Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
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ABSTRACT |
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Unilateral ureteral obstruction (UUO) leads to
interstitial fibrosis of the obstructed kidney, and transforming growth
factor-1 (TGF-
1) is thought to play an important role in this
process. Although increased TGF-
1 mRNA expression in the obstructed
kidney has been demonstrated, the source of the increased TGF-
1
remains to be elucidated. To determine the precise localization of
TGF-
1 in the obstructed kidney, we examined TGF-
1 mRNA expression
using in situ hybridization and competitive RT-PCR in rats with UUO. In
situ hybridization demonstrated that TGF-
1 mRNA expression was
preferentially increased in tubular epithelial cells and to a lesser
degree in infiltrating macrophages in obstructed kidneys. Quantitative
analysis using competitive RT-PCR in microdissected nephron segments
revealed that levels of TGF-
1 mRNA in obstructed kidneys relative to
control kidneys increased significantly in proximal tubules, thick
ascending limbs of Henle, and distal convoluted tubules, whereas those
in glomeruli and collecting ducts did not change significantly. Of the
tubular segments, the proximal tubules appeared to predominantly
contribute to increased TGF-
1 mRNA. Our findings suggest that renal
tubules, particularly proximal tubules, are the main contributors to
increased TGF-
1 mRNA expression in obstructed kidneys and to the
subsequent interstitial fibrosis.
unilateral ureteral obstruction; interstitial fibrosis; microdissection; competitive polymerase chain reaction; transforming
growth factor-1
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INTRODUCTION |
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INTERSTITIAL FIBROSIS IS
A common consequence of progressive renal disease. Because
previous studies have revealed that tubulointerstitial changes
correlate with decrements in glomerular filtration rate in a variety of
renal diseases (20), interstitial fibrosis is considered
to play a major role in progression of chronic renal disease.
Unilateral ureteral obstruction (UUO) is a well-characterized model of
experimental hydronephrosis, which results in tubulointerstitial fibrosis of the obstructed kidney (14, 19). In general,
fibrosis develops as a result of an imbalance between extracellular
matrix (ECM) synthesis and matrix degradation. Several investigators have examined the mechanisms underlying fibrosis in the UUO model, and
numerous cytokines have been implicated as mediators of interstitial fibrosis in this model (15). Among these cytokines,
transforming growth factor-1 (TGF-
1) is recognized as a major
regulator of ECM deposition and fibrogenesis (16).
TGF-
1 stimulates ECM protein synthesis and inhibits matrix
degradation by increasing the activity of protease inhibitors and
decreasing the amount of proteases (25). In addition,
TGF-
1 stimulates synthesis of matrix protein receptors such as
integrins and osteopontin (17).
In the UUO model, increased TGF-1 expression in the obstructed
kidney has been reported by a number of investigators (7, 13,
32). However, the intrarenal localization of increased TGF-
1
expression remains to be clearly identified. Therefore, we designed
this study to determine which sites mainly express TGF-
1 in the
obstructed kidneys of rats with UUO. For this purpose, we examined the
expression of TGF-
1 mRNA using in situ hybridization in kidney
tissue sections and competitive RT-PCR in microdissected nephron
segments. In preliminary experiments, we noted a progressive development of interstitial fibrosis from the onset of ureteral ligation, which often made microdissection of the nephron segments from
the obstructed kidneys difficult. Previous studies have reported that
the TGF-
1 mRNA level in obstructed kidneys increased significantly compared with that in control kidneys as early as 3 days post-ureteral ligation, and, in addition, the level was as high as the maximal level
during UUO (13). Thus we performed the main experiments at
3 days after the initiation of UUO and assessed TGF-
1 expression.
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MATERIALS AND METHODS |
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Experimental protocol. Male Sprague-Dawley (SD) rats, weighing 200-250 g, were used in the present study. After induction of general anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt), rats were subjected to either UUO or sham operation. Ureteral obstruction was performed by ligating the left ureter with 4-0 silk through a small suprapubic incision (19). Sham operation consisted of a similar suprapubic incision and visualization of the left ureter without further manipulation. Sham-operated animals were used to obtain control kidneys. Rats were killed at 3, 7, and 14 days after operation for histological examination of kidney tissue. A group of rats was killed 3 days after an operation for microdissection of nephron segments. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Kyushu University.
