (22). This is in contrast to the pancreatic ß cell, however, where GLUT2 is decreased in experimental and spontaneous diabetes and increased during hyperglycemic glucose infusion (23).
This is the first study to describe GLUT5 mRNA levels in the diabetic kidney. GLUT5 is exclusively localized in PSTs (7) and may serve to transport fructose and glucose (24). Our study shows that renal GLUT5 mRNA levels are increased in chronic diabetes in correlation with the sharp increase in GLUT2 mRNA levels. This observation agrees with a recent study of increased GLUT5 mRNA and protein in the small intestine of 45-day diabetic rats (22). Renal fructose metabolism has not been well characterized, but the enzymes necessary for fructose metabolism are localized in proximal tubules (4, 10) and are linked to renal gluconeogenesis. Gluconeogenesis is elevated in ketotic animals (4, 10), and it is possible that GLUT5 serves to transport fructose into proximal tubule cells for entry into gluconeogenic pathways. On the other hand, fructose is a product of the sorbitol pathway, which is increased in the chronic diabetic rat kidney (10, 25), and thus, it is possible that GLUT5 may serve to transport fructose out of the cell.
This also is the first study to evaluate renal tubular GLUT4 mRNA levels in diabetes. A recent study (26) used RT-PCR to quantitate GLUT4 mRNA levels in dissected glomeruli and microvessels from rat kidneys after 1 week of STZ-DM and reported a decrease of approximately 70%. GLUT4 immunoreactivity and glucose transport also were decreased in this microdissected tissue. GLUT1 was not investigated in this previous study, though the present data, showing significantly decreased GLUT1 mRNA levels in the renal cortex, suggests that decreases in GLUT1 may contribute to the effects seen in the Marcus et al. study. Furthermore, muscle and adipocyte GLUT4 levels generally are decreased in STZ-DM and increased with insulin treatment (reviewed in Ref.23); thus, our finding that GLUT4 mRNA levels are transiently increased in early diabetes, demonstrates a renal tubular specific pattern of regulation of GLUT4 expression in STZ-DM. We have shown previously that GLUT4 mRNA and protein are focally localized in TALs in the IS/OM (7). The TALs have the highest level of Na+/K+ ATPase activity of any segment of the nephron and preferentially use glucose to fuel their pump activity (4, 27). Renal sodium reabsorption is increased in diabetes (18), and the timing of GLUT4 mRNA elevation in the diabetic TAL correlates with increases in Na+/K+ ATPase activity observed in TALs after 2 days of STZ-DM (28). Thus, it seems that TAL-specific increases in GLUT4 expression may serve to increase glucose uptake to fuel the heightened Na+/K+ ATPase in this segment. We have shown previously that VP positively regulates renal GLUT4 mRNA levels (7), and VP is increased in uncontrolled diabetes (29); hence, VP may stimulate the increases in TAL GLUT4 mRNA levels in early STZ-DM.
The observation that GLUT 1 and 4 gene expression did not normalize at the 2-day point in the insulin-treated group is probably caused by the fact that the animals had only received a single injection of insulin at this point and had not yet metabolically stabilized. In support of this view, the fact that the early changes in GLUTs 1 and 4 are totally normalized with insulin treatment by day 7 is a strong argument against a general toxic effect of STZ. Furthermore, if the changes in gene expression were caused by a toxic effect of STZ, it would be difficult to explain why only GLUTs 1 and 4 were affected immediately after exposure, whereas GLUTs 2 and 5 were affected weeks later.
In summary, this study has shown that STZ-DM is accompanied by complex, nephron segment-specific, temporally dependent changes in steady-state mRNA levels for each of the four facilitative GLUTs found in the rat kidney. The high-affinity transporters, GLUTs 1 and 4, demonstrate parallel increases in mRNA levels in the metabolically highly active renal medulla during the first week of diabetes, presumably facilitating basolateral epithelial glucose import for use as substrate to fuel increased active transport functions. The low-affinity transporters, GLUTs 2 and 5, demonstrate parallel increases in steady-state mRNA levels in proximal tubules during the chronic phase of diabetes, presumably to support the increased requirements for basolateral epithelial glucose export. At the same time, in the same proximal tubules, GLUT1 mRNA levels are reduced, illustrating the complexity of facilitative GLUT regulation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received September 25, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glutamyl transpeptidase in rat kidney with
double-peroxidase immunocytochemistry. Diabetes 41:766–770[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  V. Douard and R. P. Ferraris Regulation of the fructose transporter GLUT5 in health and disease Am J Physiol Endocrinol Metab, August 1, 2008; 295(2): E227 - E237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. S. Freitas, G. F. Anhe, K. F. S. Melo, M. M. Okamoto, M. Oliveira-Souza, S. Bordin, and U. F. Machado Na+-Glucose Transporter-2 Messenger Ribonucleic Acid Expression in Kidney of Diabetic Rats Correlates with Glycemic Levels: Involvement of Hepatocyte Nuclear Factor-1{alpha} Expression and Activity Endocrinology, February 1, 2008; 149(2): 717 - 724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Schmidt, K. Hocherl, and M. Bucher Regulation of renal glucose transporters during severe inflammation Am J Physiol Renal Physiol, February 1, 2007; 292(2): F804 - F811. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Keembiyehetty, R. Augustin, M. O. Carayannopoulos, S. Steer, A. Manolescu, C. I. Cheeseman, and K. H. Moley Mouse Glucose Transporter 9 Splice Variants Are Expressed in Adult Liver and Kidney and Are Up-Regulated in Diabetes Mol. Endocrinol., March 1, 2006; 20(3): 686 - 697. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Rahmoune, P. W. Thompson, J. M. Ward, C. D. Smith, G. Hong, and J. Brown Glucose Transporters in Human Renal Proximal Tubular Cells Isolated From the Urine of Patients With Non-Insulin-Dependent Diabetes Diabetes, December 1, 2005; 54(12): 3427 - 3434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Grover, D. Buckley, A. R. Buckley, and W. Cacini Reduced Expression of Organic Cation Transporters rOCT1 and rOCT2 in Experimental Diabetes J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 949 - 956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Marks, N. J C Carvou, E. S Debnam, S. K Srai, and R. J Unwin Diabetes increases facilitative glucose uptake and GLUT2 expression at the rat proximal tubule brush border membrane J. Physiol., November 15, 2003; 553(1): 137 - 145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R. Merriman-Smith, A. Krushinsky, J. Kistler, and P. J. Donaldson Expression Patterns for Glucose Transporters GLUT1 and GLUT3 in the Normal Rat Lens and in Models of Diabetic Cataract Invest. Ophthalmol. Vis. Sci., August 1, 2003; 44(8): 3458 - 3466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Odetti, C. Pesce, N. Traverso, S. Menini, E. P. Maineri, L. Cosso, S. Valentini, S. Patriarca, D. Cottalasso, U. M. Marinari, et al. Comparative Trial of N-Acetyl-Cysteine, Taurine, and Oxerutin on Skin and Kidney Damage in Long-Term Experimental Diabetes Diabetes, February 1, 2003; 52(2): 499 - 505. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mogyorosi and F. N. Ziyadeh GLUT1 and TGF-{beta}: the link between hyperglycaemia and diabetic nephropathy Nephrol. Dial. Transplant., December 1, 1999; 14(12): 2827 - 2829. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
