GLUT-1 Overexpression
Link Between Hemodynamic and Metabolic Factors in
Glomerular Injury?
Luigi Gnudi, GianCarlo Viberti, Leopoldo Raij, Veronica Rodriguez, Davina Burt,
Pedro Cortes, Barry Hartley, Stephen Thomas, Sabrina Maestrini, Gabriella Gruden
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Abstract—Mesangial matrix deposition is the hallmark of hypertensive and diabetic glomerulopathy. At similar levels of
systemic hypertension, Dahl salt-sensitive but not spontaneously hypertensive rats (SHR) develop glomerular
hypertension, which is accompanied by upregulation of transforming growth factor ␤1 (TGF-␤1), mesangial matrix
expansion, and sclerosis. GLUT-1 is ubiquitously expressed and is the predominant glucose transporter in mesangial
cells. In mesangial cells in vitro, GLUT-1 overexpression increases basal glucose transport, resulting in excess
fibronectin and collagen production. TGF-␤1 has been shown to upregulate GLUT-1 expression. We demonstrated that
in hypertensive Dahl salt-sensitive (S) rats fed 4% NaCl (systolic blood pressure [SBP]: 236⫾9 mm Hg), but not in
similarly hypertensive SHR (SBP: 230⫾10 mm Hg) or their normotensive counterparts (Dahl S fed 0.5% NaCl, SBP:
145⫾5 mm Hg; and Wistar-Kyoto, SBP: 137⫾3 mm Hg), there was an 80% upregulation of glomerular GLUT-1
protein expression (Pⱕ0.03). This was accompanied by a 2.7-fold upregulation of TGF-␤1 protein expression in
glomeruli of DSH compared with DSN rats (P⫽0.02). TGF-␤1 expression was not upregulated and did not differ in the
glomeruli of Wistar-Kyoto and SHR rats. As an in vitro surrogate of the in vivo hemodynamic stress imposed by
glomerular hypertension, we used mechanical stretching of human and rat mesangial cells. We found that after 33 hours
of stretching, mesangial cells overexpressed GLUT-1 (40%) and showed an increase in basal glucose transport of similar
magnitude (both Pⱕ0.01), which could be blocked with an anti TGF-␤1–neutralizing antibody. These studies suggest
a novel link between hemodynamic and metabolic factors that may cooperate in inducing progressive glomerular injury
in conditions characterized by glomerular hypertension. (Hypertension. 2003;42:19-24.)
Key Words: hypertension, experimental 䡲 mesangium 䡲 stress 䡲 transforming growth factors
I
n hypertensive glomerular injury, mesangial expansion
with extracellular matrix accumulation is one of the harbingers of glomerulosclerosis.1,2 Similarly, mesangial expansion is a central feature of diabetic glomerulopathy, in which
both hemodynamic and metabolic perturbations play a crucial
role in the pathogenesis of this disease.3
Mesangial cells express primarily 2 types of glucose
transporters: the facilitative brain type glucose transporter
GLUT-1, the predominant isoform, and the sodium-coupled
glucose transporter.4 GLUT-1 is ubiquitously expressed, and
its cellular distribution resides mainly in the cellular plasma
membrane, where it plays a major role in the basal rate of
glucose transport into the cell.5 This is particularly relevant
for glucose metabolism of cells such as mesangial cells in
which glucose uptake is relatively insulin-independent.4 As
with all the other members of the facilitative glucose transport family, GLUT-1 is a high-affinity, low-capacity glucose
transporter and is at or near saturation at physiological
glucose levels. Therefore, changes in GLUT-1 expression,
translocation, or intrinsic activity are necessary for cells to
significantly increase their basal glucose uptake.6
Overexpression of GLUT-1 in rat mesangial cells cultured in
normal glucose conditions results in increased basal glucose
transport accompanied by induction of fibronectin and of collagen expression, mimicking the effect of high glucose.7 Conversely, overexpression of an antisense GLUT-1 mRNA in
mesangial cells leads to reduction of glucose uptake and prevention of glucose-induced fibronectin.8
In hypertensive as well as in diabetic glomerulopathy,
similar signaling pathways and cytokines are upregulated,
notably transforming growth factor ␤1 (TGF-␤1).9,10
TGF-␤1 has been shown to upregulate GLUT-1 mRNA and
protein expression and basal glucose transport in mesangial
cells in culture.11
Received December 9, 2003; first decision December 26, 2002; revision accepted April 29, 2003.
