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* Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan;
Department of Pathology, Otowa Hospital, Yamashina, Kyoto 607-8062, Japan;
Lilly Research Laboratories, Indianapolis, Indianapolis 46285, USA; and
§ Research Division, Joslin Diabetes Center and Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02215, USA
1Correspondence: Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan. E-mail: haneda{at}belle.shiga-med.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: transforming growth factor ß (TGF-ß) • fibronectin • type IV collagen • diabetic nephropathy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Asian countries.
Clinical studies such as the Diabetes Control and Complications Trial
(DCCT) in subjects with type 1 diabetes (2)
and UK
Prospective Diabetes Study (UKPDS) in subjects with type 2 diabetes
(3)
clearly link hyperglycemia to vascular complications,
including diabetic nephropathy. Furthermore, the maintenance of
normoglycemia for 10 years by pancreatic transplantation regressed
glomerular pathological changes in eight subjects with type 1 diabetes
(4)
. However, the intensive treatment in both DCCT and
UKPDS failed to maintain normoglycemia in most subjects with diabetes.
Therefore, an understanding of the hyperglycemia-related molecular
pathogenesis of diabetic nephropathy is needed to provide further
insight into therapeutic strategies for diabetic kidney disease.
Multiple biochemical mechanisms by which the diabetic state
(hyperglycemia) causes glomerular dysfunction have been proposed,
including activation of protein kinase C (PKC) extracellular-regulated
protein kinase (ERK) pathway (5
6
7
8)
, enhanced polyol
pathway (9
, 10)
related to myo-inositol
depletion (10
, 11)
, altered redox state, and oxidative
stress (12
, 13)
, overproduction of advanced glycation end
products (14)
, and enhanced growth factor and cytokine
production (15)
. The inappropriate activation of PKC has
been implicated as a putative mediator in the pathogenesis of diabetic
nephropathy based on evidence in both in vivo experimental
animal models for type 1 diabetes and in vitro studies in
cultured glomerular cells (5
6
7
8)
. An increase in de
novo synthesis of diacylglycerol generated from glycolytic
intermediates and the resulting activation of PKC, followed by the
activation of ERK, were found in renal glomeruli (7
, 16
17
18
19
20)
of streptozotocin (STZ)-induced diabetic animals as well
as in vascular cells such as glomerular mesangial cells (18
, 21
, 22)
. Furthermore, a variety of glomerular and mesangial cell
dysfunction caused by diabetes or high glucose was mimicked by phorbol
esters, which directly activate PKC, and abrogated by PKC inhibitors,
implicating PKC activation in the pathogenesis of glomerular and
mesangial dysfunction in diabetes (7
, 23
, 24)
. We have
recently shown that short-term treatment with orally administered PKC
ß inhibitor (LY333531) can prevent early diabetes-induced glomerular
dysfunction such as glomerular hyperfiltration, albuminuria, and
enhanced mRNA expression of transforming growth factor ß1 (TGF-ß1)
and extracellular matrix (ECM) proteins in STZ-induced,
insulin-deficient diabetic rats, a rodent model for type 1 diabetes
(18
, 19)
. However, the effect of long-term PKC inhibition
on diabetes-induced glomerular pathology in preclinical models of
diabetes remains to be clarified.
The chronic renal response to diabetes is characterized by histological
abnormalities such as glomerular hypertrophy, basement membrane
thickening, and mesangial expansion. Of these, the progression of
mesangial expansion is considered to be responsible for the
obliteration of capillary lumen leading to glomerulosclerosis and ESRD
(25
26
27)
. We examined whether chronic administration of a
PKC ß inhibitor could prevent the glomerular mesangial expansion in a
preclinical model of diabetes. Since type 2 diabetes is the most
prevalent clinical form not only in Asian countries but also in Western
societies, we used diabetic db/db mice, a rodent model for type 2
diabetes. Our findings provide evidence for the beneficial effect of
PKC ß inhibition on glomerular mesangial expansion as well as on
other glomerular dysfunction such as albuminuria in diabetic db/db
mice, a rodent model for type 2 diabetes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of glomeruli and the measurement of glomerular PKC
activity
Sixteen weeks after treatment with the PKC ß inhibitor (25 wk
of age), renal glomeruli were isolated by sieving with stainless steel
and nylon meshes, as described previously (28)
. In brief,
bilateral kidneys were dissected, homogenized in ice-cold RPMI1640
media containing 20 mM HEPES (pH 7.4) after removing the capsules, and
passed through sieves of various sizes to isolate the glomeruli.
