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NDT Advance Access originally published online on January 18, 2006
Nephrology Dialysis Transplantation 2006 21(5):1212-1222; doi:10.1093/ndt/gfk076
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Experimental Nephrology

Effects of low molecular weight heparin in obstructed kidneys: decrease of collagen, fibronectin and TGF-ß, and increase of chondroitin/dermatan sulfate proteoglycans and macrophage infiltration

Inah M. D. Pecly2, Rômulo G. Gonçalves1, Ednei P. Rangel1, Christina M. Takiya3, Fernanda S. Taboada2, Cesônia A. Martinusso1, Mauro S. G. Pavão2 and Maurilo Leite, Jr1

1 Serviço de Nefrologia, Departamento de Clínica Médica, Hospital Universitário Clementino Fraga Filho, 2 Laboratório de Tecido Conjuntivo, Instituto de Bioquímica Médica, Programa de Glicobiologia and 3 Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil

Correspondence and offprint requests to: Maurilo Leite Jr, Rua Ministro Otávio Kelly 296, 302 Niterói, R.J. CEP 24220-301, Brazil. Email: mleitejr{at}hucff.ufrj.br



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Heparin exerts beneficial effects in different experimental models of nephropathy, as observed by the preservation of the structural morphology of the kidney after heparin therapy. Here we investigate molecular and cellular events involved in the protective effects of heparin in the progression of renal disease after unilateral ureteral obstruction.

Methods. Thirty-six rats were divided into six groups: group C (control) was not subjected to any surgical manipulation; group S (sham) was subjected to surgical manipulation but without ureteral ligation; group UUO was subjected to ureteral obstruction and received no treatment; group UUO + S was subjected to ureteral obstruction and received saline subcutaneously (s.c.) once daily; group UUO + H was subjected to ureteral obstruction and received low molecular weight heparin (LMW-Hep; 4 mg/kg) s.c. once daily; and group C + H was not subjected to any surgical manipulation and received LMW-Hep (4 mg/kg) s.c. once daily. After 14 days, the content of collagen, fibronectin, total glycosaminoglycans (GAGS), chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs), transforming groth factor-ß (TGF-ß) and cellular infiltration were determined in the kidneys by immunohistochemical and biochemical techniques.

Results. Collagen, fibronectin, total GAGS, CS/DSPGs, TGF-ß and cellular infiltration increased significantly in group UUO. LMW-Hep treatment reduced collagen, fibronectin and TGF-ß, but induced an increase in the content of total GAGS, CS/DSPGs and macrophage infiltration in group UUO + H when compared with group UUO.

Conclusions. LMW-Hep diminishes fibrosis in obstructed kidneys by downregulating the synthesis of collagen, fibronectin and TGF-ß. The mechanisms underlying the overproduction of CS/DSPGs and the increase in cellular infiltration upon LMW-Hep administration remain to be elucidated.

Keywords: animal model; collagen; glycosaminoglycans; low molecular weight heparin; unilateral ureteral obstruction



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The pathogenesis of progressive renal diseases leading to renal failure has been the subject of intense investigation. Some features influencing renal inflammation and fibrosis have been well recognized, such as matrix deposition, infiltration of blood mononuclear cells, proliferation of interstitial mesenchymal cells, and apoptosis of tubular epithelial cells [1–7].

L-, P- and E-selectin mediate the initial events involving the migration of leukocytes across the endothelium in inflamed tissues [9–13]. Heparin and heparin-like glycosaminoglycans (GAGS) bind to L- and P-selectin and it has been shown that inhibition of these selectins accounts for the potent anti-inflammatory effect of heparin [14]. Heparin has been used successfully as a therapeutic agent in different animal models of nephropathy. Subcutaneous (s.c.) injection of non-anticoagulant heparin reduces glomerulosclerosis in rats [15], and ameliorates the progression of renal disease in rats with subtotal renal ablation [16]. In addition, it has also been demonstrated that heparin inhibits macrophage infiltration and transforming growth factor-ß (TGF-ß) synthesis in puromycin glomerulosclerosis [17] and that low molecular weight heparin (LMW-Hep) drastrically reduces macrophage infiltration in a kidney transplant model [18].

