Simvastatin attenuates renal inflammation, tubular transdifferentiation and interstitial fibrosis in rats with unilateral ureteral obstruction

José Mauro Vieira, Jr, Eduardo Mantovani, Leonardo Tavares Rodrigues, Humberto Dellê, Irene Lourdes Noronha, Clarice Kazue Fujihara and Roberto Zatz

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



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The pleiotropic actions of statins have been largely explored. These drugs have been tested in several models of progressive renal disease, most of them accompanied by hypertension. We sought to investigate more closely the effects of simvastatin on renal interstitial fibrosis due to unilateral ureteral obstruction (UUO).

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. RT–PCR 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 {alpha}-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. {alpha}-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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
HMG-CoA reductase inhibitors, a class of drugs known as statins and initially described as lipid-lowering drugs, have anti-inflammatory, antiproliferative and immunomodulatory actions in vitro and in vivo [1,2]. Their effects include the blockade of the transcription factor NF{kappa}-B signalling pathway and therefore statins prevent the expression of downstream inflammatory mediators [3]. Besides their actions on atherosclerosis and cardiovascular diseases [4], several beneficial effects of statins have been described in experimental models of renal disease, most of them accompanied by hypertension and vascular injury [5,6]. Recently a statin was used successfully to decrease renal interstitial fibrosis in a model related to cyclosporine nephrotoxicity [7]. However, it is well known that this model encompasses arteriolar injury and renal ischemia as well [7,8].

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 [9–11]. 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.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Male Munich-Wistar rats weighing ~250 g, obtained from a local colony at the University of São Paulo, were used in this study. Rats were maintained with free access to regular food and water, at 22±1°C under a 12/12 h light/dark cycle. All experimental procedures were conducted according to our institutional guidelines.

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 Duboscq–Brazil 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 2–3 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, {alpha}-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 {alpha}-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-{alpha}-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 {alpha}-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. {alpha}-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.

RT–PCR
Total RNA was extracted by guanidinium thiocyanate–phenol–chloroform 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 [Tris–HCl 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 Tris–HCl 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.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
There was no difference between groups concerning the gain of weight of rats. Body weight at the end of study was 260±2 g and 264±4 g in the VH and SIMV groups, respectively. Cholesterol serum levels did not differ between groups (74.1±3.9 in VH vs 72.9±3.5 mg/dl in SIMV). Contralateral (CL) kidneys were pooled since there was no morphological, cellular or molecular difference between contralateral kidneys of both VH- and SIMV-treated rats (data not shown).

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).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Semiquantitative analysis of (A) interstitial fibrosis (%IF) and quantification of (B) ED1 positive cells (macrophage) and (C) CD3 positive cells (lymphocyte) in the interstitial renal cortex. CL kidneys presented low interstitial expansion and showed rare inflammatory cells, whereas VH-treated rats subjected to UUO showed a striking interstitial fibrosis and a marked cell infiltration. SIMV significantly attenuated IF and inflammatory cell infiltration. *P<0.01 vs VH. #P<0.05 vs VH.

 


View larger version (150K):
[in this window]
[in a new window]
 
Fig. 2. Representative photomicrographs of renal morphology and immunostaining for macrophage (ED1) and lymphocyte (CD3) (A) obstructed kidney from VH-treated rat demonstrating interstitial fibrosis compared to (B) an obstructed kidney from SIMV-treated rat. Representative sections of (C) macrophage and (D) lymphocyte renal influx patterns from immunostaining in an obstructed kidney from a VH-treated rat. (E) Macrophages were seen disrupting tubular basal membrane (tubulitis) (arrows) and were identified even within tubular lumens (arrows, F) of obstructed kidneys. [Original magnification: (A–D), 200x; (E and F) 400x].

 
Inflammatory findings: MCP-1, macrophages and lymphocytes
CL kidneys displayed rare cells positive for either macrophages or lymphocytes antigen (2.7±0.3 and 3.3±0.4 cells/0.5 mm2, respectively) (Figure 1). Ureteral obstruction induced an inflammatory cell influx in renal cortex, which predominated in the interstitial compartment. Interestingly, we observed peritubular inflammatory cells disrupting tubular basal membrane, and macrophages were even seen invading tubular lumen (Figure 2E). SIMV treatment led to a significant attenuation on the macrophage (14.8±0.7 vs 21.5±0.8 cells/0.5 mm2, P<0.01) and lymphocyte (10.1±1.5 vs 16±1.5 cells/0.5 mm2, P<0.05) recruitment compared to non-treated animals (Figures 1 and 2). Analysis of MCP-1 mRNA expression in the kidneys showed a marked induction of this chemokine in the VH-treated animals compared with contralateral kidneys (64.3±3.1 vs 19.2±7.5 OD, P<0.05), which was significantly downnregulated with SIMV treatment (48.9±2.5 vs 64.3±3.1. OD, P<0.01) (Figure 3).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3. Representative gel and semiquantitative analysis of RT–PCR of MCP-1. MCP-1 mRNA is clearly overexpressed in obstructed kidneys of VH-treated rats, which is downregulated by SIMV treatment. b2-microglobulin was used as housekeeping gene to assure equal RNA content between samples. *P<0.01 vs VH.

