Internal Medicine, Renal Division, University of São Paulo, São Paulo, Brazil
Correspondence and offprint requests to: José Mauro Vieira Jr, Internal Medicine, Renal Division, University of São Paulo, São Paulo, Brazil. Email: josemvjr{at}usp.br
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Abstract |
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Methods. Munich-Wistar rats were submitted to UUO and studied after 14 days. Animals were divided into two groups: vehicle (VH) or simvastatin (SIMV) 2 mg/kg b.i.d. by gavage. At sacrifice kidneys were harvested for morphology, mRNA and protein analysis. RTPCR was done to assess expression of collagen I and III, fibronectin, MCP-1, TGF-ß1 and bFGF. Protein expression was assessed by western blot (TGF-ß) and immunostaining (macrophage, lymphocyte, PCNA, vimentin and -smooth muscle actin). Contralateral kidneys (CL) were used as controls.
Results. SIMV-treated animals had less severe renal inflammation. MCP-1 was markedly expressed in obstructed kidneys and diminished with SIMV (48.9± 2.5 vs 64.3±3.1 OD in VH, P<0.01). Interstitial fibrosis (IF) was significantly attenuated with SIMV (8.2±1.3 vs 13.2±0.6%, P<0.01 SIMV vs VH), which was confirmed by a decrease in collagen I and fibronectin renal expression. Vimentin, a marker of dedifferentiation, was expressed in tubular cells of VH and decreased with SIMV treatment. -SMA, a marker of myofibroblast-type cells, was increased in renal interstitium of VH rats and SIMV significantly reduced its expression. PCNA was increased in the UUO kidneys, but SIMV did not decrease tubular or interstitial proliferating cells. TGF-ß1, which was highly induced in the obstructed kidneys, decreased at the post-transcriptional level with SIMV treatment (5.35±0.75 vs 13.10±2.9 OD in VH, P<0.05). bFGF mRNA was also overexpressed in the obstructed kidneys, although SIMV treatment did not significantly decrease its expression.
Conclusions. SIMV had an evident protective effect on renal interstitial inflammation and fibrosis. It is conceivable that by attenuating inflammation, SIMV prevented tubular activation and transdifferentiation, two processes largely involved in the renal fibrosis of the UUO model.
Keywords: inflammation; interstitial fibrosis; MCP-1; simvastatin; transdifferentiation; unilateral ureteral obstruction
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Introduction |
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In this study we sought to determine the effects of simvastatin in a model of pure tubulointerstitial renal injury, the one related to unilateral ureteral obstruction (UUO). In order to explain the mechanisms of action of simvastatin (SIMV) we studied the expression of monocyte chemoattractant protein-1 (MCP-1), one of the most important mediators of the inflammatory process within the kidney, and the resulting renal infiltration by inflammatory cells.
Furthermore, it has recently been demonstrated that progressive renal disease is accompanied by epithelial-myofibroblast transdifferentiation (EMT), a pathophysiological mechanism that helps to explain renal fibrosis [911]. It appears that in several models of renal disease, tubular cells are activated and undergo dedifferentiation, followed by migration and acquisition of a phenotype of myofibroblast. EMT could account for the large number of myofibroblasts in renal progressive diseases, particularly in the UUO model [11]. We investigated the effects of SIMV on surrogate markers of EMT, and the impact of this treatment on the expression of profibrogenic cytoquines TGF-ß and bFGF, two possible mediators of EMT and the resulting renal fibrosis.
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Subjects and methods |
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Experimental design
Rats were randomly assigned to receive either vehicle (VH, n = 7), or SIMV (n = 7), for 2 days prior to surgical procedure. Then the UUO was performed, after anaesthesia with 50 mg/kg i.p. sodium pentobarbital injection, through a ventral laparotomy. The left ureter and kidneys were exposed and the ureter was then ligated next to the uretero-pelvic junction. After abdominal closure rats were allowed to recover and returned to cages to follow VH or SIMV treatment for 14 days after obstruction. The right contralateral kidneys of rats from both groups were harvested to function as controls (CL). SIMV 2 mg/kg (Merck, Rahway, NJ, USA) was administered twice a day by gavage and was dissolved in a 1% solution of carboxymethyl cellulose (Sigma, St Louis, MA, USA). VH-treated rats received carboxymethyl cellulose 1 ml/kg p.o. twice a day. This dosage and method of SIMV administration were chosen according to previous reports showing that SIMV at this dosage did not alter blood lipid levels [12].