In the present work, we investigate molecular and cellular events involved in the protective effect of LMW-Hep in obstructed kidneys in rats. The results indicate that LMW-Hep produced different effects: it attenuates collagen and fibronectin deposition, and decreases TGF-ß, but it increases the content of GAGs, chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs) and cellular infiltration of macrophages.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals and experimental protocol
All animal work was carried out in accordance with the Brazilian Animal Protection Law. The study was performed in 36 adult male Wistar rats weighing 180–220 g. Rats were kept in a 12 h light/dark cycle at 25°C and fed a standard rat chow, and water ad libitum. Ureteral obstruction was conducted as follows: rats were anaesthetized s.c. with ketamine (35 mg/kg) and xylazine (9 mg/kg). Under sterile conditions, an abdominal midline incision was done; the left ureter was exposed and ligated using 4-0 silk at two points. The ureter was then sectioned between the ligatures. The skin was sutured for approximation and the rats kept in regular cages. The rats were divided into six groups. A group of six rats served as control and was not subjected to any surgical manipulation (group C). The group S (sham-operated animals, n = 6) was subjected to the surgical procedures, except that the left ureter was manipulated without ligation and sectioning. The group UUO (n = 6) was subjected to ureteral obstruction and were given no treatment. The group UUO + S (n = 6) was subjected to ureteral obstruction and was given 0.30 ml of saline s.c. once daily. Another group of six rats was subjected to ureteral obstruction and was given LMW-Hep (Enoxiparin sodium, anti-Xa activity, 98 IU/mg; and anti-IIa activity, 26.6 IU/mg; 4 mg/kg/day, dissolved in 0.30 ml of saline) s.c. once daily (group UUO + H). Finally, a group of six rats was not subjected to any surgical manipulation and was given 0.30 ml of LMW-Hep (4 mg/kg/day)-containing solution s.c. once daily (group C + H).

Tissue preparation
At the end of 14 days, the rats were sacrificed under anaesthesia and the kidneys were removed. Animals were perfused with citrate solution (3.5%) via the left cardiac ventricle for 20 min. Kidneys were removed and sectioned mid-frontally into two pieces. One fragment was immersed in a 10% buffered formaldehyde solution and embedded in paraffin for histological examination, while the other was immersed in acetone for hydroxyproline and GAG analysis. Kidney paraffin sections of 7 µm were stained with haematoxylin–eosin (HE), with 1% alcian blue (8GX, Sigma, St Louis, MO), pH 1.0, in a 0.1 M HCl solution for 30 min for GAG staining, and a modified Sirius red technique [19,20] for collagen staining. For detection of macrophages, a mouse monoclonal antibody against rat ED-1 (Serotec, USA) was used. Fibronectin was detected by a rabbit anti-human fibronectin antibody (Dako, USA), and CS/DSPGs by a mouse anti-chondroitin sulfate (CS) DDi-4S monoclonal antibody (Seikagaku, Japan). For TGF-ß analysis, a pan-specific anti-TGF-ß antibody (R&D System, USA) was used. Antibodies were revealed with the kit Dako LSAB® 2 system HRP (Dako, USA), using diaminobenzidine as the chromogen substrate (Liquid DAB, Dako, USA).

Measurement of interstitial area
Renal interstitium was defined as the renal space not occupied by glomeruli, tubules or vessels. The interstitial area measured was taken as an estimate of the interstitial volume. To measure the renal volume, paraffin sections stained with HE were used. Under high magnification (400x), consecutive non-overlapping fields were photographed from each section of kidney. A total of 50 fields per animal were counted. A standard point counting method was used to quantify the volume of the renal interstitium. Briefly, a grid containing 117 (13 x 9) sampling points was superimposed on each photograph and a total of 1404–2691 points were evaluated in each kidney. The number of points comprising the space surrounded by tubular basement membrane, the glomerular structures and vessels, were excluded from the total count. The results were expressed as the ratio of the interstitial area over the total area.

