Renal-Electrolyte and Hypertension Division and Penn Center for Molecular Studies of Kidney Diseases, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6144
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ABSTRACT |
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Inhibition of
gene expression by antisense oligodeoxynucleotides (ODNs) relies on
their ability to bind complementary mRNA sequences and prevent
translation. The proximal tubule is a suitable target for ODN therapy
in vivo because circulating ODNs accumulate in the proximal tubule in
high concentrations. Because increased proximal tubular transforming
growth factor- 1 (TGF-
1) expression may mediate diabetic renal
hypertrophy, we investigated the effects of antisense TGF-
1 ODN on
the high-glucose-induced proximal tubular epithelial cell hypertrophy
in tissue culture and on diabetic renal hypertrophy in vivo. Mouse
proximal tubular cells grown in 25 mM D-glucose and exposed
to sense ODN as control (1 µM) exhibited increased
3[H]leucine incorporation by 120% and total
TGF-
1 protein by 50% vs. culture in 5.5 mM D-glucose.
Antisense ODN significantly decreased the high-glucose-stimulated
TGF-
1 secretion and leucine incorporation. Continuous infusion for
10 days of ODN (100 µg/day) was achieved via osmotic minipumps in
diabetic and nondiabetic mice. Sense ODN-treated
streptozotocin-diabetic mice had 15.3% increase in kidney weight, 70%
increase in
1(IV) collagen and 46% increase in fibronectin mRNA
levels compared with nondiabetic mice. Treatment of diabetic mice with
antisense ODN partially but significantly decreased kidney TGF-
1
protein levels and attenuated the increase in kidney weight and the
1(IV) collagen and fibronectin mRNAs. In conclusion, therapy with
antisense TGF-
1 ODN decreases TGF-
1 production and attenuates
high-glucose-induced proximal tubular cell hypertrophy in vitro and
partially prevents the increase in kidney weight and extracellular
matrix expression in diabetic mice.
transforming growth factor-1; nephropathy; proximal tubule; glucose; collagen type IV; fibronectin; osmotic mini-pumps
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INTRODUCTION |
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DIABETIC RENAL HYPERTROPHY is a consistent finding in the early stage of type 1 diabetes mellitus in humans and in the streptozotocin-diabetes model (23, 25). Increased steady-state mRNA levels encoding type IV collagen and fibronectin in the kidney are also features of the early stages of diabetic nephropathy (9, 14, 22). Diabetic renal hypertrophy is predominantly due to tubular epithelial cell hypertrophy, which accounts for the increase in whole kidney weight (16, 19, 20). Hyperglycemia and other metabolic and hemodynamic factors underlying the diabetic state are the principal causes for the development of diabetic nephromegaly (19, 25). In fact, recent in vitro studies provided evidence that high ambient glucose per se can induce proximal tubular epithelial cell hypertrophy (29, 33, 34) and increase the synthesis of extracellular matrix components in these cells (3, 34).
Several key pieces of evidence have implicated the TGF- system as an
etiologic agent in the genesis and maintenance of diabetic renal
hypertrophy (11, 30). In an in vitro system, we have previously
reported that proximal tubular epithelial cells express significant
increments in TGF-
1 mRNA levels and bioactivity when cultured in
high glucose concentration (17). Furthermore, we and others have
demonstrated that TGF-
1 mRNA and protein levels are significantly
increased in the kidney cortex after only a few days of overt
hyperglycemia in animal models of type 1 diabetes mellitus (14, 21,
24). Direct evidence implicating the TGF-
system in the pathogenesis
of experimental diabetic renal hypertrophy has derived from the results
of a short-term study whereby a neutralizing monoclonal antibody
directed against all three mammalian isoforms of TGF-
was
administered intraperitoneally to streptozotocin-diabetic mice (22).
Treatment with the antibody totally prevented glomerular hypertrophy,
reduced the increment in kidney weight by ~50%, and significantly
attenuated the increase in renal mRNA levels of
1(IV) collagen and
fibronectin. However, the individual contribution of the TGF-
1
isoform alone in mediating the renohypertrophic effects of the diabetic
state could not be deciphered from these studies.
