1 Departments of Medicine and 2 Cellular and Molecular Physiology and 3 Department of Surgery, Milton S. Hershey Medical Center and the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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Antisense oligonucleotide inhibition of angiotensinogen and ANG II type 1 receptor (AT1) mRNA translation in rat proximal tubules (PT) was examined to provide direct evidence for a role of the renin-angiotensin system (RAS) in upregulated osteopontin expression observed following mechanical cell stretch. Male Sprague-Dawley rats underwent unilateral ureteral obstruction (UUO) under Brevital anesthesia. In situ hybridization and Western blot analysis demonstrated angiotensinogen mRNA and angiotensin converting enzyme (ACE) protein localized to PTs and upregulated in obstructed kidneys, respectively, confirming an increased expression of renal RAS in vivo. In vitro studies were performed to provide mechanistic insight into ANG II-dependent osteopontin expression following mechanical cell stretch, which putatively mimics the increased PT luminal pressure post-UUO. A cationic transfection method was used to introduce either angiotensinogen or AT1 antisense oligonucleotide into cultured rat PT cells prior to 1 h of cyclic mechanical cell stretch. Northern blot analysis revealed that PT cells subjected to cyclic mechanical stretch with/without prior transfection with a sense oligonucleotide exhibited increased osteopontin mRNA expression compared with unstretched cells. Blockade of either angiotensinogen or AT1 mRNA translation by antisense oligonucleotide inhibition prior to cell stretch was found to significantly decrease osteopontin mRNA levels 2.4-fold (P < 0.004) and 1.6-fold (P < 0.001), respectively, compared with values observed in control unstretched cells. This study provides evidence that stretch-induced upregulation of osteopontin mRNA expression is mediated, in part, via production of ANG II. These results lend insight into upregulation of osteopontin via a local PT RAS leading to macrophage infiltration in the tubulointerstitium in experimental hydronephrosis.
ureteral obstruction; angiotensin II; transfection; renin-angiotensin system; cyclic cell stretch
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INTRODUCTION |
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UNILATERAL URETERAL OBSTRUCTION (UUO) has been shown to elicit a florid macrophage infiltration of the kidney, which in turn leads to interstitial fibrosis. The degree of interstitial fibrosis has been correlated experimentally and clinically with the extent of renal functional impairment. We have previously demonstrated increased expression of osteopontin, a secreted acidic glycoprotein with macrophage chemoattractant ability, in a rodent model of experimental hydronephrosis (7) and provided evidence for a role of ANG II produced by the proximal tubule epithelium in the increased synthesis of osteopontin in the obstructed kidney (8).
Osteopontin is a highly acidic, phosphorylated, and secreted
glycoprotein that is a cell adhesion and migration molecule due to an
adhesive Arg-Gly-Asp sequence that binds to
v
1 and
v
5 integrins, CD44, and extracellular matrix proteins including type I
collagen and fibronectin (4, 13, 38). Osteopontin can induce monocyte
infiltration and readily bind to macrophages (35). Recent clinical and
experimental studies have shown a close association between macrophage
infiltration and osteopontin expression in the development of a range
of glomerular and tubular interstitial diseases (9, 18, 19, 22, 41).
A myriad of biological insults, including production of ANG II, cytokines, ischemic injury, proteinuria, and membrane stretch as a result of obstruction, can elicit injury to the proximal tubular epithelial cell resulting in macrophage recruitment and renal perturbations (29). We have previously demonstrated increased osteopontin mRNA and protein expression by controlled cyclic mechanical cell stretch, an in vitro tool that putatively mimics the early hemodynamic changes occurring to the proximal tubules after ureteral obstruction (8). This stretch-induced increase in osteopontin expression was decreased following pretreatment with an ANG II type 1 receptor (AT1) antagonist. These results suggest that ANG II and AT1 stimulation may play a critical role in osteopontin stimulation by proximal tubular epithelial cells (8).