Histomorphometry
Histomorphometry was performed using an imaging analysis system constituted by a digital camera (Coolpix 990, Nikon, Japan) coupled to a light microscope (Eclipse 400, Nikon, Japan). Fifteen fields of renal cortex and of medulla from Sirius red- and ED-1-stained sections, as well as sections stained with anti-fibronectin, anti-CS/DSPG and anti-TGF-ß were captured from each animal, using a 40x magnification objective lens. Quantification was estimated on captured high quality images (2048x1536 pixels buffer) by considering the percentage of stained areas in the total histological fields using the Image Pro Plus 4.5.1 (Media Cybernetics, Silver Spring, MD). Results were expressed as the surface area (µm2), percentage of surface density or as number of macrophages/fields.

Determination of hydroxyproline
The amount of hydroxyproline (the hydroxylated form of the collagen-specific amino acid proline) in the renal tissue was estimated by a modified method described by Stegemann and Stalder [21]. Briefly, the samples were immersed in acetone for 24 h at 4°C and dried in an oven at 60°C. About 30 mg of the dried material was subjected to acid hydrolysis with 6 N HCl at 107°C for 18 h. Then, HCl was removed by evaporation and the hydrolysed material mixed with 200 ml of buffer (5% citric acid·1H2O, 1.2% acetic acid, 12% sodium acetate·3H2O and 3.4% sodium hydroxide, pH 6.0) diluted 1:10. The mixture was incubated with 1 ml of chloramine-T solution for 20 min at room temperature and 1 ml of aldehyde/perchloric acid solution was added and incubated for another 15 min at 60°C. The absorbance at 570 nm was recorded after 20 min. The concentration was estimated by a standard curve using a pure solution of hydroxyproline.

Isolation and quantification of GAGs
The dried renal samples (~1 g) were individually suspended in 20 ml of 0.1 M sodium acetate buffer (pH 5.5), containing 100 mg papain, 5 mM EDTA and 5 mM cysteine, and incubated at 60°C for 24 h. The mixtures were centrifuged (2000 g for 10 min at room temperature). Another 100 mg of papain in 20 ml of the same buffer, containing 5 mM EDTA and 5 mM cysteine, was added to the precipitate. The mixture was then incubated for another 24 h. The clear supernatants from the two extractions were combined, and the GAGs precipitated with a solution of cetylpyridinum chloride (0.5% final concentration), followed by 2 vols of 95% ethanol and maintained at 4°C for 24 h. The precipitate formed was collected by centrifugation (2000 g for 10 min at room temperature), freeze-dried and dissolved in 2 ml of distilled water. The amount of GAGs in the renal samples was estimated by the content of hexuronic acid, using the carbazole reaction [22].

Agarose gel electrophoresis
The intact GAGs extracted from the different renal samples were analysed by agarose gel electrophoresis, as described previously [23]. Briefly, ~1.5 µg (as uronic acid) of the glycans, and a mixture of standard GAGs, containing CS, dermatan sulfate (DS) and heparan sulfate (HS; 1.5 µg as uronic acid of each), were applied to a 0.5% agarose gel in 0.05 M 1,3-diaminopropane/acetate (pH 9.0), and run for 1 h at 110 mV. After electrophoresis, the GAGs were fixed with aqueous 0.1% cetylmethylammonium bromide solution and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, by vol.). The relative proportions of the GAGs were estimated by densitometry of the metachromatic bands on a Bio-Rad densitometer, following agarose gel electrophoresis.

Statistics
Data are given as mean±SD or median (maximum and minimum), whichever was more convenient. Comparisons between groups were done by one-way analysis of variance (ANOVA), or Kruskal–Wallis and post-tested by Tukey test, and were considered significant when P<0.05.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Histomorphological changes in obstructed kidneys
The effect of s.c. administration of LMW-Hep on the progression of renal disease was examined in rats after 14 days of UUO. Evident morphological changes were observed in obstructed kidneys including tubular changes, interstitial oedema, mononuclear cell infiltration and collagen deposition. Administration of LMW-Hep to obstructed rats increased the interstitium, due to oedema, promoted dilation of the tubular structures (an aspect also seen in group C + H animals) but attenuated the tubular atrophy. Inflammation, on the other hand, seemed to persist as in the UUO group. Control animals (groups S and C) did not exhibit modification of renal structures.

