The effect of simvastatin and erythropoietin on renal fibrosis in rats with unilateral ureteral obstruction

Abstract

Prevention of fibrosis is a very important therapeutic strategy in the treatment of obstructive nephropathy (ON). The aim of this study is to show and compare the actions of Simvastatin (Simv) and Erythropoietin (Epo) in renal expression of nuclear factor kappa B (NFκB), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), platelet-derived growth factor B (PDGF-B), fibronectin and development of interstitial fibrosis in rats with unilateral ureteral obstruction (UUO). A total of 48 Sprague–Dawley rats were allocated to 4 groups of sham, Epo, Simv and control. Unilateral ureteral ligation was performed on all rats except the Sham group. For interstitial fibrosis Masson’s trichrome stain and for the expression of TGF-β, PDGF-B, bFGF, NFκB and fibronectin, immunohistochemical methods were used. In the Epo and Simv groups, expression of TGF-β and fibronectin and staining with Masson’s trichrome were less compared to the control group. In addition, fibronectin expression in the Epo group was less than the Simv group. Unlike the Simv group, NFκB and bFGF expression in the Epo group were less when compared to the control group. Consequently, it was seen that both Epo and Simv prevented fibrosis in ON. Epo was superior in this effect by suppressing the expressions of NFκB and bFGF more effectively than Simv. Based on this finding, Epo might be a better agent than Simv in the prevention of fibrosis in ON.

• Erythropoietin
• experimental
• fibrosis
• obstructive nephropathy
• simvastatin

Introduction

Obstructive nephropathy (ON) is one of the most important causes of chronic kidney disease in children and infants.1,2 In recent decades, studies have exhibited that urinary tract obstruction affects renal morphogenesis, maturation and renal growth during early development. In the long term, chronic tract obstruction leads to renal interstitial fibrosis in the obstructed kidney.3 In recent years, recovery of renal morphology following the relief of unilateral ureteral obstruction (UUO) has been examined in neonatal rats. Interestingly, it has been demonstrated that progressive tubulointerstitial and glomerular damage persisted in the obstructed and contralateral kidney and a decrease in glomerular filtration rate (GFR), and an increase in proteinuria occurred at the end of 1 year after relief of UUO.4–7 Consequently, new therapy approaches are needed to prevent progression of renal injury along with surgical intervention. Therefore, concomitant treatment with an antifibrotic agent at the time of relief of UUO may prevent deterioration of renal function due to fibrosis.

Renal fibrosis, characterized as a progressive connective tissue deposition on the expanded tubulointerstitial field is a harmful process leading to renal function deterioration. Renal fibrosis pathophysiology has been investigated in many studies. As a result, nuclear factor kappa B (NFκB) or transforming growth factor-β (TGF-β) were found to be important treatment targets in renal fibrosis.8–10,11 Urinary tract obstruction leads to activation of the intrarenal renin–angiotensin system (RAS).12 Following activation of RAS, angiotensin II activates NFκB, which is a transcription factor that promotes the expression of proinflammatory genes. Many experimental studies have revealed that inhibition of NFκB activation decreases interstitial fibrosis in rats with UUO.13 Another important process in the renal fibrosis pathogenesis of ON is epithelial-to-mesenchymal transition (EMT).9,14 Epithelial-to-mesenchymal transition (EMT), in which tubular epithelial cell phenotype is lost and mesenchymal phenotype is acquired, is a reversible process in fibrogenesis. Transforming growth factor-β (TGF-β) is the major regulator for inducing EMT and it also regulates platelet-derived growth factor B (PDGF-B) and basic fibroblast growth factor (bFGF), which are other important growth factors in EMT induction. Inhibition of NFκB and TGF-β activation prevents apoptosis and interstitial fibrosis in UUO.9

