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Introduction
A large body of experimental evidence has suggested
that transforming growth factor (TGF)-b1 is an important
mediator of chronic fibrosis in a range of disease states,
including the development of glomerulosclerosis in chronic
renal failure (CRF)[1_4]. TGF-¦Â1 is a 25 kDa multifunctional
homodimeric polypeptide growth factor with a wide range of
biological effects[5,6]. It regulates biological processes, such
as cell proliferation, differentiation, and immunological
reaction[1,7]. An important function of
TGF-¦Â1 is the acceleration of tissue healing by increasing the synthesis of the
extracellular matrix and suppressing its degradation by reducing the
activity of metalloproteinases and increasing the
production of the tissue inhibitors of these
proteinases[8].
Studies have shown that the injured glomeruli express
more TGF-¦Â1 mRNA, synthesize more TGF-¦Â1 protein, and
produce more extracellular matrixes compared to normal
glomeruli[9]. Although there are successful trials that have shown
that the production of TGF-¦Â1 in glomerulosclerosis models
is suppressed by antibodies against TGF-¦Â1, so far, there
have been no reports on the assessment of renal function
following the blockade of TGF-¦Â1 activity in these models.
Furthermore, the use of such antibodies in humans is not
feasible because they may cause serum sickness or immune
complex formation when administered over prolonged periods
of time[10]. Therefore, there is a need for alternative
options to be explored. One possible alternative could be the design
and use of antisense oligodeoxynucleotides (ODN) to
TGF-¦Â1, which have sequences complementary to
TGF-¦Â1 mRNA at the translation initiation region. It has been suggested that
the TGF-¦Â1 antisense ODN is able to block the abnormal
expression of the TGF-¦Â1 gene[11,12] by binding to a selected
length of the mRNA, thereby leading to a reduction in
TGF-¦Â1 activity. Thus, the present study was designed to
investigate the effectiveness of the TGF-¦Â1 antisense ODN in
ameliorating the deterioration of kidney function in a rat model
of CRF based on a functional and morphological evaluation.
Materials and methods
General preparations All of the experiments were
approved by the local Animal Ethical Care Committee and
conformed to national guidelines. Male Wistar Kyoto rats
(150_220 g) were unilaterally nephrectomized 3 weeks prior to the
commencement of the study. Four groups were prepared as
follows: Group A (control group; n=14) received a
subcutaneous (sc) injection of saline (1 mL/kg) for 8 weeks; Group B
(n=19), C (n=13), and D (n=8) received sc injections of
puromycin (20 mg/kg) for the same period of time to induce
CRF[13]. The TGF-¦Â1 antisense ODN (Alta Bioscience, University of
Birmingham, UK) and scrambled ODN (1 mg/kg,
intravenously administered; diluted in glucose) was given to rats in
groups C and D, respectively. In these rats, the
TGF-¦Â1 antisense ODN and the scrambled ODN was given 24 h after
the 6th, 7th, and 8th puromycin injections. Puromycin was
given as a sc injection while the ODN were given
intravenously via the tail vein of the animals that were briefly
anaesthetized with a halothane/O2 mixture. The sequence of
the TGF-¦Â1 antisense ODN was
5´-CGAGGGCGGCATGGG-3´ while the sequence of the scrambled ODN was
5´-GCATGGGCGAGGGGC-3´[14]. Two other groups (groups A
and B) were given glucose intravenously instead of ODN.
At the end of the 8 week period, the rats were anaesthetized
with pentobarbitone sodium (60 mg/kg, intraperitoneally
administered) for the acute study.
Acute study After tracheostomy, the left jugular vein
and carotid artery were cannulated for continuous infusion
of anesthesia (12.5
mg·kg_1·h_1 at 3 mL/h in 140 mmol/L NaCl)
and the measurement of blood pressure,
respectively[15_17]. The left femoral artery was cannulated for blood collection.
The left kidney was exposed and the ureter was cannulated
for urine collection. An electromagnetic flow probe (Carolina
square wave electromagnetic flowmeter and EP 100 series
probe; Carolina Medical, NC, USA) was fitted around the
renal artery for renal blood flow measurement. Upon
stabilization, 4 30 min clearances were taken. Arterial blood
samples were taken at the beginning and at the end of every
2 clearances. At the end of the acute study, the animals were
killed by a rapid intravenous injection of 1 mL sodium
pentobarbital, and the kidney was collected for the
morphological study.
Chemical analysis The urine protein concentration was
measured using a bicinchoninic acid protein assay kit
(Bio-Rad, USA). Inulin clearance was calculated from urine and
plasma inulin concentrations, measured as previously
described[19], and used as a measure of glomerular filtration
rate. Urine and plasma electrolytes were measured by flame
photometry. The expression of TGF-¦Â1 in the kidney of
the rats was analyzed by measuring TGF-¦Â1 protein levels
in the kidney cortex and medulla using an ELISA-based
method (TGF-¦Â1 BD OptEIA ELISA; Pharmingen,
USA)[20].
