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From the Division of Artificial Kidneys*
and
the Department of Geriatric Medicine,
Faculty
of Medicine, Kyoto University, Sakyo-ku, Kyoto; the Chugai
Pharmaceutical Company, Limited,
Shizuoka;
and the Department of Laboratory Medicine,
School of Medicine, The University of Tokushima, Tokushima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle
actin (-SMA) compared to the normal group. The treatment of OCT or
1,25(OH)2D3 significantly reduced the
expression of these proteins. The mRNA of the glomeruli of anti-thy-1
model expressed significantly higher levels of type I and type IV
collagens, and -SMA at day 8 compared to normal rats.
Treatment with OCT or 1,25(OH)2D3
inhibited the mRNA expressions of type I and type IV collagens,
as well as that of -SMA. These data demonstrate that OCT inhibits
mesangial cell proliferation and extracellular matrix expansion with a
low calcemic activity. Disease control rats showed significantly
increased levels of transforming growth factor-ß1 protein in the
glomeruli, but treatment with OCT or
1,25(OH)2D3 markedly reduced this
expression. The levels of mRNA in glomeruli were also consistent with
these protein levels. Therefore, the suppressive effect of OCT
may be mediated by inhibition of transforming growth factor-ß1. The
present results suggest that OCT has potential for use in therapeutic
strategy for the treatment of glomerulonephritis without inducing
hypercalcemia.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, 1,25(OH)2D3 was shown to have a preventive effect in progressive glomerular damage in a renal ablation model.5 However, 1,25(OH)2D3 has an adverse effect in that its use leads to hypercalcemia and hyperphosphatemia, which eventually represent risk factors for the progression of renal injury. It is, therefore, difficult to use 1,25(OH)2D3 for the practical treatment of patients at the predialysis stage. One of the vitamin D analogs, 22-oxa-calcitriol (OCT), is a potent regulator of cell differentiation6,7 and of immunological function,8 and has a low activity in relation to hypercalcemia. We recently demonstrated that OCT markedly regulated mesangial cell proliferation in vitro compared to 1,25(OH)2D3 in a transforming growth factor (TGF)-ß mediated manner.9 In the present study, we report an investigation of the effect of OCT in vivo using a model of mesangial proliferative glomerulonephritis induced by anti-thy-1 glomerulonephritis. This glomerular lesion is characterized by mesangial cell proliferation and subsequent mesangial matrix expansion by the accumulation of extracellular matrix protein. The findings herein show that OCT inhibits mesangial cell proliferation and extracellular matrix expansion, and prevents albuminuria with low calcemic activity in anti-thy-1 glomerulonephritis. The effect of OCT could be mediated by the inhibition of TGF-ß1 in this model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Wistar rats weighing 
250 g were used for all experiments.
The rats were maintained on standard chow and water ad
libitum. OCT was synthesized by Chugai Pharmaceutical Co., Ltd,
Tokyo, Japan.
1,25(OH)2D3 was purchased
from Philips Duphar Co. (Amsterdam, The Netherlands). Four groups of 20
rats each were used. Group 1 contained normal control rats that were
given an intravenous injection of 0.1 ml of phosphate-buffered saline
(PBS) (pH 8.0) containing 0.2% ethanol and 0.01% Tween 20 (PBS-ET) on
alternate days. Group 2 was a disease control group, that received an
injection of 0.1 ml of PBS-ET on alternate days intravenously from the
next day, after an intravenous injection of monoclonal anti-Thy-1.1
antibody (OX-7), 1 mg/kg body weight, diluted with PBS (pH 7.4) as
described previously.10
Group 3 was treated intravenously
with OCT at 0.4 µg/kg body weight diluted with PBS-ET (total volume
0.1 ml) on alternate days from the next day after an intravenous
injection of OX-7. Group 4 was treated with
1,25(OH)2D3 at 0.4 µg/kg
body weight diluted with PBS-ET (total volume, 0.1 ml) on alternate
days after an intravenous injection of OX-7. Urine samples were
collected at day 8. All rats were sacrificed under ether anesthesia by
heart puncture at day 8, day 14, and day 28. Blood samples were
collected and both kidneys were removed. Three independent sets of
experiment were performed. The food intakes and body weights were
examined at day 8.
