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Regular Articles |
From the Departments of Nephrology and Medicine,*
Monash
Medical Centre, Monash University, Clayton, Australia; the Department
of Nephrology,
The First Hospital, Sun Yat-Sen
University of Medical Sciences, Guangzhou, China; and the Division of
Nephrology,
University of Washington Medical
School, Seattle, Washington
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75–85%,
P < 0.001) and tubules (45–60%,
P < 0.001). The reduction in OPN expression was
associated with significant inhibition of macrophage accumulation and
progressive renal injury. In vitro, the addition
of IL-1 to the normal rat tubular epithelial cell line NRK52E
up-regulated OPN mRNA and protein levels, an effect that was
dose-dependent and inhibited by the addition of IL-1 receptor
antagonist, thus demonstrating that IL-1 can act directly to
up-regulate renal OPN expression. In conclusion, this study
provides in vivo and in vitro evidence
that IL-1 up-regulates OPN expression in experimental kidney disease
and support for the argument that inhibition of OPN expression is one
mechanism by which IL-1 receptor antagonist treatment suppresses
macrophage-mediated renal injury.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 integrin (vitronectin receptor), CD44, collagen type I, and
fibronectin.1-3
A wide range of cell types
including osteoclasts, some epithelia, macrophages, T cells, smooth
muscle cells, and some tumors has been shown to express OPN in a
constitutive or inducible fashion.2-9
The
adhesive functions of OPN are thought to be involved in diverse
biological activities such as bone absorption, tumor metastasis, and
inhibition of renal stone formation.2,9-11
A
functional role for OPN in monocyte infiltration at sites of
inflammation has recently been established. OPN, which binds avidly to
macrophages, induces prominent monocyte infiltration when injected
subcutaneously in mice.12
In addition, macrophage
accumulation induced by intradermal injection of the chemoattractant
N-formyl-met-leu-phe in rats is inhibited by administration
of a neutralizing anti-OPN antibody.13 Macrophage infiltration is thought to play an important role in mediating renal injury in both immune and nonimmune forms of kidney disease.14 A clear association between up-regulation of OPN expression and macrophage infiltration has been described in a wide range of experimental models of glomerular and interstitial nephritis.15-22 A functional role for OPN in promoting macrophage-mediated renal injury has recently been demonstrated in rat crescentic anti-glomerular basement membrane (GBM) glomerulonephritis.23 Administration of a neutralizing anti-OPN antibody during the induction phase of the disease (days 0 to 7) significantly inhibited glomerular and interstitial macrophage and T cell infiltration and the associated glomerular injury (proteinuria), loss of renal function, and histological damage including glomerular crescent formation and tubulointerstitial lesions. Furthermore, delaying administration of the anti-OPN antibody until disease was established caused a partial reversal of renal injury and damage.23
Having established the functional importance of OPN in promoting macrophage-mediated renal injury, the next issue is to identify the factors that up-regulate OPN expression within the kidney. We postulate that the cytokine interleukin-1 (IL-1) may be an important inducer of renal OPN expression, based on two observations. First, IL-1 has been shown to up-regulate OPN gene expression in chrondocytes and osteoblasts.24,25 Second, blocking IL-1 activity inhibits macrophage infiltration and renal injury in rat anti-GBM disease.26,27 Therefore, the current study examined whether IL-1 is an important inducer of OPN expression in experimental crescentic glomerulonephritis.
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This study used archival material from two previous studies.26,27 Accelerated autologous anti-GBM glomerulonephritis was induced in inbred male Sprague-Dawley rats (150–200 g) by subcutaneous immunization with 5 mg normal rabbit IgG in Freund's complete adjuvant, followed 5 days later (termed day 0) by an intravenous injection of 10 ml/kg rabbit anti-rat GBM serum (12.5 mg IgG/ml). In the first experiment, disease was induced in two groups of six rats which were treated from day -1 until being killed at day 14 (induction phase of disease) by a constant infusion of either human recombinant IL-1 receptor antagonist (IL-1ra) (Amgen, Boulder, CO) or saline by means of an Alzet 2002 miniosmotic pump implanted under the skin of the back. In the second experiment, disease was induced in three groups of six rats each. One group was killed on day 7 with no treatment. The other two groups were treated with IL-1ra or saline starting on day 7 and maintained until the animals were killed on day 21 (established disease). One group of six normal rats was also studied.
