Nephrol Dial Transplant (2002) 17: 985–991
Original Article
Progression of tubulointerstitial injury by osteopontin-induced macrophage recruitment in advanced diabetic nephropathy of transgenic (mRen-2)27 rats Darren J. Kelly1, Jennifer L. Wilkinson-Berka2, Sharon D. Ricardo3, Alison J. Cox1 and Richard E. Gilbert1 1
Department of Medicine, University of Melbourne, St Vincent’s Hospital, Fitzroy, 2Department of Physiology, University of Melbourne, Parkville and 3Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia
Abstract Background. Osteopontin is a macrophage chemotactic protein that has been pathogenetically linked to tissue injury in non-diabetic kidney disease. Methods. To examine osteopontin expression and macrophage accumulation in diabetic nephropathy, diabetes was induced with streptozotocin (STZ) in the transgenic (mRen-2)27 rat, a rodent model which develops the structural and functional features of its human counterpart when rendered diabetic. Non-diabetic rats were randomly selected to receive either (STZ) or citrate buffer. Diabetic rats were further randomly selected to receive either the angiotensin-converting-enzyme inhibitor, perindopril (6 mgukguday), or the vehicle only for 12 weeks. Results. When compared with control animals, diabetes was associated with a 10-fold increase in the gene expression of osteopontin. Increased transcript and immunostainable osteopontin were detected in tubular epithelial cells in association with extensive macrophage accumulation. Treatment with perindopril significantly ameliorated the overexpression of osteopontin in association with attenuation of macrophage accumulation. Conclusions. These findings suggest that osteopontin expression and macrophage accumulation may play a role in the tubulointerstitial injury in diabetic nephropathy, and that inhibition of osteopontin expression may be one of the mechanisms by which blockade of the renin-angiotensin system confers a renoprotective effect. Keywords: diabetes; kidney; macrophage; osteopontin
Correspondence and offprint requests to: Dr Darren J. Kelly, Department of Medicine, St Vincent’s Hospital, Fitzroy, Victoria, 3065, Australia. Email:
[email protected] #
Introduction While pathological changes within glomerulus, and in particular the mesangium, have been a major focus in diabetes, tubulointerstitial injury is also a major feature of diabetic nephropathy. Indeed, as with other primary glomerular diseases, the extent of tubulointerstitial injury in the diabetic kidney correlates closely with long-term renal function and is an important predictor of renal impairment [1–3]. Recently, the cellular and molecular mechanisms that lead to the tubulointerstitial pathology in progressive renal disease have begun to be elucidated [4]. Experimental evidence has consistently demonstrated a role for macrophages in the development of both glomerular and tubulointerstitial injury [5]. However, while the presence of macrophages has been noted in both the glomerulus and tubulointerstitium in human diabetic nephropathy [6,7], the mechanisms which lead to their infiltration in diabetes are not well understood. Furthermore, whether the renoprotective effects of angiotensin-converting-enzyme (ACE) inhibition in diabetes [8] may be in part related to a reduction in macrophage infiltration has also not been explored. In non-diabetic renal disease, recent studies do suggest that blockade of the renin-angiotensin system (RAS) is associated with attenuation of the macrophage infiltration [9]. However, macrophage infiltration is not a feature of many rodent models of experimental diabetic nephropathy, which develop only mild structural changes and preserved GFR [10]. In the present study, a transgenic diabetic (mRen-2, 27) rat, which develops the structural, functional and molecular characteristics of human diabetic nephropathy [11,12], was used to assess macrophage infiltration and the expression of the chemokine osteopontin in the tubulointerstitium. The effects of a blockade of the RAS using ACE inhibition on these changes was also examined.