Biochemical Measurements
Urinary albumin excretion was measured by using Nephrat II enzyme-linked immunosorbent assay kit (Exocell, INC., PA) and urine creatinine was determined using the picric acid method (Creatinine test Wako; Wako, Osaka, Japan). Urine albumin/creatinine ratio was used to estimate quantitation of albuminuria.11,12 Rats were sacrificed under ether anesthesia by heart puncture. Blood samples were collected via heart puncture and both kidneys were removed. Serum creatinine, blood urea nitrogen (BUN), calcium, and phosphate were determined with a Hitachi Model 736 autoanalyzer.
Light Microscopy
The kidneys were fixed in methyl Carnoy’s solution and embedded in paraffin for light microscopic examination and 2-µm-thick sections were stained with hematoxylin and eosin, periodic acid Schiff’s reagent, and periodic silver methenamine. The total number of nuclei per glomerular cross-section was determined and glomerulosclerosis was assessed on all glomeruli. The severity of sclerosis for each glomerulus was graded from 0 to 4+ based on the modified method of Raij and colleagues.13 A whole kidney sclerosis index was obtained by averaging the scores from all glomeruli in each section.
Morphometric Analysis
The profile areas of 100 glomerular tufts were measured on the periodic acid Schiff’s reagent-stained sections with a digitized graphic pad and the NIH image. Both superficial cortical and juxtamedullary glomeruli were measured. The mean glomerular volume was derived from the mean equatorial area of the glomeruli of each specimen, as approximated by the method of DeHoff and Rhines14 for the mean size of particles with a similar shape. The mean area of each class (A1, A2,... ... ..An.) was calculated, to determine the harmonic mean (z) of the profile areas (z = 1/n [1/A1 + 1/A2 +... . . 0.1/An]. The mean equatorial area (a[mean]) was estimated from equation a[mean] = K2k4/z where k2 and k4 are dimensional measures to be estimated, and the shape or the particles. The value of k2 was set at 1 and that of k4 at 3.14/2, on the assumption that the glomeruli correspond to spheres or ellipsoids.
Immunohistochemistry
Four-µm-thick cryostat sections were processed by an indirect
immunoperoxidase technique. The following primary antibodies were used:
an IgG fraction of polyclonal guinea pig anti-rat type I
collagen,15
an IgG fraction of polyclonal rabbit anti-rat
type IV collagen,15
and an IgG fraction of polyclonal
rabbit TGF-ß1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). An
immunofluorescence detection of glomerular -smooth muscle actin
(-SMA) was performed on 4-µm-thick cryostat sections using an
indirect immunofluorescence procedure. Monoclonal antibody anti--SMA
(Sigma Chemical Co., St Louis, MO) was used as the primary antibody,
followed by a mouse IgG (Tago Inc., Camarilb, CA) and fluorescein
isothiocyanate conjugated with streptavidin (Zymed Laboratories, Inc.,
South San Francisco, CA). To evaluate the staining for type I and type
IV collagens or TGF-ß1 or -SMA each glomerulus was graded
semiquantitatively as described previously.16
The
specificities of the stainings of type I collagen and TGF-ß1 were
confirmed by the absorption experiment, which were examined
preincubating each antibody with excess of the corresponding specific
antigen (rat collagen type I, C7661; Sigma Chemical Co.; and TGF-ß1
peptide, sc146P; Santa Cruz Biotechnology, Inc.).
Quantitation of Glomerular Cell Proliferation
Proliferation cell nuclear antigen (PCNA) was identified by modified an immunohistology method described previously.17 Methylcarnoy-treated paraffin sections were deparaffinized with xylene and graded ethanol. After blocking endogenous peroxidase with 3% hydrogen peroxidase, the sections were treated with blocking kit for endogenous biotin (Vector Laboratories, Burlingame, CA). They were incubated with anti-PCNA for 60 minutes (DAKO, Burlingame, CA), with biotinylated anti-mouse IgG for 30 minutes (Vector), and avidin-biotin complex (Vector) followed by diaminobenzidine peroxide and counterstained with methyl green. For each sample, 50 glomeruli were sequentially selected and PCNA-positive cells were quantitated.