Isolated glomeruli were washed twice with RPI1640 media containing 20
mM HEPES (pH 7.4) and once with a salt solution (137 mM NaCl, 5.4 mM
KCl, 0.3 mM sodium phosphate, 0.4 mM potassium phosphate, 5.5 mM
glucose, 10 mM MgCl2, 25 mM ß-glycerophosphate,
5 mM EGTA, 2.5 mM CaCl2, and 20 mM HEPES, pH
7.4). PKC activity was determined by a modified in situ PKC
assay using isolated glomeruli that was validated as described
previously (29)
. The glomeruli were incubated with a salt
solution for 15 min in the presence or absence of 100 µM PKC-specific
substrate, RTLRRL, after the addition of 5 mg/ml digitonin (final
concentration, 50 µg/ml) and 1 mM ATP (final concentration, 100 µM)
mixed with 
-[32P]ATP (<1500 cpm/pmol). The reaction
was stopped by 5% trichloroacetic acid (TCA). Aliquots of the reaction
were spotted on 2.5 x 2.5 cm phosphocellulose paper (Whatman P81,
Maidstone, U.K.) and washed in three changes of 75 mM phosphoric acid
and once with 75 mM sodium phosphate (pH 7.5). Radioactivity of
phosphorylated substrate was determined by liquid scintillation
counting. Protein content of each sample was measured by the method of
Bradford (30)
. Glomerular PKC activity was normalized by
the corresponding protein content.
Sixteen weeks after treatment with the PKC ß inhibitor (25 wk of age), 24 h urine samples were collected in metabolic cages for 2 consecutive days. Albumin in urine was measured by a competitive ELISA (Albuwell M, Exocell Inc., Philadelphia, Pa.) according to the manufacturer’s instruction.
Histological and morphometric procedures
Sixteen weeks after treatment with the PKC ß inhibitor (25 wk
of age), mice were deeply anesthetized by an intraperitoneal injection
of 50 mg/kg body weight pentobarbital sodium (Abbott laboratories,
Chicago, Ill.). Inferior vena cava and abdominal aorta were exposed,
and 24 gauge needles were inserted into the former and the latter
caudal to the renal vessels, respectively. After perfusion with
ice-cold Ringer solution, the kidneys were perfused with 10% buffered
formalin (FM), excised, decapsulated, weighed, and immersed in 10% FM.
Both kidneys were embedded in paraffin and sections of 4 µm thickness
were cut perpendicular to the long axis of the kidney for morphometric
and immunohistochemical analysis.
For morphometric analysis of the glomeruli, sections were stained with periodic acid-Sciff (PAS). To quantify mesangial expansion, sections were coded and read by an observer unaware of the experimental protocol applied. In each animal of the four experimental groups, 20 glomeruli cut at their vascular pole were used for a morphometric analysis. The extent of increase in mesangial matrix (defined as mesangial area) was determined by the presence of PAS-positive and nuclei-free area in the mesangium; the glomerular area was also traced along the outline of capillary loop using a computer-assisted color image analyzer LUZEX F (Nikon, Tokyo, Japan).