From the Department of Diabetes, Endocrinology, and Internal Medicine, GKT School of Medicine, Guy’s Hospital (L.G., G.C.V., V.R., D.B., S.T.,
G.G.), King’s College London, UK; the Nephrology and Hypertension Division, Department of Veterans Affairs Medical Center and University of Miami
School of Medicine (L.R.), Miami, Fla; the Division of Nephrology and Hypertension, Henry Ford Hospital (P.C.), Detroit, Mich; the Department of
Histopathology, St James Hospital, Leeds University (B.H.), Leeds, UK; and the Department of Metabolism and Diabetes, Istituto Auxologico Italiano
(S.M.), Milano, Italy.
Correspondence to Luigi Gnudi, MD, PhD, Department of Diabetes, Endocrinology, and Internal Medicine, King’s College, 5th Floor, Thomas Guy
House, Guy’s Hospital, London SE1 9RT, UK. E-mail [email protected]
© 2003 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000075949.19968.EF
19
20
Hypertension
July 2003
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Animal models and humans with combined hypertension
and diabetes show the most severe glomerular lesions3,12–14;
therefore, reduction of glomerular pressure, with agents that
interrupt the renin-angiotensin system, downregulates
TGF-␤1 and specifically delays the progression toward renal
failure.15,16 Whether intraglomerular pressure and increased
glucose uptake operate independently or work in synergy to
produce glomerular damage is unknown. We hypothesized
that the injurious effect of the hemodynamic stress of glomerular hypertension could be mediated, at least in part, by
upregulation of GLUT-1 expression resulting in a pathologically increased glucose uptake.
We tested this hypothesis by comparing GLUT-1 expression in hypertensive Dahl salt-sensitive rats and spontaneously hypertensive rats (SHR) matched by age and severity of
hypertension. We have previously shown that in Dahl saltsensitive rats but not in SHR, systemic hypertension is
accompanied by glomerular hypertension and injury.17 In
addition, in studies in vitro, we investigated whether, in
mesangial cells, mechanical stretch upregulates GLUT-1,
leading to an increased transport of glucose.
Methods
Chemicals and Reagents
All chemicals were purchased from Sigma unless otherwise specified. Fetal calf serum was purchased from Gibco BRL and [3H]
2-deoxyglucose from Amersham. Animals were provided by Harlan
SD.
Animals
Male Dahl salt-sensitive (DS) rats, SHR, and Wistar-Kyoto (WKY)
rats were studied between 11 and 12 weeks of age. DS rats were fed
from the age of 4 weeks a low salt (0.5%) diet, DSN normotensive
control, or a high salt (4%) diet, DSH hypertensive animals.18
DSH were used as a model of systemic and intraglomerular
hypertension19,20; SHR served as a model of systemic hypertension
without intraglomerular hypertension.21 The WKY was used as the
normotensive control for the SHR.
All rats were maintained in a 24 °C environment, with a 12 hour
light/dark cycle, and fed ad libitum with water and standard chow
diet.
Before the animals were killed, systolic blood pressure was
measured in unanesthetized rats with a tail noninvasive blood
pressure determination system, and proteinuria was quantified in
24-hour urine collections from rats individually kept in metabolic
cages, using a BioRad protein assay kit.18
Immediately after death, frozen kidney tissue for immunohistochemistry analysis and isolated glomeruli were obtained. Glomeruli
were obtained by sequential sieving from kidneys of each individual
animal.22 After sieving, the glomeruli were collected in a sterile
universal container and washed 3 times with cold PBS to preclude
tubuli contamination.
Cell Culture
Human mesangial cells (HMC) were obtained from glomeruli isolated from
tumor-free portions of tumor nephrectomy specimens or from donor
kidneys unsuitable for transplantation by serial sieving of renal cortex
followed by collagenase digestion, as previously reported.23 Rat mesangial
cells (RMC) were obtained from a cloned cell line (16KC2) derived from
the outgrowth of rat glomeruli and previously characterized.24 Cells were
cultured in RPMI 1640 medium, 7 mmol/L glucose, supplemented with
insulin-transferrin-selenium (ITS), 20% FCS, 100 U/mL penicillin, and
100 ␮g/mL streptomycin in a humidified 5% CO2 incubator at 37°C and
the experiment performed in cells grown on collagen type I.23
Subconfluent mesangial cells were serum-starved (0.5% serum)
for 12 hours and subsequently exposed to mechanical stretch (60
cycles/min) at 10% average cell elongation for 12, 24, 30, and 33
hours (Flexercell 3000 Strain Unit).23,25,26 Control unstretched cells
were studied in parallel. In HMC, experiments were conducted in the
presence of a neutralizing anti–TGF-␤1 antibody (200 ng/mL) (R&D
System) or control nonimmune IgG.25
Immunohistochemistry
Kidney tissue was frozen in isopenthane, cooled in liquid nitrogen,
and stored at ⫺80°C. GLUT-1 immunoperoxidase staining was
carried out on 4-␮m frozen tissue sections fixed in acetone at 4°C for
10 minutes. Tissue sections were washed in 0.2% Tween Tris buffer
saline (TTBS), pH 7.4, followed by peroxidase blocking step with
0.03% hydrogen peroxide for 10 minutes. The sections were blocked
for 30 minutes with 10 % goat serum in TTBS, and this was followed
by 1-hour incubation at room temperature with primary rabbit
polyclonal anti GLUT-1 antibody (1:600 dilution)(Alpha Diagnostic). Sections were subsequently incubated with a peroxidase-labeled
polymer conjugated to a goat anti-rabbit IgG (Dako) for 30 minutes.