In recent years, various agents have been studied to investigate their antifibrotic properties on kidneys in experimental models of UUO. However, no effective therapies to preclude renal fibrosis have been found as yet. The effects of Erythropoietin (Epo) and Simvastatin (Simv) on fibrosis in various experimental models have been previously studied. Those studies revealed that Epo, in addition to its hematopoietic effects, had a renoprotective effect in toxic renal injury induced by cisplatin and cyclosporine A, and ischemia-reperfusion renal injury.15–17 A study from Korea revealed that Epo also decreased renal fibrosis in mice with ureteral obstruction by inhibiting TGF-β-induced EMT.18 However, there are limited data as to whether Epo has an inhibitory effect on NFκB, which is a very important factor in fibrosis development. Similarly, many experimental studies have revealed that statins have a renoprotective effect in toxic renal injury caused by cyclosporine A and diabetic nephropathy by blocking the NFκB signaling pathway, janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and TGF-β-induced EMT.19–21

The aim of this study was to evaluate and compare the actions of Simv and Epo on NFκB, TGF-β, bFGF, PDGF-B and fibronectin expression and the development of interstitial fibrosis in rats with experimentally induced UUO.

Materials and methods

Experimental design

A total of 48 adult male Sprague–Dawley rats (weight: 200–250 g) were used in this study. The animals were supplied by Ondokuz Mayis University Experimental Surgical Research and Application Center. The experimental protocol was approved by the Ondokuz Mayis University Animal Ethics Committee (2007-28). The animals were kept in a 12:12 h light:dark cycle at 22 ± 2 °C and fed with standard rat chow and water ad libitum. The rats were divided into four groups as sham (n:12), Epo (n:12), Simv (n:12) and control (n:12). Under ketamine and xylazine anesthesia, the left ureter was ligated next to the ureteropelvic junction at two points and cut between the ligatures in all rats except the sham group through the ventral incision. In the sham group operation, the ureter was exposed and repositioned without further manipulation. After abdominal closure, the rats were allowed to recover in a heated environment and were returned to their cages. Group Epo was given 1000 U/kg epoetin alpha (Santa-Farma, Turkey) intraperitoneally every other day, Group Simv was given 2 mg/kg Simv, which was dissolved in a 1% solution of carboxymethyl cellulose (Sigma, St. Louis, MO), by gavage twice a day, Group control was given carboxymethyl cellulose 1 mL/kg by gavage twice a day. All rats were sacrificed and the left kidneys were harvested at 14 days after surgery.

Morphology

For histological studies, the kidneys were placed in saline to clear blood-borne cells and then fixed in neutral formaldehyde (10%) for 24 h. The kidneys were incised on the coronal plane and embedded in paraffin after standard tissue protocol. The 4-µm-thick sections were prepared and stained by hematoxylin & eosin (H&E) and Masson’s trichrome for the examination of tissue injury and collagen deposition, respectively.

Immunohistochemistry

The kidney sections were deparaffinized in graded alcohol and immersed in methanol containing 3% hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity. After rinsing in phosphate-buffered saline (PBS), sections were treated with sodium citrate to facilitate antigen retrieval in 60 min for TGF-β, 15 min for proteinase K, 30 min for NFκB, 60 min for bFGF, 35 min for PDGF-B and 30 min for fibronectin. After that, sections were incubated with blocking solution for 30 min. Sections were incubated with primary antibodies at room temperature. Monoclonal mouse antibody against TGF-β (1:50 dilution, 90 min) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal rabbit antibody against NFκB (1:200 dilution, 60 min) (Gene Tex), monoclonal mouse antibody against bFGF (1:25 dilution, 180 min) (Gene Tex, Irvine, CA), monoclonal rabbit antibody against PDGF-B (ready for use, 15 min) (Thermo Scientific), and polyclonal rabbit antibody against fibronectin (1:50 dilution, 90 min) (Gene Tex) were used as primary antibodies. After rinsing, the slides were incubated with biotinylated link anti-mouse and anti-rabbit secondary antibodies (Gene Tex) for 30 min and then with streptavidin-horseradish peroxidase (Gene Tex) for 20 min. The slides were then developed with diaminobenzidine for 10 min as a chromogen. All sections were finally counterstained with Harris’ hematoxylin, and mounted. The sections without primary antibody were used as negative controls.