Routine histological examination. Kidney tissues for light microscopy were fixed in neutral formalin and embedded in paraffin, and 2-µm-thick sections were stained with periodic acid-Schiff reagent.
Serial section analysis.
To confirm TGF-1 protein expression, we carried out a serial section
analysis of TGF-
1 mRNA and TGF-
1 protein. In addition, to
determine whether the TGF-
1-expressing cells were infiltrating macrophages, we carried out another serial section analysis of TGF-
1
mRNA and macrophage cell marker antigen. Kidney tissues were fixed with
4% paraformaldehyde in 0.1 M phosphate buffer overnight, dehydrated
with a graded ethanol series and chloroform, and embedded in paraffin.
Two serial 3-µm-thick sections were prepared and mounted on 2%
3-amino-propyl-triethoxysilane-coated slides. All treatments were
performed under ribonuclease-free conditions.
Immunohistochemical staining.
Kidney sections were deparaffinized, immersed in methanol containing
0.3% H2O2 for 30 min, and digested with
proteinase K (5 µg/ml) (Boehringer Mannheim Biochemica, Mannheim,
Germany) at 37°C for 5 min (TGF-1) or 15 min (ED-1).
Sections were then immunostained for TGF-
1 with TGF-
1(V), a
rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, Santa Cruz,
CA), using biotinylated goat anti-rabbit IgG, or immunostained for
macrophages with ED-1, a mouse monoclonal IgG antibody (Serotec,
Oxford, UK), using biotinylated rabbit anti-mouse IgG. Sections were
developed using 3,3'-diaminobenzidine (Nichirei, Tokyo, Japan) as the
chromogen and were counterstained with 1% methyl green solution. To
quantify interstitial macrophage infiltration, numbers of ED-1-positive
cells in the cortex were counted in 20 consecutive fields and averaged
(cells/high-power field).
In situ hybridization.
Digoxigenin-labeled single-strand RNA probes were prepared using the
DIG RNA labeling kit (Boehringer Mannheim) according to the
instructions provided by the manufacturer. A 404-bp fragment was
excised from a 1.6-kb TGF-1 cDNA (provided by Dr. T. Nakamura, Osaka
University) with SmaI and PstI and subcloned into
the SmaI-PstI site of pBluescript II SK(
)
transcription vector that contains promoters for T7 and T3 RNA
polymerase (Stratagene, La Jolla, CA). Antisense and sense probes were
obtained by in vitro transcription of this plasmid linearized with
BamHI or EcoRI with the use of T7 or T3 RNA
polymerase, respectively. The specificity of the 404-bp cDNA was
verified by DNA sequencing.
Microdissection of nephron segments. Rats were anesthetized intraperitoneally with pentobarbital sodium 50 mg/kg body wt. The kidneys were perfused in situ via the aorta with ice-cold microdissection solution (Hanks' balanced-salt solution, pH 7.4; GIBCO-BRL, Grand Island, NY), containing 1 mg/ml collagenase type I (Sigma) and 1 mg/ml BSA. The left kidney was removed and cut into 1-2-mm-thick coronal slices. The kidney slices were incubated at 37°C for 30 min in the dissection solution containing collagenase and BSA. The incubated tissues were washed with cold dissection solution containing 10 mM vanadyl ribonucleoside complex (GIBCO-BRL) to inhibit RNA degradation (27).