Recent advancements in biotechnology have made possible the application
of antisense oligodeoxynucleotide (ODN) therapy in vivo to inhibit the
function of a specific protein or enzyme (4-6, 27). Inhibition of
gene expression by antisense ODNs relies on their ability to bind
complementary mRNA sequences and the prevention of protein translation
(26). The proximal tubule is a suitable target for ODN therapy in vivo
because it has been previously demonstrated that circulating ODNs
accumulate in the kidney, mainly in the proximal tubule, in
concentrations that are higher than in any other organ (6, 18, 31). For
example, therapy with antisense ODNs against the sodium-phosphate
cotransporter suppressed phosphate uptake into the brush-border
membrane of the proximal tubule (13). Because increased proximal
tubular TGF-1 expression may mediate diabetic renal hypertrophy, we
investigated the effects of therapy with antisense TGF-
1 ODN on the
high-glucose-stimulated protein synthesis in proximal tubular cell
culture and on diabetic renal hypertrophy in vivo. This allowed us to
test the hypothesis that increased activity of the renal TGF-
1
system in early diabetes mellitus in mice contributes to renal
hypertrophy in this disease.
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METHODS |
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Proximal tubular cell culture. MCT is a murine proximal tubular cell line that has been propagated in culture by SV-40 virus transformation (7). The cells demonstrate stable structural and functional features that are characteristic of differentiated proximal tubular epithelial cells (7, 28). Cells were cultured at 37°C in a humidified incubator with 5% CO2-95% air and passaged twice every week by light trypsinization. The growth media was DMEM (GIBCO-BRL) containing 5.5 mM D-glucose, 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine.
Preparation of ODNs.
We designed and synthesized 19-mer ODNs that were phosphorothioate
modified to increase stability (Cancer Center, Univ. of Pennsylvania).
The antisense ODN encompasses the ATG of the murine TGF-1 gene and
has the sequence
5'-AGGGCG
GGGGAGGC-3' (6.13 mg/µmol). The sense ODN, used as control, has the sequence
5'-GCCTCCCCCATGCCGCCCT-3' (5.75 mg/µmol). Lyophilized
ODNs were resuspended in distilled water and then diluted to give the
required final concentration.
3[H]leucine incorporation.
Incorporation of 3[H]leucine into
acid-precipitable proteins was used as a sensitive index of de novo
protein synthesis in MCT cells (29, 33, 34). Cells were plated at 5 × 105 cells/well in 24-well plates (Nunc) in the
growth media. The next day, cells were rested in serum-free medium for
24 h and then exposed for 48 h to either normal (5.5 mM) or high (25 mM) D-glucose. Sense or antisense ODNs (1 µM) were added
to some of the wells. For the last 12 h of culture, cells were pulsed
with 5 µCi 3[H]leucine (142 Ci/mmol,
Amersham). Cell monolayers were washed twice with ice-cold
phosphate-buffered saline, precipitated in ice-cold 10% TCA,
redissolved in 500 µl of 0.5 M NaOH+0.1% Triton X-100, and counted
for emissions. Additional plates of cells exposed to the same
experimental conditions were used to obtain cell counts in a Coulter
counter. Leucine incorporation was expressed as counts per minute per
106 cells.
Diabetic mice.
Diabetes was induced in 8-wk-old C57/bl female mice by two consecutive
intraperitoneal injections of streptozotocin (200 mg · kg1 · day
1;
Sigma Chemical) dissolved in 10 mM Na citrate, pH 5.5 (22). Nondiabetic
mice were injected with buffer alone. Once glycosuria was detected,
regular insulin (0.1-0.2 U) was administered subcutaneously every
day to prevent ketonuria and maintain a moderately elevated blood
glucose concentration (22-28 mM). Osmotic minipumps (model 2002, Alza) were implanted subcutaneously in the flank of each mouse under
sterile conditions. The next day, diabetic and nondiabetic mice were
weight-matched to receive either sense or antisense ODNs. The ODN
delivery rate through the pump was 100 µg/day. At the end of the
experimental period, individual 24-h urine collections were obtained.
The animals were then weighed, blood was collected, and the kidneys
were harvested, weighed, and immediately frozen in liquid nitrogen for
RNA and protein extraction. Kidney protein extracts were determined in
duplicate (Bio-Rad).
TGF-1 ELISA.