A growing body of evidence suggests that a local renin-angiotensin system (RAS) plays an important role in tubular injury and macrophage infiltration in obstructive nephropathy. In a series of studies, Pimental et al. (27, 28) and El-Dahr et al. (10) have demonstrated that acute UUO results in increased synthesis of ANG II in the obstructed kidney within hours post-UUO. They observed that RAS genes are induced with consequent increments in peptide levels and activities as early as 1-2 h post-UUO (28). Evidence continues to accumulate in support of a local, independent proximal tubular RAS (12, 16). ANG II binding sites are found in high concentrations in proximal tubules, and the presence of renin, angiotensinogen, and angiotensin converting enzyme (ACE) has been identified (3). ACE is found in greatest concentrations in the kidney in the proximal tubules with smaller amounts in arterioles and glomeruli (33). ACE is present in the proximal tubular brush border and could convert ANG I to ANG II.
The present study will first delineate the expression and localization of ACE and angiotensinogen following experimental hydronephrosis in the rat. With these components being upregulated, we further postulate that proximal tubular ACE facilitates the epithelial generation of ANG II that then stimulates, in an autocrine manner, the production of macrophage recruitment factors, such as osteopontin, by these cells. Expression of markers of the RAS, namely renin, will be investigated in vitro to determine whether renin is increased following mechanical stretch of proximal tubular cells. Oligonucleotide antisense therapy, a novel strategy designed to bind specifically and efficiently to the complementary sequence of a targeted mRNA and lead to translational arrest, has been used extensively in both in vivo and in vitro systems. Interference of both the initial and terminal sites of the RAS cascade will be achieved by blockade of angiotensinogen and AT1 mRNA translation using antisense oligonucleotide therapy in cultured rat proximal tubular epithelial cells undergoing mechanical cell stretch, thereby providing mechanistic insight into osteopontin's increased synthesis in the obstructed kidney cortex.
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METHODS |
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Experimental animals. Male Sprague-Dawley rats (Charles River
Breeding Laboratories, Wilmington, MA), weighing 150-200 g, were
used in this study. Animals were fed standard rodent chow (Purina
Chows, St. Louis, MO) and were given water ad libitum. Following
intraperitoneal Brevital (50 mg · kg1 · body
wt
1 ip; E. Lilly, Indianapolis, IN),
anesthetized animals (n = 10 per time point) underwent either
left proximal ureteral ligation or a sham operation. Both the
obstructed kidney and the contralateral unobstructed kidney (CUK) as
well as normal kidneys from sham-operated animals were harvested from
UUO rats at 6, 12, 24, and 96 h postureteral ligation or a sham procedure.
RNA extraction and Northern hybridization. The kidney cortex was removed and homogenized in ice-cold GIT buffer solution D containing 4 M guanidinium thiocyanate, 0.5% sarkosyl, 1 M sodium citrate, and 0.1 M mercaptoethanol. Total cellular RNA was extracted using the acid guanidinium thiocyanate-phenol chloroform method (5). For Northern analysis, total RNA (5 µg/lane) was denatured and electrophoresed through 1.2% agarose gels containing 0.66 M formaldehyde and transferred to nylon filters (Nytran; Schleicher and Schuell, Keene, NH) by capillary blotting. RNA was immobilized by baking at 80°C for 30 min. The blots were examined under ultraviolet light in the presence of ethidium bromide to determine the position and integrity of the 28S and 18S ribosomal RNA bands. The blots were hybridized as previously described (30) using a 2B7 cDNA probe for osteopontin (kindly provided by Dr. C. Giachelli, University of Washington, Seattle, WA) or a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (Clontech, Palo Alto, CA), which yields 1.6-kb and 1.3-kb mRNA transcripts, respectively. Following hybridization, blots were washed in conditions of increasing stringency. and mRNA signals on autoradiographs were quantified by laser densitometry and evaluated with an IBM-AT-compatible computer (Quantity One; PDI, Huntington, NY). GAPDH was used as a reference probe to correct for variations in loading of RNA samples. The mRNA levels for osteopontin were expressed as ratios of the optical density of osteopontin to that of GAPDH. The peak optical density of reading of each band on the autoradiograph is arbitrarily reported as densitometric units.