Morphological study After fixing in formalin for at
least 24 h, the kidney tissues were processed using an
automatic tissue processor (Leica, Germany). Paraffin
blocks with individual kidney tissue embedded in each block
were produced. Thin 4_5 mm kidney tissue sections were
obtained from the paraffin blocks and stained with
hematoxylin-eosin. All of the slides were examined under a light
microscope with the help of a certified pathologist who was
unaware of the experiment protocols applied to avoid any
bias.
Statistical analysis Data were expressed as mean±SEM
and were analyzed using two-way ANOVA followed by Bonferroni/Dunn (all means) post-hoc test (Superanova;
Abacas, Berkeley, CA, USA). Experimental differences were
considered significant if P<0.05.
Results
There was a gain in body weight in all groups
throughout the entire 8 week study. However, all the CRF rats,
including those treated with either the antisense or scrambled
ODN, showed a slight decline in body weight toward the
end of the experimental period. The weight of the animals at
the end of the 8 week period and at the start of the acute
study is given in Table 1. Blood pressure at the start of the
experimental protocol was similar in all experimental groups
and is shown in Table 1.
Renal hemodynamic function Renal blood flow was
significantly lower in the CRF group, approximately 50%
compared to the control group (1.76±0.26 vs 3.44±0.27 mL·
min-1·g-1 kidney weight; P<0.05; Figure 1), but decreased by only 17% in the CRF rats treated with the
TGF-¦Â1 antisense ODN (2.09±0.36
mL·min_1· g_1 kidney weight, P<0.05). By contrast, the renal blood flow in the group treated with
scrambled ODN was also lower to a degree compared to
the untreated CRF group at 1.76±0.26
mL·min-1·g-1 kidney
weight (Figure 1).
It is evident that the glomerular filtration rate (GFR) in the
CRF group was only 30% of that of the saline-treated
animals (1.52±0.24 vs 5.02±0.53
mL·min_1·kg_1, P<0.05). However, following the TGF-¦Â1 antisense ODN treatment,
the GFR was only 29% lower than that of the saline control
group (3.55±0.43
mL·min_1·kg_1, P<0.05). In contrast, the scrambled ODN treatment did not have any significant effect,
so the GFR levels were not different from those of the CRF
rats (2.54±0.59 mL·
min_1· kg_1, Figure 2).
Renal excretory function The urine flow rate was
markedly lower in the control rats as compared to that in the CRF
and CRF-scrambled ODN-treated groups (P<0.05; Table 1).
Urinary protein excretion in the acute study was
approximately 3-fold higher in the CRF rats compared to the control
group (0.90±0.12 vs 0.30±0.05
mg·min_1·kg_1, P<0.05; Figure 3). Following the TGF-¦Â1 antisense ODN treatment, the
increase in urinary protein excretion in the CRF rats was
attenuated (0.46±0.09
mg·min_1·kg_1, P<0.05), but in the CRF group treated with the scrambled ODN, urinary protein
excretion was comparable to the CRF rats at
0.99±0.34
mg·min_1·kg_1.
There was no difference in absolute
Na+ excretion rates in any of the groups in the study. Interestingly, fractional
Na excretion was as much as 6-fold higher in the CRF rats as
compared to the control group (3.95±1.15 vs 0.61%±0.18%; P<0.05). This increment was attenuated by the
TGF-¦Â1 antisense ODN (0.82%±0.33%, P<0.05), but not by the
scrambled ODN (4.09%±1.69%) treatment (Figure 4).
Kidney weight The CRF rats had a 78% higher kidney
weight compared to that of the control rats. The kidney
weight of the CRF rats treated with the TGF-¦Â1 antisense
ODN was only 19% more than the controls and it was also
significantly lower than the untreated CRF rats (all P<0.05; Table 1). The kidney weight of the CRF rats treated with the
scrambled ODN was not significantly different from that of
the untreated CRF rats (P>0.05).
Morphological study Focal segmental
glomerulosclerosis (FSGS) was observed in the kidneys of both the untreated
and scrambled ODN-treated CRF groups (Figure 5). Periglomerular fibrosis and occasionally pinkish
hyalinization around the glomeruli were also observed in the kidneys
from these 2 groups. However, these changes around the
glomeruli were not seen in the kidneys of the
TGF-¦Â1 antisense ODN-treated group.
