Departments of 1 Nephrology and 3 Pathology, Saitama Medical College, Saitama 350-04, Japan; and 2 Nephrology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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In this study, we have shown that intravenously administered antisense oligodeoxynucleotide (ODN) was demonstrated to be taken up by tubular epithelium, after which it blocked mRNA expression of target genes in normal and nephritic rats. Therefore, we injected osteopontin (OPN) antisense ODN to Goodpasture syndrome (GPS) rats every second day between days 27 and 35, the time when renal OPN expression increased and interstitial monocyte infiltration was aggravated. In parallel to blockade of tubular OPN expression, this treatment significantly attenuated monocyte infiltration and preserved renal plasma flow in GPS rats at day 37, compared with sense ODN-treated and untreated GPS rats. No significant changes were observed in OPN mRNA level by RT-PCR and histopathology of the glomeruli after ODN treatment, which was compatible with an absence of differences in the urinary protein excretion rate. In conclusion, OPN expressed by tubular epithelium played a pivotal role in mediating peritubular monocyte infiltration consequent to glomerular disease.
antisense oligodeoxynucleotide; glomerular and interstitial histopathology; proteinuria; glomerular filtration rate; renal plasma flow
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INTRODUCTION |
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IT HAS LONG BEEN NOTED that the degree of renal tubulointerstitial alteration secondary to glomerular diseases correlated better than glomerular alteration itself with the prognosis of the disease (27). A number of investigators have confirmed this thesis in human glomerular diseases (1, 31). However, it still remains unclear how glomerular disease leads to such tubulointerstitial alterations, e.g., interstitial cell infiltration, tubular atrophy, and fibrosis (7, 20, 22, 25, 28, 32).
Chemoattractants and cell adhesion molecules derived from renal cells
are postulated to play an important role in tubulointerstitial alteration not only by recruiting inflammatory cells but also by
anchoring them to interstitial areas (7, 20, 22, 25, 28). Among them,
chemokines such as MCP-1 (JE), interleukin-8, MIP-1,
and RANTES, which recruit inflammatory cells attaching to activated
vascular endothelium and migrating into extravascular interstitium,
have been intensively investigated (28). Because an initial event in
progressive interstitial alteration in most of the experimental models
of renal diseases is often monocyte infiltration of the interstitial
compartment, this is particularly important (20, 35). Furthermore,
transforming growth factor-
(TGF-
) is one of the key cytokines
contributing to renal fibrosis which causes permanent loss of renal
function, and monocyte/macrophages are the main source of TGF-
at
sites of inflammation (20, 32).
In this study, we have focused on osteopontin (OPN), because OPN is thought to be involved in renal tubulointerstitial disease as a leukocyte adherence protein (8, 9, 11, 23, 24, 35-37). A marked upregulation of OPN was demonstrated in tubular epithelial cells in experimental models, where OPN expression corresponded closely to the sites and the degree of monocyte accumulation (8, 11, 23, 24, 35-37). However, thus far there has been no definitive evidence for a role of OPN in mediating such accumulation of monocytes in tubulointerstitial diseases. Since systemic treatment with neutralizing antibodies affects both glomerulus and tubulointerstitium, in situ roles of target molecules expressed by tubular epithelium have remained unproven (10, 28, 37). Therefore, to test that OPN expressed by tubular epithelium can directly induce accumulation of monocytes in the interstitium, we applied OPN antisense oligodeoxynucleotide (ODN) to Goodpasture syndrome (GPS) rats (12) to block OPN expression exclusively in tubular cells. This treatment substantially decreased monocyte infiltration and attenuated renal functional impairment.
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METHODS |
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Cell culture. The rat renal proximal tubular epithelial cell
line, RPTC (kindly provided by Dr. J. Ingelfinger, Harvard Medical School), was maintained in culture in DMEM supplemented with 5% FCS,
100 U/ml penicillin, and 100 µg/ml streptomycin. To stimulate OPN
synthesis in RPTC, the cells were grown to confluence and treated with
10 ng/ml TGF-1 (R & D Systems) in media with 0.5% FCS, and RNA were
extracted after 0, 3, 6, 12, and 24 h of incubation (17). Because the
highest signal was obtained after 6 h of incubation, RPTC cells were
incubated with 0, 1.0, 3.0, 10.0, and 30.0 ng/ml TGF-
1, and RNA was
isolated for Northern blot analysis after 6 h.