After intermediate washes with TTBS, visualization of GLUT-1 was
obtained with 3,3⬘-diaminobenzidine (brown staining). The sections
were counterstained with hematoxylin to visualize nuclei, and finally
were mounted in mounting medium. Negative controls were prepared by either omitting the anti–GLUT-1 antibody or by the
addition of specific GLUT-1– blocking peptide (Alpha Diagnostic).
Fifteen randomly selected glomeruli were analyzed for each
animal by a masked observer; the calculated average GLUT-1
staining was then used for statistical analysis. The proportional area
occupied by immunoreactive GLUT-1 was calculated by using a
computer-assisted image analysis system KS-300 (Zeiss) connected
to a BX60 microscope (Olympus) and a KY-F55B (JVC) color
videocamera. Glomeruli boundaries were defined by external perimeter of the capillary loops as previously described.27 Determinations
were made at the same light microscope intensity.
GLUT-1 and TGF-␤1 Protein
Expression Determination
Total protein from both rat glomeruli and mesangial cells were
extracted with the use of a lysis buffer containing 50 mmol/L
Tris-HCl, pH 7.6, with 1% Triton, 2 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L dithiothreitol (DTT), 1 mmol/L
phenylmethylsulfonil-fluoride (PMSF), and 100 mmol/L sodium
chloride. Lysates were then sonicated for 45 seconds at 4°C. Each
lane was loaded with 60 ␮g total cell lysate.
GLUT-1 and TGF-␤1 protein expression was studied by Western
immunoblotting with specific GLUT-1 antiserum (1:2500) (Alpha
Diagnostic) and TGF-␤1 antisera (1:6000) (Santa Cruz Biotechnology,
Inc). Both antisera specifically bind, respectively, to GLUT-1 and
TGF-␤1 from rat tissues, and they have been widely validated in
previous work.28 –30
Equal loading was confirmed by Ponceau staining of the proteins
transferred onto the nitrocellulose membrane. After enhanced chemiluminescence, band intensity was quantified by densitometry.25
Glucose Transport
Glucose transport assay in mesangial cells in vitro was performed as
previously described,31 with few modifications. In brief, basal transport
was started by the addition of 100 ␮mol/L 2-deoxyglucose (2-DOG)
with ⬇0.4 ␮Ci/ well of [3H] 2-DOG in glucose-free minimal essential
media. Transport was performed at 37°C for 5 minutes and stopped with
the addition of 1 mL phloretin solution in PBS (82 mg/L) (preliminary
experiments showed a linear 2-DOG uptake up to 15 minutes, data not
shown). 2-DOG incorporation into cells was measured with a ␤-counter.
2-DOG uptake was calculated after subtraction of 2-DOG–nonspecific
uptake in parallel samples incubated in the presence of 10 ␮mol/L
cytochalasin B. Results were normalized for cell number determined in
parallel wells.
Statistical Analysis
Differences among groups were analyzed by repeated-measures
ANOVA. Post hoc pairwise comparisons were performed by using
least significant differences and Dunnett test methods. A Student t
Gnudi et al
Mechanical Stretch and GLUT-1
21
in the SHR (8.5⫾3 mg/24 h, n⫽6) was lower than the DSH;
normotensive WKY did not show significant proteinuria.
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Figure 1. GLUT-1 immunoperoxidase staining of kidney cortex
from DSH, DSN, SHR, and WKY rats. Intense GLUT-1 staining
(brown) was seen in DSH rat glomeruli but not in DSN, SHR, or
WKY rats (magnification ⫻40; see text for statistics).
test was used to compare DSH or SHR with their respective controls,
specifically for immunohistochemistry and immunoblotting determinations. Statistical significance was accepted at a level of P⬍0.05.
Results are reported as mean⫾SEM unless otherwise stated.