Examination of TGF-β, NFκB, PDGF-B and bFGF

The stained slides were assessed by a pathologist who was blind to the groups. Ten consecutive non-overlapping fields in the inner cortex and corticomedullary regions were evaluated for each slide. Immunohistochemical staining was semi-quantitatively evaluated according to the extent and intensity of staining and then combined scores were calculated. Staining extent and intensity were quantified with point counting methodologies.22 A light microscope with a 40×-field objective was used (Olympus Bx51, Olympus Optical Company, Tokyo, Japan) and each field was carried over to screen of computer, which was fixed on a graticule with 32 equidistant intersecting lines, by Olympus DP 71 camera (Tokyo, Japan).

The extent of staining for each grid field was assessed as the percentage of stained cells area on a 0–4 scale (0: no expression; 1: 1–25%, 2: 26–50%, 3: 51–75%, 4: 76–100). Then the total percentage was calculated according to the formula below:

Staining intensity was scored on a 0–3 scale; 0 = no specific staining, 1 = weakly positive (mild cytoplasmic staining and no nuclear staining), 2 = nuclear moderately positive (moderate cytoplasmic staining and present or absent nuclear staining), 3 = strongly positive (severe cytoplasmic and nuclear staining). Each intensity score value was multiplied by their number of grid intersections and then added. After that, the result was divided by the total number of grid intersections.

Examination of fibronectin and Masson’s trichrome

Fibronectin and Masson’s trichrome staining were evaluated by staining extent (% positive staining area). Ten consecutive non-overlapping fields were photographed using an Olympus DP71 camera, which was fixed on an OlympusB51 microscope (×40 objective), in the inner cortex and corticomedullary regions for each slide. These digital images were transferred to Image J program (NIH Image). The positive stained area (%) was calculated after all tubules, glomerulus and vessels were marked and subtracted.

Statistical analysis

Data were given as the median number (min–max). To compare groups, Kruskal–Wallis analysis of variance was used and p values <0.05 were considered significant. The Mann–Whitney U test with Bonferroni correction was used to evaluate differences between two groups and p values <0.01 were considered significant. Data were analyzed using the SPSS 15.0 software.

Results

Morphological assessment

Nonobstructed kidneys from Group sham did not show any morphological alteration on H&E. The kidneys of rats exposed to ureteral obstruction showed obvious tubulointerstitial injury consisting of tubular dilatation and atrophy and interstitial expansion. However, glomeruli were well preserved (Figure 1). As shown by Figure 1, Masson’s trichrome staining revealed collagen deposition in the interstitium. Similarly, immunohistochemical staining revealed fibronectin deposition in the tubules and interstitium. However, immunohistochemical staining of TGF-β, NFκB, PDGF-B and bFGF revealed staining in the tubules as shown in Figure 1.

Figure 1. Representative photomicrographs (×40 magnifications) of histochemical staining for Masson’s trichrome demonstrating collagen accumulation in the obstructed rat kidneys. Immunostaining for TGF-β, NFκB, PDGF-B, bFGF and fibronectin are seen in tubular cytoplasms, in addition NFκB staining seen in tubular nuclei and fibronectin staining also seen in interstitium. All staining is the densest in group control and faint in group sham.