The tissue fixed with 10% FM was used for an immunohistochemical study of fibronectin, type IV collagen, and transforming growth factor (TGF)-ß using specific antibodies: a polyclonal anti-mouse fibronectin antibody; A852/R5H (Biogenesis, Poole, England); a polyclonal anti-mouse collagen IV antibody (Becton Dickinson Labware, Bedford, Pa.); and a polyclonal anti-TGF-ß antibody that recognizes all of the TGF-ß variants (R&D Systems Inc., Minneapolis, Minn.). Immunostaining was performed by the streptavidin-biotin immunoperoxidase method using a histofine SAB-PO kit (Nichirei, Tokyo, Japan) according to the instructions of manufacturer. Immunoreactive products were visualized using diaminobenzidine as a chromogen and counterstained with either methyl green or hematoxylin. Control staining was performed using nonimmune serum and the appropriate secondary antibody. To check the specificity of immunohistochemical findings for fibronectin, type IV collagen, and TGF-ß, absorption tests were performed; sections were preincubated in the serum containing corresponding antibodies in the presence of fivefold molar excess of the peptide used for immunization, then processed for further immunohistochemical staining as described above.
To evaluate the immunostaining for fibronectin, type IV collagen, and
TGF-ß, a total of 20 randomly chosen glomeruli per mouse were coded
and graded semiquantitatively in a double-blind manner by two
independent observers. The degree of fibronectin and type IV collagen
expression in five mice from each group was graded as follows: 0,
absent staining to 5%; 1, 5 to 25%; 2, 25 to 50%; 3, 50 to 75%, 4,
>75% (31)
. The TGF-ß expression in five mice from each
group was semiquantified as follows: 0, all glomerular cells and
mesangial matrix negative; 1, weak staining of 1 or 2 cells and/or
<25% of the glomerular tuft weakly positive; 2, intense staining of 1
or 2 cells, and/or weak staining of three or more cells and 25 to 50%
of the glomerular tuft positive; 3, intense cytoplasmic staining of
three or more cells and/or >50% of the glomerular tuft positive
(32)
.
Statistical analysis
All data are presented as mean ± standard deviation.
Comparisons among four experimental groups were analyzed by one-way
analysis of variance (ANOVA), followed by either Scheffe’s test or
Bonferroni/Dunn’s test to evaluate statistical difference between two
groups. P values less than 0.05 were defined as
statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Throughout the experimental periods (9–25 wk of age), blood glucose
levels were consistently higher in db/db mice than in db/m mice. Mean
blood pressure measured at the age of 16 wk by a tail cuff was not
affected by treatment with a PKC ß inhibitor: 117 ± 11 mmHg in
db/m (n=5) vs. 125 ± 6 mmHg in db/m treated with a PKC
ß inhibitor (n=5), not significant; 110 ± 8 mmHg in
db/db (n=4) vs. 104 ± 10 mmHg in db/db treated with a
PKC ß inhibitor (n=5), not significant. Body weights were
also greater in db/db mice than in db/m mice. Kidney weights were
slightly, but not significantly, increased in db/db mice as compared to
db/m mice. Kidney-to-body weight ratio differed significantly between
nondiabetic db/m and diabetic db/db mice (data not shown), since the
diabetic db/db mice exhibited greater body weights. Long-term oral
treatment with LY333531, a PKC ß inhibitor, at a dose of 10 mg/kg
body weight/day did not affect blood glucose levels, body weight, or
kidney weight in either nondiabetic db/m or diabetic db/db mice (Table 1)
.
|
Since glomerular PKC activity has been shown to be increased in the
glomeruli of STZ-induced, insulin-deficient diabetic rats, a rodent
model for type 1 diabetes (7
, 16
17
18
19
, 29)
, we tested
whether glomerular PKC activity was increased in db/db mice, a model
for type 2 diabetes. At 25 wk of age, PKC activity in the glomeruli of
the diabetic db/db mice was 180% relative to that observed in the
nondiabetic db/m mice (Fig. 1
). Treatment with PKC ß inhibitor reduced PKC activity to normal
levels (Fig. 1)
. However, administration of the PKC ß inhibitor did
not affect glomerular PKC activity in the nondiabetic db/m mice.
|
Urinary albumin excretion, which is one of parameters of glomerular
dysfunction in diabetes, was also measured. Urinary albumin excretion
was significantly increased in db/db mice as compared with that in db/m
mice at the age of 25 wk (Fig. 2
). Administration of the PKC ß inhibitor significantly abrogated the
increased urinary albumin excretion rates in db/db mice (Fig. 2)
.