 
Extracellular matrix compounds, TGF-ß and bFGF
We compared mRNA expression of some extracellular matrix compounds in the obstructed kidneys of VH- and SIMV-treated rats in order to confirm the results obtained with trichrome staining. The UUO model is notoriously characterized by a striking upregulation of EMC renal expression (Figure 4). Nevertheless, the expression of collagen I and fibronectin in the SIMV-treated rats was significantly downregulated compared to VH-treated animals (collagen I, 32.5±5.9 vs 53.1±1.9 OD; fibronectin 52±2.1 vs 66.9±5.5 OD, P<0.05 for both parameters). Collagen III expression, also increased in the UUO compared to CL, showed a trend toward decrease with SIMV but did not reach significance (Figure 4). The renal expression of TGF-ß mRNA was upregulated in the obstructed kidneys compared to CL (81.2±1.2 vs 3.8±0.5 OD, P<0.0001), although it was not changed with SIMV treatment with either 3 days (14.8±.0.8 vs 16.2±1.3 OD, P = NS) or 14 days of ureteral obstruction (77.1±1.7 vs 81.2±1.2 OD, P = NS) (Figure 4). TGF-ß is a cytokine described to be involved in the renal fibrosis of the UUO model, thus we analysed the renal expression of TGF-ß at the post-transcriptional level by western blot. We confirmed the upregulation of TGF-ß in the obstructed kidneys of VH-treated animals (13.10±2.9 vs 0.45±0.1 OD, P<0.001). However, at the protein level SIMV significantly decreased TGF-ß expression in the renal tissue (5.35±0.75 vs 13.10±2.9 OD, P<0.05) (Figure 5A). bFGF was also upregulated in the kidneys of UUO animals compared to CL kidneys (21.1±5.0 vs 3.2± 0.4 OD, P<0.01). SIMV treatment determined a trend toward downregulation of renal bFGF expression that, however, did not reach significant difference (11.2±2.7 vs 21.1±5.0 OD, P = 0.1) (Figure 5B).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4. mRNA renal expression for collagen I and III, fibronectin and TGF-ß. (A) Representative RT–PCR gels and (B) semiquantification of optical densities (OD). Collagen I and fibronectin expression in the obstructed kidneys was significantly decreased in SIMV-treated animals compared with VH-treated rats. TGF-ß renal mRNA expression, highly induced in the obstructed kidneys, did not change with SIMV treatment. ß2-microglobulin was used as a housekeeping gene to assure equal RNA content between samples. *P<0.05 vs VH.

 


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5. TGF-ß and bFGF renal expression. (A) Representative blot from western analysis of TGF-ß1. TGF-ß1 protein is markedly overexpressed in obstructed kidneys from VH-treated rats. SIMV treatment significantly decreases TGF-ß1 protein expression in the obstructed kidney. (B) bFGF mRNA renal expression. bFG mRNA is induced in the UUO model although SIMV treatment did not significantly decreased its expression. *P<0.01 vs VH; #P<0.05 vs VH.

 
Vimentin, {alpha}-SMA and PCNA
CL kidneys normally expressed vimentin in epithelial visceral cells and smooth muscle cells of arterioles. This basal constitutive expression was not influenced by UUO. In contrast, tubular cells, which were negative for vimentin in contralateral kidneys, showed a dramatic overexpression for this protein in obstructed kidneys of VH-treated rats. In the SIMV group, the upregulated vimentin expression in tubular cells significantly decreased (17.6±2.0 vs 29.4±2.5 positive tubules/200x field, P<0.005) (Figures 6 and 7). {alpha}-SMA, constitutively expressed in smooth muscle cells of renal arterioles, was rarely seen in the interstitium of contralateral non-obstructed kidneys (0.69±0.07% of interstitial cortical area), whereas ureteral obstruction caused a striking increase in the expression of {alpha}-SMA in the interstitium compartment. SIMV significantly reduced interstitial {alpha}-SMA expression at day 14 (8.0±0.76 vs 12.34±0.23% in the VH group, P<0.001) (Figures 6 and 7). CL kidneys demonstrated only scant proliferating tubular and interstitial cells. However, UUO induced a severe tubular and interstitial proliferative response that occurred as early as 3 days (tubular cells 5.2±0.2 in VH vs 2.5±0.6 cells/0.5 mm2 in CL; and interstitial cells 6.8±3.5 in VH vs 1.99±0.8 cells/0.5 mm2 in CL, P<0.05 for both parameters) and was maintained at day 14 (tubular cells 3.4±0.5 cells/0.5 mm2 in VH; interstitial cells 9.8±1.8 cells/0.5 mm2 in VH). Nevertheless, SIMV treatment did not significantly change proliferation at any timepoint (tubular cells 4.4±0.1 and 4.2±0.9 cells/0.5 mm2, at 3 and 14 days, respectively. Interstitial cells 5.8±0.5 and 6.1±1.5 cells/0.5 mm2, at 3 and 14 days, respectively, P = NS) (Figure 7).



