1 Nagoya University Daiko Medical Center, 1-1-20, Daiko-minami, Higashi-ku, Nagoya, 2 Kureha Chemical Industry, Tokyo, and 3 Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
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Abstract |
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Methods. Two weeks after 5/6-nephrectomy, 10 rats were divided into pairs such that both rats in each pair exhibited almost the same levels of serum creatinine, blood urea nitrogen and creatinine clearance. One rat from each pair was assigned to a control uraemic group, the other to a uraemic group which received AST-120 everyday for 11 weeks. The localization of indoxyl sulphate was studied by immunohistochemistry using a monoclonal anti-indoxyl sulphate antibody we had developed. The renal cortical gene expression was studied by using northern blotting.
Results. Rats treated with AST-120 showed decreased levels of serum creatinine, blood urea nitrogen and urinary protein as well as increased levels of creatinine clearance as compared with control uraemic rats. AST-120 markedly decreased indoxyl sulphate levels in both serum and urine. Immunohistochemistry demonstrated that indoxyl sulphate was localized in the renal proximal tubular epithelial cells, especially of dilated tubules, and that AST-120 markedly reduced the tubular staining of indoxyl sulphate. AST-120 attenuated interstitial fibrosis, tubular injury as well as glomerular sclerosis, and reduced the renal gene expression of TGF-ß1, TIMP-1 and pro-1(I)collagen.
Conclusions. AST-120 reduces the gene expression of TGF-ß1, TIMP-1 and pro-1(I)collagen in the kidneys, and delays the progression of CRF, at least in part, by alleviating the overload of indoxyl sulphate on remnant proximal tubular epithelial cells.
Keywords: chronic renal failure; indoxyl sulphate; oral sorbent; progression; TGF-ß
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Introduction |
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We have demonstrated that indoxyl sulphate is one of the circulating uraemic toxins accelerating the progression of CRF [710]. Indoxyl sulphate is derived from dietary protein. A part of protein-derived tryptophan is metabolized into indole by tryptophanase in intestinal bacteria such as Escherichia coli. Indole is absorbed into the blood from the intestine, and is metabolized to indoxyl sulphate in the liver. Indoxyl sulphate is normally excreted into urine. In uraemia, however, reduced renal clearance of indoxyl sulphate leads to elevated serum levels of indoxyl sulphate. In fact, the serum levels of indoxyl sulphate are markedly increased in both uraemic rats and patients [7,1113]. AST-120 reduces the serum and urine levels of indoxyl sulphate in the uraemic rats and patients by adsorbing indole in the intestines, and consequently stimulating its excretion into faeces [3,7,11,12]. The administration of indoxyl sulphate to 5/6-nephrectomized rats promoted the progression of CRF [7,8] accompanied by increased gene expression of transforming growth factor (TGF)-ß1, tissue inhibitor of metalloproteinase (TIMP)-1 and pro-1(I)collagen [9,10]. These findings support the notion that indoxyl sulphate is one of the uraemic toxins stimulating the progression of CRF by increasing the renal expression of these fibrosis-related genes.
The aims of the present study are to determine the effects of AST-120 on the localization of indoxyl sulphate in uraemic rat kidneys, and to examine whether the administration of AST-120 reduces the renal cortical gene expression of TGF-ß1, TIMP-1, and pro-1(I)collagen, and ameliorates glomerular and tubulointerstitial injuries in uraemic rats.
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Subjects and Methods |
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The levels of serum creatinine, serum and urine indoxyl sulphate, BUN, creatinine clearance, and urine protein were determined just before, and 2, 6 and 10 weeks after starting the administration of AST-120. The remnant kidneys were removed after perfusion at the end of experiment for histopathological and gene expression studies.
Analytical procedures
BUN and creatinine levels were measured using a Beckman BUN analyser (Fullerton, CA, USA) and a Beckman creatinine analyser (model 2), respectively. Serum levels of indoxyl sulphate were measured using high-performance liquid chromatography [13].