Western blot analysis. Specimens from renal cortex containing
~500 mg of protein were sonicated in extraction buffer containing 10 mM Tris · HCl (pH 7.5), 1 mM EDTA, and a
minicomplete protease inhibitor cocktail tablet
(Boehringer Mannheim, Indianapolis, IN) made up to a 10-ml
volume. Following extraction, the homogenates were stored at
70°C after boiling for 10 min. The protein concentration of
the homogenates was determined with the Bio-Rad assay. Samples containing 10 µg of protein were diluted in SDS-PAGE Laemmli buffer and loaded onto a precast gradient gel. For Western blotting, the gel
was removed and placed on a Hybond transfer membrane (Amersham, Cleveland, OH) using filter paper saturated with 300 mM Tris buffer (pH
10.4) in 5% methanol, then covered with additional filter paper
saturated with 25 mM Tris, 40 mM glycine, and 20% methanol. This
transfer unit was placed in a MilliBlot-SDE transfer system (Millipore,
Bedford, MA) and run for 30 min. Transfer was judged by the standard
bands (New England Biolabs, Beverly, MA) on the transfer membrane. The
gel was stained in 0.1% Ponceau solution (Sigma Chemical, St. Louis,
MO) in 5% acetic acid to determine the transfer efficiency. The
membrane was washed in 20 mM Tris · HCl buffer (pH
7.6) with 137 mM NaCl, 0.1% Tween-20, and 5% wt/vol nonfat dry milk
for 1 h at room temperature before incubation with either osteopontin,
ACE, or renin antibody. A monoclonal mouse anti-rat osteopontin
antibody (MPIIIB10 at 1:4,000 dilution; Developmental Studies Hybridoma
Bank, Univ. of Iowa) was incubated for 1 h in horseradish
peroxidase-conjugated anti-mouse antibody (1:2,000 dilution, Amersham)
at room temperature to demonstrate a 66-kDa osteopontin protein. A
rabbit anti-goat ACE monoclonal antibody (1:12,000 dilution; kindly
provided by Dr. J. Ingelfinger, Massachusetts General Hospital, Boston,
MA) was used to demonstrate a 62-kDa ACE protein, and a rabbit anti-rat
IgG antibody (1:4,000 dilution; kindly provided by Dr. T. Inagami,
Vanderbilt Univ. School of Medicine, Nashville, TN) was used to
demonstrate the 60-kDa and 61-kDa renin doublet. A mouse anti-rat
monoclonal antibody obtained from Chemicon (Temecula, CA) was used to
demonstrate the 49-kDa AT1. The membrane was washed in an
Amersham ECL detection mixture layered on the surface of the membrane.
To measure differences between samples, the bands on the membrane were
scanned for optical density and compared with other bands. Densimetric
quantification with statistical analysis was performed on
autoradiographs from at least three Northern and Western blots using
the same sample batch.
In situ hybridization. A 35S-labeled antisense cRNA for angiotensinogen was prepared using the Promega transcription protocol for synthesis of high specific activity RNA probes with T7 RNA polymerase and 35S-UTP (NEN, Boston, MA), following linearization with EcoR I. An angiotensinogen sense cRNA was transcribed from Hind III-cut angiotensinogen cDNA using SP6 RNA polymerase.
Six, 12, 24, and 96 h after obstruction, UUO and CUK kidney specimens
were immersion fixed in 10% neutral buffered formalin for 3 h,
processed and embedded in paraffin, and sectioned at 4 µm. Sections
were alcohol dehydrated and incubated with prehybridization solution
containing, 1.2 M NaCl, 0.02 M Tris, 0.04% Ficoll, 0.04% BSA, 0.04%
polyvinylpyrrolidone, 0.002 M EDTA, 0.1% salmon sperm DNA, and 0.1 mg/ml of yeast tRNA (final concentrations). Tissue sections were hybridized at 50°C overnight in an identical
solution, containing, 25% formamide, 10 mM DTT, 0.1% SDS, and the
35S-labeled cRNA probe at a specific interstitial activity
of 4 × 104
counts · min1 · µl
1.