Urinary albumin excretion rates in db/db mice treated with the PKC ß
inhibitor were nearly comparable with those in nondiabetic db/m mice
(Fig. 2)
. The PKC ß inhibitor did not affect urinary albumin
excretion in db/m mice.
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PKC ß inhibitor significantly ameliorates mesangial expansion and
prevents the enhanced expression of ECM proteins and TGF-ß
One of the most striking characteristics of diabetic nephropathy
is mesangial expansion, which results from accumulation of ECM proteins
(33
34
35)
. Since mesangial expansion is strongly associated
with a decrease in filtration surface and ultimately ESRD in human
diabetics (25
26
27)
, we tested whether the long-term
treatment with a PKC ß inhibitor could reduce mesangial expansion in
diabetic db/db mice. The glomerular appearance in db/db mice showed
accelerated mesangial expansion characterized by an increase in
PAS-positive mesangial matrix area relative with that observed in db/m
mice at 25 wk of age (Fig. 3
, panel A vs. panel C). This mesangial expansion
in db/db mice was prevented by the treatment with the PKC ß inhibitor
for 16 wk (Fig. 3D
). The PKC ß inhibitor did not alter the
glomerular appearance in db/m mice (Fig. 3B
). Mesangial
expansion was further quantitated by a morphometrical analysis. The PAS
positive and nuclei-free mesangial area in the glomeruli of db/db mice
was 441% of that in db/m mice (Fig. 4A
). The treatment with the PKC ß inhibitor reduced the
extent of mesangial expansion by 48% in db/db mice, but did not affect
mesangial area in db/m mice (Fig. 4A
). Total glomerular area
by tracing along the outline of capillary loop was also increased in
db/db mice compared with that in db/m mice, but PKC ß inhibitor did
not affect this parameter (Fig. 4B
). Thus, the relative
mesangial area calculated by mesangial area/total glomerular area ratio
was increased by 320% in db/db mice as compared with db/m mice.
Administration of the PKC inhibitor significantly ameliorated the
increase in the relative mesangial area in db/db mice compared with
that in untreated db/db mice (Fig. 4C)
.
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Consistent with the results of mesangial expansion, the diabetic db/db
mice demonstrated overexpression of ECM components such as fibronectin
and type IV collagen as compared with db/m mice (Fig. 5A
and Fig. 6A
vs. Fig. 5C
and Fig. 6C
,
respectively). Administration of the PKC ß inhibitor also reduced the
overexpression of fibronectin and type IV collagen in the glomeruli of
db/db mice to levels comparable to those observed in db/m mice (Fig. 5D
and Fig. 6D
vs. Fig. 5A
and Fig. 6A
, respectively). Expression of TGF-ß, one of the
possible mediators responsible for the overexpression of ECM proteins
in diabetes (15
, 36)
was also enhanced in the glomeruli of
db/db mice (Fig. 7
C). Treatment with the PKC ß inhibitor reduced TGF-ß
overexpression in db/db mice (Fig. 7D
). Semiquantitative
scores for fibronectin, type IV collagen, and TGF-ß expression
increased from 1.4 ± 0.5, 1.3 ± 0.5, 1.3 ± 0.5 in
db/m mice to 3.4 ± 0.7, 3.6 ± 0.5, 2.8 ± 0.4 in db/db
mice, respectively (db/m vs. db/db, n=5,
P<0.05). Treatment with the PKC ß inhibitor again reduced
scores for fibronectin, type IV collagen, and TGF-ß expression to
1.8 ± 0.5, 1.8 ± 0.5, 1.5 ± 0.7, respectively (db/db
vs. db/db treated with a PKC ß inhibitor, n=5,
P<0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 35
, 37)
. Progression of the latter is
considered to be responsible for the obliteration of capillary lumen,
which leads to glomerulosclerosis and the decline of glomerular
filtration rates (27
, 34)
. Although numerous in
vivo studies in glomeruli of diabetic animals as well as in