Interstitial area
At day 14, quantitative histomorphometry of HE-stained sections (Figure 1A and B) from obstructed kidneys revealed an ~1.7-fold increase in the interstitial area, when compared with control kidneys. The interstitial area (µm2) was 0.50±0.07 (n = 50) in obstructed kidneys (group UUO) vs 0.28±0.06 (n = 50, P<0.05) in control kidneys (group C). No difference was observed in HE-stained sections from control and sham-operated kidneys (0.30±0.07). Daily s.c. administration of LMW-Hep for 14 days to rats after UUO, significantly increased the interstitial area of obstructed kidneys (0.57±0.08, n = 50) compared with all other groups (P<0.05). In group C + H (0.34±0.05, n = 50), the interstitial area was significantly higher than in groups C and S, but lower than in groups UUO + H (P<0.05) and UUO (P<0.05) (Figure 1A and B).


Figure 1
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  		Fig. 1. Measurement of the interstitial area on sections of kidneys from rats submitted to different treatments. (A) The interstitial area was used to estimate the interstitial volume of kidneys obtained from animals of group C (control kidney), group S (sham-operated kidney), group UUO (obstructed kidney with no treatment), group UUO + H (obstructed kidney treated with 4 mg/kg/day of LMW-Hep for 14 days) and group C + H (control kidney treated with 4 mg/kg/day LMW-Hep for 14 days). Results are expressed as mean±SD, n = 50. (B) Haematoxylin-stained sections of kidneys from rats of groups C, UUO and UUO + H. Magnification 40x. Arrows indicate interstitial area.

 
Macrophage infiltration
To investigate the effect of LMW-Hep on the infiltration of macrophages, sections from control and obstructed kidneys were stained with a mouse monoclonal antibody against rat ED-1. Thirteen fields of renal cortex and medulla from ED-1-stained sections were captured from each animal and the results expressed as the number of macrophages per field. ED-1-positive cells increased from 0.1±0.4 in control group C to 21.9±11.1 in group UUO (P<0.0001) (Figure 2A and B). Surprisingly, administration of LMW-Hep to rats after UUO further increased the infiltration of ED-1-positive cells on group UUO + H to the value of 29.4±10.3 (P<0.001) (Figure 2A and B). There was no statistical difference between groups S (0.1±0.3), C and C + H (0.1±0.4). Also, no difference was found between groups UUO and UUO + S (21.1±5.1).


Figure 2
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  		Fig. 2. Macrophage infiltration into the kidney of rats submitted to different treatments. (A) Macrophage infiltration was estimated by the content of ED-1-positive cells on sections from kidneys of rats from group C (control kidney), group S (sham-operated kidney), group UUO (obstructed kidney with no treatment), group UUO + H (obstructed kidney treated with 4 mg/kg/day of LMW-Hep for 14 days) and group C + H (control kidney treated with 4 mg/kg/day LMW-Hep for 14 days). Results are expressed as mean±SD, n = 6. (B) ED-1-stained sections from kidneys of rats of groups C, UUO and UUO + H. Magnification 40x. Arrows indicate ED-1-positive cells.

 
Changes in the extracellular matrix in obstructed kidneys
To investigate the effect of LMW-Hep in the extracellular matrix of obstructed kidneys, the changes in the content of collagen, fibronectin, total GAGs and CS/DSPGs in the kidneys of rats after UUO were assessed.