Immunohistochemistry
Immunohistochemical staining for OPN protein and macrophage accumulation was performed on formalin-fixed, paraffin-embedded sections using a microwave antigen retrieval method.22,23,28 Sections were dewaxed and treated with 10 minutes of microwave oven heating in 400 ml of 0.01 mol/L sodium citrate, pH 6.0, at 2450 MHz and 800W. After preincubation with 10% fetal calf serum (FCS) and 10% normal goat serum in PBS for 20 minutes, sections were drained and then labeled with mouse mAbs to rat OPN (MPIIIB10, obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA)29,30 or rat macrophages (ED1)31,32 for 60 minutes, washed 3 times in phosphate-buffered saline (PBS) and endogenous peroxidase inactivated by incubation in 0.3% H2O2 in methanol. Sections then were washed in PBS, incubated with peroxidase-conjugated goat anti-mouse IgG, washed in PBS, incubated with mouse peroxidase antiperoxidase complexes, and developed with 3,3-diaminobenzidine to produce a brown color.
Double immunostaining was used to detect OPN and macrophages within the same section. After staining with the ED1 mAb was completed, as described above, sections were given a second round of microwave oven heating to block antibody cross-reactivity and enhance detection of OPN. Following precincubation as above, sections were incubated with the MPIIIB10 mAb, then incubated sequentially with alkaline phosphatase-conjugated goat anti-mouse IgG and mouse alkaline phosphatase anti-alkaline phosphatase complexes and developed with Fast Blue BB Salt (Ajax Chemicals, Melbourne, Australia). No staining was seen in negative control sections using the 73.5 IgG1 (anti-human CD45R) irrelevant isotype control mAb. All peroxidase- and alkaline phosphatase-conjugated antibodies and complexes were purchased from Dakopatts (Glostrup, Denmark).
Probes
A 1-kb cRNA probe was generated from the rat smooth muscle osteopontin cDNA clone 2B7.8 Sense and antisense cRNA probes were labeled with either [35S] or digoxigenin (DIG)-UTP using a RNA polymerase kit (Boehringer Mannheim, Mannheim, Germany). A 358-bp cRNA antisense riboprobe for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also DIG-labeled. Probe labeling was determined by liquid scintillation counting (35S-labeled probes) or by dot blotting (DIG-labeled probes).
In Situ Hybridization
In situ hybridization was performed on 4-µm paraffin sections of formalin-fixed tissues using a radioactive method as described previously.33 Tissue sections were hybridized with 300,000 cpm/section of sense or anti-sense OPN cRNA probe at 55°C. After washing, the hybridized probe was detected by emulsion photography. Only low levels of background hybridization were seen using the sense probe (0 to 2 grains/cell).
Quantitation of Immunohistochemistry and in Situ Hybridization Staining
Positively stained cells were quantitated in tissue sections as previously described.22 Briefly, the number of cells labeled with the antisense OPN cRNA probe (defined as >5 grains per cell) or the different mAbs were counted under high power in at least 50 glomerular cross-sections (gcs) per animal. The number of tubules labeled with the antisense OPN cRNA probe or the different mAbs were scored under high power in at least 1000 cortical tubules. All scoring was performed on coded slides. Data are expressed as the mean ± SE for groups of six animals.
Cell Culture
A normal rat kidney epithelial-derived cell line, NRK52E, was
obtained from the American Type Culture Collection (Manassas, VA) and
cultured in Dulbecco's minimal essential medium (DMEM)
containing 2% fetal calf serum (FCS). Cells were grown to confluence
in 125-cm2
plastic tissue culture flasks, the
medium was changed to serum-free, and the cells were then cultured with
or without recombinant mouse IL-1 (1, 10, or 20 ng/ml) for up to 5
days. Preincubation of cells with IL-1ra (20µg/ml) for 30 minutes was
used specifically to block IL-1 stimulation of cultured cells.
Northern Blot Analysis
Northern blotting was performed as previously described.22,34 Briefly, total cellular RNA from cultured NRK52E cells was extracted using the RNAzol reagent (Gibco BRL, Gaithersburg, MD) and 20-µg samples denatured with glyoxal and dimethylsulfoxide, size-fractionated on 1.2% agarose gels, and capillary-blotted onto positively charged nylon membranes (Boehringer Mannheim). Membranes were hybridized overnight at 68°C with DIG-labeled cRNA probes in DIG Easy Hyb solution (Boehringer Mannheim). Following hybridization, membranes were washed finally in 0.1 x SSC/0.1% sodium dodecyl sulfate (SDS) at 68°C. Bound probes were detected using sheep anti-DIG antibody (Fab) conjugated with alkaline phosphatase and development with CPD-Star enhanced chemiluminescence (Boehringer Mannheim). Chemiluminescence emissions were captured on Kodak XAR film and densitometry analysis performed using the Gel-Pro Analyzer program (Media Cybernetics, Silver Spring, MD).