TGF-β, NFκB, PDGF-B, bFGF, fibronectin and Masson’s trichrome staining assessment

All immunohistochemical staining and Masson’s trichrome staining were significantly lower in Group sham than in the other groups (p < 0.01). Transforming growth factor-β expression was found to be significantly lower in Group Epo and Group Simv than Group control (p < 0.01), whereas, no significant difference was found between Group Simv and Group Epo (Table 1 and Figure 2). No significant difference was found in NFκB and bFGF expressions either between Group Simv and Group control or between Group Simv and Group Epo. However, NFκB and bFGF expressions were found to be significantly lower in Group Epo than in Group control (Table 1; Figures 3 and 4). Fibronectin and Masson’s trichrome percentage of staining area were found to be significantly lower in Group Simv and Group Epo than in Group control (Table 1; Figures 5 and 6). In comparison of Group Epo and Group Simv, there was no difference in the percentage of Masson’s trichrome staining, whereas the percentage of fibronectin staining was found to be lower in Group Epo than in Group control (p < 0.01) (Table 1; Figures 5 and 6). In PDGF-B expression, there was no significant difference between Groups Simv, Epo and control (Table 1 and Figure 7).

Figure 2. The effect of Epo and Simv on TGF-β Expression. *p < 0.01 group sham versus group Epo, group Simv and group control, **p < 0.01 group control versus group Epo and group Simv.

Figure 3. The effect of Epo and Simv on NFκB expression. *p < 0.01 group sham versus group Epo, group Simv and group control, **p  <  0.01 group control versus group Epo.

Figure 4. The effect of Epo and Simv on expression of PDGF-B. *p <  0.01 group sham versus group Epo, group Simv and group control.

Figure 5. The effect of Epo and Simv on expression of bFGF. *p <  0.01 group sham versus group Epo, group Simv and group control, **p  <  0.01 group control versus group Epo.

Figure 6. The effect of Epo and Simv on expression of fibronectin. *p <  0.01 group sham versus group Epo, group Simv and group control. **p <  0.01 group control versus group Epo and group Simv. ***p <  0.01 group Epo versus group Simv.

Figure 7. The effect of Epo and Simv on Masson’s trichrome staining. *p <  0.01 group sham versus group Epo, group Simv and group control, **p <  0.01 group control versus group Epo and group Simv.

Table 1. Combined score of TGF-β, NFκB, PDGF B, bFGF and positive staining area % of fibronectin and Masson’s trichrome.

	Group Sham median (min–max)	Group Epo median (min–max)	Group Simv median (min–max)	Group control median (min–max)	p
TGF-β	3.00 (2.00–3.00)	5.00 (4.00–7.00)	5.00 (4.00–6.00)	7.00 (6.00–8.00)	<0.01*
NFκB	3.00 (3.00–5.00)	4.00 (3.00–6.00)	5.00 (4.00–8.00)	6.00 (5.00–6.00)	<0.01‡
PDGF-B	2.50 (2.00–3.00)	6.00 (4.00–8.00)	6.00 (4.00–6.00)	6.00 (4.00–6.00)	<0.01†
bFGF	2.00 (2.00–3.00)	5.00 (3.00–6.00)	6.00 (4.00–6.00)	6.00 (4.00–7.00)	<0.01§
Fibronectin	5.35 (1.10–9.50)	11.30 (6.20–25.70)	20.25 (8.70–35.40)	29.85 (16.30–83.40)	<0.01¥
Masson’s trichrome	4.40 (2.20–8.40)	19.75 (10.70–35.70)	24.40 (15.80–38.10)	42.05 (21.40–62.80)	<0.01£

Notes: *Group sham versus group Epo, group Simv and group control, *group control versus group Epo and group Simv.

‡Group sham versus group Epo, group Simv and group control, ‡group control versus group Epo.

†Group sham versus group Epo, group Simv and group control.

§Group sham versus group Epo, group Simv and group control, §group control versus group Epo.

¥Group sham versus group Epo, group Simv and group control, ¥group control versus group Epo and group Simv, ¥ group Epo versus group Simv.

£Group sham versus group Epo, group Simv and group control, £group control versus group Epo and group Simv.

Discussion

According to recent studies, one of the key strategies for the treatment of ON should be the prevention and reversal of interstitial fibrosis as well as surgical relief of the obstruction.4–6 Therefore, many agents have been investigated for their antifibrotic effect on ON. Statins and Epo are two of these agents. In this study, the effects of Simv and Epo on fibrogenesis in ON were compared. To the best of our knowledge this is the first study comparing the effects of Simv and Epo on fibrogenesis in ON. In addition, the effect of Simv on NFκB and PDGF-B expression and the effect of Epo on NFκB, PDGF-B and bFGF expression in ON were studied for the first time in this study.