Collagen
The content of collagen was estimated by measuring hydroxyproline in renal tissues and by histomorphometry of Sirius red-stained sections, as described in Materials and methods. Determination of the hydroxyproline content (µg/mg of tissue dry weight) (Figure 3A) revealed an ~3.1-fold increase in obstructed kidneys (group UUO) (7.78±0.91), when compared with non-ligated kidneys (group C) (2.56±0.47) (P<0.05). No significant difference was observed between group C and group S (2.17±0.31). Daily s.c. administration of LMW-Hep for 14 days to rats after UUO, significantly reduced collagen deposition in the interstitial space of obstructed kidneys (group UUO + H) to the value of 2.50±0.62, as compared with group UUO (P<0.05) (Figures 3A). No significant reduction in collagen deposition was observed in obstructed kidneys of rats receiving saline (group UUO + S) (6.68±0.16). In addition, no significant change was observed in the content of collagen in non-ligated kidneys of rats receiving LMW-Hep (group C + H) (2.46±0.11) when compared with groups C and S (Figure 3A).


Figure 3
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  		Fig. 3. Content of collagen in kidneys from rats submitted to different treatments. (A) Hydroxyproline (µg/mg of dry weight) was used to estimate the content of collagen in kidneys of rats from group C (control kidney), group S (sham-operated kidney), group UUO (obstructed kidney with no treatment), group UUO + H (obstructed kidney treated with 4 mg/kg/day of LMW-Hep for 14 days) and group C + H (control kidney treated with 4 mg/kg/day LMW-Hep for 14 days). Results are expressed as mean±SD, n = 6. (B) Histomorphometry of Sirius red-stained sections of kidneys from rats of groups C, UUO and UUO + H. Horizontal bars represent medians; boxes represent the 25th and 75th percentiles, and vertical bars represent ranges, n = 30. (C) Sirius red-stained sections of kidneys from rats of groups C, UUO and UUO + H. Magnification 40x.

 
The histomorphometry of Sirius red-stained sections, expressed in µm2 (Figure 3B and C), revealed an ~7.12-fold increase in collagen deposition in the interstitium of obstructed kidneys (group UUO) (3526.2±1527.9) when compared with group C (494.90±330.2) (P<0.05). No significant difference was observed between groups C, C + H (417.9±577.7) and S (454.7±432.3), and between groups UUO and UUO + S (3151.9±1153.8). The group UUO + H (1930.5±1343.0) revealed a lower collagen deposition than groups UUO and UUO + S (P<0.05) and higher than groups C, C + H and S (P<0.05), representing an ~1.8-fold reduction when compared with obstructed kidneys of rats not receiving LMW-Hep (P<0.001).

Glycosaminoglycans
The content of GAGs was estimated by the hexuronic acid-specific method using carbazole [22], expressed as µg/mg of dry tissue, and a qualitative analysis performed by agarose gel electrophoresis of the papain-extracted glycans, as described in Materials and methods. Total GAGs increased ~2.6-fold in obstructed kidneys of rats after UUO (group UUO), compared with the control group C (Figure 4A). The content of GAGs in group C was 0.30±0.06 (n = 5) vs 0.78±0.11 (n = 4) in group UUO (P<0.05) and 0.79±0.16 (n = 4) in group UUO + S (P<0.05). Daily s.c. administration of LMW-Hep for 14 days to rats after UUO significantly increased the content of GAGs in group UUO + H (0.98±0.17, n = 6, P<0.05), and induced an ~ 3.1-fold increase in the content of GAGs in control group C + H (0.92±0.13, n = 4, P<0.05). Groups S (0.30±0.07) and C were not statistically different, and neither was the comparison between groups C + H and UUO and between groups UUO + S and C + H (Figure 4A). Analysis of the GAGs after UUO by agarose gel electrophoresis revealed a qualitative change in the expression of the glycans in obstructed kidneys (Figure 4B). In control kidneys, HS was the only GAG detected by the method used, as indicated by a unique metachromatic band migrating as standard HS, which completely disappeared after incubation with nitrous acid (data not shown) (Figure 4B). After UUO, in addition to the HS band, two additional metachromatic bands migrating as standard DS and CS became evident in the obstructed kidney (Figure 4B). These bands disappeared after incubation with chondroitin ABC- and AC-lyases, respectively (data not shown). Administration of LMW-Hep to rats after UUO increased the expression of DS and CS in obstructed kidneys to a greater extent, when compared with non-treated animals after UUO (Figure 4B). Analysis of alcian blue (AB)-stained sections revealed that the GAGs increased in the interstitium of obstructed kidneys from rats after UUO, when compared with kidneys from control animals (Figure 4C). Administration of LMW-Hep to rats after UUO increased the GAGs at the basal membrane and in some areas of the interstitium, as indicated by AB-stained sections from obstructed kidneys (Figure 4C).