Total (latent + active) TGF-
1 levels were measured in
MCT-conditioned culture media, mouse plasma, urine, and kidney protein extracts by using an ELISA kit (Genzyme), as previously described (8,
22). Quiescent MCT cells were cultured as described above. Cells were
exposed for 48 h to DMEM containing either 5.5 or 25 mM
D-glucose and incubated in the presence of sense or
antisense TGF-
1 ODNs. At the end of the incubation period
conditioned media were collected, centrifuged, and stored in
70°C. Samples of media, plasma, or urine were acid activated
to convert latent into active TGF-
1 and to record detectable levels
of TGF-
1. Samples were activated with 1 N HCl for 60 min at 4°C
followed by neutralization with 1 N NaOH. Samples were plated on
microtiter plates coated with mouse monoclonal anti-human TGF-
1
antibody and incubated at 37°C for 60 min. After being washed five
times, wells were incubated with a second horseradish
peroxidase-conjugated polyclonal anti-TGF-
1 antibody, and then the
peroxidase reaction was initiated. A standard curve was constructed by
using serial dilutions of human TGF-
1 (Genzyme), and TGF-
1 levels
in samples were compared with known standards and read as nanograms per
106 cells. TGF-
1 levels in urine samples were expressed
per milligram of urinary creatinine, measured by a colorimetric assay
(Sigma Chemical). Total kidney levels of TGF-
1 protein were
expressed per milligram protein of tissue extract.
Northern hybridization.
Cultured MCT cells or frozen kidneys were lysed and denatured in 4 M
guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl,
and 0.1 M 2-mercaptoethanol (24, 32). RNA extraction was performed as
previously described. RNA from individual samples (25 µg) were
electrophoresed on a 1.2% agarose gel containing 0.67 M formaldehyde,
transferred by capillary blotting to Gene-Screen nylon membrane (NEN
Research Products), and ultraviolet crosslinked. The cDNA probes
encoding murine 1(IV) collagen, fibronectin, and ribosomal protein
mrpL32 were as previously reported (12, 22). Hybridization and washing
were performed as previously described (8, 32). The membranes were
autoradiographed with intensifying screens (Kodak, Wilmington, DE) at
70°C for 1-4 days. Exposed films were scanned with a
laser densitometer (Hoefer Scientific Instruments), and mRNA levels
were calculated relative to corresponding mrpL32. Measurements of
density in normal glucose media (5.5 mM) or in nondiabetic mice treated
with sense ODN were assigned a relative value of 100%.
Statistical analysis. Data are presented as the means ± SE with n as the number of different experiments or the number of animals per group. Groups were compared by analysis of variance, and an unpaired t-test was used to compare individual groups. A P value < 0.05 was considered significant.
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RESULTS |
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Response of proximal tubular cells.
Total TGF-1 protein was measured by ELISA to test whether antisense
TGF-
1 ODN was able to reduce TGF-
1 synthesis in MCT cells. As
control, exposure of MCT cells to high ambient glucose concentration
(25 mM) for 48 h without treatment with ODN, stimulated total TGF-
1
protein secretion into the culture media by ~50% compared with
normal glucose concentration (5.5 mM) (Fig.
1). Addition of 1 µM antisense
TGF-
1 ODN effectively reduced the high-glucose-induced increase in
TGF-
1 (Fig. 1). However, and as would be expected, there was no
inhibitory effect on TGF-
1 production when MCT cells were exposed to
sense ODN (Fig. 1).
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Response of diabetic hypertrophy.
Table 1 displays the body and
kidney weights and the blood glucose concentration after 10 days of ODN
infusion in the four groups of mice. Diabetic mice treated with either
sense or antisense ODNs had equivalently elevated blood glucose
concentration at the end of the study period (Table 1). Body weight was
similarly reduced in the diabetic mice treated with either sense or
antisense ODNs. None of the animals died during the study. Total kidney weight increased by 15.3% in diabetic mice treated with sense TGF-1
ODN compared with nondiabetic control mice (240 ± 7 vs. 276 ± 14 mg, respectively, P < 0.05). In contrast, kidney weight in
diabetic mice treated with antisense TGF-
1 ODN was increased by
6.5% only, compared with antisense ODN-treated nondiabetic mice (Table
1). Thus the increment in total kidney weight in antisense ODN-treated
diabetic mice was less than one-half that of sense ODN-treated diabetic
mice. These results suggest that autocrine production of TGF-
1 is
responsible, at least in part, for the acute stimulatory effect of
diabetes on kidney weight in mice.
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Response of the renal TGF-1 system in diabetic mice.
Plasma levels of TGF-
1 were not significantly different between the
normal and diabetic groups, suggesting that the short duration of the
diabetic state did not increase circulating TGF-
1 (data not shown),
confirming our previous report (22). In contrast, urinary levels of
total TGF-
1 protein (latent + active fractions) were markedly
stimulated in diabetic mice (sense ODN-treated diabetic mice: 6.7 ± 1.3 ng/mg creatinine vs. sense ODN-treated nondiabetic controls: 1.1 ± 0.3 ng/mg creatinine; P < 0.05). Treatment of diabetic
mice with antisense ODN reduced urinary TGF-
1 levels only slightly
and not significantly (5.9 ± 1.2 ng/mg creatinine). However, Fig.