After hybridization, the slides were rinsed in a series of washes, including RNase posttreatment. The final wash was in 2× SSC for 2 h at 60°C. The slides were exposed to BioMax autoradiography film
(Eastman Kodak, Rochester, NY) and then dipped in diluted Kodak NTB-2
emulsion and stored at 4°C for 1-2 wk. Sections were developed
and counterstained with hematoxylin.
Controlled cyclic mechanical cell stretch. Continuous cycles of stretch/relaxation utilizing a rat plasmid-transformed immortalized proximal tubule cell line (Ref. 37; kindly provided by Dr. J. Ingelfinger, Harvard Medical School, Boston, MA) were performed. This specific cell line was chosen since it has all the components of the RAS to locally generate ANG II as well as AT1 (37). Rat proximal tubule cells were plated at a density of 3-5 × 104/ml in DMEM containing 5% FBS using 6-well plates with a flexible (experimental stretch/relaxation)-bottomed type I collagen-coated membrane (Flexcell, McKeesport, PA) according to our previously published methods (8). Cells were grown to confluence over 72 h and then subjected to cyclic stretch (i.e., stretch and relaxation/cycle) at 15 cycles/min for 1 h using a computer-driven, vacuum-operated, stress-providing instrument (Flexcell Strain Unit FX-2000, Flexcell). An applied vacuum of 20 kPa was used resulting in an elongation of the membrane by ~15%. One 6-well plate constituted an n = 1. Studies were performed in triplicate. As a control, rat proximal tubule cells were grown to confluence on the similar type I collagen-coated flexible-bottomed plates but were not subjected to repetitive cycles of stretch/relaxation, although these cells were cultivated under identical conditions and durations to the cyclic/stretch counterparts.
Angiotensinogen and AT1 antisense oligonucleotide delivery to rat proximal tubular cells. For angiotensinogen, transfection was performed using phosphorothioate angiotensinogen antisense oligonucleotide synthesized on an automated solid phase synthesizer using standard phosphoramide chemistry. The sequence of antisense oligonucleotide used was 30-mer human preangiotensinogen starting from position 934 to 963 (5'-CAC-TGA-GGT-GCT-GTT-GTC-CAC-CCA-GAA-CTC-3') and a control scrambled sequence (20). The sequence of this region is homologous in humans and rats (20). The AT1 antisense oligonucleotide was obtained from Chemicon in the form of a phosphorothioate DNA sodium salt. The control consisted of a CG-matched randomized-sequence phosphorothioate oligonucleotide.
A cationic transfection method was used to introduce the antisense and scrambled oligonucleotides into rat proximal tubular cells plated on type I collagen-coated flexible plates as described above. Two micrograms of DNA were added to 100 µl of Opti-MEM serum-free media (GIBCO, Life Technologies, Grand Island, NY). This solution was then added to either 16 µl or 8 µl of Lipofectamine (GIBCO, Life Technologies, Grand Island, NY) for angiotensinogen or AT1, respectively, in 100 µl of Opti-MEM and incubated for 45 min at room temperature. After DNA-liposome complexes were been formed, the subconfluent cells were washed and 0.8 ml of Opti-MEM added to each of the culture wells before the addition of the DNA solution. Cells were incubated with the antisense and sense scrambled oligonucleotides for 24 h before the addition of growth medium and initiation of cell stretch. Two six-well plates constituted an n = 1 for the mechanical cell stretch experiments as described above. Oligonucleotide uptake, efficiency, the desired DNA:Lipofectamine ratio, and incubation times for transfection, were determined using a fluorescein isothiocyanate (FITC)-labeled oligonucleotide at the 5'-amine to estimate cellular fluorescein uptake (FluoReporter Oligonucleotide Phosphate labeling kit; Molecular Probes, Eugene, OR). RNA and protein were extracted from the cells for Northern and Western blot analysis, respectively.