ODN preparation. Phosphorothioate-capped ODNs were synthesized by an automated synthesizer. After deprotection, ODN were dissolved in water, extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and redissolved in water. The OPN sense ODN sequence comprised 5'-CACTGCCAGTCTCATGGT-3', the antisense ODN sequence comprised 5'-ACCATGAGACTGGCAGTG-3', and the mutated antisense ODN sequence comprised 5'- AGGATCACAGTCCGACTC-3'; these sequences were chosen from the rat OPN gene (21) as likely to corrupt ribosomal docking by their location near the initiation site. In addition, 3'-FITC-labeled ODN were generated to demonstrate ODN uptake by renal tubular epithelium.
ODN application in vitro by streptolysin-O permeabilization.
The method for streptolysin-O (SL-O) permeabilization was essentially that of Clark and colleagues (30). Briefly, after washing in serum-free
medium, 5 × 106 RPTC cells were exposed to
dithiothreitol (DTT)-activated SL-O at 150 U/ml and 30 µM ODN in 200 µl of permeabilization buffer (137 mM NaCl, 100 mM PIPES, pH 7.4, 5.6 mM glucose, 2.7 mM KCl, 2.7 mM EGTA, 1 mM sodium ATP, and 0.1% BSA).
After incubation for 20 min at 37°C, 1 ml of growth medium was used
to reseal the cells. Twenty minutes later, the cells were transferred
to culture dishes containing 10 ml growth medium. Four hours later,
stimulation with TGF-1 was started. Untreated cells were
permeabilized with SL-O without ODN treatment and stimulated with
TGF-
1, and the control cells were also permeabilized with SL-O
without ODN treatment but not followed by stimulation with TGF-
1.
Northern blot and RT-PCR analysis. Total RNA was extracted with TRIzol (Life Technologies) from cultured cells, whole kidneys, or glomeruli isolated by a sieving method. The purity of glomeruli in the samples was determined by direct counting under microscope, and the samples containing tubules more than 5% were discarded. RNA (30 µg/lane) was loaded onto a 1% RNase-free agarose gel in 2.2 M formaldehyde and transferred to GeneScreen membrane (New England Nuclear, Boston, MA) in 10× SSC buffer. The murine OPN cDNA (generous gift from Dr. S. Nomura, Osaka University) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were labeled with random primers and [32P]dCTP. Northern blot analysis was carried out according to the method reported previously (33), and hybridization was performed at the highest stringency in 0.1× SSC, 0.1% SDS at 65°C.
The GeneAmp RNA PCR Kit (Perkin-Elmer Cetus, Norwalk, CT) was used for cDNA synthesis and PCR amplification, following the method of Zohar et al. (38). In the PCR reaction, the primers for OPN, 5'-CTCGCGGTGAAAGTGGCTGA and 3'-GACCTCAGAAGATGAACTCT, were used to amplify an 871-bp target, which incorporates nearly all of the coding sequence for rat OPN in combination with primers for the housekeeping gene GAPDH, yielding a 515-bp fragment. The primers for GAPDH were 5'-AATGCATCCTGCACCACCAA, and 3'-GTAGCCATATTCATTGTCATA. PCR was carried out for 25 cycles under the following conditions: denaturation at 94°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min in one cycle. Amplification products were analyzed by electrophoresis in a 2% agarose gel in 0.5× Tris-borate/EDTA buffer (TBE) and visualized with 0.5 µg/ml ethidium bromide.
Quantitative densitometry was performed on autoradiograms using a computer-based measurement system (Mac SCOPE, Ver. 2.5; Mitani, Hukui, Japan). The expression of the OPN gene was corrected by dividing the signal density by that obtained for GAPDH.
