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Hyonam Kidney Laboratory, Soon Chun Hyang University, Seoul, Korea.
Correspondence to Dr. Hi Bahl Lee, Hyonam Kidney Laboratory, Soon Chun Hyang University, 657 Hannam Dong, Yongsan Ku, Seoul 140-743, Korea. Phone: +82 2 709 9171, +82 2 792 6657; Fax: +82 2 792 5812; E-mail: hblee{at}korea.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown that the inhibition of 3-hydroxy-3-methylglutaryl CoA
(HMG CoA) reductase with lovastatin is associated with amelioration of
albuminuria and glomerulosclerosis in 5/6 nephrectomized rats
(11,12),
obese Zucker rats (13), and
streptozotocin-induced diabetic uninephrectomized rats
(14). Because lovastatin also
reduces serum cholesterol levels in these experimental models, the beneficial
effect of lovastatin has generally been attributed to a decrease in
circulating levels of cholesterol. There is evidence, however, that the effect
of lovastatin on glomerular structure and function may result, at least in
part, from a direct action at the cellular level. Lovastatin has been shown to
inhibit mesangial cell proliferation
(15) and induce apoptosis in
cultured mesangial cells (16).
Lovastatin has been shown to inhibit NF-
B activation
(17) and monocyte
chemoattractant protein-1 production
(18), and modulate the
plasminogen activator-plasmin system
(19). We have demonstrated
that lovastatin effectively suppresses TGF-ß1 mRNA expression in diabetic
rat glomeruli 2 d after induction of diabetes
(20). These studies suggest
that the beneficial effect of lovastatin on diabetic nephropathy may be due to
its direct effect on TGF-ß and ECM synthesis. However, there is no
long-term study showing that lovastatin prevents the renal manifestations of
diabetic nephropathy or suppresses TGF-ß expression and accumulation of
ECM in diabetic glomeruli. Therefore, we explored the effects of lovastatin on
the expression of TGF-ß1 in both streptozotocin-induced diabetic rat
glomeruli and in rat mesangial cells cultured under high glucose concentration
and assessed the effect of lovastatin on the expression of fibronectin in
these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum Cholesterol and Creatinine and Urine Albumin Excretion
Serum cholesterol was measured by an enzyme-linked (cholesterol
hydrolase/cholesterol oxidase/peroxidase) colorimetric method using
Cholesterol-E kit (Yeong Dong Pharmaceutical Co., Kyunggi, Korea). Serum
creatinine was measured by modified Jaffe method using Creatinine-E kit (Yeong
Dong Pharmaceutical Co.). Twenty-four-hour urine samples were obtained from
animals in metabolic cages with access to drinking water only. Urine albumin
was measured by a quantitative reaction with bromcresol green.
Mean Glomerular Volume
Glomerular volume (VG) was calculated according to the
method of Weibel (21) as
described previously (22). In
brief, surface areas of 75 to 100 glomeruli selected randomly were measured
with Image-pro (version 1.2; Media Cybernetics, Silver Spring, MD) software
program on periodic acid-Schiff-stained tissue sections, and
VG was calculated using the equation:
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Rat Glomeruli Isolation and Mesangial Cell Culture
Rat glomeruli were isolated from 8-wk-old male rat kidney according to the
sieving method (24). Mesangial
cells were obtained from the isolated glomeruli after treatment with
collagenase and identified by typical stellate morphology, positive staining
for the intermediate filaments, desmin, and vimentin
(24). Rat mesangial cells were
cultured in Dulbecco's modified Eagle's medium (Life Technologies, Grand
Island, NY) containing 10% fetal bovine serum, 100 U/ml penicillin, 100
µg/ml streptomycin, and 0.5 µg/ml fungizone as described previously
(5). As the cells reached 80%
confluence in 100-mm culture dishes, cells were incubated in serum-free media
for 2 d to arrest and synchronize cell growth. Cells were then treated with
serum-free media containing either control (5.5 mM) or high (30 mM) glucose.
At the beginning of glucose treatment, lovastatin (10 µM) or mevalonate
(100 µM) (Sigma Chemical Co.) was added to the culture alone or together,
and the cells were incubated for 24 to 48 h. After incubation, culture
supernatant was centrifuged to remove cell debris and was used in Western blot
analysis for secreted TGF-ß and fibronectin proteins. Total RNA was
prepared from the cultured cells for Northern analysis of TGF-ß1 and
fibronectin mRNA.