Western Blot Analysis
NRK52E cells were grown in 125-cm2 flasks with or without IL-1 and analyzed by Western blotting as previously described.35 Cells were washed in PBS and then lysed in 1 ml of 1% Nonidet P-40, 25 mmol/L Tris-HCl, 150 mmol/L NaCl, 10 mmol/L EDTA, pH 8.0, containing a 1:50 dilution of a protease inhibitor cocktail (P2714, Sigma-Aldrich Co., Castle Hill, Australia) for 30 minutes on ice. Samples were centrifuged at 14,000 g for 5 minutes to pellet cell debris. Samples (20 µg) were mixed with SDS polyacrylamide gel electrophoresis sample buffer, boiled for 5 minutes, electrophoresed on a 10% SDS polyacrylamide gel, and electroblotted onto Hybond-ECL nitrocellulose membrane (Amersham International, Buckinghamshire, UK). The membrane was blocked in PBS containing 5% skimmed milk powder, 1% FCS, and 0.02% Tween 20 and then incubated for 1 hour with 5µg/ml of MPIIIB10 mAb diluted in the above buffer. After washing, the membrane was incubated with a 1:20,000 dilution of peroxidase-conjugated goat anti-mouse IgG in PBS containing 1% normal goat serum and 1% FCS. The blot was then developed using the ECL detection kit (Amersham) to produce a chemiluminescence signal which was captured on X-ray film.
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In situ hybridization and immunostaining identified OPN
mRNA and protein expression by some glomerular parietal epithelial
cells in normal rat kidney (Figure 1A)
.
In addition, constitutive OPN expression was seen in the thick
ascending limbs of the loop of Henle, accounting for less than 5%
(3.3 ± 1.8%) of cortical tubules (Figures 1A, 2A, and 2B)
.
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There was marked up-regulation of OPN mRNA and protein expression
on day 14 of rat anti-GBM glomerulonephritis in saline-treated rats. In
glomeruli, both podocytes and infiltrating macrophages (shown by double
immunostaining) expressed OPN, whereas crescents showed strong OPN mRNA
and protein staining (Figures 1B, 2A, 2B, and 3A)
. There was also a striking increase
in tubular OPN expression (50.6 ± 3.2% OPN-positive cortical
tubules), most particularly in proximal tubular epithelial cells, which
was associated with focal infiltration of ED1-positive macrophages and
tubular damage (Figures 1B, 2B, and 3A)
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. This was associated with reduced macrophage
accumulation within the glomeruli (10 ± 2.1 versus
4.8 ± 1.9 cells/gcs in saline and IL-1ra treated rats,
respectively; P < 0.01) and in the cortical
tubulointerstitium (170 ± 41 versus 29 ± 2.5
cells/mm2; P < 0.01). As
previously described,26
IL-1ra treatment
suppressed proteinuria (332 ± 80 versus 260 ±
62 mg/24hrs; P < 0.05), prevented a decline in
creatinine clearance (0.66 ± 0.1 versus 0.96 ±
0.25 ml/min; P < 0.01), and reduced histological
injury, including glomerular crescent formation (13 ± 11.2%
versus 1.2 ± 1.6%, P < 0.01). IL-1ra Treatment Suppresses OPN Expression in Established Rat Crescentic Glomerulonephritis (Experiment 2)
The ability of IL-1 to up-regulate OPN expression during the
progressive phase of established renal injury was examined in
Experiment 2. Crescentic disease was established by day 7 after
anti-GBM serum administration and was associated with significant
up-regulation of glomerular and tubular OPN expression (Figures 1D, 2C, and 2D)
. Saline treatment from days 7 to 21 saw a further increase in
glomerular and tubular OPN mRNA and protein expression (Figures 1E, 2C, 2D, and 3C)
in association with a further decline in creatinine
clearance and a progressive increase in proteinuria as previously
described.27
Double immunohistochemistry showed a
tight association between OPN expression and macrophage accumulation in
areas of severe tissue damage, such as glomerular crescent formation
and tubulointerstitial lesions (Figure 3C)
.
IL-1ra treatment of established anti-GBM disease from days 7 to 21
caused a significant reduction in glomerular and tubular OPN mRNA and
protein expression (Figures 1F, 2C, 2D, and 3D)
. Indeed, the number of
glomerular OPN-positive cells was reduced to levels below that seen on
day 7 of disease, before the induction of IL-1ra treatment (Figure 2C)
.