Glomerular RNA Preparation and Quantitative Polymerase Chain Reaction (PCR)
Total RNA was prepared from glomeruli that had been isolated by
the graded sieving procedure. Quantitative PCR was performed using real
time Taqman TM technology18
and analyzed on a Model 7700
sequence detector (Applied Biosystems, Foster City, CA).19
Five independent samples from each group were measured. Primers and the
TaqMan probe for 18s ribosomes were purchased from Perkin-Elmer Applied
Biosystems. Table 1
shows the sequences
for the TaqMan probes and primers used. AmpliTaqMan DNA polymerase
extended the primer and displaced the TaqMan probe through its 5' to 3'
exonuclease activity. The probes were labeled with a reporter
fluorescent dye 6-carboxy-fluorescein or
2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein at the 5' end and a
quencher fluorescent dye (6-carboxy-tetramethyl-rhodamine) at the 3'
end. Nuclease degradation of the hybridization probe removed the
quenching effect of the 6-carboxy-tetramethy-rhondamine from the
6-carboxy-fluorescein or
2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein fluorescent emission,
thus increasing the peak fluorescent emission at 517 and 554 nm,
respectively. No signal was emitted when the probe was intact. The
conditions of one-step reverse transcriptase-PCR were as follows: 2
minutes at 50°C, 3 minutes at 60°C, 5 minutes at 95°C followed by
40 cycles of 20 seconds at 94°C and 1 minute at 60°C. The assay
used an instrument capable of measuring fluorescence in real time (ABI
Prism 7700 sequence detector, Perkin-Elmer Applied Biosystems). Signals
were detected according to the manufacturer’s instructions. A computer
algorithm compared the amount of reporter dye emission (R) with the
quenching dye emission (Q) during the PCR amplification, generating an
Rn value as follows:
Rn =
(Rn+)-(Rn-), where
Rn+ is (the emission intensity of the
reporter)/(the emission intensity of the quencher at any given time in
the reaction tube), and Rn- is (the emission
intensity of the reporter)/(the emission intensity of the quencher
measured before PCR amplification in the same reaction tube). The
Rn value represents the emission of
the reporter over the starting background fluorescence and was used for
construction of amplification plots.
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All values are expressed as the mean ± SE and analyzed by Mann-Whitney nonparametric analysis, or one-way analysis of variance with a modified t-test, performed using the Bonferroni correction. Statistical significance was defined as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The urine albumin excretion, which was corrected for urine
creatinine concentration as denominator, is demonstrated in Figure 1
. The disease control group (group 2)
showed markedly increased amounts of urine albumin, compared to the
normals. The OCT-treated group and the
1,25(OH)2D3-treated group
(groups 3 and 4) had a significantly lower urine albumin to creatinine
ratio than the disease control.
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The serum concentration of creatinine, BUN, calcium, and phosphate
are represented in Figure 2
. There were
no differences in serum creatinine levels between the disease control
group and the OCT or the
1,25(OH)2D3 groups.
BUN values were significantly lower in boththe OCT-and
1,25(OH)2D3-treated groups,
compared tothe disease control group, however, BUN values werenot
different between the OCT-treated group and
1,25(OH)2D3-treated group.
Serum calcium was significantly higher in the
1,25(OH)2D3 group, compared
to the disease control group. On the other hand, no difference in serum
calcium between the disease control group and the OCT group was
detected. The serum phosphate in both the OCT and
1,25(OH)2D3 groups were
significantly lower compared to the disease control group.
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Figure 3
shows a representative
light microscopic picture in each group. The disease control group
showed a markedly increased level of glomerular proliferation, on the
other hand, the OCT- or
1,25(OH)2D3-treated groups
ameliorated glomerular cell proliferation (Figure 4)
.Proliferating cells, detected by
PCNA staining, weremarkedly increased in disease controls, and OCT-
or 1,25(OH)2D3-treated
groups had significantly decreased PCNA-positive cells. The disease
control group showed increases in the mesangial matrix. The
glomerulosclerosis index was higher in the disease control group,
compared to the normal control group (Figure 4)
. Both the OCT- and
1,25(OH)2D3-treated groups
significantly decreased the glomerulosclerosis index. However, no
differences were found in the mean glomerular volume in each group
(Figure 4)
. The mesangiolysis was observed at day 8 in the disease
control group, and the lesion was also found in both the OCT- and
1,25(OH)2D3-treated groups.