Animal manipulation and experimental protocols. Male Wistar
rats of 160-200 g were purchased from Clea Japan (Tokyo, Japan). Animal care and treatment were in conformity with the institutional guidelines. All animals were housed in a constant-temperature room with
a 12-h dark/12-h light cycle, fed a standard diet and given water ad
libitum. To generate GPS in rats, animals were immunized with 25 µg
of 3(IV) NC1 collagen (12) subcutaneously in complete Freund's
adjuvant followed by a booster injection 2 wk later in incomplete
Freund's adjuvant, as we reported for the mouse model (12).
At first, GPS rats (n = 25) and the control rats (n = 25) were used to characterize the GPS model. At days 21, 28, 35, 42, and 49, four rats of each group were placed in metabolic cages for 24 h, to determine daily urinary protein excretion, and were then killed for evaluation of renal histopathology and OPN expression.
Second, sense or antisense ODN were injected intravenously at a concentration of 1.0 mg/kg on day 27 (GPS rats with sense ODN, n = 3; those with antisense ODN, n = 3; GPS rats untreated with any ODNs, n = 3; control rats; n = 3), and the rats were killed 12 h later to assess the effect of ODN on renal OPN expression.
Third, because of an abrupt increase in interstitial infiltrating mononuclear cells from day 28 to day 35 in GPS rats, ODN were injected at days 27, 29, 31, 33, and 35 to determine how OPN antisense ODN affected this mononuclear cell infiltration. The experimental groups of rats included OPN antisense ODN-treated GPS rats (OPN-AS; n = 8), OPN sense ODN-treated GPS rats (OPN-S; n = 8), untreated GPS rats (n = 8), and the normal control rats (n = 6). Kidney tissues were collected from each rat at the time of death on day 37. One kidney was used for RNA and protein extraction. Tissues from the other kidney were fixed in 4% paraformaldehyde in PBS at room temperature overnight. One half of each fixed tissue was processed into paraffin blocks for histopathology and immunohistochemistry. The other half was rinsed in serial concentrations of sucrose solutions, and then snap-frozen for in situ hybridization.
Histopathology and immunohistochemistry. Four-micrometer paraffin sections were stained with hematoxylin and eosin (HE) and Masson's Trichrome (MT). Glomerular hypercellularity and interstitial mononuclear cell infiltration on HE-stained sections, as well as interstitial fibrosis area in blue on MT-stained sections, were quantitatively evaluated with a computer-assisted image analysis system (Mac SCOPE) in 30 glomeruli and 30 high-power (×200) cortical fields, respectively. Each histopathological index was calculated as the mean cell number per 10 glomeruli or per one high-power cortical field, and the mean percentage area of fibrosis in blue per one high-power cortical field. Immunohistochemistry was carried out according to the method reported previously (33) with a slight modification. In this study, the sections were boiled in citrate buffer under microwave in addition to the treatment with proteinase K for unmasking antigenicities. Murine monoclonal antibodies, MPI-IIB10 (obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa) and ED-1 (Serotec, Oxford, UK), were used as the primary antibodies to detect OPN and monocyte/macrophages, respectively. Biotinylated goat anti-mouse IgG (American Qualex) was then applied as the secondary antibodies. ED-1-positive monocyte/macrophages in the glomeruli and the interstitium were counted as described above. Negative controls for the immunostaining consisted of replacing each of the primary antibodies with equivalent concentrations of a murine IgG.
Western blot analysis. The kidney tissues were sonicated in a cold RIPA lysis buffer (1% Nonidet P-40, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 2 µg/ml aprotinin, 2 µg/ml antipain, and 2 µg/ml leupeptin in PBS), and the homogenates were centrifuged for 5 min at 4°C. The protein concentration in the supernatant was determined using a BCA protein assay kit (Pierce, Rockford, IL). Thirty micrograms of proteins were examined by Western blot analysis as reported previously (33). The blot was incubated at 4°C overnight with mouse anti-OPN (1:1,000), followed by incubation for 1 h with alkaline phosphatase-conjugated anti-mouse IgG (1:5,000) (Sigma). The signals were visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium.