The light microscopic examination also showed that there
was a loss of kidney architecture in the kidneys from the
untreated and scrambled ODN-treated CRF groups. The
histology of the kidneys obtained from these groups showed
infiltration of a number of acute inflammatory cells
(neutrophils) within the glomerular tufts and chronic
inflammatory cells (lymphocytes) in the interstitium (data not
shown). Dilatation of tubules in the focal area and some
blood vessels with thick walls were also observed. In contrast,
in the CRF rats treated with TGF-¦Â1 antisense ODN, even
though there was some loss in the architecture of the kidney,
the tissue still resembled that of a normal rat. The infiltration
of neutrophils in the glomerular tuft was still present and the
interstitium showed mild and scattered infiltrate of chronic
inflammatory cells predominantly lymphocytes. However,
the overall changes were less severe in nature compared to
the untreated and scrambled ODN-treated groups (Figure 5).
Expression of TGF-¦Â1 protein levels in the kidney
cortex and medulla It was found that the
TGF-¦Â1 protein levels in the kidney cortex lysates were not significantly different
between the puromycin-induced CRF rats, either untreated
(2.567±0.278 pg/mg protein) or treated with the
TGF-¦Â1 antisense (2.404±0.264 pg/μg protein) or scrambled
ODN (2.155±0.218 pg/μg protein; all P>0.05). Similarly, no
significant difference was detected in the lysates of the kidney
medulla amongst all these groups: untreated
puromycin-induced CRF (2.980±0.336 pg/μg protein), puromycin-induced
CRF treated with TGF-¦Â1 antisense ODN (3.286±0.283
pg/μg protein), and puromycin-induced CRF treated with scrambled
ODN (3.495±0.410 pg/μg protein; all P>0.05).
Discussion
In the present study, puromycin was used to induce
chronic renal failure in rats. Even though the pathogenesis
of FSGS following administration of puromycin remains
unclear, it has been reported that the level of
TGF-¦Â1 was increased in this model of
CRF[18]. It has been suggested that
TGF-¦Â1 could be one of the mediators which plays a
major role in the process of kidney repair following injury.
Thus, repeated tissue injury (consequent to repeated
dosing with puromycin) could lead to the continuous
production of TGF-¦Â1, a progressive deposition of extracellular
matrixes, which would eventually result in tissue fibrosis.
This abnormal continuous production of TGF-¦Â1
production would convert the repair process into a form of CRF.
Therefore, a strategy was developed in an attempt to
suppress the overproduction of TGF-¦Â1 by administering the
TGF-¦Â1 antisense ODN in order to block the translation of
TGF-¦Â1 mRNA and thus reducing the deleterious action of
TGF-¦Â1.
An interesting observation of this study was that, in the
CRF rats, in spite of obvious changes in the renal functional
parameters to the treatment with TGF-¦Â1 ODN, there was no
change in the level of TGF-¦Â1 in the kidney tissues. It is
important to point out that we chose to utilize a particular
dosing regimen, that is, 1 dose of antisense ODN per week,
and this was done 24 h after the puromycin challenge. It may
well be a possibility that a more effective suppression of the
puromycin-induced renal functional and morphological
changes could be achieved using different dosing protocols
and such a different dosing protocol may accompany a
possible change in the level of TGF-¦Â1 in the kidney tissues.
Nonetheless, the results derived clearly showed that there
might have suppression in the level of TGF-¦Â1, perhaps too
small to be detected. However, the relationship between the
effect of the puromycin damage and the ability of the
TGF-¦Â1 antisense ODN to suppress these responses was very clear,
but would need to be explored further with some other dose
regimen of the TGF-¦Â1 antisense ODN.
Our observations showed that the weight of the kidneys
from CRF rats was higher than that of the control rats (Table
1). This is consistent with the overproduction of
extracellular matrixes induced by the continuous production of
TGF-¦Â1 in response to the repetitive injury caused by puromycin.
Indeed, TGF-¦Â1 has been reported to be a prime candidate in
mediating the development of renal
hypertrophy[10]. Even though TGF-¦Â1 is typically known to be an inhibitor of cell
growth, it actually prevents only the synthesis of DNA and
cell proliferation, but promotes mRNA and protein synthesis, therefore enhancing cellular
hypertrophy[10]. The treatment of the CRF rats with the
TGF-¦Â1 antisense ODN to a large degree prevented the hypertrophy of the kidneys, as
reflected by the attenuation of the increased kidney weight
compared to the untreated CRF group (Table 1). One
possibility is that this might be due to the suppression of the
extracellular matrix accumulation in the kidney following the
TGF-¦Â1 antisense ODN treatment, thus decreasing the
cellular hypertrophy. Our findings are supported by an earlier
reported finding in which it is stated that the
TGF-¦Â1 antisense ODN administration could reduce the kidney
weight in diabetic mice with kidney
hypertrophy[21].