View larger version (92K):
[in this window]
[in a new window]
 
Fig. 6. Representative photomicrographs of renal immunohistochemistry for {alpha}-SMA (A, B and C) and vimentin (C, D and E). (A) CL kidneys showed positive arteriolar {alpha}-SMA and a weak staining in the interstitium. UUO induced a marked {alpha}-SMA interstitial expression in the VH-treated rats (B), which was reduced with SIMV treatment (C) [original magnification: (A–C), 200x). (D) Vimentin expression normally occurred in the arterioles (arrow) and visceral epithelial cells (arrowhead) as seen in the CL kidney (100x). No tubular staining was found in CL kidneys. (E) Abnormal tubular expression was markedly induced with ureteral obstruction in VH-treated rats (400x), which was attenuated with SIMV. (F) Larger view of the undifferentiated tubular cells expressing vimentin in an obstructed kidney from a VH-treated rat (original magnification 1000x).

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7. Semiquantitative analysis of (A) {alpha}-SMA, (B) vimentin and (C and D) PCNA immunostaining. (A) {alpha}-SMA is greatly expressed in the obstructed kidney, and SIMV treatment significantly attenuates its interstitial expression. (B) There is no expression of vimentin in normal, non-obstructed tubular cells. UUO leads to a high expression of this marker of undifferentiated state. SIMV partially prevented this de novo expression. (C) Tubular proliferation (PCNA staining). UUO induced a marked tubular proliferation that occurred as early as day 3 after UUO. However, SIMV did not prevent tubular cell proliferation. (D) PCNA positive interstitial cells were progressively increased in the obstructed kidneys. SIMV groups showed a trend toward a decrease in proliferation of interstitial cells, although without reaching significance. #P<0.001 vs VH. *P<0.005 vs VH.

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Several features of HMG-CoA reductase inhibitors, better known as statins, have been revealed, most of them regarding anti-inflammatory actions [1,3,4]. Moreover, these effects, which appear to depend largely on the inhibition of the transcription factor NF-{kappa}B, are independent of their lipid-lowering properties [4,12,17]. Initially described in the cardiovascular scenario, these effects of statins have also been tested in renal diseases. Others have shown a protective effect of statins on renal injury associated with hypertension, either renin- or salt-dependent [5,6]. Moreover, statins showed beneficial biological effects in the Heymann passive nephritis, a model of nephrotic syndrome [12]. More recently, the anti-inflammatory effects of statins were shown to attenuate progressive renal disease in a model of diabetes, and the fibrosis associated with cyclosporine nephrotoxicity [7,18].

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{kappa}-B activation, and that SIMV treatment decreases NF-{kappa}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 [9–11]. 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 {alpha}-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.