Morphology
At the end of the study (day 14), rats were anaesthetized with 50 mg/kg i.p. sodium pentobarbital injection. The abdominal wall was sectioned and the left kidney was then prepared for analysis by either of two methods. (i) For paraffin embedding, half of the left kidney, obtained through a midcoronal section, was fixed with DuboscqBrazil solution for 30 min, followed by a postfixation in buffered 10% formaldehyde solution. The material was then embedded in paraffin for assessment of renal cortical interstitial injury and for immunohistochemical studies. (ii) The other half of left kidneys were snap-frozen in liquid nitrogen, and stored at 80°C for further mRNA and protein analysis (see below).
Paraffin-embedded renal tissue was dewaxed using standard sequential techniques, and 23 µm-thick sections were stained with Masson's trichrome technique. The fraction of renal cortex occupied by interstitial tissue (IF) was quantified in trichrome-stained sections by a point-counting technique as described previously [13,14]. Briefly, the percentage of the renal cortical area occupied by interstitial tissue (%INT) was estimated in Masson-stained sections by a point counting technique in 25 consecutive microscopic fields, at a final magnification of 100x, under a 176-point grid. Morphological measurement was performed blindly by a single observer. Before sacrifice, blood was drawn to measure cholesterol serum levels.
Immunohistochemistry
The inflammatory infiltrate was analysed by assessing the number of macrophages and lymphocytes in the cortex of the renal tissue. In addition, the expression of vimentin, a marker of dedifferentiated mesenchymal cells, -SMA, a protein expressed in fibroblast-like cells, and proliferating cell nuclear antigen (PCNA) were also investigated. The inflammatory cells and the expression of vimentin, PCNA and
-SMA were identified by immunohistochemistry using standard techniques as described previously [14,15]. Briefly, 4 µm-thick sections obtained from paraffin-embedded tissue were dewaxed and mounted using conventional techniques. Sections were then subjected to microwave irradiation in citrate buffer to enhance antigen retrieval and preincubated with 5% normal rabbit or horse serum (Vector Labs, Burlingame, CA, USA) in Tris-buffered saline (TBS). The renal tissue was then incubated with 1:200 monoclonal anti-ED1 antibody (Serotec, Oxford, UK) for macrophage detection, 1:100 anti-CD3 (Harlan Seralab, Oxford, UK) for lymphocyte, 1:200 anti-vimentin (Santa Cruz Biotechnology, CA, USA), 1:800 anti-
-SMA (Dako, Copenhagen, Denmark), and 1:100 anti-rat PCNA (Dako). The incubation of primary antibodies was carried out overnight at 4°C in a humidified chamber. Sections were then incubated with appropriate species-specific secundary antibodies for 45 min at room temperature. To complete the detection of antigens, we used the complex of alkaline phosphatase anti-alkaline phosphatase method (Dako) for ED1 or the avidin-biotinylated horseradish peroxidase technique (Dako) for CD3, vimentin, PCNA and
-SMA [14,15]. Negative control experiments were routinely performed by (i) omitting the incubation with the primary antibody, and (ii) replacing the primary antibody with unspecific rat IgG.
The quantification of ED1, CD3 positive cells and PCNA was carried out in a blinded fashion under 200x magnification and expressed as cells/0.5 mm2. Vimentin expression was quantified by counting positive tubules (at least one positive cell/tubule profile) per 200x magnification field. -SMA expression in the interstitium was assessed by a point-counting technique as for the trichome quantification. For each section at least 30 consecutive renal cortical 200x fields were examined.
RTPCR
Total RNA was extracted by guanidinium thiocyanatephenolchloroform method [16]. The cDNA was synthesized from 2 µg of total RNA using an oligo dT primer (Promega, San Luis, USA) and the MMLV-RT enzyme (Promega). PCR was carried out to amplify the following specific cDNAs: collagen I (primers: sense 5'-CTT CGT GTA AAC TCC CTC C-3' and antisense 5'-CAC TGG TTT TTG GTT TTC AC-3', product of 224 bp), collagen III (primers: sense 5'-GCG GCT TTT CAC CAT ATT AG-3' and antisense 5'-GCA TGT TTC TCC GGT TTC-3', product of 266 bp), fibronectin (primers: sense 5'-TTA TGA CGA CGG GAA GAC CTA-3' and antisense 5'-GGC TGG ATG GAA AGA TTA CTC-3', product of 295 bp), transforming growth factor-ß1 (TGF-ß1) (primers: sense 5'-GGA CTA CTA CGC CAA AGA AG-3' and antisense 5'-TCA AAA GAC AGC CAC TCA GG-3', product of 293 bp), basic fibroblast growth factor (bFGF) (primers: sense 5'-AAG CAG AAG AGA GAG GAG TTG T-3' and antisense 5'-TTA GCA GAC ATT GGA AGA AAC-3', product of 270 bp), MCP-1 (primers: sense 5'-TAT GCA GGT CTC TGT CAC GC-3' and antisense 5'-AAG TGT TGA ACC AGG ATT CAC A-3', product of 570 bp) and the housekeeping gene ß2-microglobulin (primers: sense 5'-GAG TGA CGT GTT TAA CTC TGC AAG C-3' and antisense 5'-CTC CCC AAA TTC AAG TGT ACT CTC G-3', product of 249 bp).