Preparation of Lovastatin and Mevalonate for Use in Cell Culture
Inactive lactone forms of lovastatin and mevalonate were converted to their
active forms as described before
(15,25).
In brief, 40 mg of the inactive lovastatin was dissolved in 1 ml of absolute
ethanol, and 0.1 M NaOH was added, heated at 50°C for 2 h, and neutralized
with 0.1 M HCl to pH 7.2. The final volume was adjusted to 20 ml with
distilled water. Mevalonate (32.5 mg) was dissolved in 2 ml of absolute
ethanol, 23 ml of RPMI 1640 (Life Technologies) was added, and pH was adjusted
to 7.4. The prepared lovastatin and mevalonate were stored at -20°C until
use.
Isolation of Total RNA and Northern Blot Analysis
Total RNA from the isolated glomeruli was prepared using TRI-Reagent
(Molecular Research Center, Cincinnati, OH) according to the manufacturer's
description after homogenization with motor-driven pellet pestle (Sigma
Chemical Co.). From the cultured mesangial cells, total RNA was obtained by
using RNeasy mini total RNA kits (Qiagen, Valencia, CA) according to the
manufacturer's instructions. For Northern blot analysis, an equal amount of
total RNA (approximately 15 to 20 µg) was applied on 1.0% agarose gel
containing 2.2 M formaldehyde after denaturation. After gel electrophoresis,
fractionated RNA was transferred onto nylon membranes and cross-linked by
irradiation with ultraviolet light. Rat TGF-ß1 cDNA (American Type
Culture Collection, Rockville, MD) and partial cDNA for rat fibronectin and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cloned after reverse
transcription-PCR and confirmed by DNA sequencing were used for the respective
DNA probes. Prehybridization was performed at 65°C for 2 h. Hybridization
was carried out at 65°C for 16 h with the DNA probe labeled radioactively
using 5 µCi of 32P-
-dCTP (3000 Ci/mmol) and Ready-To-Go
random DNA labeling kit (Amersham Pharmacia Biotech, Buckingham-shire, United
Kingdom). After hybridization, the nylon membrane was washed at 65°C for
15 min with 2x SSC (20x SSC: 3 M NaCl, 0.3 M sodium citrate, pH
7.0) containing 0.5% sodium dodecyl sulfate (SDS) followed by washing with
0.5x SSC containing 0.1% SDS under the same conditions. At the final
step, the membrane was washed with 0.1x SSC containing 0.1% SDS at
50°C for 10 min. After washing, air-dried membrane was autoradiographed
using Kodak x-ray film with intensifying screen at -70°C for appropriate
times. For the subsequent rehybridization with a different DNA probe, the blot
was stripped at 80°C with stripping solution (0.1x SSC plus 0.5%
SDS). Quantification of mRNA signals was carried out by densitometry using
GS-700 imaging densitometer (Bio-Rad, Hercules, CA) and normalized with GAPDH
mRNA signal.
Western Blot Analysis
TGF-ß and fibronectin secreted from cultured mesangial cells were
analyzed by Western blot. The respective supernatant containing an appropriate
amount of protein was subjected to SDS-polyacrylamide gel electrophoresis (15%
gel for TGF-ß and 8% gel for fibronectin). After completion of
electrophoresis, proteins were transferred onto nitrocellulose membrane
(Hybond N, Amersham Pharmacia Biotech) in transfer buffer (50 mM Tris-HCl, pH
7.0, 380 mM glycine, and 20% methanol). The blots were blocked in 5% nonfat
dry milk dissolved in phosphate-buffered saline with 0.1% Tween 20. The
membranes were incubated for 1 h with polyclonal rabbit pan-specific
anti-TGF-ß antibody (R&D Systems, Minneapolis, MN) or rabbit anti-rat
fibronectin antibody (Chemicon International, Temecula, CA). The secondary
antibody was peroxidase-conjugated goat anti-rabbit IgG antibody (Cappel,
Durham, NC). The luminescence detection of peroxidase was performed with the
enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to
the manufacturer's instructions. The relative amount of positive
immunoreactive proteins was quantified with densitometric analysis using a
GS-700 imaging densitometer (Bio-Rad).