Histopathological analysis
Paraffin-embedded sections were stained with periodic acid-Schiff (PAS) and Masson's Trichrome. Morphological analyses were performed with an observer blind to the animal experiment. Glomerular sclerosis was assessed by a standard semiquantitative analysis with glomerular sclerosis index (GSI) by Raij et al. [14]. Fifty glomeruli per rat in PAS-stained sections were graded as 0, 1, 2, 3 or 4, according to normal, less than 25, 2550, 5075, or over 75% cross-sectional sclerosis, respectively. The sclerosis index for each rat was calculated from the formula (N1x1+N2x2+N3x3+N4x4)/n, where N1, N2, N3, and N4 represent the numbers of glomeruli exhibiting grades 1, 2, 3, and 4, respectively, and n is the number of glomeruli assessed (n=50) [2].
The extent of tubulointerstitial injury was estimated using a standard point counting method [15]. Under high power magnification (x300) light microscopy, 10 random and non-overlapping fields per each rat were photographed from each Masson's Trichrome-stained section of renal cortex. A grid containing 121 (11x11) sampling points was superimposed on each photograph, and a total of 1210 points were evaluated in each rat. Points falling on (i) interstitial volume (interstitial fibrosis), (ii) interstitial non-fibrotic area (peritubular capillaries), (iii) normal tubular cells, (iv) dilated tubular cells, (v) atrophic tubular cells, or (vi) tubular lumen were counted, and their percentages were calculated. The renal interstitial volume was determined according to the method by Ishidoya et al. [16]. The Masson's Trichrome method stains collagen fibres in the tubular basement membrane (TBM), glomeruli, and interstitial space. Points overlying TBM and interstitial space were counted as interstitial volume. Points falling on large vessels, glomeruli or Bowman's capsules were excluded from the total count.
Preparation of monoclonal anti-indoxyl sulphate antibody
An epoxy group was introduced into indoxyl sulphate to prepare 1-(2',3'-epoxypropyl)indoxyl sulphate, and a sulphhydryl (SH) group was introduced into bovine serum albumin (BSA) and human transferrin. 1-(2',3'-epoxypropyl)indoxyl sulphate was reacted with either BSASH or transferrinSH to make indoxyl sulphateBSA or indoxyl sulphatetransferrin. Ten-week-old female BALB/c mice were given intramuscular injection of indoxyl sulphateBSA (0.3 mg) with Titer MaxTM (CytRx, Norcross, GA, USA). The equivalent amount of indoxyl sulphateBSA was injected twice at 23 week intervals. Finally, indoxyl sulphateBSA (1.5 mg) was intravenously administered to the mice. Fusion of the splenic cells with myeloma cells and their culture were performed according to the methods of Köhler and Milstein.
Hybridoma cells secreting anti-indoxyl sulphate antibodies were screened by enzyme-linked immunosorbent assay (ELISA) using goat anti-transferrin IgG (ICN Pharmaceuticals, Aurora, OH, USA). Supernatant isolated from culture (0.1 ml) was incubated at room temperature for 1 h on a microplate attached with indoxyl sulphatetransferrin. After washing with phosphate-buffered saline (PBS) containing 0.05% Tween 20, 0.1 ml of peroxidase-labelled goat F(ab')2 fragment to mouse IgG(Fc) was added into the microplate, followed by incubation at room temperature for 1 h. After washing with PBS containing 0.05% Tween 20, 0.2 ml of o-phenylenediamine hydrochloride (1 mg/ml) containing 0.0124% H2O2 was added to the microplate, and then incubated at room temperature for 30 min. The reaction was terminated with 1.3 M H2SO4. The absorption at 492 nm was measured.