Nuclear factor kappa B, which is one of the most important transcription factors involved in many cellular processes, is activated in innate immune response.23 This system is activated by the factors causing cellular stress such as growth factors, vasoactive agents, cytokines, bacterial lipopolysaccharides, microbial and viral pathogens and oxidative stress. NFκB, when activated, increases inflammation and fibrosis by regulating the transcription of genes encoding many proteins involved in inflammatory response.24

Recent studies have revealed that a decrease in NFκB activity by AT1 inhibition led to attenuation of fibrosis in rats with UUO.25,26 Although the effect of Simv on NFκB expression has been previously studied in cerebral ischemia and atherosclerosis, it has not been studied in ON.27,28 Vieira et al.29 reported that Simv attenuated interstitial fibrosis due to UUO by decreasing the level of TGF-β, MCP-1, vimentin and α-SMA. On the other hand, in recent studies it has been indicated that Epo decreased NFκB expression in ischemia-reperfusion injury, traumatic brain injury and peritoneal fibrosis.30–32 However, the effect of Epo on NFκB activity in ON has not been studied before. In this study, no significant difference was found between Group Simv and Group control in NFκB expression whereas NFκB expression was observed to be significantly lower in Group Epo than in Group control. Therefore, it was concluded that Epo was more effective in inhibiting NFκB expression than Simv in ON.

The TGF-β signal pathway is another important pathway in fibrogenesis in ON. It has been reported that in rats with UUO, inhibition of the TGF-β signal pathway prevented fibrosis.33 In this study, a significant decrease in TGF-β expression was also shown in both Group Simv and Group Epo. In a similar study, Vieira et al.29 reported that Simv did not alter the level of TGF-β mRNA compared to controls while it significantly decreased the level of TGF-β at posttranscriptional level. Accordingly, Simv affects the phase of TGF-β protein production from mRNA. Park et al.18 showed that Epo prevented fibrosis by inhibiting TGF-β expression and EMT in ON. On the other hand, Epo and its non-erythropoietic derivative, carbamylated-epo, were investigated for the purpose of renoprotective effect on interstitial fibrosis in ON. According to that study, Epo did not significantly alter TGF-β level while its derivative inhibited TGF-β expression.34 The results of the current study, identical with Park’s study, showed that Epo inhibited TGF-β expression in ON.

The formation of interstitial fibrosis and tubular atrophy are the most important consequences and prognostic indicators in ON, as with all chronic kidney disease.35 In the tubulointerstitial area, the accumulation of fibronectin and blue stained areas by Masson’s trichrome are indicators of irreversible fibrosis. In the current study, fibronectin expression and Masson’s trichrome staining were shown to be lower in both the Epo and Simv treated rats than in the controls. This implies a significant antifibrotic effect for both of these agents in ON. In addition, fibronectin expression was less in Group Epo than in Group Simv. Therefore, it was concluded that Epo was more effective than Simv in preventing fibrosis. More effective inhibition of NFκB and bFGF expression by Epo might have led to this difference.

It can be concluded from the results of this study that, both Epo and Simv decreased fibrosis in rats with UUO. They reduced EMT by inhibiting TGF-β expression effectively and Epo also significantly reduced expressions of NFκB and bFGF. Epo seems to be superior to Simv in preventing fibrosis in rats with UUO, because of more effective inhibition of NFκB and bFGF. Therefore, it was concluded that Epo is a more effective agent than Simv in the prevention of fibrosis in ON. Further studies evaluating the effect of these drugs on fibrosis are needed.

Declaration of interest

The authors report no conflict of interest.

Acknowledgements

The present study was supported by Ondokuz Mayis University Research Fund (Samsun, Turkey) (Grant No: T.559).