Statistical Analyses
Results were expressed as mean ± SEM. ANOVA was used to assess the
differences between multiple groups. If the F statistic was significant, the
mean values obtained from each group were then compared by the Fisher least
significant different method. A P value <0.05 was used to indicate
a statistically significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of Lovastatin on Urine Albumin Excretion and Mean Glomerular
Volume in Diabetic Rats
Changes in 24-h urine albumin excretion (UAE) were monitored at 1, 3, 6,
and 12 mo after induction of diabetes
(Figure 1). A progressive
increase in UAE with time was found in untreated diabetic rats. UAE in
untreated diabetic rats was sevenfold higher than in control rats at 12 mo of
diabetes. In contrast, UAE was significantly lower in lovastatin-treated rats
than in untreated diabetic rats and was not significantly different from that
in control rats at 6 and 12 mo. The changes in mean glomerular volume from the
three experimental groups are illustrated in
Figure 2. The mean glomerular
volume of untreated diabetic rats was 37 and 47% larger than that of control
rats at 6 and 12 mo of diabetes, respectively. Lovastatin effectively
prevented the glomerular hypertrophy associated with diabetes, and the mean
glomerular volume of lovastatin-treated rats was not different from that of
control rats at 6 and 12 mo.
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Effect of Lovastatin on TGF-ß1 mRNA Expression in Diabetic Rat
Glomeruli
Total RNA was obtained from the glomeruli at 4 d, 1 wk, 4 wk, and 12 wk
after induction of diabetes, and Northern blot analysis was performed for
TGF-ß1 mRNA. There was a 1.3-fold (4 d) to 1.9-fold (12 wk) increase in
glomerular TGF-ß1 mRNA in untreated diabetic rats compared with control
rats. Lovastatin effectively suppressed glomerular TGF-ß1 mRNA expression
in diabetic rats as shown in Figure
3. There were significant differences in TGF-ß1 mRNA
expression between untreated and control and between untreated and
lovastatin-treated diabetic rats in all periods (P < 0.05).
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Effect of Lovastatin on TGF-ß1 mRNA Expression and Protein
Synthesis in Cultured Rat Mesangial Cells
To confirm the effect of lovastatin on TGF-ß1 mRNA expression and
protein synthesis, rat mesangial cells were cultured under control (5.5 mM) or
high (30 mM) glucose concentration with or without lovastatin (10 µM). In
addition, activated mevalonate (100 µM) was added in the culture alone or
together with lovastatin (Figure
4). High glucose upregulated TGF-ß1 mRNA expression in
mesangial cells, and this upregulation was effectively blocked by lovastatin.
Lovastatin suppressed TGF-ß1 mRNA expression in mesangial cells cultured
under both control and high glucose concentrations. The suppressive effect of
lovastatin on TGF-ß1 mRNA expression was partially reversed by
mevalonate. Western blot for TGF-ß in the culture supernatant revealed a
unique signal at an apparent molecular weight of about 55 kD, but not at 12.5
kD, the TGF-ß monomer under the reducing condition
(Figure 5). The amount of this
protein changed in parallel with the TGF-ß1 mRNA level in response to
lovastatin and mevalonate. Although the 12.5-kD TGF-ß monomer was not
detected in our Western blot presumably because of low concentration,
TGF-ß activity as measured by mink lung epithelial cell growth inhibition
assay was detected in culture media in the absence of treatment to activate
the TGF-ß produced. The activity in media containing high glucose was
slightly higher than that in media containing control glucose (data not
shown). However, lovastatin was highly toxic to mink lung epithelial cells
and, therefore, TGF-ß activity could not be assayed in culture
supernatant containing lovastatin. The toxic effect on mink lung epithelial
cells was not abolished completely even after dialysis of media with
phosphate-buffered saline for 4 d (data not shown).