3 demonstrates that the markedly elevated
TGF-
1 protein level in the kidney extracts of diabetic mice was
significantly attenuated by treatment with antisense ODN (Fig. 3; 29%
reduction in the increment of renal TGF-
1 protein compared with
sense ODN-treated diabetic mice). These studies are consistent with the
notion that the diabetic state is characterized by increased renal
production of TGF-
1 and that systemic administration of antisense
TGF-
1 ODN is effective in attenuating this increment.
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Response of matrix overexpression in diabetic kidney.
Figure 4, A and B, shows
that the mRNA encoding 1(IV) collagen in the kidney of diabetic mice
treated with sense ODN was increased by 70% compared with sense
ODN-treated nondiabetic mice after 10 days of diabetes. Inhibition of
renal TGF-
1 with antisense ODN treatment was associated with
significantly decreased overexpression of
1(IV) collagen mRNA; the
increment was only 24% in antisense ODN-treated mice compared with
sense ODN-treated mice (Fig. 4, A and B). Figure
5, A and B, demonstrates
similar changes in renal fibronectin mRNA expression; the 46% increase
in mRNA level in sense-treated diabetic mice was significantly reduced
by antisense ODN treatment, nearly to the level found in nondiabetic
mice treated with sense ODN. Thus inhibiting TGF-
1 activity in the
early phase of streptozotocin-induced diabetes markedly attenuates the
stimulation in gene expression of the extracellular matrix molecules,
type IV collagen and fibronectin, in the mouse kidney. However, we cannot exclude that other isoforms of TGF-
and/or non-TGF-
pathways can be partly responsible for increased matrix expression in
the kidney.
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DISCUSSION |
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Increased activity of the renal TGF- system has been implicated as
an etiologic factor in the genesis and maintenance of diabetic renal
hypertrophy (22). We sought to test this hypothesis by examining the
effects of therapy with antisense TGF-
1 ODN on the
high-glucose-mediated tubular epithelial cell hypertrophy in vitro and
on diabetic renal hypertrophy in vivo. Antisense ODN suppressed the
high-glucose-stimulated TGF-
1 secretion and leucine incorporation in
mouse renal proximal tubular cells in culture. Short-term continuous
infusion of antisense ODN in streptozotocin-induced diabetic mice
significantly decreased renal TGF-
1 protein levels and attenuated
the increase in kidney weight and extracellular matrix mRNA levels.
Our previous investigations in tissue culture have shown that high
glucose concentration in the culture media of proximal tubule cells
inhibits cell proliferation (33, 34), induces cell hypertrophy (29,
34), and increases TGF-1 mRNA level and bioactivity (17). High
glucose concentration also stimulates collagen gene transcription and
secretion (33, 34). A neutralizing anti-TGF-
antibody that is
directed against TGF-
1 and TGF-
2 prevents the inhibitory effect
of high glucose on cell proliferation (17). Using specific antisense
TGF-
1 ODN in the present study, we demonstrate that autocrine
production of TGF-
1 is responsible, at least in part, for the
stimulatory effect of high glucose on protein synthesis and matrix
expression in proximal tubular cells. Our studies in
tubular cells extend the results of Kolm et al. (10), who used
antisense TGF-
1 ODN in cultured glomerular mesangial cells to
inhibit the high-glucose-mediated stimulation of fibronectin and
heparan sulfate proteoglycan production.
The experiments utilizing systemic infusion of antisense TGF-1 ODN
in diabetic mice are the first to exploit this technique. The tissue
distribution after administration of ODN through subcutaneous, intradermal, or intraperitoneal routes, is similar to that of intravenous injections (1). We administered ODN through a single subcutaneous implantation of miniosmotic pumps in mice to release ODNs
over a 10-day period. The ODNs were phosphorothioate modified to
increase stability and to prevent rapid degradation. The results of
these experiments provide direct evidence implicating TGF-
1 specifically in the pathogenesis of diabetic renal hypertrophy. The
increment in total kidney weight in antisense ODN-treated diabetic mice
was only one-half that of sense ODN-treated diabetic mice.