The mesangiolysis was decreased at day 14 in both the OCT- and
1,25(OH)2D3-treated groups
and it was completely resolved at day 28 (Figure 3)
.
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Type IV collagen was clearly visible along the glomerular basement
membrane as well as weakly in the mesangial area in the normal control
group (Figure 5E)
. Type I collagen and
-SMA were not found in the normal glomeruli (Figure 5, A and I)
. In
the disease control group these proteins were strongly positive in the
expanded mesangial area (Figure 5; F, B, and J
). Both the OCT- and
1,25(OH)2D3-treated groups
had a significantly reduced expression of these proteins in glomeruli
(Figure 5
and Table 2
). A weak level of
expression of TGF-ß1 proteins was found in normal glomeruli, but was
diffuse and strongly positive in diseased glomeruli. OCT and
1,25(OH)2D3 reduced the
expression of TGF-ß1 in glomeruli (Figure 5
and Table 2
). The
specificities of the staining of type I collagen and TGF-ß1 was
performed with the absorption study using excess amounts of each
specific antigen, and the stainings of both type I collagen and
TGF-ß1 were completely blocked (Figure 6)
.
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Table 3
shows the mean mRNA
concentrations of type I and type IV collagens, -SMA, and TGF-ß1,
which were corrected for the level of 18s RNA. To normalize for the
differences in the amount of total RNA added to each reaction, the 18s
ribosome was selected as an endogenous RNA control. The mRNA
expressions of type I and IV collagens as well as -SMA were
significantly increased in the diseased glomeruli compared to normals.
OCT and 1,25(OH)2D3 led to
a reduction in the level of expression of these mRNAs. The expression
of TGF-ß in glomeruli showed significantly an approximate twofold
increase in the disease control group, and OCT and
1,25(OH)2D3 led to a
significant decrease in mRNA expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-SMA in this model.
The underlying mechanism of this suppression is related to the action
of TGF-ß. The compatible mRNA data in the glomeruli were also
obtained by a specific real-time PCR technique. In conclusion, these
results indicate that OCT has considerable potential for use as a
therapeutic strategy in the treatment of progressive glomerular
disease.
1,25(OH)2D3 is recognized,
not only as an important factor in calcium homeostasis, but also as a
potential therapeutic agent for hyperproliferative
disorders20-22
and immune
dysfunction.23
1,25(OH)2D3 also suppresses
the growth of mesangial cells24
and proximal tubular
cells in vitro.25
The administration of
1,25(OH)2D3 led to a
significant reduction in proteinuria and suppressed the autoantibodies
of active Heymann nephritis.26
Recently,
1,25(OH)2D3 has been
reported to inhibit glomerular cell proliferation and
glomerulosclerosis, and to prevent albuminuria in subtotally
nephrectomized rats.5
However
1,25(OH)2D3 produces a
significant risk for hypercalcemia and hyperphosphatemia, which are
believed to be strong risk factors in the progression of chronic renal
failure. In these aspects, it is not practical to treat the patients
clinically with
1,25(OH)2D3. On the other
hand, OCT, a vitamin D analog, could overcome these problems and has a
promising potential for clinical use. It had a strong biological
activity, including anti-proliferativeactivity and immunosuppressive
activity. Its antiproliferative effect and potent cell differentiation
activity are 10-fold, and immunosuppressive effect is 50-fold
that of 1,25(OH)2D3. The
mechanism of the difference is partly to because of the following
points, including different binding activity of OCT to vitamin D
receptor and vitamin D-binding protein (DBP) and different delivery and
metabolism of OCT to target organ, compared to
1,25(OH)2D3. However, OCT
was only 1/100 as active as
1,25(OH)2D3 in inducing
hypercalcemia.27
The precise mechanism of low activity of
hypercalcemia is still unknown, however all published data showed rapid
metabolic clearance caused by low affinity with binding proteins and
low intestinal absorption through calcium-binding proteins.