Results
In Vivo Studies
Rat Characteristics
Body weight was similar in all groups of rats studied (DSH,
349.3⫾63 g; DSN, 332⫾53 g; SHR, 285⫾33 g; WKY,
293⫾53 g, n⫽4). Systolic blood pressure was similar between DSH (236⫾9 mm Hg, n⫽4) and SHR (230⫾
10 mm Hg, n⫽4) but significantly elevated when compared
with their respective normotensive controls DSN
(145⫾5 mm Hg, n⫽4) and WKY (137⫾3 mm Hg, n⫽4) rats.
Twenty-four-hour urine protein excretion in the DSH
(147⫾23 mg/24 h, n⫽4) was 5-fold higher than in the
normotensive DSN rats (36.6⫾11 mg/24 h, n⫽4). Proteinuria
GLUT-1 Is Upregulated in Glomeruli of DSH Rat by
Immunohistochemistry in Renal Cortex and by
Immunoblotting in Isolated Glomeruli
By immunohistochemistry renal cortex analysis, the percent
change in staining for GLUT-1 per glomerular area was
significantly higher in DSH than DSN rats (182⫾19.7%
versus 100⫾22.1%, respectively, P⫽0.03), whereas no difference was found between SHR and WKY rats
(109.6⫾5.8% versus 100⫾6.4%, respectively). The staining
pattern in the glomeruli of DSH rats suggested GLUT-1
upregulation in different glomerular cell types, including
the mesangium. GLUT-1 immunostaining was present in the
tubules of all animals. No staining was observed when the
first antibody was omitted or blocking peptide added. Figure
1 shows representative glomeruli from the 4 animal groups.
Consistent with this, glomerular GLUT-1 protein expression was 80% greater in the DSH rats than in DSN rats
(P⫽0.004) as assessed by Western blotting, whereas no
difference was seen between SHR and WKY (Figure 2).
TGF-␤1 Is Upregulated in Isolated Glomeruli of DSH Rat
Immunoblotting with specific anti–TGF-␤1 antiserum in total
cell lysate of isolated glomeruli from the 4 animal groups
showed that TGF-␤1 expression was 2.7-fold higher in DSH rats
compared with DSN rats. By contrast, expression of TGF-␤1
was virtually undetectable in both WKY and SHR rats, and there
were no differences between the 2 groups (Figure 3).
In Vitro Studies
Mechanical Stretch Upregulates GLUT-1 Protein Level in
Human Mesangial Cells
We studied the effect of mechanical stretch on GLUT-1
protein expression in HMC stretched for 12, 24, and 33 hours.
Exposure to mechanical stretch for 33 hours significantly
increased GLUT-1 protein level by 40% over control animals
(P⫽0.01) (Figure 4a), whereas no significant changes were
observed at earlier time points (arbitrary units percentage
Figure 2. GLUT-1 protein levels in glomeruli
isolated from WKY, SHR, DSN, and DSH
rats. Upper panel shows representative
Western immunoblotting; lower panel shows
densitometry analysis for GLUT-1 expressed
as percentage change over control (WKY
and DSN, respectively). *P⫽0.004, DSH vs
DSN (n⫽4 to 5 animals per group).
22
Hypertension
July 2003
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Figure 3. TGF-␤1 protein level in glomeruli isolated from WKY, SHR, DSN, and DSH rats. Upper panel shows representative Western
immunoblotting; lower panel shows densitometry analysis for TGF-␤1 expressed as percentage change over control (WKY and DSN,
respectively). *P⫽0.02, DSH vs DSN (n⫽4 animals per group).
change over control⫽100%, at 12 hours, 119⫾14%; at 24
hours, 127⫾14%; P⫽NS).
Mechanical Stretch Upregulates Glucose Transport in
Human and Rat Mesangial Cells
To investigate whether GLUT-1 upregulation was paralleled
by an increase in cellular glucose uptake, 2-DOG basal
glucose transport assay was performed after 33 hours of
stretch when GLUT-1 upregulation was observed. There was
a significant 60% increase (P⫽0.002) in 2-DOG uptake in
HMC exposed to stretch as compared with control nonstretched cells (Figure 4b). A similar significant increase,
although less in magnitude, was observed in stretched RMC
(arbitrary unit % change over control nonstretched
cells⫽100%, at 30 to 33 hours, 122.5⫾4.3%, P⫽0.005),
indicating that this phenomenon is common in both species.