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Subjects and methods Animals Six-week-old female, heterozygous Ren-2 rats (non-diabetic), weighing 125"5 g were randomly selected to receive either 55 mgukg of streptozotocin (STZ) (Sigma, St Louis, MO, USA) diluted in 0.1 M citrate buffer, pH 4.5, or citrate buffer alone by tail vein injection following an overnight fast. Diabetic Ren-2 rats (ns6) were treated either with an ACE inhibitor, perindopril (Servier Laboratories, Paris, France) (average dose, 6 mgukguday), for 12 weeks post-STZ, or with the vehicle only. All rats were housed in a stable environment (maintained at 22"18C with a 12 h lightudark cycle) and allowed free access to tap water and standard rat chow (GR2, Clark-King & Co., Gladesville, NSW, Australia). Each week, rats were weighed and blood glucose (non-diabetic, 4–8 mmolul; diabetic, 18–22 mmolul) was determined using an AMES glucometer (Bayer Diagnostics, Melbourne, Australia). Diabetic rats received a daily injection of insulin (2–4 units i.p.; Ultratard, Novo Nordisk, Denmark) to promote weight gain and to reduce mortality. Experimental procedures adhered to the guidelines of the National Health and Medical Research Council of Australia’s Code for the Care and Use of Animals for Scientific Purposes, and were approved by the Bioethics Committee of the University of Melbourne.
Tissue preparation Rats were anaesthetized (Nembutal 60 mgukg body weight, i.p.; Boehringer-Ingelheim, Australia) and the abdominal aorta cannulated with an 18 G needle. Perfusionexsanguination commenced at 180–220 mmHg via the abdominal aorta with 0.1 M phosphate-buffered saline (PBS), pH 7.4 (20–50 ml) to remove circulating blood and the inferior vena cava adjacent to the renal vein was simultaneously severed allowing free-flow of the perfusate. After clearance of circulating blood, 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 was perfused for a further 5 min (100–200 ml of fixative) to fix the tissues [13]. Kidneys were removed from the animals, decapsulated and sliced transversely. Kidneys were post-fixed in the same fixative for 2 h, routinely processed, embedded in paraffin and sectioned.
Tubulointerstitial injury A modified Masson’s trichrome stain was performed on kidney sections to identify matrix deposition within the interstitium as previously described [12].
In situ hybridization The 1.1 kb cDNA probe for rat osteopontin was cloned into Bluescript (pBSSK, Stratagene, La Jolla, CA, USA) and linearized with Bam HI to generate a 33P-labelled riboprobe (33P from Geneworks, Adelaide, SA, USA) using T7 RNA polymerase [14]. In situ hybridization was performed on 4 mm paraffin sections, which were hybridized with anti-sense probes to osteopontin. Tissue sections were dewaxed in histosol, hydrated through graded ethanol and immersed in distilled water. In brief, tissue sections were dewaxed, rehydrated and microwaved, then digested with Pronase E (125 mguml in 50 mM Tris-HCl, pH 7.2, 5 mM EDTA,
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pH 8.0) at 378C for 10 min. After two 2 min rinses in 0.1 M phosphate buffer, pH 7.2, sections were fixed in 4% paraformaldehyde, pH 7.4 for 10 min at room temperature and rinsed again in 0.1 M phosphate buffer. Sections were then hybridized with 33P-labelled anti-sense and sense specific probes (5 3 105 cpmu25 ml hybridization buffer) in hybridization buffer (300 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM Na2HPO4, pH 6.8, 5 mM EDTA, pH 8.0, 1 3 Denhardt’s solution, 0.8 mguml yeast RNA, 50% deionized formamide and 10% dextran sulphate), heated to 858C and 25 ml added to the sections. Coverslips were placed on the sections and the slides incubated in a humidified chamber (2 3 SSC, 50% formamide) at 608C for 14–16 h. Slides were then washed in 2 3 SSC (0.3 M NaCl, 0.33 M Na3C6H5O7.2H2O) containing 50% formamide at 508C to remove the coverslips. The slides were washed with 2 3 SSC, 50% formamide for 1 h at 558C. Sections were then rinsed three times in RNase buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 0.5 M NaCl) at 378C, treated with 150 mguml RNase A in RNase buffer for a further 1 h at 378C, and washed with 2 3 SSC at 558C for 45 min. Finally, sections were dehydrated through graded ethanol, air dried and exposed to Kodak BioMax MR Autoradiography film for 4 days at room temperature. Slides were coated with Ilford K5 emulsion (Ilford, 1 : 1 with distilled water), stored with desiccant at 48C for 21 days, developed in Ilford Phenisol, fixed in Ilford Hypam and stained with haematoxylin and eosin [14].