Figure 4
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  		Fig. 4. Content of GAGs in kidneys from rats submitted to different treatments. (A) Hexuronic acid (µg/mg of dry weight) was used to estimate the content of GAGs in kidneys of rats from group C (control kidney), group S (sham-operated kidney), group UUO (obstructed kidney with no treatment), group UUO + H (obstructed kidney treated with 4 mg/kg/day of LMW-Hep for 14 days) and group C + H (control kidney treated with 4 mg/kg/day LMW-Hep for 14 days). Results are expressed as mean±SD, n = 6. (B) Agarose gel electrophoresis of GAGs (~1.5 µg as hexuronic acid) from kidneys of rats from groups C, UUO and UUO + H, and a mixture of standard GAGs, containing chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) were applied to a 0.5% agarose gel in 0.05 M 1,3 diaminopropane/acetate. (C) Alcian blue-stained sections from kidneys of rats of groups C, UUO and UUO + H. Magnification 40x.

 
Chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs)
Immunohistochemical studies using an anti-DDi-4S monoclonal antibody were done to evaluate the expression of CS/DSPGs in the kidneys. The histomorphometry of stained sections, expressed as a percentage of sulface density, showed an ~10-fold increase in the content of CS/DSPGS in the extracellular matrix of obstructed kidneys (group UUO) (13.6±7.5), when compared with groups C (1.4±2.6, P<0.001) and S (2.4±2.4, P<0.001) (Figure 5A and B). LMW-Hep treatment induced an ~2- and ~19-fold increase in the content of CS/DSPGs in group UUO±H (26.3±13.0), when compared with groups UUO and C (P<0.001) (Figure 5A and B). There was no statistical difference between groups S, C and C±H. Also, no difference was found between groups UUO and UUO±S.


Figure 5
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  		Fig. 5. Content of CS/DSPGs in kidneys from rats submitted to different treatments. (A) The content of CS/DSPGs was estimated using an anti-DDi-4S monoclonal antibody, which recognizes specific CS/DS epitopes left on protein cores of CS/DSPGs after exhaustive treatment with Chase ABC. Histomorphometry of anti-DDi-4S-stained sections of kidneys from rats of group C (control kidney), group S (sham-operated kidney), group UUO (obstructed kidney with no treatment), group UUO + H (obstructed kidney treated with 4 mg/kg/day of LMW-Hep for 14 days) and group C + H (control kidney treated with 4 mg/kg/day LMW-Hep for 14 days). Results are expressed as a percentage of the surface density (mean±SD, n = 30). (B) Anti-DDi-4S-stained sections from kidneys of rats of groups C, UUO and UUO + H. Magnification 40x.

 
Fibronectin
The expression of fibronectin in the kidneys was indirectly evaluated by immunohistochemistry using an anti-fibronectin antibody, as described in Materials and methods. The histomorphometry of stained sections, expressed as a percentage of surface density, revealed an ~50-fold increase in the content of fibronectin in obstructed kidneys of rats after UUO (group UUO) (20.19±11.1), when compared with groups C (0.41±0.5, P<0.001) and S (0.46±0.6, P<0.001) (Figure 6A and B). The group UUO±H (9.29±6.6) showed a much lower fibronectin content than groups UUO and UUO±S (20.22±11.1) (P<0.001) (Figure 6A and B). There was no statistical difference between groups S, C and C±H. Also, no difference was found between groups UUO and UUO±S.