The occurrence of proteinuria in the CRF rats is likely
due to the damage of the glomerular basement membrane
and the epithelial cells elicited by the puromycin administration;
this is also a feature of chronic renal failure. Under such
circumstances, to promote the wound-healing process,
TGF-¦Â1 production would induce the deposition of extracellular
matrixes and also their own production. However, repeated
puromycin challenges would lead to continuous injury of
the tissue and a positive feedback mechanism whereby a
vicious circle of TGF-¦Â1 stimulation is created. This would
result in an overproduction of extracellular matrix and fibrotic
tissue, thereby preventing the normal repair mechanism of
the glomerular basement membrane and epithelial cells to
take place adequately. The level of proteinuria was reduced
in the CRF rats treated with the TGF-¦Â1 antisense ODN (Figure
3). This observation would suggest that in the presence of
the TGF-¦Â1 antisense ODN, the overproduction of
TGF-¦Â1 was probably blunted, resulting in decreased extracellular
matrix and fibrotic tissue accumulation, thus enabling the
repair mechanism of the glomerular basement membrane and
epithelial cells to adequately take place.
The reduction in renal blood flow in the CRF group may
indicate that an increase in intrarenal arterial resistance has
occurred. The treatment of CRF rats with the
TGF-¦Â1 antisense ODN prevented the reduction in renal blood flow
(Figure 1), suggesting that the resistance of the renal
vasculature was lower in the TGF-¦Â1 antisense ODN-treated group
as compared to the untreated group. Another finding from
our study was that the CRF rats had a lower GFR compared
to the control group (Figure 2). This might be due to fibrosis
developing within the glomeruli leading to a decrease in the
glomerular surface area, thereby resulting in a fall in
ultrafiltration coefficient,
Kf[19]. The treatment of the CRF rats with
the TGF-¦Â1 antisense ODN attenuated the percentage
reduction of GFR in the CRF rats consistent with the argument
that the TGF-¦Â1 antisense ODN tends to suppress the
vicious cycle of TGF-¦Â1 and fibrotic tissue overproduction.
This suggestion is supported by the observation that no
glomerulosclerosis were observed in the kidneys from the
CRF rats treated with the TGF-¦Â1 antisense ODN (Figure 5).
The fractional Na+ excretion was higher in the CRF group
compared to the control group (Figure 4), probably as a
consequence of the decreased reabsorptive ability of the tubules.
This could have resulted from the continuous damage of the
tubules caused by repeated insults from the puromycin
challenges related to the abnormal repair of the tubular cells.
The magnitude of rise in fractional Na+ excretion in the CRF
rats was significantly attenuated following treatment with
the TGF-¦Â1 antisense ODN. This observation may suggest
that the tubules were adequately repaired in the presence of
the TGF-¦Â1 antisense ODN, thereby enabling the
reabsorption of Na+ to take place appropriately.
Regarding the effects TGF-¦Â1 antisense on the tubular
function of the CRF rats, a similar conclusion that the
impaired tubular reabsorptive ability caused by
puromycin-induced nephrotoxic insult, ameliorated by the antisense
treatment, came from the data obtained from the urine flow
rate. It was observed that in the CRF rats, puromycin caused
marked diuresis, which was brought almost to a normal level
by treatment with the TGF-¦Â1 antisense ODN.
In conclusion, the administration of the TGF-¦Â1 antisense
ODN to CRF rats could reduce the severity of proteinuria
and improve the renal blood flow as well as GFR and plasma
inulin concentration. It also improved the reabsorptive
function of tubules, as reflected by the attenuation of the raised
fractional Na+ excretion. Moreover, the increased kidney
weight in the CRF rats was partly prevented following the
TGF-¦Â1 antisense ODN treatment. These improvements in
the kidney function of the CRF rats following the
TGF-¦Â1 antisense ODN treatment could be due to the suppression
of extracellular matrix and fibrotic tissue overproduction, as
demonstrated by the light microscopic examination of the
kidneys. Most importantly, the TGF-¦Â1 antisense ODN
brought all these changes virtually without the expression
of renal TGF-¦Â1. It is important to note that with a single
weekly dose, the TGF-¦Â1 antisense ODN has very strong
ameliorative activity in correcting the compromised renal
function and morphological changes in puromycin-induced
CRF rats.
Taken together, these findings suggest that the
TGF-¦Â1 antisense ODN has the potential to ameliorate the severity
of the deterioration of kidney function in rats with CRF
induced by repeated puromycin administration. The results
also indicate that the TGF-¦Â1 antisense ODN in part
prevented the inappropriate repair mechanism following
puromycin-induced injury to progress to a state of chronic
fibrotic disease, resulting in improved renal function.
Acknowledgements
We would like to thank the Departments of
Pharmacology and Allied Health Science, Faculty of Medicine,
University of Malaya, Kuala Lumpur, Malaysia for the provision of
their facilities.
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