Immunohistochemistry Three micron sections were placed into histosol to remove the paraffin wax, hydrated in graded ethanol and immersed into tap water before being incubated for 20 min with normal goat serum (NGS) diluted 1 : 10 with 0.1 M PBS at pH 7.4. Sections were then incubated for 18 h at 48C with specific primary polyclonal antisera to osteopontin (1 : 8000) and the monoclonal rat macrophage marker (ED-1, 1 : 200 Serotec, Raleigh NC, USA). Sections that were incubated with 1 : 10 NGS instead of the primary antiserum served as the negative control. After thorough washing with PBS (three 5 min changes), the sections were flooded with a solution of 5% hydrogen peroxide, rinsed with PBS (two 5 min washes) and incubated either with biotinylated goat anti-rabbit IgG (Dakopatts, Glostrup, Denmark) diluted 1 : 200, or with goat anti-mouse IgG (Dakopatts) diluted 1 : 200 with PBS. Sections were rinsed with PBS (two 5 min washes) and incubated with an avidin-biotin peroxidase complex (Vector, Burlingame, CA, USA) diluted 1 : 200 with PBS. Following rinsing with PBS (two 5 min washes), sections were incubated with 0.05% diaminobenzidine and 0.05% hydrogen peroxide (Pierce, Rockford, IL, USA) in PBS at pH 7.6 for 1–3 min, rinsed in tap water for 5 min, counterstained in Mayer’s haematoxylin, differentiated in Scott’s tap water, dehydrated, cleared and mounted in Depex [12].
Quantitation of histological parameters The expression of osteopontin mRNA and protein was quantitated by several different established methodologies. Autoradiographs from six kidneys per group in control, diabetic and perindopril-treated diabetic rats were digitized and the relative optical density, measured by image analysis [13]. For quantitation of in situ hybridization and immunohistochemistry, five randomly chosen fields per kidney
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section (ns6 per group) from control, diabetic and perindopril-treated diabetic rats were captured and digitized using a Fujix HC-2000 digital camera (Fuji, Tokyo, Japan). Regional gene expression and protein were quantitatively measured to determine the proportion of each area occupied by autoradiographic grains or positive DAB staining, respectively, as previously described [13]. For Masson’s trichrome stained sections, the proportional areas of blue stain (representing collagen deposition) from five randomly chosen fields from control, diabetic and perindopril-treated rats (ns6 per group) were also measured using the image analysis system [13]. All estimates were measured using computer assisted image analysis (Analytical Imaging Software, AIS, Ontario, Canada) on a Pentium III computer. Macrophage number was estimated by counting the number of macrophages in six sections per animal from each group.
Statistics Data are expressed as mean"SEM unless otherwise stated. Statistical significance was determined by a two-way ANOVA with a Fisher’s post-hoc comparison. Analyses were performed using Statview IIqGraphics package (Abacus Concepts, Berkeley, CA, USA) on an Apple Macintosh G3 computer (Apple Computers Inc., Cupertino, CA, USA). A P-value -0.05 was regarded as statistically significant.
Results Rats that had received STZ all became diabetic (Table 1). Diabetes was associated with reduced body mass and GFR when compared with control animals.
Tubulointerstitial injury Increased collagen deposition and numerous damaged and degenerated cortical tubules were apparent in diabetic Ren-2 rats when compared with the controls (P-0.01), while perindopril treatment was associated with reduced tubular collagen deposition and injury (P-0.001; Figure 1, Table 2).
Table 1. Body weight, plasma glucose, glomerular filtration rate and systolic blood pressure in rats that were either control, diabetic or perindopril-treated diabetic
Control Diabetic Perindopril
Body weight (g)
Plasma glucose (mmolul)
GFR (mlumin)
Blood pressure (mmHg)
286"4 213"91 215"51
5.9"0.2 22.4"1.02 21.5"0.82
3.26"0.2 1.64"0.21 2.45"0.11,3
211"8 216"12 143"61,3
Data are expressed as mean"SEM. GFR, glomerular filtration rate; 1 P-0.05 and 2P-0.01 when compared with controls; 3P-0.05 when compared with controls. ns6 per group.
Fig. 1. Masson’s Trichrome stained section of kidney from transgenic Ren-2 rats. In both control (A) and diabetic rats treated with perindopril (C) there is very little collagen within the interstitium and no tubular pathology, while diabetes (B) was associated with tubular dilation (arrows) and collagen deposition (lighter stained areas). Original magnification 3 340.