Characterization of monoclonal anti-indoxyl sulphate antibody
The reactivity of the produced monoclonal antibodies with indoxyl sulphate, indole, tryptophan, indoxyl phosphate, indoleacetic acid, 5-hydroxy-indoleacetic acid, sulphalinic acid or benzenesulphonic acid was measured by using a competitive ELISA. The sample solution (40 µl) was incubated with the monoclonal anti-indoxyl sulphate antibody (40 µl) at room temperature for 1 h in an indoxyl sulphatetransferrin attached microplate. After washing with PBS containing 0.05% Tween 20, 0.1 ml of peroxidase-labelled goat F(ab')2 fragment to mouse IgG(Fc) was added into the microplate, followed by incubation at room temperature for 1 h. After washing with PBS containing 0.05% Tween 20, 0.2 ml of o-phenylenediamine hydrochloride (1 mg/ml) containing 0.0124% H2O2 was added to the microplate, and then incubated at room temperature for 30 min. The reaction was terminated with 1.3 M H2SO4. The absorption at 492 nm was measured. Figure 1 shows the reactivity of the monoclonal anti-indoxyl sulphate antibody with indolic compounds and sulphate compounds, demonstrating that the antibody specifically recognizes only indoxyl sulphate, but not the other indolic or sulphate compounds.
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Immunohistochemical analysis
Immunostaining of indoxyl sulphate in renal tissue sections was performed using the streptavidinbiotinylated peroxidase complex (SABC) method. The tissue specimens were divided into thin sections (1-µm thick) that were then deparaffinized. The sections were washed three times with distilled water for 5 min. The sections were treated with Protease K (1 : 20000; Merck, Darmstadt, Germany) in distilled water at 37°C for 15 min, and washed three times with PBS for 10 min. Endogenous peroxidase activity was blocked by incubating the sections with 0.3% H2O2 in methanol for 20 min at room temperature. The sections were washed three times with PBS for 5 min. The sections were incubated with 10% rabbit serum at 37°C for 60 min to reduce the non-specific background staining, and washed three times with PBS for 5 min. Then, the sections were incubated with a monoclonal anti-indoxyl sulphate antibody (7 µg/ml) dissolved in PBS containing 3% BSA and 0.1% NaN3 at 4°C overnight, and washed three times with PBS for 10 min; followed by incubation with a biotinylated rabbit antibody against mouse IgG+IgA+IgM (10 µg/ml) (Nichirei, Tokyo, Japan) at 37°C for 40 min. The sections were washed three times with PBS for 5 min, and then incubated with peroxidase-labelled streptavidin (Nichirei, Tokyo, Japan) at 37°C for 30 min. After washing three times with PBS for 10 min, the reaction was completed by the addition of diaminobenzidineH2O2 solution for 15 min, and washed three times with distilled water for 5 min, then the slides were counter-stained with methylgreen.
The immunostaining of indoxyl sulphate was quantified using an image analyser V10LAB (Toyo Boseki, Tokyo, Japan) by evaluating the positively stained area of the sections under the same light intensity for microscopy. The intensity of colour component for red, green or blue was graded from 0 to 256°. Areas which showed intense brown colour were extracted from the microscopic fields (number of fields for each tissue sample, six fields; magnification on the display: x300) under the following conditions; red component ranging from 104 to 158°, green component from 81 to 129°, and blue component from 70 to 123°.
The primary anti-indoxyl sulphate antibody (1 : 100) was incubated with indoxyl sulphate (10 mg/ml) at 4°C overnight. After centrifuging the mixture at 10 000 g for 30 min, the supernatant was used as the primary antibody solution followed by the usual SABC method. There was no positive staining in the renal cortex when the primary antibody was pre-incubated with indoxyl sulphate.
There was also no positive immunostaining in the renal cortex when the primary anti-indoxyl sulphate antibody was omitted from the protocol.