TGF-␤1 Mediates Stretch-Induced GLUT-1 and Basal
Glucose Transport Upregulation
To elucidate the mechanism of stretch-induced GLUT-1 and
basal glucose transport upregulation in HMC, we studied
GLUT-1 expression and basal 2-DOG glucose uptake in
mesangial cells stretched either in the presence or absence of
a specific TGF-␤1–neutralizing antibody.25 TGF-␤1 blockade
significantly blunted by ⬇80% both the stretch-induced
GLUT-1 protein expression (P⫽0.04) and basal glucose
transport (P⫽0.006), observed at 33 hours. No change was
detected when the control IgG was added (Figures 4a and 4b).
Thus, stretch-induced GLUT-1 overexpression and basal
glucose transport appear largely mediated by TGF-␤1.
Discussion
In this study, we have demonstrated that in hypertensive DSH
rats but not in similarly hypertensive SHR there is 80%
increase in glomerular GLUT-1 both by immunohistochemistry and by Western blot; GLUT-1 levels in normotensive
WKY and DSN were similar to those in SHR.
These findings suggested that GLUT-1 upregulation might
be a consequence of the hemodynamic stress imposed by
glomerular hypertension. It has been previously shown that
Figure 4. GLUT-1 protein expression (a)
and basal glucose transport (b) in 33
hours stretched (s) and nonstretched (ns)
HMC in the presence or absence of anti–
TGF-␤1–neutralizing antibody or control
IgG. Data are expressed as percentage
change over nonstretched cells (experiments were n⫽5 performed in duplicate
for glucose transport, and n⫽5 for
GLUT-1 protein). *P⬍0.05 for stretch
control IgG vs other conditions; †P⬍0.01
for stretch control IgG vs other conditions.
Representative Western immunoblotting
for GLUT-1 protein is shown in (a).
Gnudi et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
transmission of the elevated systemic blood pressure to the
glomerular circulation in the DSH rat20,32 results in glomerular hypertension, mesangial matrix expansion, and development of glomerulosclerosis.17 In contrast, in the SHR, increased preglomerular resistances prevent glomerular
capillary pressure from rising in response to systemic hypertension, and glomerular damage is modest and delayed.17,21,33
When the preglomerular resistances are reduced such as in
uninephrectomized or in the diabetic SHR, the development
of glomerular capillary hypertension is associated with the
development of accelerated proteinuria and an increased
mesangial expansion and glomerulosclerosis.3,34
Previous studies have reported an increased expression of
TGF-␤1 in the glomeruli of hypertensive Dahl salt-sensitive
rats treated with a high salt diet.9,35 In contrast, no differences
have been observed in TGF-␤1 expression in SHR rats
between 7 and 16 weeks of age when compared with
normotensive WKY rats.35,36
In this study, we confirmed that TGF-␤1 is upregulated in
isolated glomeruli of DSH rats, suggesting that the upregulation of TGF-␤1 may account for the increase of GLUT-1
expression that we have observed.
This interpretation of the in vivo findings is consistent with
our results in vitro, which show that mesangial cells, an
important target for mechanically induced glomerular injury,37 when subjected to mechanical stretch, upregulate
GLUT-1 protein expression as well as basal glucose transport
through a TGF-␤1– dependent mechanism.
These data are in accord with previous observations both in vivo
and in vitro. In DHS rats, reduction of intraglomerular pressure with
an angiotensin II antagonist prevents glomerular TGF-␤1 upregulation.38 Further systemic treatment with anti–TGF-␤1 antibodies in
hypertensive Dahl rats reduces blood pressure as well as proteinuria
and the severity of glomerulosclerosis.39
In this context, the finding that diabetic animals with
incipient diabetic nephropathy have increased urinary excretion of TGF-␤1, which is associated with greater abundance of
renal cortical GLUT-1 protein, is notable.40
In cultured mesangial cells mechanical stretch upregulates
TGF-␤1 levels and activity.25,26,41,42 Furthermore, addition of
TGF-␤1 to mesangial cells in culture increases basal glucose
transport by stimulating GLUT-1 expression both at the
mRNA and protein levels.11
In mesangial cells, high glucose also upregulates GLUT-1,43 an
event believed to represent one of the mechanisms of glomerular
injury in diabetes. This glucose-induced GLUT-1 upregulation is, at
least in part, mediated by TGF-␤1.11 On the other hand, GLUT-1
overexpression in mesangial cells in normal glucose medium leads
to excess extracellular matrix production.7
This is the first demonstration that mechanical stretch in
normal ambient glucose concentration leads to increased
glucose transport through TGF-␤1– dependent upregulation of
GLUT-1. Collectively, our findings suggest a possible mechanism of interaction between mechanical forces and glucosemediated pathways in the pathogenesis of glomerular injury.