PCR was performed mixing 2 µl of RT reaction product (cDNA), 10 µl of 10x buffer [TrisHCl 20 mM pH = 8.0, potassium chloride 100 mM, EDTA 0.1 mM, DTT 1 mM, 50% glycerol, 0.5% Tween-20 and 0.5% Nonidet-P40], 2 µl of 10 mM dNTP mix, 10 µM of each primer and 5 U of Taq DNA polymerase (Promega) and water up to 100 µl. Magnesium chloride concentration was adjusted for each pair of primers, and cDNAs were amplified according to the following conditions: 94°C for 50 s, 55°62°C (the annealing temperature varied for each pair of primers) for 60 s, and 72°C for 2 min. The number of cycles was predetermined for each pair of primers in order to avoid the PCR plateau phase. The PCR products were analysed in 2% agarose gel along with a 100 bp DNA ladder, and the bands were were semiquantified using a software (ImageMaster version 2.0; Pharmacia Biotech, Buckinghamshire, UK). Negative controls were routinely done by omitting the primers in the PCR. All PCRs resulted in the amplification of a single product of the predicted size. The results are expressed as optical density (OD), and the ß2-microglobulin expression was used as a control of the reaction to assure equal cDNA among samples.
Western blot
Kidney sections stored at 75°C were homogenized in lysis buffer (10 mM HEPES pH 7.6, 25 mM potassium chloride, 3 mM magnesium chloride, 5 mM EDTA, 10% glycerol, 1 mM DTT, 0.1% SDS and 1:100 protease inhibitor cocktail; Sigma) and centrifuged at 10 000 g. The supernatant was recovered and the protein concentration determined by the Bradford method (Bio-Rad, California, USA).
Thirty micrograms of total protein was loaded in a stacking polyacrylamide gel and then resolved on 12% polyacrylamide gel. The gel was stained with Coomassie blue to assess the equal loading of protein. Then samples were wet-transferred to a 0.2 µ nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, USA). Thereafter blots were blocked for 1 h with 5% nonfat dry milk in TBST buffer (20 mM TrisHCl pH 7.6, 0.8% NaCl and 0.05% Tween-20) and incubated overnight at room temperature with a 1:500 mouse anti-TGF-ß1 primary antibody (Genzyme, Cambridge, USA). After washing in TBST, the blots were incubated with secondary anti-mouse IgG-horseradish peroxidase conjugate antibody at 1:5 000 (Santa Cruz Biotechnology, CA, USA) for 1 h. Blots were then washed again in TBST and bands detected by enhanced chemiluminescence (Amersham Bioscience, Buckinghamshire, UK). ODs for quantification were obtained using a software (ImageMaster version 2.0; Pharmacia Biotech, Buckinghamshire, UK). A prestained-protein marker (New England BioLabs Inc., Beverly, USA) was used as molecular weight standard.
Statistical analysis
Data are reported as mean±SEM. Comparisons between groups were done by one-way analysis of variance (ANOVA) followed by the Bonferroni's multiple comparison post-test. The level of statistical significance was P<0.05.
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Results |
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Renal morphology
Nonobstructed contralateral kidneys from rats of both groups did not show any morphological alteration. The kidneys of rats subjected to ureteral obstruction developed a conspicuous tubulointerstitial injury consisting of tubular dilatation and atrophy, interstitial inflammation and a marked interstitial fibrosis. Glomeruli and vessels were well preserved. Analysis of interstitium by the trichrome staining showed that SIMV significantly attenuated interstitial fibrosis (8.2±1.3 vs 13.2±0.6%, P<0.01 SIMV vs VH) (Figures 1 and 2).
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Discussion |
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In this study, we investigated the effects of SIMV on a model of renal fibrosis that is independent of hypertension, vascular injury, renal insufficiency or the systemic oxidative stress that accompanies diabetes. To that end, we chose the UUO model, which is characterized by intense renal inflammation and striking fibrosis limited to the tubulointerstitial compartment [19]. We clearly demonstrated that SIMV treatment attenuated the inflammation and the resulting interstitial fibrosis in the obstructed kidneys.