Tubular segments were microdissected under a stereomicroscope using fine forceps. We obtained the following structures: glomerulus (Glm), proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thick ascending limb of Henle (mTAL), cortical thick ascending limb of Henle (cTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). In general, 30 glomeruli or 5-10 mm of tubule segments were pooled to constitute one sample. The time period for dissection was limited to within 1 h after digestion with collagenase. The microdissected samples were rinsed, snap-frozen, and stored atRNA extraction and RT. Total RNA was extracted from the microdissected tissues or whole kidney tissues using the acid guanidinium thiocyanate-phenol-chloroform extraction method (6) with Isogen (Nippon Gene, Tokyo, Japan). The final RNA pellets from the tissues were resuspended in diethylpyrocarbonate-treated water and quantified spectrophotometrically with the absorbance at 260 nm (A260). First-strand cDNA was synthesized from 100 ng of total RNA using 200 U of Moloney murine leukemia virus RT (Superscript II; GIBCO-BRL) in a total volume of 20 µl. The reaction mixture consisted of (in mM) 50 Tris · HCl (pH 8.3), 75 KCl, 3 MgCl2, 2.5 dithiothreitol, and 1 each of dNTPs as well as 1 U/µl RNase inhibitor and 2.5 µM oligo(dT)12-18 primer. The mixture was incubated at room temperature for 10 min and at 42°C for 45 min, and the reaction was terminated by heating to 70°C for 15 min. After being chilled on ice, 2 U RNaseH (GIBCO-BRL) were added and incubated at 37°C for 20 min to remove the RNA template. The synthesized cDNA was then used in a PCR amplification procedure.
Oligonucleotide primers.
For rat TGF-1, the specific primers used were sense
5'-GGACTACTACGCCAAAGAAG-3' (bp 715-734) and antisense
5'-TCAAAAGACAGCCACTCAGG-3' (bp 989-1,008). The anticipated
PCR product was 294 bp in length. For rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the specific primers used were sense 5'-TGAGA- ATGGGAAGCTG-3' (bp 251-266) and antisense 5'-TTGGG- GGTAGGAACAC-3' (bp
766-781). The anticipated PCR product was 531 bp in length. To
verify the authenticity of the PCR products, the amplified products
were gel purified, subcloned into pBluescript II SK(
), and validated by DNA sequencing using the dideoxyribonucleotide sequencing method.
Competitive mutant template preparation.
Competitive mutant DNA templates were prepared as described previously
(5). The 254-bp template was generated from rat TGF-1
cDNA (24) by deleting a 40-bp segment using the
following primers: sense 5'-GGACTACTACGCCAAAGAAG-3' and
antisense 5'-TCAAAAGACAGCCACTCAGGCAGGAATTGTTGCTATATTT-3'. The 450-bp
template was generated from rat GAPDH cDNA (31) by deleting an 81-bp segment by using the following primers: sense 5'-TGAGAATGGGAAGCTG-3' and antisense
5'-TTGGGGGTAGGAACACTGATGTTCTGG- GCTGC-3'. Mutant templates shared the
same primer template with the target cDNA sequence but contained a
smaller intervening sequence. Mutant fragments were gel purified and
quantified spectrophotometrically. The authenticity of the PCR
amplified products was verified by DNA sequencing.
Competitive PCR.
To quantify TGF-1 mRNA, competitive PCR was performed as described
previously (10). Six equal aliquots (1 µl) of sample cDNA were co-amplified with the dilution series of mutant template prepared for TGF-
1 in a total volume of 50 µl. The reaction
mixture comprised 20 pmol of each primer, 10 mM Tris · HCl (pH
8.3), 50 mM KCl, 2 mM MgCl2, 200 µM each of dNTPs, and
1.25 U of Taq DNA polymerase. After the mixture was heated
at 95°C for 5 min, the PCR reaction was run for 35 cycles
with a denaturing phase at 95°C for 1 min, an annealing
phase at 54°C for 1 min, and an extension phase at
72°C for 1 min. The reaction was followed by a final
elongation step at 72°C for 7 min. Controls, with no
added RT, were run in parallel to rule out genomic contamination. No
negative controls generated PCR products (data not shown). The
amplified products were size fractionated by electrophoresis on 3%
NuSieve-agarose gels (FMC Bioproducts, Rockland, ME), and product bands
were visualized by ultraviolet fluorescence after ethidium bromide
staining. The intensity of the luminescence was measured by a
charge-coupled device image sensor and quantified (29).
Statistical analysis. Data are expressed as means ± SE. Differences between groups were assessed by the unpaired t-test. Statistical significance was defined as P < 0.05.
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RESULTS |
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Morphological findings after initiation of UUO.