Additionally, treatment of diabetic mice with antisense ODN partially
but significantly decreased kidney TGF-
1 protein levels and
attenuated the increase in the mRNA levels encoding
1(IV) collagen
and fibronectin in the kidney. Thus it can be concluded that the
increased production of TGF-
1 in the diabetic kidney is responsible,
at least in part, for the acute stimulatory effect of the diabetic
state on renal hypertrophy and the accompanying overexpression of
extracellular matrix proteins. The persistently elevated urinary
TGF-
1 levels, despite antisense ODN treatment (as opposed to the
significant decrement in total kidney TGF-
1 levels), may relate to
the possibility that urinary TGF-
1 originates from multiple sources
(the circulation, glomeruli, tubules), whereas ODN therapy targets
effectively only tubules.
In a previous short-term in vivo study we demonstrated that
intraperitoneal administration of a neutralizing monoclonal antibody directed against the three mammalian isoforms of TGF- (TGF-
1, TGF-
2, and TGF-
3) into streptozotocin-diabetic mice prevented glomerular hypertrophy, reduced the increment in kidney weight, and
significantly attenuated the increase in renal mRNA levels of
1(IV)
collagen and fibronectin (22). It was unclear what the contribution was
of each of the three isoforms to the diabetic changes in the kidney
(22). Using specific reagents such as antisense ODN directed at the
TGF-
1 sequence, as was done in the present study, has provided
evidence that increased production of TGF-
1 in the diabetic kidney
is responsible, at least in large part, for mediating diabetic renal hypertrophy.
The present study supports the feasibility and the efficacy of
delivering specific ODNs to the kidney. Inhibition of gene expression
by antisense ODN relies on its ability to bind complementary mRNA
sequences and prevent translation (4-6, 27). The proximal tubule
is a suitable target for ODN therapy in vivo because circulating ODNs
accumulate in the kidney, mainly in the proximal tubule, in
significantly high concentrations (6, 18, 31). Because increased
proximal tubular TGF-1 expression may mediate diabetic renal
hypertrophy, systemic infusions of ODN are expected to accumulate in
the proximal tubule where they can target TGF-
1 production. Using
intravenous administration of 32P-labeled ODN, Rappaport et
al. (15) readily detected the presence of ODN in Bowman's space, the
proximal tubular lumen, and tubular epithelial cells where the ODN
accumulated. In addition, Oberbauer et al. (13) demonstrated that
antisense ODN directed at the sodium-phosphate cotransporter suppressed
phosphate uptake across the brush-border membrane in the proximal
tubule. Targeting other cells of the kidney may require different
delivery systems. Gene transfer of TGF-
1 antisense ODN to the
glomerulus has been previously attempted by the hemagglutinating virus
of Japan liposome method delivered through the arterial supply of the
kidney (2); a successful reduction of matrix molecule overexpression in
the glomerulus was therefore achieved in a model of glomerulonephritis. Targeting TGF-
1 production in the glomerulus rather than the tubules
in the diabetic state may require a similar approach as that used by
Akagi and co-workers (2).
The partial effects of antisense ODN observed in this study may have
many explanations, including the relatively low dose (which should
avoid nonspecific effects), the role of other isoforms of TGF-
and/or non-TGF-
pathways in mediating diabetic hypertrophy, and the
nonhomogeneous distribution of ODN in the kidney, which preferentially
favors tubular uptake.
In conclusion, antisense TGF-1 ODN attenuates the
high-glucose-induced hypertrophy of proximal tubular cells in vitro. In addition, short-term treatment with antisense TGF-
1 ODN given in
continuous infusion via osmotic minipumps is efficacious in decreasing
renal TGF-
1 production and attenuating kidney hypertrophy and the
increase in extracellular matrix expression in diabetic mice.
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ACKNOWLEDGEMENTS |
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This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-44513, DK-45191, DK-54608, and training grant DK-07006 and the Juvenile Diabetes Foundation International. Drs. D. C. Han and S. W. Hong are visiting scholars at the University of Pennsylvania. Dr. D. C. Han is supported by the Korean Research Foundation and the Hyonam Kidney Laboratory at Soon Chun Hyang University Hospital, Seoul, Korea. Dr. S. W. Hong is supported by Konsei University, Seoul, Korea. Dr. B. B. Hoffman is supported by an Individual National Research Service Award from the National Institutes of Health.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: F. N. Ziyadeh, Renal-Electrolyte and Hypertension Division, Univ. of Pennsylvania, 700 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6144 (E-mail: ziyadeh{at}mail.med.upenn.edu).
Received 26 April 1999; accepted in final form 24 November 1999.
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