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Effect of Lovastatin on Fibronectin mRNA and Protein Expression in
Cultured Rat Mesangial Cells
The expression of fibronectin increased at the protein level as well as the
mRNA level under high glucose concentration, as shown in Figures
6 and
7. This increase was markedly
reduced by lovastatin. The suppressive effect of lovastatin on fibronectin
expression was almost completely reversed by mevalonate. The effects of
lovastatin and mevalonate were observed in cells cultured in control glucose
as well. The changes in fibronectin expression in response to lovastatin and
mevalonate were comparable to those of TGF-ß1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Western blot analysis, a single protein with apparent molecular weight of 55 kD was specifically reactive with polyclonal TGF-ß antibody, and the amount of this protein changed in parallel with TGF-ß1 mRNA levels in response to lovastatin and mevalonate. The activation of TGF-ß1 requires cleavage of pro-TGF-ß1 (390 amino acid protein) to produce small latent complexes composed of latency-associated protein and 12.5-kD mature TGF-ß1 (112 amino acid protein). It has been suggested that the protease responsible for this cleavage might be KEX/furin-like proteases, which recognize multibasic sites in secreted proteins (27). The pro-TGF-ß1 has an apparent molecular weight of 45 to 56 kD depending on glycosylation (28) and is usually produced from various cells in serum-free culture media because of deficiency in proteases. Therefore, it is suggested that the 55-kD protein observed in this study is an unprocessed pro-TGF-ß.
The possible cellular targets of lovastatin are membrane-bound small GTP binding proteins including Ras and Rho family proteins since isoprenoids such as FPP and GGPP are also metabolites of the mevalonate pathway. These small GTP binding proteins must be bound to membrane (through the protein prenylation [29] such as farnesylation and geranylgeranylation) to become functional in the signaling pathway. Therefore, the inhibition of the mevalonate pathway affects the lipidation and localization of the small GTP binding proteins (30) by limitation of FPP and GGPP. Ras protein is involved in the signaling pathway for cell growth, differentiation, and development. The major signaling pathway following Ras activation is the mitogen-activated protein (MAP) kinase pathway leading to expression of early responsive genes such as c-fos and c-jun. The heterodimer of c-Fos and c-Jun proteins, so called activator protein-1 (AP-1), is a transcription factor for many genes encoding proteins involved in cell proliferation. Interestingly, there are three different AP-1 binding sites in the promoter region of human TGF-ß1 gene (31), and these AP-1 binding sites are the major responsible elements in tetradecanoylphorbol acetate activation and TGF-ß1 autoinduction of TGF-ß1 gene expression. A recent study showed that Smad3 and Smad4, intracellular signaling molecules involved in TGF-ß signaling, cooperate with c-Fos/c-Jun to mediate TGF-ß-induced transcription efficiently (32). Therefore, it is reasonable to assume that the reduction of farnesylated Ras by inhibition of the mevalonate pathway with lovastatin possibly suppresses AP-1 (c-Fos/c-Jun) synthesis and results in the suppression of TGF-ß1 expression. It is well known that TGF-ß is a key mediator of deposition of ECM proteins, including fibronectin under various conditions such as high glucose (5,33). In this context, it is suggested that the suppressive effect of lovastatin on TGF-ß expression through the reduction in Ras activity may result in the suppression of ECM synthesis including fibronectin in diabetic glomeruli. Recently, Essig et al. (19) proposed that lovastatin may enhance the degradation of ECM proteins by modulating plasminogen activator/plasmin system. In rat proximal tubular cells, limited geranylgeranylation of Rho proteins rather than farnesylation of Ras after lovastatin treatment led to increases in tissue-type plasminogen activator (tPA) and urokinase (uPA) and decreases in plasminogen activator inhibitor-1 (PAI-1) through reorganization of cytoskeleton (19). This was the first demonstration that lovastatin increases ECM-degrading protease activity.
Our in vivo and in vitro results suggest that HMG CoA reductase inhibition by lovastatin delays the onset and progression of diabetic glomerular injury in experimental diabetes by suppressing glomerular TGF-ß and fibronectin expression. Although the precise mechanism involved in the lovastatin effect on ECM proteins at the cellular and molecular level is not known, it appears from this and other studies that lovastatin decreases lipidation of Ras and/or Rho proteins leading to suppression of TGF-ß1 and PAI-1 expression and an increase in ECM-degrading enzymes such as tPA and uPA which, in turn, reduce accumulation of ECM proteins.
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Acknowledgments |
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,
where VG is glomerular volume, 1.38 represents the shape
coefficient, and 1.01 is the size distribution coefficient. Obtained values
are presented as mean ± SEM, n is the number of glomeruli
examined, and N represents the number of rats used in the
morphometric study. Abbreviations as in