Light microscopy of periodic acid-Schiff-stained renal sections
from the obstructed kidneys of rats with UUO showed that the cortical
and medullary interstitial spaces progressively increased due to edema
with infiltration of mononuclear cells after the initiation of UUO, as
reported previously (19, 26). A large number of renal
tubules were dilated, and in some the epithelium was flattened. In
contrast, sections from the control kidneys of sham-operated rats
appeared normal. Immunohistochemical labeling revealed that the number
of ED-1-positive macrophages increased 3 days after UUO in kidneys with
ureteral obstruction, suggesting that the infiltrating mononuclear
cells were, at least in part, ED-1-positive macrophages.
Quantitatively, the number of macrophages in the obstructed kidneys was
8.4-fold greater than that in control kidneys (Table
1).
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Localization of TGF-1 mRNA by in situ hybridization.
In control kidneys of sham-operated rats, TGF-
1 mRNA expression was
detected in glomeruli and tubules, predominantly in distal tubules
(Fig. 1A). Glomerular TGF-
1
mRNA expression was localized mainly in mesangial cells and glomerular
epithelial cells (Fig. 1B). In obstructed kidneys of rats at
3 and 7 days after UUO, increased TGF-
1 mRNA expression was
observed. TGF-
1 mRNA was predominantly expressed by renal tubular
cells in the cortex (Fig. 1, C and E) and outer
medulla (Fig. 1D) but was also present in the peritubular
interstitial cells. After 2 wk, although many collapsed tubules were
observed, the predominance of tubular expression of TGF-
1 mRNA was
still evident (Fig. 1F). A control experiment with a sense
probe did not show any tissue staining (Fig. 1G).
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Localization of TGF-1 protein by immunohistochemistry.
To confirm TGF-
1 protein expression, we performed immunolabeling
with anti-TGF-
1 antibody in the consecutive section of in situ
hybridization. Immunohistochemical staining showed that the nephron
segments expressing TGF-
1 mRNA were also positive for TGF-
1
protein in both control kidneys of sham-operated rats (Fig.
2, A and B) and
obstructed kidneys of rats at 3 days after UUO (Fig. 2, C
and D).
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Identification of interstitial cells expressing TGF-1 mRNA.
To characterize the fewer cells expressing TGF-
1 in the
interstitium, we performed immunolabeling for macrophages with ED-1 in
the consecutive section of in situ hybridization. Immunohistochemical staining showed that, although infiltrating macrophages also expressed TGF-
1 mRNA (Fig. 2, E and F), the intensity of
the signal in macrophages was not as strong as that in renal tubular
cells. Therefore, the majority of TGF-
1 mRNA-expressing cells in
obstructed kidneys were renal tubular cells.
Appearance of microdissected renal tubules.
It was considerably more difficult to dissect tubules in obstructed
kidneys compared with control kidneys, suggesting that UUO induced
interstitial fibrosis in obstructed kidneys as early as 3 days after
ureteral ligation as reported previously (28). The
proximal tubules (PCT and PST) and DCT from obstructed kidneys were
slightly dilated in diameter compared with those from control kidneys,
and the epithelia of these tubules were slightly flattened. The
collecting tubules (CCD, OMCD, and IMCD) from obstructed kidneys were
markedly dilated in diameter, and the epithelia of these tubules were
also flattened compared with those from control kidneys (Fig.
3). In contrast, there was no evidence of
dilatation in the thick ascending loops of Henle from obstructed
kidneys. Glomeruli appeared to be unchanged after UUO.
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Quantification of TGF-1 mRNA along nephron segments.
For quantification of TGF-
1 mRNA expression in microdissected
nephron segments and whole kidney, quantitative competitive RT-PCR was
performed. Two- or threefold dilutions of DNA templates (termed
"competitor") were used to compete for PCR amplification of
reverse-transcribed sample cDNAs (termed "target"). A plot of the
ratio of competitor to target product vs. the known concentration of
input competitor was linear when plotted on a log-log scale (Fig.
4). The r-value for all
competitive PCR linear regression curves ranged from 0.92 to 0.99. At
the point where competitor and target product are in equivalence (i.e.,
competitor-to-target ratio = 1.0), the starting amount of TGF-
1
cDNA is equal to the known starting amount of the competitor. Final
quantitative data were arbitrarily expressed as the ratio of TGF-
1
to GAPDH derived from the same cDNA samples (Fig.