Hypertension is a major risk factor in the progression of
renal disease in general44 and diabetic nephropathy in paticular.45,46 It is known that agents that interrupt the renin-angiotensin system are particularly renoprotective.47– 49 These
Mechanical Stretch and GLUT-1
23
agents reduce systemic as well as glomerular hypertension
and, by downregulating TGF-␤1,15,16,38 are likely to interrupt
the TGF-␤1–GLUT-1 axis. Our present work describes a
novel pathophysiological mechanism that may be operative in
renal disease accompanied by glomerular hypertension.
Perspectives
The studies reported herein, by linking mechanical stretch with
glucose transport/metabolism, suggest a novel pathophysiological mechanism of injury in hypertensive glomerular diseases.
Clinically, these studies may lead to the development of new
therapeutic strategies in hypertensive glomerulopathies.
Acknowledgments
This work was supported by Diabetes UK Grants RD/98/0001608
(R.D. Lawrence Fellowship to Dr Gruden), RG/98/0001862 (equipment grant to Dr Gnudi), RD98/0001759 (project grant for Miss
Burt), and RD98/0001861 (project grant for Dr Rodriguez); and a
grant from Department of Veterans Affairs to Dr Raij. We thank Mr
Brian Johnston and Mr Mark Blades for technical assistance.
References
1. Raij L. Nitric oxide in hypertension: relationship with renal injury and left
ventricular hypertrophy. Hypertension. 1998;31:189 –193.
2. Raij L, Azar S, Keane W. Mesangial immune injury, hypertension, and
progressive glomerular damage in Dahl rats. Kidney Int. 1984;26:137–143.
3. Cooper ME. Interaction of metabolic and haemodynamic factors in mediating
experimental diabetic nephropathy. Diabetologia. 2001;44:1957–1972.
4. Wakisaka M, He Q, Spiro MJ, Spiro RG. Glucose entry into rat mesangial
cells is mediated by both Na(⫹)-coupled and facilitative transporters.
Diabetologia. 1995;38:291–297.
5. Mueckler M. Facilitative glucose transporters. Eur J Biochem. 1994;219:
713–725.
6. Heilig CW, Brosius FC, Henry DN. Glucose transporters of the glomerulus and the implications for diabetic nephropathy. Kidney Int. 1997;
52:S91-S99.
7. Heilig CW, Concepcion LA, Riser BL, Freytag SO, Zhu M, Cortes P.
Overexpression of glucose transporters in rat mesangial cells cultured in
a normal glucose milieu mimics the diabetic phenotype. J Clin Invest.
1995;96:1802–1814.
8. Heilig CW, Kreisberg JI, Freytag S, Murakami T, Ebina Y, Guo L, Heilig K,
Loberg R, Qu X, Jin Y, Henry D, Brosius FC III. Antisense GLUT-1 protects
mesangial cells from glucose induction of GLUT-1 and fibronectin expression.
Am J Physiol Renal Physiol. 2001;280:F657–F666.
9. Tamaki K, Okuda S, Nakayama M, Yanagida T, Fujishima M. Transforming growth factor-beta 1 in hypertensive renal injury in Dahl saltsensitive rats. J Am Soc Nephrol. 1996;7:2578 –2589.
10. Ziyadeh FN, Hoffman BB, Han DC, Iglesias-De La Cruz MC, Hong SW,
Isono M, Chen S, McGowan TA, Sharma K. Long-term prevention of
renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming
growth factor-beta antibody in db/db diabetic mice. Proc Natl Acad Sci
U S A. 2000;97:8015– 8020.
11. Inoki K, Haneda M, Maeda S, Koya D, Kikkawa R. TGF-beta 1 stimulates glucose uptake by enhancing GLUT1 expression in mesangial
cells. Kidney Int. 1999;55:1704 –1712.
12. Microalbuminuria Collaborative Study Group, United Kingdom. Risk
factors for development of microalbuminuria in insulin dependent
diabetic patients: a cohort study. BMJ. 1993;306:1235–1239.
13. Lurbe E, Redon J, Kesani A, Pascual JM, Tacons J, Alvarez V, Batlle D.
Increase in nocturnal blood pressure and progression to microalbuminuria
in type 1 diabetes. N Engl J Med. 2002;347:797– 805.
14. Poulsen PL, Hansen KW, Mogensen CE. Ambulatory blood pressure in
the transition from normo- to microalbuminuria: a longitudinal study in
IDDM patients. Diabetes. 1994;43:1248 –1253.