Statistics. All values are expressed as means ± SD. When multiple groups were compared, one-way ANOVA was performed initially to confirm the presence of significant differences. Then individual comparisons were performed with Student's t-test, and multiple pairwise comparisons were according to the method of Bonferroni as appropriate. Statistically significant differences between groups were defined as P < 0.05.
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RESULTS |
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Expression of components of the RAS in UUO. To discern the role
of the RAS in proximal tubular osteopontin expression in experimental hydronephrosis, both the obstructed and CUK specimens from UUO rats
were examined for ACE and angiotensinogen protein and mRNA expression,
respectively. On Western blot analysis there was a significant increase
of ACE protein in the renal cortex of obstructed kidneys in comparison
to the CUK specimens (0.78 ± 0.10 vs. 0.25 ± 0.10, P < 0.002). Figure 1 is a representative
Western blot analysis demonstrating an increased 62-kDa protein
corresponding to ACE, in the renal cortex of 96 h obstructed kidneys in
comparison to the CUK specimens from the same animals. This ACE
antibody was previously tested on rat lung and testis to ensure
specificity to ACE protein (not shown).
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In situ hybridization using a rat angiotensinogen riboprobe revealed
localization of angiotensinogen mRNA to the rat proximal tubule
epithelium of only the obstructed kidney, as early as 6 h post-UUO in
comparison to the CUK from the same animals. Figure 2 demonstrates angiotensinogen expressed in
the proximal tubules of the renal cortex as evidenced by localization
of the 35S-labeled antisense probe (Fig. 2, A and
B). Renal cortical arterioles also demonstrated angiotensinogen
expression, whereas glomerular tufts demonstrated relatively low
angiotensinogen expression. A marked decrease in the number of silver
granules representing mRNA transcription was observed in the
corresponding CUK section (Fig. 2D). The specificity of the
antisense probe hybridization signal was demonstrated by the lack of
label in sections hybridized to the sense probe (Fig. 2C) or
following predigestion of tissue sections with RNase.
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Cyclic mechanical cell stretch. To gain mechanistic insight
into osteopontin's increased synthesis in the obstructed kidney cortex, we evaluated the response of cultured proximal tubular epithelial cells to controlled cyclic mechanical stretch in specific regards to increased renin expression. We have previously reported that
rat proximal tubule cells subjected to cyclic mechanical cell stretch
for 1 h exhibited a 2.1-fold increment in osteopontin mRNA levels,
which was decreased to control values following pretreatment with
losartan (8). The present study demonstrated that proximal tubules
subjected to mechanical cell stretch for 1 h exhibited a significant
3.0-fold increase in renin protein expression compared with unstretched
cells at the same time point (0.13 ± 0.04 vs. 0.04 ± 0.01, P < 0.007; Fig. 3). This data
provides evidence that mechanical cell stretch can induce upregulation
of components of a local, independent renin-angiotensin cascade within
the proximal tubular epithelial cell.
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Oligonucleotide delivery to proximal tubule epithelial cells.
To investigate whether stretch-induced osteopontin upregulation is
dependent on the renin-angiotensin cascade, the impediment of
angiotensinogen and AT1 mRNA translation was performed by
antisense oligonucleotide delivery to cultured proximal tubular
epithelial cells. Figure 4 shows
fluorescent micrographs of FITC-labeled angiotensinogen and
AT1 antisense oligonucleotides in proximal tubular
epithelial cells. Rapid uptake of the oligonucleotides was observed
after incubation of the liposome-DNA complexes for 12 h. From
12-24 h strong fluorescent signal was observed in the nucleus and
perinuclear organelles (Fig. 4). Following incubation, the cells were
cultured for 24 h in serum-containing media before initiation of
mechanical cell stretch.