5).
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DISCUSSION |
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The main finding of the present study is that the increased
TGF-1 mRNA expression after UUO is predominantly localized to renal
tubular cells in the obstructed kidney. The in situ hybridization study
clearly demonstrated that UUO increased TGF-
1 mRNA expression in the
obstructed kidney, which was associated with TGF-
1 protein expression, and that the main sites of increased TGF-
1 expression in
obstructed kidneys were renal tubules in the cortex and outer medulla.
Although increased TGF-
1 expression in obstructed kidneys has been
reported by many investigators (13, 32), the intrarenal localization of TGF-
1 in obstructed kidneys is still controversial. In previous reports, Diamond et al. (7) noted highly
significant increments in renal cortical TGF-
1 mRNA levels in
obstructed kidneys and localized TGF-
1 protein to peritubular cells
of the renal interstitium by immunolabeling, suggesting the
infiltrating renal interstitial macrophage as a cellular source for
increased TGF-
1 expression. In contrast, other investigators
reported that the increased TGF-
1 in obstructed kidneys was confined
to renal tubular cells, using immunohistochemistry (35) or
RT-PCR (13). With the use of in situ hybridization, we
detected TGF-
1 mRNA expression in the peritubular interstitial cells
in addition to renal tubular cells. Thus, to confirm TGF-
1 mRNA
expression in infiltrating macrophages as reported in the former study,
we performed immunolabeling for macrophages with ED-1 and in situ
hybridization of TGF-
1 in the serial section. Our results showed
that TGF-
1 mRNA expression was also localized to a lesser degree to
ED-1-positive infiltrating macrophages but that the predominant
localization was in renal tubular cells. This finding confirms that the
main cells expressing TGF-
1 mRNA in the obstructed kidney are not the interstitial infiltrating macrophages but renal tubular cells.
To determine the main segment contributing to increased TGF-1 mRNA
expression, we applied a competitive RT-PCR method to microdissected
nephron segments. In control kidneys of sham-operated rats, TGF-
1
mRNA was found to be constitutively expressed all along the nephron
segments, consistent with our previous report (1).
Quantitative data showed that the abundance of TGF-
1 mRNA in
proximal tubules was lower than that in the other nephron segments,
which was consistent with our results using in situ hybridization. In
rats with UUO, our results showed that TGF-
1 mRNA levels in PCT and
PST from obstructed kidneys were markedly increased and that those in
mTAL, cTAL, and DCT from obstructed kidneys were moderately increased
compared with those in the corresponding segments from control kidneys.
In contrast, TGF-
1 mRNA levels in glomeruli, CCD, OMCD, and IMCD
from obstructed kidneys did not change significantly. Although the
degree of TGF-
1 mRNA increase in proximal tubules was most striking,
the expression level in this segment was not higher than in any other
tubular segments, as the baseline level of TGF-
1 mRNA in proximal
tubules was relatively low. Regardless of this result, proximal tubular
cells may contribute predominantly to increased TGF-
1 mRNA
expression in obstructed kidneys. The relative kidney tissue volume
occupied by proximal tubules (PCT and PST) has been reported as ~66%
of whole kidney tissue in normal rats (21). If the
relative volume for the proximal tubules does not change during UUO,
the increase in TGF-
1 (normalized by GAPDH) mRNA after UUO in the
proximal tubules amounted to ~11.8 × 10
3, which
can be estimated to occupy over half that in the whole kidney
(~22.8 × 10
3) (Fig.
6). This indicates, therefore, that the
proximal tubular cells predominantly contribute to the increase in
TGF-
1 mRNA expression in obstructed kidneys of rats with UUO. This
finding also implies that the major tubules highly expressing TGF-
1
in the cortex and outer medulla could be PCT and PST, respectively.
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In this study, we used a competitive RT-PCR method to quantify TGF-1
mRNA. Although RT-PCR is a powerful tool for detecting small amounts of
RNA, it provides only qualitative results due to the exponential nature
of the amplification process. To generate quantitative data, the
concept of competitive RT-PCR, which is not cycle dependent, has been
developed (2, 10, 33). The principle of this method is
based on the coamplification of the sequence to be quantified (the
target) with a known amount of another sequence (the competitor), which
is flanked by the same primers as the target. The competitor is added
to the sample as cRNA before the RT step or as cDNA after the RT step.