15. Cao Z, Cooper ME, Wu LL, Cox AJ, Jandeleit-Dahm K, Kelly DJ,
Gilbert RE. Blockade of the renin-angiotensin and endothelin systems on
progressive renal injury. Hypertension. 2000;36:561–568.
16. Perico N, Remuzzi G. Angiotensin II receptor antagonists and treatment
of hypertension and renal disease. Curr Opin Nephrol Hypertens. 1998;
7:571–578.
24
Hypertension
July 2003
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
17. Hayakawa H, Raij L. Nitric oxide synthase activity and renal injury in
genetic hypertension. Hypertension. 1998;31:266 –270.
18. Hayakawa H, Coffee K, Raij L. Endothelial dysfunction and cardiorenal
injury in experimental salt-sensitive hypertension: effects of antihypertensive therapy. Circulation. 1997;96:2407–2413.
19. Rapp JP. Dahl salt-susceptible and salt-resistant rats. Hypertension. 1982;
4:753–763.
20. Azar S, Limas C, Iway J, Weller D. Single nephron dynamics during high
sodium intake and early hypertension in Dahl rats. Jpn Heart J. 1979;
20(suppl 1):138 –140.
21. Arendshorst WJ, Beierwaltes WH. Renal and nephron hemodynamics in
spontaneously hypertensive rats. Am J Physiol. 1979;236:F246 –F251.
22. Keane WF, Raij L. Relationship among altered glomerular barrier permselectivity, angiotensin II, and mesangial uptake of macromolecules. Lab
Invest. 1985;52:599 – 604.
23. Gruden G, Thomas SM, Burt D, Lane S, Chusney GD, Sacks S, Viberti
GC. Mechanical stretch induces VEGF expression in human mesangial
cells: mechanisms of signal transduction. Proc Natl Acad Sci U S A.
1997;94:12112–12116.
24. Dumler F, Cortes P. Uracil ribonucleotide metabolism in rat and human glomerular epithelial and mesangial cells. Am J Physiol. 1988;255:C712–C718.
25. Gruden G, Zonca S, Hayward A, Thomas S, Maestrini S, Gnudi L, Viberti
GC. Mechanical stretch-induced fibronectin and transforming growth
factor-beta1 production in human mesangial cells is p38 mitogen-activated protein kinase-dependent. Diabetes. 2000;49:655– 661.
26. Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D,
Narins RG. Cyclic stretching force selectively up-regulates transforming
growth factor-beta isoforms in cultured rat mesangial cells. Am J Pathol.
1996;148:1915–1923.
27. Osterby R, Gundersen HJ, Nyberg G, Aurell M. Advanced diabetic
glomerulopathy: quantitative structural characterization of nonoccluded
glomeruli. Diabetes. 1987;36:612– 619.
28. Haspel HC, Rosenfeld MG, Rosen OM. Characterization of antisera to a
synthetic carboxyl-terminal peptide of the glucose transporter protein.
J Biol Chem. 1988;263:398 – 403.
29. Masumi A, Akamatsu Y, Kitagawa T. Modulation of the synthesis and glycosylation of the glucose transporter protein by transforming growth factor-beta 1
in Swiss 3T3 fibroblasts. Biochim Biophys Acta. 1993;1145:227–234.
30. Chandrasekar B, Troyer DA, Venkatraman JT, Fernandes G. Dietary
omega-3 lipids delay the onset and progression of autoimmune lupus
nephritis by inhibiting transforming growth factor beta mRNA and
protein expression. J Autoimmunol. 1995;8:381–393.
31. Gnudi L, Frevert EU, Houseknecht KL, Herhardt P, Kahn BB. Adenovirus mediated gene transfer of dominant negative ras-asn17 in 3T3L1
adipocytes does not alter insulin stimulated PI3-kinase activity or glucose
transport. Mol Endocrinol. 1997;11:67–76.
32. Takenaka T, Forster H, De Micheli A, Epstein M. Impaired myogenic
responsiveness of renal microvessels in Dahl salt-sensitive rats. Circ Res.
1992;71:471– 480.
33. Azar S, Johnson MA, Scheinman J, Bruno L, Tobian L. Regulation of
glomerular capillary pressure and filtration rate in young Kyoto hypertensive rats. Clin Sci (Colch). 1979;56:203–209.
34. Dworkin LD, Feiner HD. Glomerular injury in uninephrectomized spontaneously hypertensive rats: a consequence of glomerular capillary hypertension. J Clin Invest. 1986;77:797– 809.
35. Nagase M, Kaname S, Nagase T, Wang G, Ando K, Sawamura T, Fujita T.
Expression of LOX-1, an oxidized low-density lipoprotein receptor, in experimental hypertensive glomerulosclerosis. J Am Soc Nephrol. 2000;11:
1826–1836.