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Effect of the blockade of angiotensinogen and AT1 mRNA
translation on osteopontin expression. By Northern blot analysis,
rat proximal tubular cells exhibited a 2.0-fold increase in osteopontin mRNA expression following 1 h of cyclic mechanical cell stretch, compared with unstretched cells grown on the same substratum for an
identical duration (0.10 ± 0.00 vs. 0.05 ± 0.00, P < 0.001), as we have previously described (8). As shown in Fig.
5, osteopontin mRNA expression was
significantly decreased 2.4- and 1.6-fold in stretched proximal tubular
epithelial cells following transfection with antisense oligonucleotide
for either angiotensinogen (0.042 ± 0.02 vs. 0.10 ± 0.00, P < 0.004) or AT1 (0.10 ± 0.00 vs. 0.16 ± 0.00, P < 0.001), respectively, compared with stretched cells with/without prior transfection with the control scrambled
oligonucleotide.
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On Western blot analysis, proximal tubular epithelial cells transfected
with angiotensinogen antisense oligonucleotide exposed to cyclic
mechanical cell stretch exhibited osteopontin protein levels comparable
to unstretched cells at the same time point. These stretched proximal
tubular epithelial cells transfected with antisense oligonucleotide
demonstrated a significant decrease in osteopontin protein expression
compared with cells stretched for an hour (0.16 ± 0.08 vs. 0.42 ± 0.11, P < 0.01; Fig.
6). Figure 7
is a representative Western blot demonstrating osteopontin and AT1 protein in cultured rat proximal tubular epithelial
cells following the introduction of the AT1 antisense or
scrambled oligonucleotide. Proximal tubular epithelial cells
transfected with the AT1 antisense oligonucleotide had a
significant 2.2-fold decrease in osteopontin protein (0.68 ± 0.10 vs.
0.31 ± 0.19, P < 0.01) and 4.0-fold less AT1
(0.22 ± 0.02 vs. 0.053 ± 0.01; P < 0.001) protein
expression compared with cultured stretched cells at the same time
point. These data suggest that the antisense oligonucleotide to
AT1 receptor mRNA was effectively taken up by the proximal
tubular cells resulting in a significant decrease (0.68 ± 0.10 vs.
0.31 ± 0.19, P < 0.01) in osteopontin protein
expression in cultured stretched cells transfected with AT1
antisense oligonucleotide, compared with stretched cells at the same
time point.
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DISCUSSION |
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Our studies document increased cortical ACE expression and localization of angiotensinogen to proximal tubules in the renal cortex of the obstructed kidney as early as 6 h after UUO. A significantly increased renin expression was reported in cultured proximal tubule cells exposed to mechanical cell stretch. Angiotensinogen and AT1 oligonucleotide antisense oligonucleotide delivery to cultured rat proximal tubule cells provides direct evidence that ANG II may mediate, in part, the activation of osteopontin following cell stretch. This data underscores the importance of the RAS in regulating proximal tubule osteopontin expression following the mechanical injury as a result of urinary tract obstruction.
Many recent clinical and experimental studies have clearly demonstrated
that one of the initial events taking place in the process of
progressive renal injury is monocytic infiltration of the glomerular
and tubulointerstitial compartments. Osteopontin is a macrophage
chemotactic and adhesion molecule that may be involved in the
macrophage infiltration of the renal interstitium. A number of
inflammatory molecules including ANG II, transforming growth factor
(TGF)-, epidermal growth factor (EGF), and interleukin (IL)-1, can
enhance osteopontin transcription in tubular epithelium (5, 11, 23, 26,
41). In a model of focal tubulointerstitial injury with ANG II
infusion, Giachelli et al. (11) noted an elevated expression of
osteopontin occurring early followed by monocyte/macrophage influx. The
macrophages localized almost exclusively to sites of tubular
osteopontin, suggesting that elevated expression of osteopontin by
kidney tubules may be an important chemoattractant mechanism in
directing the inflammatory response (11). Pichler et al. (26)
demonstrated that the degree of osteopontin expression in chronic
cyclosporin nephropathy is associated with osteopontin expression
macrophage accumulation and with TGF-
expression. More recently, Yu
et al. (41) demonstrated that IL-1 can directly upregulate osteopontin
expression in glomerular crescentic formation and tubulointerstitial
fibrosis in a rat anti-glomerular basement membrane (anti-GBM)
glomerulonephritis. Our laboratory has previously noted elevated
osteopontin expression predominantly in proximal tubular epithelium as
early as 12 h after unilateral ureteral ligation (7). Studies using
isolated proximal tubules provided evidence that ANG II may regulate
osteopontin expression due to a mechanical disturbance in the proximal
tubules occurring shortly after ureteral ligation as a result of the
transient hydrodynamic perturbations involving increased proximal
tubular pressure and membrane stretch (8).