In situ hybridization. OPN mRNA transcripts were detected in paraformaldehyde-fixed frozen tissues by in situ hybridization with OPN cRNA probes generated from the mouse OPN cDNA, following the method of Kohri et al. (13). The hybridization solution contained 50% deionized formamide, 10% dextran sulfate, 1× Denhardt's solution, 600 mM NaCl, 10 mM DTT, 0.25% SDS, 250 µg/ml Escherichia coli tRNA, and ~0.5 µg/ml digoxigenin-labeled cRNA probe. Fifty microliters of hybridization solution was placed on each section, which was covered with Parafilm and incubated at 50°C overnight in a humidified chamber. After hybridization, the Parafilm was removed by washing with 5× SSC briefly and with 50% formamide and 2× SSC for 30 min at 50°C. RNase A treatment (10 µg/ml) was carried out at 37°C for 30 min. The sections were washed with 2× SSC and 0.2× SSC for 15 min twice at 50°C. Hybridized digoxigenin-labeled probes were detected by a nucleic acid detection kit (Boehringer Mannheim Biochemica). At the step of color development, we added 1 mM levamisole into the developing solution. After color reaction, the slides were rinsed with 10 mM Tris · HCl, pH 8.0, 1 mM EDTA and mounted without counterstaining.
Urinary protein and renal function. Urinary protein was measured using a protein assay kit (Pierce). The method for renal function study was previously reported (34). Briefly, on day 37, the left external jugular vein was cannulated with a PE-50 polyethylene tube for the administration of saline containing 1% BSA, 1% inulin, and 0.1% p-aminohippurate (PAH). The infusion was started immediately after jugular cannulation at a rate of 100 µl/min and continued throughout the experiments. In addition, the right femoral artery was also cannulated for blood collection. With laparotomy, another cannula was placed in the bladder for urine collection. An equilibrium period of 30 min was then allowed before urine and blood sampling. Urine volume was measured gravimetrically, and urine flow rate was factored per gram kidney weight. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were calculated as the ratio of the urine/plasma concentration of inulin and PAH multiplied by the urine flow rate, respectively. Urinary protein excretion rate was also calculated as the ratio of urinary protein per minute divided by the GFR.
Data analysis. Values were presented as means ± SE. Statistical differences between groups were evaluated by analysis of variance, followed by Duncan's multiple range test (34), with P < 0.05 used as the requirement for significance.
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RESULTS |
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Expression of the OPN gene in RPTC cells and application of
antisense ODN in vitro. RPTC cells were treated with TGF-1 (10 ng/ml) for various durations, and as shown in Fig.
1A, OPN mRNA extracted from the
cells increased time dependently over 12 h. It was highest at 6 and 12 h of incubation with TGF-
1, showing the effect of TGF-
1 was
short-lived. Among various concentrations of TGF-
1, 10 ng/ml
resulted in the highest level of OPN mRNA expression after 6-h
incubation (Fig. 1B). Treatment with OPN antisense ODN
prevented the increase in the OPN mRNA level of SL-O-permeabilized RPTC
cells after 6-h incubation with TGF-
1 (10 ng/ml), whereas sense ODN
and mutated antisense ODN had no significant effects (Fig. 1, C
and D). This confirms the specificity of the OPN antisense ODN
employed.
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Characterization of GPS in rats and expression of OPN in the
nephritic kidneys. All rats immunized with 3(IV)
NC1 domains developed significant proteinuria (223 ± 54 mg/24 h) on
day 21, and the level increased further to a peak (475 ± 89 mg/24 h) around day 35 (Fig. 2).
Rat IgG was found along the glomerular and alveolar basement membranes
on day 21, but there were no significant crescentic formation
and pulmonary hemorrhage (data not shown). Focal crescentic glomerulonephritis with glomerular hypercellularity became apparent on
day 28 (Fig. 3A). On
day 35, crescent formation grew more apparent in up to 100% of
the glomeruli, and interstitial mononuclear cell infiltration became
more noticeable (Fig. 3B). Possibly because of renal
insufficiency and/or pulmonary hemorrhage, those rats started dying
from day 49.
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OPN mRNA expression of the whole nephritic kidney was upregulated from
day 28 and reached a peak on days 35 and 42 (Fig. 4, A and B). OPN
protein expression was found to localize in tubular epithelium in the
cytoplasmic pattern with luminal surface accentuation on day 35 by immunohistochemistry (Fig. 3C). There was no significant OPN
protein expression detected in the glomeruli.