Figure 6
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  		Fig. 6. Content of fibronectin in kidneys from rats submitted to different treatments. (A) Histomorphometry of anti-fibronectin-stained sections of kidneys from rats of group C (control kidney), group S (sham-operated kidney), group UUO (obstructed kidney with no treatment), group UUO + H (obstructed kidney treated with 4 mg/kg/day of LMW-Hep for 14 days) and group C + H (control kidney treated with 4 mg/kg/day LMW-Hep for 14 days). Results are expressed as a percentage of the surface density (mean±SD, n = 30). (B) Anti-fibronectin-stained sections from kidneys of rats of groups C, UUO and UUO + H. Magnification 40x.

 
Immunohistochemistry for TGF-ß
TGF-ß plays a crucial role in the progression of renal fibrosis associated with the model of UUO. The histomorphometry of stained sections, expressed as a percentage of surface density, revealed, as expected, an ~2.9-fold increase, showing a uniform distribution within cortical and medullary tubules, in obstructed kidneys of rats after UUO (group UUO) (22.5±45.8), when compared with groups C (2.51±2.58) and S (2.41±2.4) (P<0.001) (Figure 7A and B). The group UUO±H (7.84±5.2) showed a much lower content of TGF-ß when compared with groups UUO and UUO±S (15.9±6.4) (P<0.001) (Figure 7A and B). There was no statistical difference between groups S, C and C±H. Also, no difference was found between groups UUO and UUO±S.


Figure 7
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  		Fig. 7. Content of TGF-ß in kidneys from rats submitted to different treatments. (A) Histomorphometry of anti-TGF-ß-stained sections of kidneys from rats of group C (control kidney), group S (sham-operated kidney), group UUO (obstructed kidney with no treatment), group UUO + H (obstructed kidney treated with 4 mg/kg/day of LMW-Hep for 14 days) and group C + H (control kidney treated with 4 mg/kg/day LMW-Hep for 14 days). Results are expressed as a percentage of the surface density (mean±SD, n = 30). (B) Anti-TGF-ß-stained sections from kidneys of rats of groups C, UUO and UUO + H. Magnification 40x.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
UUO is an experimental model widely used to study the pathogenesis of tubulointerstitial fibrosis because it is highly reproducible and induces similar pathogenetic events to those observed in human renal fibrosis [5,6,24]. In agreement with previous studies [4,25], the results obtained in the present work showed that rats responded to UUO by increasing the synthesis of collagen, fibronectin, GAGs and CS/DSPGs, as well as TGF-ß. We could also demonstrate that daily treament with LMW-Hep for 14 days attenuates interstitial fibrosis in UUO kidneys, as indicated by a significant reduction of collagen and fibronectin. LMW-Hep also reduced the expression of TGF-ß in obstructed kidneys but, surprisingly resulted in an upregulation of total GAGS and CS/DSPGs, and in an increase of macrophage infiltration.

The protective effect of heparin and LMW-Hep in different experimental models of nephropathy has been reported previously [16,26–30]. The reduction of collagen and fibronectin deposition in the tubulointerstitial matrix of obstructed kidneys after LMW-Hep, reported in the present work, can be associated with the downregulation of TGF-ß, which is overproduced in obstructed kidneys and is responsible for the pathological accumulation of extracellular matrix components [31]. It has been reported that heparin inhibits TGF-ß synthesis in a puromycin glomerulosclerosis [17].

Surprisingly, we observed that LMW-Hep did not reduce the increased GAG and CS/DSPG deposition in obstructed kidneys. On the contrary, there was a significant increase in the content of GAGs, mainly CS and DS, and CS/DSPGs. This could be explained by the increased infiltration of CS/DS-containing inflammatory cells that was observed in obstructed kidneys after LMW-Hep administration. However, LMW-Hep also induced an increase in the GAGs in the kidneys of control rats, which was not accompanied by an increase in cellular infiltration. Overall, these results suggest that LMW-Hep has different effects on extracellular matrix components, probably by modulating the activity of different growth factors.