The vasoactive effects of intrarenal ANG II in altered renal hemodynamics have been demonstrated in a variety of models. Following UUO, there is a progressive increase in renal blood flow (RBF) in the obstructed kidney which peaks at ~2 h after ureteral obstruction, followed by progressive vasoconstriction of glomerular arterioles, principally due to ANG II, leading to a marked decline in glomerular filtration rate (GFR; 17). Studies by Pimental et al. (27, 28) and El-Dahr et al. (10) have demonstrated that acute UUO results in profound changes in the expression of genes that encode for components of the RAS. It is accepted that an intrarenal RAS exists that modulates renal function by the paracrine and autocrine effects of ANG II synthesized locally (24). ANG II is synthesized by the kidneys independently of plasma levels (25). Angiotensinogen is synthesized by the proximal tubule cell (16, 31). Ingelfinger et al. (15) demonstrated that the expression of angiotensinogen mRNA by Northern analysis in the renal cortex and medulla is regulated by dietary salt intake. The present study used in situ hybridization to localize angiotensinogen to the cytoplasm of the proximal tubule and cortical arterioles as early as 6 h following ureteral obstruction, compared with CUK specimens from the same animals. Localization and regulation of angiotensinogen in the proximal tubule suggest that angiotensinogen could be released into the lumen or interstitium of the obstructed kidney cortex, providing a source of substrate for the intrarenal generation of ANG I and II. Also, ACE is expressed in great abundance on the proximal tubular brush border, with smaller amounts in glomeruli and arterioles (2, 33). The present observations that ACE protein is statistically elevated in the obstructed kidney suggest that proximal tubular brush border ACE may facilitate epithelial generation of ANG II, leading to osteopontin synthesis and secretion by these cells.
The present study used oligonucleotide antisense transfection targeted at both the initial and terminal portions of the RAS, namely angiotensinogen and AT1, respectively, to provide direct evidence for ANG II-mediated osteopontin expression. Two main ANG II receptors have been cloned, AT1 and AT2 (6). All known actions of the RAS are mediated by AT1, belonging to the superfamily of G protein-coupled receptors it is the predominant receptor subtype found in adult rat kidneys (40). The role of AT2 receptors and their signaling mechanisms are unclear; however, it has been speculated that the AT2 receptor may counterbalance the actions of AT1 (36). The present study used antisense oligonucleotides to hybridize AT1 or angiotensinogen to lead to translational arrest of the specific protein in cultured rat proximal tubule epithelial cells. Our observations with fluorescence microscopy confirm that the oligonucleotides were taken up by the proximal tubular cells optimally by 24 h of culture. Li et al. (21) used an AT1 oligonucleotide to determine the cellular uptake in bovine adrenal cells. In dose-response studies of both uptake and receptor inhibition, they noted a close correlation between uptake and effect; however, characteristic of antisense inhibition, there was never a 100% decrease in binding (20). Following mechanical cell stretch, the present study showed a significant decrease, but not complete inhibition, of osteopontin expression following AT1 and angiotensinogen transfection of proximal tubular cells at both the RNA and protein level. The use of antisense oligonucleotide therapy for the incomplete blockade of gene expression may be important therapeutically, as the attempt of targeting antisense oligonucleotides to specific genes is to inhibit overactive systems rather than inhibit the physiological activity completely.