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Application of OPN antisense ODN in vivo and its effects. To
demonstrate the delivery of ODNs into renal tubular epithelium, FITC-conjugated ODN was injected intravenously into normal rats and GPS
rats on day 27 at a concentration of 1 mg/kg. Because of the
significant increase in expression of OPN observed in GPS rat kidney on
day 28, at 12, 24, and 48 h after injection, their kidneys were
harvested and evaluated with fluorescence microscopy. Kidneys obtained
from both groups of rats showed significant accumulation of ODN in the
tubular epithelium 12 h after injection (Fig.
5, A and B). This remained
for 24 h and disappeared 48 h later (data not shown). A single
administration of OPN sense ODN had no effects (Fig.
6, A-C), but antisense ODN
decreased OPN mRNA levels in the whole nephritic kidney obtained from
day 28 GPS rat 12 h after injection (Fig. 6, A and
B). Such an inhibitory effect localized in the tubular
epithelium (Fig. 6D). On the other hand, RT-PCR disclosed that
the level of OPN mRNA in the glomeruli of day 28 GPS rats was
increased compared with that of control rats, and sense and antisense
ODNs did not affect the glomerular OPN mRNA (Fig. 6F). This is
consistent with our observation that intravenously injected
FITC-conjugated ODN at the relevant dose was accumulated exclusively in
the tubular epithelium of normal rats and day 28 GPS rats.
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Modification of renal morphological and functional deterioration in
GPS. Because of the significant increase in OPN mRNA level and the
aggressive progression in interstitial mononuclear cell infiltration,
ODN treatment was initiated in GPS rats from day 27 and
continued every 2 days to day 35. The interval between each
injection was determined on the basis of the finding that FITC-conjugated ODN remained in the kidney for 24 h after injection but
subsequently disappeared by 48 h. On day 37, renal functional parameters were assessed prior to death by the method described above.
OPN expression in the kidney was significantly inhibited at the protein
level in the OPN-AS group (Fig. 7,
B and C) but remained in the kidney of the OPN-S group
at the same level as the untreated group (Fig. 7, A and
C). The glomerular hypercellularity (Fig.
8, A and B) and the number
of ED-1-positive monocyte/macrophages in the glomeruli (Fig. 8,
D and E) of both groups increased to a similar degree
(Fig. 8, C and D). The incidence of glomerular crescent
formation was also mostly similar between these groups (90 ± 5% in
the OPN-S vs. 87 ± 5% in the OPN-AS). However, the number of
mononuclear cells, especially ED-1-positive monocytes, infiltrating the
interstitium was significantly lower in the OPN-AS group than in the
OPN-S group (Fig. 8, A-F). The percentage area of
interstitial fibrosis was less than 5% in both groups, and the
difference was not significant (data not shown). The degrees of lung
histopathology were not affected by ODN administration, and there
seemed to be no differences between the OPN-AS and the OPN-S groups
(data not shown).
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There was no significant difference in urinary protein excretion rate
between the two groups on day 37 (Fig.
9A). However, the decrease in RPF
was attenuated significantly in the OPN-AS group compared with the
OPN-S group, and the decrease in GFR also tended to be
suppressed in the former (Fig. 9, B and C).
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DISCUSSION |
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In this study, we have demonstrated that OPN expressed by tubular
epithelium has an essential role in mediating secondary interstitial
monocyte infiltration associated with primary glomerular diseases. A
number of humoral factors has been found to be produced in tubular
epithelium, and it has been speculated that they may be associated with
the primary and secondary forms of tubulointerstitial diseases (7, 20,
25, 28). Among them, OPN has been focused on in this study. OPN is a
multipotent molecule and has independent, different roles at various
sites in the body (3-5, 15). In the kidney under physiological
conditions, descending thin limb and papillary surface epithelium
constitutively express OPN (15, 16), whereas in a rat model of stone
formation, distal tubular epithelium is also positive for OPN. In this
case OPN is presumably involved in urinary stone formation as the stone
matrix (13). OPN can bind to monocyte/macrophages, and subcutaneous
injection of OPN induces prominent monocyte infiltration in mice (29). In the case of inflammatory renal diseases, experimental observation led to a notion that OPN is likely to act as a chemoattractant for
monocytes, because the degree of OPN expression is parallel to that of
monocyte infiltration (8, 11, 23, 24, 35-37). Some humoral factors
including angiotensin II, TGF-1, epidermal growth factor, and OPN
itself, directly or indirectly enhance OPN transcription in tubular
epithelium (6, 17, 37). We showed that TGF-
1 increased OPN
expression in RPTC cells, raising the possibility that OPN and TGF-
1
constitute a local positive feedback loop in accumulating monocytes.