Although the presence of CS/DS GAGs [25] and CS/DSPGs of the small leucine-rich proteoglycan (SLRP) family has been reported previously in the kidneys [4,32], in the present study, HS was the only GAG biochemically detected in the kidneys of control rats. This apparent discrepancy can be explained by the different sensitivities of the methods used to detect the GAGs. In the study of Koshiishi et al. [25], the authors used a methodology in which GAGs were detected by disaccharide analysis after degradation of tissue slices with CS/DS-specific lyases (chondroitinase ABC and ACII). Despite the higher sensitivity of this method, when compared with that used in our work, it would not detect HS.

UUO induced an increase in the synthesis of CS and DS, as observed by biochemical analysis of the papain-extracted glycans. Similar results were obtained recently in rats after five-sixths mass ablation of the kidney [33]. In this study, HS was also the main GAG detected in control kidneys, with trace amounts of CS which, in addition to DS, increased in the kidney after ablation. It is well known that CS/DS-containing proteoglycan members of the SLRP family, mainly decorin and biglycan, are upregulated in UUO [4]. This was indirectly observed in the present work, by immunohistochemistry, using an anti-DDi-4S monoclonal antibody, which recognizes specific CS/DS epitopes left on protein cores of CS/DSPGs after exhaustive treatment with Chase ABC. We assume that the increase in the immunostaining observed in UUO kidneys represents an increase in the amount of CS/DS-containing core proteins. This increase of CS/DSPGs could be related to a protective role for these proteoglycans that directly or indirectly modulate the activity of TGF-ß, which also increased in UUO kidneys. For example, the absence of decorin induces greater tubular damage due to enhancing apoptosis, an increase in the expression of collagen and migration of macrophages [4]. Overall, the results of the present work, showing an increase in CS and DS after UUO are in agreement with previously reported studies using experimental renal damage.

The recruitment of leukocytes from blood and lymphatic systems into tissues facilitates the response to tissue injury. The adhesion molecule members of the selectin family (E, P and L) mediate the initial events that direct the movement of leukocytes across the endothelium in inflamed tissues, by interacting with sialylated, fucosylated carbohydrate antigens related to sialyl Lewisx [Slex, Neu5Ac{alpha}2,3Galß1, 4(Fuc{alpha}1,3)GlcNAcß-] at the surface of cells [9–13]. Heparin and heparin-like oligosaccharides inhibit L- and P-selectin binding to Slex and have been shown to reduce dramatically leukocyte infiltration in thioglycollate-induced peritoneal inflammation in mice [14,15]. Previous studies in two different animal models of nephropathy reported the inhibition of macrophage recruitment by heparin and LMW-Hep [17,18]. In our work, we expected that the administration of LMW-Hep to rats after UUO would reduce the mobilization of leukocytes from blood by an inhibitory effect on the selectins. Surprisingly, we observed an increased number of macrophages in obstructed kidneys after LMW-Hep, indicating that, in this model, LMW-Hep did not prevent leukocyte recruitment. This effect could be due to activation of heparin-binding proteins such as amphoterin, expressed in circulating monocytes [34], or midkine, which has been shown to be expressed in tubular epithelial cells of the diseased kidney and to promote migration of neutrophils and macrophages [35,36]. In a previous study by Floege et al. [27], using the model of anti-Thy1.1 nephritis, heparin was able to decrease mesangial expansion but did not affect glomerular macrophage influx.

In conclusion, the results obtained in the present study showed that LMW-Hep diminishes fibrosis in obstructed kidneys by downregulating the synthesis of collagen, fibronectin and TGF-ß. The mechanisms underlying the overproduction of CS/DSPGs and the increase in cellular infiltration upon LMW-Hep administration remain to be elucidated.



   Acknowledgments
 
M.S.G.P was supported in part by the National Institutes of Health-Fogarty International Center (R03 TW05775), Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação Carlos Chagas Filho de Amparo ‘a Pesquisa do Estado do Rio de Janeiro.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 24. 6.05
Accepted in revised form: 22.12.05


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