Northern blot analysis
Total RNA was extracted from the renal cortex by the method described by Chomczynski and Sacchi [18]. The amount of mRNA for TGF-ß1, TIMP-1 and pro-1(I)collagen was determined using Northern blot analysis as follows. Total RNA (12 µg) was denatured in 0.01 M phosphate buffer (pH 7.0) with 1 M glyoxal, and fractionated by electrophoresis through 0.8% agarose gel. RNA was then transferred onto Gene Screen Plus membrane (New England Nuclear, Boston, MA, USA). The membrane was prehybridized in a solution containing 5xSSPE (900 mM NaCl, 5 mM EDTA, 50 mM sodium phosphate, pH 8.3), 5xDenhardt's solution (0.1% Ficoll (400 000 mol.wt), 0.1% polyvinylpyrrolidone (360 000 mol.wt), 0.1% BSA), 1.0% sodium dodecyl sulphate, 0.1 mg/ml of herring sperm DNA (Boehringer Mannheim, Mannheim, Germany), 50% formamide. cDNA probes were labelled with [32P]dCTP (New England Nuclear, Boston, MA, USA) using random primed DNA labelling kit (Boehringer Mannheim, Mannheim, Germany). The amount of the mRNA was determined after analysing the radioactive bands using a Fujix Bioimaging analyser (Bas 2000) (Fuji Photo Film Co., Tokyo, Japan). For autoradiography, the membranes were exposed to Kodak X-AR film at -20°C. Equal loading and integrity of RNA were monitored by assessing human glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), which is generally expressed at a similar level by all viable cells.
cDNA probes
A cDNA clone Hf677 [19] for pro-1(I)collagen was kindly provided by Dr ML Chu (University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA). A cDNA fragment for human GAPDH was prepared using the polymerase chain reaction (PCR) as described previously [20]. cDNA fragments for rat TGF-ß1 and rat TIMP-1 were prepared using PCR, as described previously [9].
Statistical analysis
Results are expressed as mean±SD. To compare values among three groups, ANOVA was applied, and Fisher's least significance difference (LSD) test was used. Results were considered statistically significant when the P value was <0.05.
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Results |
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Effects of AST-120 administration on histopathology of renal cortex
Both glomerular and tubulointerstitial injuries were noted in both control uraemic rats and AST-120- administered uraemic rats as compared with normal rats (Fig. 2AC
). To determine the effects of AST-120 on the glomerular injury, the extent of glomerular sclerosis was assessed by quantitative analysis using glomerular sclerosis index. As shown in Figure 3
, the administration of AST-120 significantly attenuated the development of glomerular sclerosis in uraemic rats.
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Effects of AST-120 administration on localization of indoxyl sulphate in renal cortex
Immunohistochemical analysis was performed to determine the localization of indoxyl sulphate in the renal cortex (Fig. 2DF
). In normal rats, only tubular epithelial cells were weakly stained by the monoclonal anti-indoxyl sulphate antibody, while glomeruli were hardly stained. In control uraemic rats, however, proximal tubular epithelial cells, especially of dilated tubules, were intensively stained by the anti-indoxyl sulphate antibody. In the AST-120-treated uraemic rats, the staining of indoxyl sulphate in tubular epithelial cells was less prominent as compared with that in the control uraemic rats. As shown in Figure 5
, the control uraemic rats showed increased indoxyl sulphate-positive (intensively stained) area in the renal cortex, whereas AST-120-treated rats showed markedly decreased indoxyl sulphate-positive area as compared to the control uraemic rats. These data demonstrate that AST-120 markedly reduces the overload of indoxyl sulphate on the remnant tubular epithelial cells.
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Effects of AST-120 administration on mRNA levels of TGF-ß1, TIMP-1, and pro-1(I)collagen in renal cortex
The effects of AST-120 on the gene expression of TGF-ß1, TIMP-1, and pro-1(I)collagen in the renal cortex were examined. The renal mRNA levels of TGF-ß1, TIMP-1, and pro-
1(I)collagen were markedly increased in control uraemic rats as compared with normal rats. However, the rats treated with AST-120 showed significantly reduced mRNA levels of TGF-ß1, TIMP-1, and pro-
1(I)collagen as compared with the control uraemic rats (Fig. 6A
and B
). In the uraemic rats (n=10), TGF-ß1 mRNA level was strongly correlated with TIMP-1 (r=0.88, P<0.001) and pro-
1(I)collagen (r=0.93, P<0.0001). The variation in the mRNA levels of TGF-ß1, TIMP-1 and pro-
1(I)collagen in both AST-120-treated and control uraemic rats are related to variation in the extent of CRF.