This was associated with recovery of normal renal function, a minor
reduction in proteinuria, and prevention of further histological
damage.27
IL-1 Up-Regulates OPN Expression by Renal Tubular Epithelial Cells in Vitro
To investigate whether IL-1 can act directly to up-regulate OPN
expression in the development of rat anti-GBM glomerulonephritis, we
examined the effect of adding IL-1 to the level of OPN mRNA expression
in the normal rat epithelial cell line NRK52E. This cell line was
chosen because tubular epithelial cells are the major site of OPN
production within the injured kidney. Northern blot analysis showed
that the NRK52E cell line constitutively expresses low levels of OPN
mRNA (Figure 4)
. Within 6 hours of
IL-1 stimulation, there was an increase in OPN mRNA levels that
peaked after 24 hours (threefold induction). The IL-1-induced
up-regulation of OPN mRNA levels was dose-dependent and was maintained
for a 5-day culture period (Figures 4 and 5)
. Preincubation of cells with an excess
of the IL-1ra completely blocked IL-1 up-regulation of OPN mRNA
expression, demonstrating specificity of the effect. Western blotting
showed that NRK52E cells constitutively express OPN protein of
approximately 68 kd, consistent with previous studies of NRK52E cells
and rat mesangial cells.36,37
IL-1 increased OPN
protein levels within NRK52E cells in a dose-dependent fashion and this
was completely inhibited by preincubation of cells with the IL-1ra
(Figure 6)
.
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This is the first demonstration that IL-1 can up-regulate OPN expression in kidney cells. The ability of IL-1 to up-regulate OPN expression in renal epithelial cells is consistent with previous studies reporting that IL-1 increases OPN mRNA expression by cultured chrondocytes and osteoblasts.24,25 However, it should be noted that regulation of OPN gene expression varies greatly between different cell types. For example, IL-1 failed to increase OPN gene expression in cultured rat mesangial cells.37 In addition, vitamin D can induce or suppress OPN mRNA levels in osteoblasts depending on their state of differentiation.38 Furthermore, platelet-derived growth factor induces OPN expression in vascular smooth muscle cells and marrow stromal cells, but not in NRK52E tubular epithelial cells.36,39,40
It was interesting that the increase in renal OPN mRNA and protein production seen in rat crescentic glomerulonephritis was very similar to the pattern of up-regulation of renal IL-1ß expression described previously in this disease model,41 suggesting that up-regulation of OPN expression within the injured kidney may be due to local IL-1 production. In particular, strong IL-1ß and OPN mRNA and protein expression were seen in glomerular and tubular epithelial cells, including glomerular crescents and areas of tubulointerstitial injury. Whereas double staining showed that some glomerular macrophages expressed OPN, consistent with in vitro studies,6 podocytes were the major cell type expressing OPN in the glomerulus.22 Therefore, the suppression of glomerular OPN expression by IL-1ra treatment is largely attributed to inhibition of podocyte OPN production, with a lesser effect due to the reduction in macrophage infiltration and macrophage OPN expression.
It was intriguing that the rapid induction of OPN mRNA expression seen following the addition of IL-1 to cultured tubular epithelial cells was sustained for 5 days, in contrast with the 2 days required to detect TGF-ß-induced up-regulation of OPN mRNA in this cell line.36 Since IL-1 can induce TGF-ß synthesis by tubular epithelial cells, it may be the case that the sustained IL-1 up-regulation of OPN expression by the cultured tubular epithelial cells reflects a secondary effect of IL-1-induced production of TGF-ß. This possibility warrants further investigation.
IL-1 plays a crucial role in inducing renal macrophage infiltration.42 Indeed, IL-1 induces a broad range of chemokines and leukocyte adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1, which, acting in concert, facilitate the extravasation of blood leukocytes and their subsequent focal accumulation within the kidney. Blocking the action of just one IL-1-inducible adhesion or chemotactic molecule can inhibit leukocyte infiltration in experimental kidney disease,43,44 thereby demonstrating the interdependence of these molecules. This is illustrated by the ability of anti-OPN antibody treatment to suppress leukocyte infiltration and subsequent renal injury in rat anti-GBM glomerulonephritis without affecting up-regulation of ICAM-1 expression.23 This implies that ICAM-1 is necessary for leukocyte-endothelial interactions but that renal OPN expression is required for migration and localization of macrophages and T cells within the kidney, resulting in local tissue damage.
In summary, this study provides in vivo and in vitro evidence that IL-1 up-regulates OPN expression in experimental kidney disease and argues that inhibition of OPN expression is one of the mechanisms by which IL-1ra treatment suppresses macrophage-mediated renal injury in experimental crescentic glomerulonephritis.
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Footnotes |
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Supported by grants from the National Health and Medical Research Council of Australia (930825, 971295), the Australian Kidney Foundation (G18/97, G8R/98), the Guangdong Science and Technology Council of the Peoples Republic of China (95015), and the United States Public Health Service (DK-47659, DK-43422, and EEC9529161).
Accepted for publication November 18, 1998.
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vß3 potentiate bone resorption. J Biol Chem 1993, 268:9901-9907This article has been cited by other articles:
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