The RNA competitor approach potentially makes it possible to determine the absolute amounts of target RNA. However, this method cannot check
any variations in RNA extraction efficiency and RNA quantification. Indeed, the initial amplifiable RNA amounts determined by
spectrophotometric analysis were rather variable among samples in our
experience, because this analysis cannot distinguish between undegraded
and degraded RNA or between genomic DNA and RNA. To circumvent these problems, we chose a DNA competitor and sequentially performed two
competitive PCRs using each DNA competitor as follows. We first
quantified the amounts of TGF-
1 mRNA, and, second, quantified the
amounts of mRNA of the "housekeeping gene" GAPDH as an internal standard; then we normalized the results of TGF-
1 mRNA
quantification to correct variations in RNA quantification and reverse
transcription. Thus our data provide only an estimate of the relative
number of copies of TGF-
1 mRNA normalized by GAPDH gene transcripts. Nevertheless, the results fulfill our purpose of determining
differences between samples from control and obstructed kidneys.
Although the present data detected the cellular source and abundance of
TGF-1 mRNA in the obstructed kidney, the mechanism of TGF-
1 mRNA
upregulation remains unclear. Recent studies in the UUO model have
revealed that the renin-angiotensin system (RAS) could play a key role
in the pathogenesis of tubulointerstitial fibrosis (8, 22,
23). It has been suggested that the intrarenal RAS is
upregulated in the obstructed kidney and that increased angiotensin II
stimulates TGF-
1 synthesis, thereby causing interstitial fibrosis.
The effect of angiotensin II on upregulation of TGF-
1 appears to be
mediated through an AT1 receptor, as administration of an
AT1-receptor antagonist blunted the increase in TGF-
1
mRNA and ameliorated the degree of interstitial fibrosis
(12) as did admininstration of the angiotensin-converting
enzyme (ACE) inhibitor (13). Indeed,
AT1 receptors are expressed all along the tubular segments
in the rat kidney, with the highest abundance in proximal tubules
(3, 18, 30). In addition, our data indicate that the
tubular segment expressing AT1 receptors most abundantly
coincides with the segment in which TGF-
1 mRNA increased most
markedly after UUO, namely, the proximal tubule. This finding suggests,
therefore, that the increase in tubular expression of TGF-
1 mRNA
could be stimulated by angiotensin II through AT1 receptors
in the obstructed kidney in the UUO model. This is supported by an in
vitro study in which angiotensin II induced TGF-
1 expression in
proximal tubular cells (34). In addition, recent evidence has identified proximal tubules as a de novo source for all components of the intrarenal RAS, i.e., angiotensinogen, renin, and ACE (4, 11). Thus angiotensin II may act in an autocrine or paracrine manner in the proximal tubules to upregulate TGF-
1. Taken together, prior reports and our new data indicate that the proximal tubules are
the main source of both intrarenal RAS and TGF-
1 in obstructed kidneys, such that they may play a pivotal role in the pathogenesis of
tubulointerstitial fibrosis in the setting of UUO.
In summary, we have clearly determined in the present study the sites
and relative abundance of TGF-1 mRNA expression along the nephron
segments in obstructed kidneys of rats with UUO. Our results suggest
that the main source for increased expression of TGF-
1 mRNA in the
obstructed kidney is the renal tubular cells, particularly proximal
tubular cells, and that these cells could thereby contribute to
subsequent interstitial fibrosis.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Nakamura, Osaka University, for providing TGF-1
cDNA and H. Noguchi for helpful technical assistance.
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FOOTNOTES |
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This study was supported by a research grant from the Glomerular Injury Research Committee of the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare, Japan.
Address for reprint requests and other correspondence: K. Fukuda, Dept. of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan (E-mail: kfukuda{at}intmed2.med.kyushu-u.ac.jp).
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. Section 1734 solely to indicate this fact.
Received 14 August 2000; accepted in final form 3 May 2001.
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