   Acknowledgments
 
This work was supported in part by FAPESP, grant no. 01/02932-0.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Sparrow CP, Burton CA, Hernandez M et al. Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol 2001; 21: 115–121[Abstract/Free Full Text]
  2. Terada Y, Inoshita S, Nakashima O et al. Lovastatin inhibits mesangial cell proliferation via p27Kip1. J Am Soc Nephrol 1998; 9: 2235–2243[Abstract/Free Full Text]
  3. Dichtl W, Dulak J, Frick M et al. HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2003; 23:58–63[Abstract/Free Full Text]
  4. Farmer JA. Pleiotropic effects of statins. Curr Atheroscler Rep 2000; 2: 208–217[Medline]
  5. Wilson TW, Alonso-Galicia M, Roman RJ. Effects of lipid-lowering agents in the Dahl salt-sensitive rat. Hypertension 1998; 31[part 2]: 225–231
  6. Park JK, Muller DN, Mervaala EM et al. Cerivastatin prevents angiotensin II-induced renal injury independent of blood pressure- and cholesterol-lowering effects. Kidney Int 2000; 58: 1420–1430[CrossRef][ISI][Medline]
  7. Li C, Yang CW, Park JH et al. Pravastatin treatment attenuates interstitial inflammation and fibrosis in a rat model of chronic cyclosporine-induced nephropathy. Am J Physiol 2004; 286: F46–F57[ISI]
  8. Burdmann EA, Andoh TF, Nast CC et al. Prevention of experimental cyclosporin-induced interstitial fibrosis by losartan and enalapril. Am J Physiol 1995; 269: F491–F499[ISI][Medline]
  9. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 2002; 110: 341–350[Abstract/Free Full Text]
  10. Lan HY. Tubular epithelial-myofibroblast transdifferentiation mechanisms in proximal tubule cells. Curr Opin Nephrol Hypertens 2003; 12: 25–29[CrossRef][ISI][Medline]
  11. Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol 2004; 15: 1–12[Abstract/Free Full Text]
  12. Zoja C, Corna D, Rottoli D et al. Effect of combining ACE inhibitor and statin in severe experimental nephropathy. Kidney Int 2002; 61: 1635–1645[CrossRef][ISI][Medline]
  13. Jepsen FL, Mortensen PB. Interstitial fibrosis of the renal cortex in minimal change lesion and its correlation with renal function. A quantitative study. Virchows Arch A Pathol Pathol Anat 1979; 383: 265–270[CrossRef][Medline]
  14. Fujihara CK, Malheiros DM, Zatz R, Noronha ID. Mycophenolate mofetil attenuates renal injury in the rat remnant kidney. Kidney Int 1998; 54: 1510–1519[CrossRef][ISI][Medline]
  15. Fujihara CK, Malheiros DMAC, Noronha IL, De Nucci G, Zatz R. Mycophenolate mofetil reduces renal injury in the chronic nitric oxide synthase inhibition model. Hypertension 2001; 37: 170–175[Abstract/Free Full Text]
  16. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156–159[CrossRef][ISI][Medline]
  17. Massy ZA, Guijarro C. Statins: effects beyond cholesterol lowering. Nephrol Dial Transplant 2001; 16: 1738–1741[Free Full Text]
  18. Usui H, Shikata K, Matsuda M et al. HMG-CoA reductase inhibitor ameliorates diabetic nephropathy by its pleiotropic effects in rats. Nephrol Dial Transplant 2003; 18: 265–272[Abstract/Free Full Text]
  19. Klahr S, Morrissey J. Obstructive nephropathy and renal fibrosis. Am J Physiol 2002; 283: F861–F875[ISI]
  20. Eddy AA, Giachelli CM. Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria. Kidney Int 1995; 47: 1546–1557[ISI][Medline]
  21. Morigi M, Macconi D, Zoja C et al. Protein overload-induced NF-{kappa}B activation in proximal tubular cells requires H2O2 through a PKC-dependent pathway. J Am Soc Nephrol 2002; 13: 1179–1189[Abstract/Free Full Text]
  22. Wolf G, Jocks T, Zahner G, Panzer U, Stahl RA. Existence of a regulatory loop between MCP-1 and TGF-ß in glomerular immune injury. Am J Physiol 2002; 283: F1075–F1084[ISI]
  23. Yamamoto T, Eckes B, Krieg T. Effect of interleukin-10 on the gene expression of type I collagen, fibronectin, and decorin in human skin fibroblasts: differential regulation by transforming growth factor-ß and monocyte chemoattractant protein-1. Biochem Biophys Res Commun 2001; 281: 200–205[CrossRef][ISI][Medline]
  24. Shimizu A, Yamada K, Sachs DH, Colvin RB. Persistent rejection of peritubular cappilaries and tubules is associated with progressive interstitial fibrosis. Kidney Int 2002; 61: 1867–1879[CrossRef][ISI][Medline]
  25. Nankivell BJ, Borrows RJ, Fung CL, O'Connell PJ, Allen RD, Chapman JR. Natural history, risk factors, and impact of subclinical rejection in kidney transplantation. Transplantation 2004; 78: 242–249[ISI][Medline]
  26. Higgins DF, Lappin DW, Kieran NE et al. DNA oligonucleotide microarray technology identifies fisp-12 among other potential fibrogenic genes following murine unilateral ureteral obstruction (UUO): modulation during epithelial-mesenchymal transition. Kidney Int 2003; 64: 2079–2091[CrossRef][ISI][Medline]
Received for publication: 22.10.04
Accepted in revised form: 6. 4.05





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
20/8/1582    most recent
gfh859v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Mauro Vieira, J.
Articles by Zatz, R.
PubMed
PubMed Citation
Articles by Mauro Vieira, J., Jr
Articles by Zatz, R.