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Discussion |
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AST-120 has been considered to exert its effects on CRF progression by removing uraemic toxins or their precursors in the digestive tract. However, the target uraemic toxins of AST-120 had not been well understood, until we have demonstrated that indoxyl sulphate is a circulating uraemic toxin stimulating glomerular sclerosis and interstitial fibrosis [710], and that AST-120 decreases the serum and urine levels of indoxyl sulphate by adsorbing its precursor, indole, in the intestine [3,7,11,12]. The present study also confirmed that the administration of AST-120 to uraemic rats reduces indoxyl sulphate levels in both serum and urine, and delays the progression of CRF. The histological analysis at the end of the study revealed that AST-120 ameliorates tubulointerstitial injury as well as glomerular sclerosis. Recent studies have revealed that primary or secondary tubulointerstitial injury is of equal or greater importance than glomerular sclerosis in determining whether progressive renal dysfunction will ensue in various renal diseases [2326]. Thus, AST-120 delays the progression of CRF by ameliorating not only glomerular sclerosis but also tubulointerstitial injury.
We have recently reported that the administration of indoxyl sulphate increases TGF-ß1 mRNA levels in renal cortex of uraemic rats [9,10]. TGF-ß1 stimulates the accumulation of extracellular matrix (ECM) by increasing the production of ECM proteins such as type I collagen, and inhibits its degradation by increasing production of proteinase inhibitors such as TIMP-1 in mesangial and tubulointerstitial cells. Thus, TGF-ß1 plays a central role in the development of glomerular sclerosis and tubulointerstitial fibrosis [27]. The administration of AST-120 significantly reduced TGF-ß1 mRNA levels in the renal cortex of uraemic rats. Since indoxyl sulphate is secreted from proximal tubular cells, it is likely that indoxyl sulphate exerts its toxic effects on proximal tubular cells. In fact, the addition of indoxyl sulphate increased the synthesis of TGF-ß1 in cultured pig proximal tubular cells (LLCPK-1) [10]. Although the activity of TGF-ß1 was not measured in the present study, the mRNA expression of TIMP-1 and pro-1(I)collagen which parallelled the activity of TGF-ß1, was also diminished in the AST-120- administered uraemic rats. These findings suggest that the administration of AST-120 reduces both production and activity of TGF-ß1 in the renal cortex of the uraemic rats.
AST-120 adsorbs some other uraemic toxins such as p-cresol and phenol [12,28]. However, nephrotoxicity of p-cresol and phenol is less probable, because their serum and urine levels are very low as compared to indoxyl sulphate in uraemic patients. Further, the administration of p-cresol did not accelerate the progression of CRF in 5/6-nephrectomized rats, even at the same dose as that of indoxyl sulphate (unpublished observation). Although AST-120 adsorbs some amino acids such as tryptophan in vitro [12], no decrease in the serum concentration of the amino acids was observed in AST-120-administered uraemic rats [29].
Taken together with our previous data and the present results, it is likely that AST-120-induced reduction of renal TGF-ß1 activity is mediated, at least partly, by reducing the overload of indoxyl sulphate on remnant tubular epithelial cells. Most studies in humans have also suggested that a low-protein diet is beneficial in retarding the progression of CRF [3033]. In rats, protein restriction was demonstrated to slow the progression of renal disease by reducing renal TGF-ß1 expression [3436]. Since a low-protein diet as well as the administration of AST-120 decreases the serum and urine levels of indoxyl sulphate [7,37], the inhibitory effect of protein restriction on progression of CRF may be mediated, at least partly, by the reduction of indoxyl sulphate levels.
In conclusion, the targets of nephrotoxicity of indoxyl sulphate are proximal tubular epithelial cells, especially of dilated tubules. AST-120 reduces the renal gene expression of TGF-ß1, TIMP-1, and pro-1(I) collagen, and ameliorates the progression of CRF, at least partly, by reducing the overload of indoxyl sulphate levels on the remnant tubular epithelial cells. These results support the protein metabolite hypothesis proposed by Niwa et al. [6,38] that endogenous protein metabolites such as indoxyl sulphate play an important role in the progression of CRF. However, we consider that indoxyl sulphate is not the sole stimulating factor for the progression of CRF, but a representative of the endogenous protein metabolites stimulating CRF progression.
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Notes |
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References |
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