vitro studies in glomerular mesangial cells cultured under high
glucose conditions have implicated the activation of PKC as an
important causative factor in diabetic nephropathy (5)
, a
long-term in vivo study to examine the effect of the PKC
inhibition on diabetic glomerular histological abnormalities has not
been undertaken. Since our previous studies indicated that the oral
administration of a PKC ß inhibitor (LY333531) ameliorated early
glomerular dysfunction such as glomerular hyperfiltration and
albuminuria in STZ-induced diabetic rats (19)
, we examined
whether this inhibitor would have beneficial effects in a long-term
in vivo study in db/db mice. We chose db/db mice to test
whether glomerular PKC activation in this model for type 2 diabetes
could be linked to glomerular abnormalities and glomerular mesangial
expansion resembling that found in human diabetes (38
, 39)
.
Similar to a previous report (39)
, the quantitative
analysis in the present study revealed a 400% increase in mesangial
matrix area in the diabetic db/db mice as compared with that in the
nondiabetic db/m mice. As shown by the immunohistochemical analysis,
mesangial expansion in db/db mice was accompanied by accumulation of
ECM proteins such as fibronectin and type IV collagen. Treatment of
db/db mice with the PKC ß inhibitor significantly reduced mesangial
expansion and attenuated the increased expression of fibronectin and
type IV collagen. An increase in the expression of mRNA for fibronectin
and type IV collagen has been shown in glomeruli of STZ-induced
diabetic animals (18
, 40
41
42
43
44)
as well as in the cortex of
db/db mice (39
, 45)
. We have recently shown that the PKC
ß inhibitor can abrogate the increased mRNA expression of ECM
proteins in parallel with the specific inhibition of glomerular PKC ß
activation in STZ-induced diabetic rats (18)
. The area
under the curve (AUC) in male mice treated with 30 mg/kg body
weight/day of PKC ß inhibitor, LY333531 and its equipotent metabolite
LY33852 was 3.472 µM/h, which yields an average daily AUC of 145 nM
(Dr. Carl Garner, personal communication). Extrapolating these data to
the 10 mg/kg body weight/day dose used in the present study would
predict an AUC of 1.15 µM/h, with an average daily AUC of 48 nM. This
plasma concentration of PKC inhibitory activity is specific for PKC ß
isoform and should not significantly inhibit other PKC isoforms
(19)
. Thus, amelioration of mesangial expansion by the PKC
ß inhibitor suggests that the reduction of diabetes-induced
overexpression of fibronectin and type IV collagen may occur through
the inhibition of glomerular PKC ß activation, although roles of
other PKC isoforms remain to be clarified.
The beneficial effect of PKC ß inhibition on the increased
accumulation of ECM proteins in the mesangial area could be due to its
direct effect on the production of ECM proteins by mesangial cells or
though the production of TGF-ß. TGF-ß has been recently implicated
to mediate diabetes- or hyperglycemia-induced overproduction of ECM
proteins, resulting in mesangial expansion (15
, 46
47
48)
.
Our present study has also demonstrated that the protein expression of
TGF-ß is enhanced in the glomeruli of db/db mice in association with
accelerated mesangial expansion consisting of fibronectin and type IV
collagen. Furthermore, we have found that this enhanced expression of
TGF-ß was inhibited by treatment with a PKC ß inhibitor, consistent
with the attenuation of ECM overexpressions. In contrast to our
findings, Cohen et al. (49)
reported that TGF-ß mRNA and
protein in renal cortex from db/db mice did not differ from those in
their nondiabetic littermate db/m mice, and urine and plasma
concentrations of immunoreactive TGF-ß1 were reduced in db/db mice.