Although the exact regulation of tubular OPN expression in inflammatory
renal diseases remains unclear, all of these factors distribute
ubiquitously and probably induce OPN and other downstream members of
the proinflammatory cascade (7, 20, 22, 25, 28, 35).
Most of the humoral factors and adhesion molecules expressed by tubular epithelium are also coexpressed in glomeruli. Recently, Lan et al. (14, 37) demonstrated that OPN is expressed by glomerular and tubular cells in experimental crescentic glomerulonephritis, and neutralizing anti-OPN antibodies can significantly inhibit its progression. This suggests that OPN is important in the pathological process (14, 37). However, it is still unclear whether this effect is due to a direct interaction with OPN expressed by tubular epithelial cells or is a result of the beneficial effect on glomerular damage with subsequent effects on the tubulointerstitium. Recent findings documented that ODN was reabsorbed by rodent renal tubular cells, especially by proximal tubular cells when administered intravenously (26). Indeed, Noiri et al. (19) reported that antisense ODN for inducible NO synthase could attenuate NO production in the renal epithelium, avoiding exacerbation of postischemic acute renal failure. Their observation revealed that reabsorbed antisense ODN was intact enough to operate in tubular epithelial cells. In the present study, we have provided evidence that antisense ODN acted in a similar fashion in the diseased kidney. Intravenously injected OPN antisense ODN lowered OPN mRNA level in the tubular epithelium of GPS rats at least up to 12 h later, and OPN protein level was also decreased after multiple injections. Not only tubular epithelium but also glomerular epithelial cells and mesangial cells can express OPN in vitro and in vivo (14, 18). In the same way as the glomerular OPN expression increased in some experimental glomerular diseases (14, 18), it was also enhanced in this GPS model, at least on day 28, as detected by RT-PCR. The administration of antisense ODN did not affect such increased levels of OPN mRNA in glomeruli, but in tubular epithelium, the OPN mRNA level was significantly lowered. There was an apparent increase in the number of monocyte/macrophages in the interstitium from day 28 to 35 in GPS rats. Administration of OPN antisense ODN during that period resulted in fewer monocytes infiltrating the renal interstitium of GPS rats. In addition to histological improvements, OPN antisense ODN preserved renal function in GPS rats. RPF was significantly preserved in GPS rats treated with OPN antisense ODN compared with the sense ODN-treated group. Treatment with OPN antisense ODN tended to result in conservation of GFR, but it did not attain statistical significance. Tubular function rescued by OPN antisense ODN may maintain tubuloglomerular feedback, resulting in preferential preservation of RPF compared with GFR, because tubuloglomerular feedback constricts preglomerular vasculature (2). Of interest, glomerular histopathology was not improved in antisense ODN-treated GPS rats. Consistent with this, our data indicate that OPN antisense ODN failed to lower urinary protein excretion rate, reflecting no improvements in glomerular or mesangial function.
In conclusion, we have proved for the first time that OPN expressed by tubular epithelium, independently of glomerular OPN expression, has a critical role in mediating monocyte infiltration into the interstitium, leading to tubulointerstitial alterations secondary to primary glomerulonephritis.
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ACKNOWLEDGEMENTS |
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We are grateful to S. Yamada and C. Hirata for technical support.
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: H. Suzuki, Dept. of Nephrology, Saitama Medical College, 38 Morohongo, Moroyama-machi, Irumagun, Saitama 350-04, Japan (E-mail: iromichi{at}saitama-med.ac.jp).
Received 7 June 1999; accepted in final form 9 August 1999.
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