We reported previously that OCT suppressed the proliferation of mesangial cells in vitro.9 In a thy-1 model, mesangial cells are injured by antibody and activated complement at the initial phase followed by the proliferation of mesangial cells of transformed phenotype. The data herein show that OCT inhibits mesangial cell proliferation and extracellular matrix expansion, and prevents albuminuria. The anti-proliferative effect of OCT might be attributed to regulation of phenotypic change after the initial insult. It is important that preceding mesangial proliferation is a prerequisite for matrix expansion in several experiments, although it is still debatable.2,3 TGF-ß is an important pathogenic factor for glomerulosclerosis and overexpression of TGF-ß is found in most glomerular sclerotic lesions in vivo that are associated with increased turnover. The possibility cannot be excluded that the inhibition of the mesangial proliferation results in a prevention of matrix expansion, however it is likely that the suppression of TGF-ß expression prevents the synthesis of mesangial matrix proteins. The mechanism to suppress TGF-ß expression by OCT is still unknown, however, recently a molecular basis for cross-talk between TGF-ß and vitamin D signal pathways was reported. The cooperative action of these molecules can be synergistic or antagonistic in a tissue-specific manner.28
The in vivo biological effects of OCT are distinguishable from those of 1,25(OH)2D3. The 1,25(OH)2D3-treated group showed lower body weight than disease control at day 8, however, the former represented the same food intake as the latter group (data not shown). The tendency of lower BUN and phosphate and normal plasma creatinine level in the 1,25(OH)2D3-treated group seems to be explained by the low food absorption especially low protein absorption because the animals developed hypercalcemia. On the other hand, OCT did not induce hypercalcemia in this experiment, therefore, the suppressive effects of BUN and plasma creatinine by OCT may be related to the direct effect of improvement of glomerular injury.
The real-time PCR method used in this study measures PCR product accumulation through a dual-labeled fluorogenic probe. The method is performed in a closed-tube system and requires no manipulation after PCR of the sample. The method analyzes during the log phase of product accumulation, which permits many different genes to be analyzed simultaneously. This supports the use of a housekeeping gene for quantitative reverse transcriptase-PCR controls. Therefore, the real-time RCR provides very accurate and reproducible quantitation of gene copies. The method guarantees the twofold difference of the gene expression in any experiments. The system also allows for a very large dynamic range.19
The biological effect of OCT is different from that of
1,25(OH)2D3 in this study.
The differences probably relate to the pharmacokinetic properties of
these analogs, which are mainly generated by their affinity for DBP.
DBP is a serum protein that functions as a carrier of
1,25(OH)2D3 and its
analogs. It has been estimated that the affinity of OCT for DBP is
1/600 that of
1,25(OH)2D3. After an
in vivo injection, the low affinity of OCT for DBP allows it
to circulate primarily as the unbound form and therefore promotes a
rapid clearance and facilitates its accessibility to target
tissues.29
Therefore, the in vivo effects of
OCT on target gene activation are primarily dependent on its
pharmacokinetic properties. These pathological effects of OCT in
vivo were confirmed by chronic model of progressive
glomerulosclerosis in subtotally nephrectomized rats.30
Further study is in progress to clarify the mechanism of gene
regulation by OCT in vivo. In conclusion, these results
indicate that OCT has powerful therapeutic potential for the treatment
of progressive glomerular disease.
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Footnotes |
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Supported by grants-in-aid from the "Research for the Future" Program of Japan Society for the Promotion of Science (JSPS-RFTF97L00805).
Accepted for publication January 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3, is a potent modulator of in vivo immunoregulating activity without inducing hypercalcemia in mice. Endocrinology 1989, 124:2645-2647
3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 1991, 88:7276-7280,25-dihydroxyvitaminn D3 on the growth of the renal carcinoma cell line. Kidney Int 1986, 29:834-840[Medline]
,25-dihydroxyvitamin D3 and synthetic analogues of vitamin D3 with an oxygen atom in their side chain. Biochem Int 1989, 19:1143-1149[Medline]
,25 dihydroxyvitamin D3. Clin Exp Immunol 1993, 94:412-417[Medline]
,25-dihydroxyvitamin D3–24-hydroxylase mRNA expression in rats by 1,25-dihydroxyvitamin D3 and its analogs. Life Sci 1998, 62:453-459[Medline]
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