MCP-1 is a protein largely produced by tubular cells in response to renal injury. Eddy and Giachelli [20] have shown that proteinuria very early elicits a proximal tubular activation and MCP-1 secretion, which could account for the interstitial inflammation that follows glomerular proteinuria. Furthermore, Morigi et al. [21] demonstrated that proximal tubule cells subjected in vitro to albumin respond with NF-B activation, and that SIMV treatment decreases NF-
B-dependent MCP-1 production [12]. In our study we demonstrate that in the UUO model there is an upregulation of the MCP-1 renal expression that is independent of proteinuria. SIMV not only clearly decreased MCP-1 mRNA levels in the kidney but also led to less severe renal interstitial inflammation, as demonstrated by a decrease in the lymphomononuclear cells in that compartment.
Recently, Wolf et al. [22] have described a regulatory loop between MCP-1 and TGF-ß in renal injury independent of macrophage recruitment. Here, the relationship between the decrease in the inflammatory cells and the resulting protection in the interstitial fibrosis afforded by SIMV could also be explained by downregulation of the profibrotic cytokine TGF-ß. Although the abundance of TGF-ß mRNA in the injured kidney was not modified with statin in this model, the expression of some extracellular matrix compounds was clearly downregulated and the regulation of TGF-ß renal expression clearly occurred at the translational level. Alternatively, it has recently been demonstrated in vitro that MCP-1 has the ability to induce fibroblast to increase production of collagen through a mechanism independent of TGF-ß [23]. Thus, it is conceivable that the MCP-1 expression could itself directly upregulate ECM compounds production by fibroblasts or activate tubular cells to transdifferentiate into fibroblast-like cells.
EMT is a process that has been described in progressive scarring renal diseases [911]. In the present study we show that SIMV interfered with the expression of surrogate markers of transdifferentiation of tubular cells, suggesting that SIMV might, either through its antinflammatory or a direct effect, decrease EMT. The overexpression of interstitial -SMA that we found in the obstructed kidneys can be partly explained by migration of dedifferentiated tubular cells through the basal membrane into the interstitium. The expression of vimentin that we demonstrated in obstructed tubular cells corresponds with an undifferentiated, mesenchymal state. This undifferentiated state might precede and allow the proliferation and migration of tubular cells to take place. Nevertheless, further in vitro studies are necessary to confirm the role of statin in preventing tubular EMT. It has been demonstrated that UUO is a model of renal disease characterized by increased proliferative activity of tubular cells [19]. We confirmed this feature in this model. However, we did not demonstrate a significant anti-proliferative action of SIMV, which argues against a direct effect of SIMV on either proliferation or apoptosis.
In contrast, SIMV treatment diminished both renal interstitial inflammation and markers of EMT. Therefore, we hypothesize that part of the beneficial effects of statin could be accounted for by a decrease in renal interstitial inflammation and the resulting tubular activation. We observed that the inflammation that follows UUO is accompanied by features of tubulitis, which might conceivably contribute to tubular activation. In experimental renal transplantation, tubulitis is a finding associated with the renal fibrosis that characterizes chronic rejection [24], and tubulitis that is found in human subclinical rejection correlates with chronic interstitial fibrosis and long-term renal dysfunction [25].
It has been demonstrated that EMT appears to be mediated by upregulation of some cytokines such as TGF-ß, bFGF and connective tissue growth factor (CTGF) [10,11,26]. We confirmed an overexpression of TGF-ß at least at the post-transcriptional level in the UUO kidneys, which was attenuated by SIMV, whereas the bFGF message was also upregulated in the obstructed VH-treated animals although not significantly decreased with SIMV. We did not discount that other cytokines such as CTGF would possibly be more relevant to transdifferentiation and renal fibrosis of the UUO model [26].
Further studies are necessary to clarify the exact role of statins on renal fibrosis and EMT, but it is possible that statins have either direct effects on the extracellular matrix production/turnover or indirect effects through their anti-inflammatory actions. The interference with renal EMT process could be an alternative explanation for the anti-fibrotic effects of this class of drugs with a marked anti-inflammatory activity.
In conclusion, in this model of renal interstitial fibrosis that is independent of vascular injury or hypertension, SIMV significantly attenuated interstitial inflammation and fibrosis. These anti-inflammatory effects are greatly explained by the downregulation in MCP-1, and the decrease in interstitial fibrosis appears to result from less severe tubular phenotypical changes, myofibroblast expression and renal TGF-ß downregulation. Beyond its role in hyperlipidaemia and atherosclerosis that accompanies several renal diseases, statins might prove to be an effective adjunctive treatment in progressive scarring renal disease.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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