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Table 2. Quantitation of immunohistochemistry, in situ hybridization and macrophage number in kidneys from rats that were either control, diabetic or perindopril-treated diabetic Osteopontin
Control Diabetic Perindopril
mRNA ROD
mRNA % grainsuarea
Protein % area
TID % area
Macrophages numberusection
1.67"0.75 20.54"7.042 1.07"0.04
0.18"0.05 23.55"6.262 0.95"0.55
0.06"0.00 15.52"1.602 0.50"0.081
8.1"1.1 18.1"3.52 3.8"0.72
1"0.8 50"152 11"53
Data are expressed as mean"SEM. TID, tubulointerstitial disease; ROD, relative optical density; 1P-0.05 and 2P-0.01 when compared with controls; 3P-0.05 when compared with diabetics. ns6 per group.
Autoradiography Kidney osteopontin mRNA was increased in diabetic rats, when compared with controls (P-0.01) and this increase was prevented with perindopril treatment (Table 2, Figure 3). In situ hybridization Kidney osteopontin mRNA was localized to proximal tubules in diabetic rats (P-0.01). In contrast, control and diabetic rats treated with perindopril had minimal osteopontin expression in the proximal tubules and interstitium (Table 2, Figure 4). Immunohistochemistry Compared with control animals, osteopontin was increased in diabetic rats and localized to proximal tubules both prior to the onset of tubulointerstitial disease and during active tubulointerstitial injury and fibrosis (P-0.01; Figure 5). Perindopril treatment dramatically reduced the diabetic increase in osteopontin immunolabelling (P-0.01; Table 2, Figure 5). In control kidney sections very few macrophages were detected, while diabetes was associated with a dramatic increase in the number of macrophages, particularly associated with regions of tubulointerstitial injury (P-0.001). The number of macrophages present within the kidney was reduced with perindopril treatment (P-0.05; Table 2, Figure 2).
Discussion The present study demonstrates several features in relation to experimental diabetic nephropathy. Firstly, macrophages are abundantly present in the tubulointerstitium of diabetic rats. Secondly, macrophage infiltration occurs in close proximity to damaged Fig. 2. Immunohistochemistry for macrophages (ED-1 positive cells) in the kidney of transgenic Ren-2 rats. In both control (A) and diabetic rats treated with perindopril (C) there were very few macrophages detected, while diabetes (B) was associated with numerous macrophages (arrows) within the interstitium, particularly in regions undergoing tubulointerstitial fibrosis. Original magnification 3 560.
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Fig. 3. Autoradiography for osteopontin mRNA in the kidney of transgenic Ren-2 rats. In both control (A) and diabetic rats treated with perindopril (C) the majority of osteopontin is localized in the medulla, while diabetes (B) was associated with a dramatic upregulation in osteopontin mRNA, in the cortex. Original magnification 3 5.
cortical tubules which express the chemokine, osteopontin. Thirdly, ACE-inhibitor treatment was associated with attenuation of osteopontin expression, macrophage infiltration and tubulointerstitial injury. In the normal kidney, macrophages are mostly restricted to the renal capsule, pelvic wall and to the adventitia of large vessels [14]. However, in glomerulonephritis, macrophage infiltration is often a prominent feature where they are believed to play a significant role in mediating injury [5]. More recently the presence of macrophages in primarily non-inflammatory renal disease, including both experimental [15] and human diabetes [7] has also been recognized. However, such studies have also been confined to the glomerulus, although cross-sectional [2,3], longitudinal [16] and intervention [17] studies all suggest a close relationship between the extent of tubulointerstitial rather than glomerular disease and declining renal function in diabetic nephropathy. In the present study, interstitial macrophage infiltration was a prominent feature. These inflammatory cells contain a range of mediators implicated in tissue injury including reactive oxygen intermediates, proteases, inflammatory cytokines and the profibrotic growth factors, platelet-derived growth factor and transforming growth factor-b (TGF-b) [5,18,19]. Indeed, like interstitial injury [1] the number of interstitial mononuclear cells also correlates closely with declining renal function in a range of renal diseases [20]. Osteopontin is a secreted acidic and phosphorylated glycoprotein with an adhesive arg-gly-asp sequence that binds to cell surface adhesion receptors (avb1, avb5), CD44, and matrix proteins (type 1 collagen, fibronectin) [21–23]. Accumulated evidence suggests that osteopontin functions as a regulator of inflammation as a consequence of its ability to regulate the function of macrophages and macrophage-derived cells [24]. While the mechanisms leading to macrophage accumulation in renal disease are incompletely understood, osteopontin is among a number of relevant chemokines that have been identified [25]. In the present study, osteopontin was abundantly expressed in the proximal tubular epithelial cells of rats with experimental diabetes but not in control animals. Considerable internephron heterogeneity in osteopontin
Fig. 4. In situ hybridization for osteopontin mRNA in the kidney of transgenic Ren-2 rats. In both control (A) and diabetic rats treated with perindopril (C) there is very little osteopontin gene expression, while diabetes (B) was associated with a dramatic upregulation in osteopontin mRNA (arrows) particularly in damaged tubules and areas undergoing active interstitial fibrosis. Original magnification 3 560.