However, Cohen et al. (49)
concluded that TGF-ß pathway
was implicated as an important mediator in the increased gene
expression of ECM by demonstrating up-regulation of TGF-ß type II
receptor mRNA and protein in db/db mice. The methods described in our
study differ from those used by Cohen et al. For instance, the
anti-TGF-ß antibody in our study recognizes all forms of TGF-ß. In
addition, we found the enhancement of TGF-ß protein expression in
glomeruli, whereas Cohen et al. examined the mRNA and protein
expression in renal cortex in db/db and db/m mice. Thus, these
differences in methodology and locus may explain the dissimilarity
between these findings. Our findings in db/db mice thus appear to
result from glomerular TGF-ß mRNA overexpression or an excess of
TGF-ß protein trapped in the glomeruli. To clarify this issue,
in situ hybridization studies for TGF-ß isoforms are
needed.
PKC activation might regulate the overexpression of TGF-ß at a
transcriptional level since its promoter contains activator protein 1
(AP-1) sites. AP-1 sites are activated by the proto-oncogenes complex,
fos-jun homodimers or heterodimers through a PKC-ERK dependent pathway
(50
, 51)
. To examine this hypothesis, we recently provided
evidence that ERK activities are enhanced in the glomeruli of
STZ-induced diabetic rats, and this enhancement was dependent on PKC
activation (7)
. Thus, diabetes-induced TGF-ß
overexpression in the glomeruli of db/db mice might result from the
enhancement of PKC-ERK activation and lead to the overexpression of ECM
proteins, finally resulting in mesangial expansion. Alternatively,
activation of PKC-ERK pathway could directly stimulate the production
of ECM proteins. For example, the AP-1 complexes were shown to be able
to increase the transcriptional activation of fibronectin gene
(52)
. Cyclic AMP-responsive elements (CRE) were found in
the promoter region of fibronectin gene (53
, 54)
, and AP-1
complexes were shown to be able to activate CRE (55)
.
Indeed, phorbol esters and serum have been reported to enhance the gene
expression of fibronectin by the activation of CRE (54)
.
Thus, it is possible that diabetes- or high glucose-induced PKC-ERK
activation could play a pivotal role in ECM overproduction in either a
direct fashion or indirectly through TGF-ß production.
Diabetic nephropathy is the leading cause of ESRD, requiring
dialysis therapy in the Western and Asian countries (1)
.
About 25–40% of patients with type 1 and type 2 diabetes develop
diabetic nephropathy 25 years after the onset of diabetes
(56
57
58
59
60)
. Based on the results of cumulative
epidemiological studies (2
, 3)
, it is evident that the
most effective therapy to prevent the development and progression of
nephropathy is to maintain normoglycemia. However, from these
epidemiological studies, it is also evident that the maintenance of
long-term normoglycemia is difficult in most subjects with type 1 or
type 2 diabetes. Pancreatic transplantation is an alternative strategy
to maintaining long-term normoglycemia. However, pancreatic
transplantation is not available to the majority of diabetic patients
worldwide. Therefore, efforts have been directed to clarify the
responsible mechanisms by which diabetes causes diabetic nephropathy
and identify the therapeutic strategies that could abrogate the
development and progression of diabetic nephropathy. Our findings in
the present study provide the first in vivo evidence that
long-term PKC inhibition by the oral treatment with a PKC ß inhibitor
can influence the development of diabetic nephropathy histologically
without causing any obvious adverse effect and independent of glycemic
control. Our results also suggest that a PKC ß inhibitor might be a
new and novel approach for the treatment of diabetic
nephropathy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 
in rat glomerular mesangial cells cultured under high glucose conditions. Diabetologia 37,838-841[Medline]
-tocopherol. J. Am. Soc. Nephrol. 8,426-435[Abstract]
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