36. Yamauchi T, Ogura T, Oishi T, Omiya T, Ota Z. The angiotensin
I-converting enzyme inhibitor, cilazapril inhibits the platelet-derived
growth factor B chain expression in glomeruli of spontaneously hypertensive rats. Ren Physiol Biochem. 1995;18:237–245.
37. Cortes P, Mendez M, Riser BL, Guerin CJ, Rodriguez-Barbero A, Hassett
C, Yee J. F-actin fiber distribution in glomerular cells: structural and
functional implications. Kidney Int. 2000;58:2452–2461.
38. Otsuka F, Yamauchi T, Kataoka H, Mimura Y, Ogura T, Makino H.
Effects of chronic inhibition of ACE and AT1 receptors on glomerular
injury in Dahl salt-sensitive rats. Am J Physiol. 1998;274:
R1797–R1806.
39. Dahly AJ, Hoagland KM, Flasch AK, Jha S, Ledbetter SR, Roman RJ. Antihypertensive effects of chronic anti-TGF-beta antibody therapy in Dahl S rats. Am J
Physiol Regul Integr Comp Physiol. 2002;283:R757–R767.
40. D’Agord SB, Lacchini S, Bertoluci MC, Irigoyen MC, Machado UF,
Schmid H. Increased renal GLUT1 abundance and urinary TGF-beta 1
in streptozotocin-induced diabetic rats: implications for the development of nephropathy complicating diabetes. Horm Metab Res. 2001;
33:664 – 669.
41. Hirakata M, Kaname S, Chung UG, Joki N, Hori Y, Noda M, Takuwa Y,
Okazaki T, Fujita T, Katoh T, Kurokawa K. Tyrosine kinase dependent
expression of TGF-beta induced by stretch in mesangial cells. Kidney Int.
1997;51:1028 –1036.
42. Riser BL, Cortes P, Yee J, Sharba AK, Asano K, Rodriguez-Barbero A,
Narins RG. Mechanical strain- and high glucose-induced alterations in
mesangial cell collagen metabolism: role of TGF-beta. J Am Soc Nephrol.
1998;9:827– 836.
43. Heilig CW, Liu Y, England RL, Freytag SO, Gilbert JD, Heilig KO, Zhu
M, Concepcion LA, Brosius FC III. D-glucose stimulates mesangial cell
Glut1 expression and basal and IGF-I-sensitive glucose uptake in rat
mesangial cell. Diabetes. 1997;46:1030 –1039.
44. Ruggenenti P, Schieppati A, Remuzzi G. Progression, remission,
regression of chronic renal diseases. Lancet. 2001;357:1601–1608.
45. Fagerudd JA, Tarnow L, Jacobsen P, Stenman S, Nielsen FS, PetterssonFernholm KJ, Gronhagen-Riska C, Parving HH, Groop PH. Predisposition to essential hypertension and development of diabetic nephropathy
in IDDM patients. Diabetes. 1998;47:439 – 444.
46. Krolewski AS, Canessa M, Warram JH, Laffel LM, Christlieb AR,
Knowler WC, Rand LI. Predisposition to hypertension and susceptibility
to renal disease in insulin-dependent diabetes mellitus. N Engl J Med.
1988;318:140 –145.
47. Bohlen L, de Court, Weidmann P. Comparative study of the effect of
ACE-inhibitors and other antihypertensive agents on proteinuria in
diabetic patients. Am J Hypertens. 1994;7:84S–92S.
48. Lewis EJ, Hunsicker LG, Clarke WR, Berl T, Pohl MA, Lewis JB, Ritz
E, Atkins RC, Rohde R, Raz I. Renoprotective effect of the angiotensinreceptor antagonist irbesartan in patients with nephropathy due to type 2
diabetes. N Engl J Med. 2001;345:851– 860.
49. Taal MW, Brenner BM. Renoprotective benefits of RAS inhibition: from
ACEI to angiotensin II antagonists. Kidney Int. 2000;57:1803–1817.
GLUT-1 Overexpression: Link Between Hemodynamic and Metabolic Factors in
Glomerular Injury?
Luigi Gnudi, GianCarlo Viberti, Leopoldo Raij, Veronica Rodriguez, Davina Burt, Pedro
Cortes, Barry Hartley, Stephen Thomas, Sabrina Maestrini and Gabriella Gruden
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Hypertension. 2003;42:19-24; originally published online May 27, 2003;
doi: 10.1161/01.HYP.0000075949.19968.EF
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2003 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://hyper.ahajournals.org/content/42/1/19
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/