Cyclic mechanical cell stretch was used to mimic the increased proximal
tubular luminal pressure as a result of the early changes following the
onset of ureteral obstruction as previously used in our laboratory (8).
Several investigators have demonstrated the role that mechanical
stretch may play in a variety of disease models and an increasing
number of reports focus on ANG II as an initial mediator
for stimuli of mechanical stretch. Harris and colleagues (1) have
extensively studied the responses of cultured rat glomerular mesangial
cells subjected to mechanical cyclic stretch based on the premise that
the increased mechanical tension, generated by elevated GFR and
glomerular capillary pressure, noted in a variety of models of
progressive renal disease, may be a primary cause of perturbed
mesangial cell biology. Interestingly, they noted that ANG II augmented
many of the mesangial cell derangements induced by mechanical cell
stretch (1). Yamazaki et al. (39) demonstrated that ANG II plays an
important role in mechanical stress-induced cardiac hypertrophy using
studies with candesartan cilexetil, an AT1 antagonist, in
cultured rat cardiac myocytes. Angiotensinogen gene expression
has been reported to be elevated following mechanical cell stretch of
rat cardiomyocytes (34). More recently, Li et al. (20) used
angiotensinogen antisense oligonucleotide transfection to demonstrate
that stretch-induced proliferation of cultured smooth muscle cells is
mediated by the RAS and a subsequent upregulation of platelet-derived
growth factor- mRNA. Further evidence for a role of
the RAS cascade comes from the present finding of increased renin mRNA
expression in proximal tubule cells following 1 h of mechanical cell
stretch. We have previously shown that mechanical perturbation of
proximal tubular cells in this cyclic stretch model downregulated
catalase mRNA expression and increased osteopontin mRNA expression,
suggesting that mechanical disturbances resulting from increased
proximal tubular pressure post-UUO may lead to amplification of the
proinflammatory state of ureteral obstruction (30). It has also been
postulated that following UUO, there is increased cortical interstitial
pressure in the obstructed kidney (32). We have recently observed that renal decapsulation of the obstructed kidney in UUO rats did not alter
the production of inflammatory chemokines or elevated expression of the
RAS, suggesting that cortical interstitial pressure does not contribute
to macrophage infiltration and the development of fibrogenesis in
experimental hydronephrosis (unpublished observations).
In summary, this study provides direct evidence for the role of a local RAS in the secretion of ANG II from cultured proximal tubular cells following cell stretch. Our data indicate that ANG II acts as an initial mediator of stretch-induced osteopontin release from proximal tubular epithelial cells. Perturbation of proximal tubule cells after ureteral ligation may increase ANG II synthesis and AT1 stimulation in an autocrine manner leading to osteopontin release. The elucidation of this mechanism provides new insights into the role of proximal tubular cell injury and the resulting chemoattractant-facilitated macrophage influx into the tubulointerstitium in experimental hydronephrosis. Further studies are needed to ascertain the importance of other cellular and molecular events resulting from the initial insult to proximal tubule cells as major codeterminants of renal scarring and fibrosis.
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ACKNOWLEDGEMENTS |
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This study was supported by a Research Grants-in-Aid and an Initiator Investigator Grant from the American Heart Association (National and Pennsylvania Affiliate), and by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-53163-01A1. A research supply award was provided by the Section of Surgical Sciences, Department of Surgery. The osteopontin monoclonal antibody in the present study was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Studies, Iowa City, IA 52242, under National Institute of Child Health and Human Development Contract NO1-HD-73263.
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FOOTNOTES |
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Present address of S. D. Ricardo and address for reprint requests and other correspondence: Dept. of Anatomy, Monash Univ., Clayton 3168, Victoria, Australia (E-mail: sdr2525{at}hotmail.com).
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. §1734 solely to indicate this fact.
Received 5 August 1999; accepted in final form 9 December 1999.
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