expression was also noted, with macrophage infiltration most closely associated with damaged cortical tubules. This is consistent with the actions of osteopontin as a potent chemotactic factor for macrophages [25]. Indeed, a similar anatomical association between tubular osteopontin expression and focal macrophage infiltration has also been observed in several models
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macrophage chemotaxis, rather than the mononuclear infiltration developing as a later consequence tubular injury. Treatment with the ACE inhibitor, perindopril, was associated with attenuation of both osteopontin overexpression and macrophage infiltration in diabetic rats. These findings suggest significant interactions between the RAS and osteopontin in renal disease. In vitro, exposure of proximal tubular epithelial cells to angiotensin II is accompanied by upregulation in osteopontin expression [30]. Furthermore, the induction of osteopontin expression can be abrogated by angiotensinogen and angiotensin receptor anti-sense oligonucleotides [31]. These in vitro findings are consistent with the induction of osteopontin expression in vivo by angiotensin II infusion [26] and its upregulation with the activation of the proximal tubular RAS that follows renal mass reduction [9,32]. Similarly, increased glucose also leads to upregulation of the intrarenal RAS in both tissue culture [33,34] and experimental diabetes [11]. However, it is also possible that indirect mechanisms may have accentuated osteopontin expression in the present study. For instance, TGF-b is also a potent inducer of osteopontin expression in cultured rat epithelial cells [35] and is overexpressed in diabetic nephropathy [36] such that the diminution in TGF-b expression by blockade of the RAS [37], may also contribute to the attenuation in osteopontin expression noted in the present study. In contrast to other rodent models with STZinduced diabetes, which are relatively resistant to the development of diabetic renal pathology, the diabetic Ren-2 rat develops structural and functional features akin to human diabetic nephropathy, including nodular glomerulosclerosis, tubulointerstitial fibrosis with a mononuclear infiltrate, albuminuria and declining renal function [11]. Such differences in animal models may underlie the absence of macrophage infiltration and tubulointerstitial injury and the paradoxical osteopontin response to ACE inhibition seen in previous studies [38]. In summary, the findings of the present study suggest that osteopontin expression and macrophage accumulation may play a role in the tubulointerstitial injury in diabetic nephropathy and that inhibition of osteopontin expression may be one of the mechanisms by which a blockade of the RAS confers a renoprotective effect. Fig. 5. Immunohistochemistry for osteopontin in the kidney of transgenic Ren-2 rats. In both control (A) and diabetic rats treated with perindopril (C) there is very little osteopontin detected. Diabetes was associated with intense osteopontin immunolabelling within cortical tubules prior to the onset of tubulointerstitial injury (B, arrows) and also within damaged cortical tubules, particularly in regions undergoing active interstitial fibrosis (C, arrows). Original magnification 3 340.
of non-diabetic experimental renal disease [9,26–29]. Moreover, in these studies, osteopontin upregulation was found to precede macrophage infiltration [9,26] suggesting that osteopontin overexpression initiates
Acknowledgements. This project was supported by grants for the Juvenile Diabetes Foundation International. Dr Darren Kelly is a recipient of a Juvenile Diabetes Fellowship and Assoc. Prof Richard Gilbert is a recipient of a Career Development Award both from the Juvenile Diabetes Foundation International. The authors thank Giao Tran for providing expert technical assistance.
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