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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 27  |  Issue : 1  |  Page : 197-204

Effect of atorvastatin and erythropoietin on renal fibrosis induced by partial unilateral ureteral obstruction in rats


1 Department of Clinical Pharmacology, Faculty of Medicine, Menoufiya University, Menufia, Egypt
2 Department of Urosurgery, Faculty of Medicine, Menoufiya University, Menufia, Egypt
3 Department of Medical Biochemistry, Faculty of Medicine, Menoufiya University, Menufia, Egypt
4 Department of Pathology, Faculty of Medicine, Menoufiya University, Menufia, Egypt

Date of Submission22-Apr-2013
Date of Acceptance22-Jul-2013
Date of Web Publication20-May-2014

Correspondence Address:
El Sayed M Abd El Salam
Department of Clinical Pharmacology, Faculty of Medicine, Menoufiya University, Shebein Elkom, Menufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.132804

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  Abstract 

Objective
This work aims at investigating the effects of atorvastatin and erythropoietin (EPO) on tubulointerstitial fibrosis induced by partial unilateral ureteral obstruction (PUUO) in rats.
Background
Renal fibrosis is the final manifestation of chronic kidney disease. It is one of the biggest problems in nephrology, indicating that patients inevitably reach end-stage renal disease. Atorvastatin has anti-inflammatory, antiproliferative, and immunomodulatory potential. EPO has been demonstrated to play an important cytoprotective role.
Methods
Fifty adult albino rats were divided into five equal groups: group 1 received saline intraperitoneally and methylcellulose orally for 14 days and served as the negative control; group 2 (sham-operated group) rats underwent the same surgical procedures as group 3 without ureteral obstruction; group 3 rats underwent PUUO without any medication; group 4 rats underwent PUUO and received atorvastatin at a dose of 50 mg/kg orally; group 5 rats underwent PUUO and received EPO at a dose of 3.000 IU/kg daily intraperitoneally. After 14 days, systolic blood pressure was measured. Blood and urine samples were also collected for measurement of serum transforming growth factor-β1, tumor necrosis factor-͍, urea and creatinine, and urinary protein and albumin levels. Renal tissue samples were collected for determination of malondialdehyde and ͍-smooth muscle actin levels, as well as for histopathological and immuonohistochemical examination.
Results
Our results showed that systolic blood pressure; serum levels of transforming growth factor-β1, tumor necrosis factor-α, and malondialdehyde; and biochemical parameters were significantly elevated. Histopathological examination of kidney tissue revealed marked degenerative changes and increased expression of α-smooth muscle actin in group 3 compared with groups 1 and 2. In contrast, biochemical, histological, and immuonohistochemical features in groups 4 and 5 showed significant improvement compared with group 3.
Conclusion
It is concluded that both atorvastatin and EPO have renoprotective effects against renal fibrosis, shown by improvement in kidney functions and using fibrosis markers.

Keywords: Atorvastatin, erythropoietin, renal fibrosis, unilateral ureteral obstruction


How to cite this article:
Abd El Salam EM, El Odemi MH, Omar AH, El-Serafy FA, Abd El Hakim TF, Samaka RM, El-Kady NM. Effect of atorvastatin and erythropoietin on renal fibrosis induced by partial unilateral ureteral obstruction in rats. Menoufia Med J 2014;27:197-204

How to cite this URL:
Abd El Salam EM, El Odemi MH, Omar AH, El-Serafy FA, Abd El Hakim TF, Samaka RM, El-Kady NM. Effect of atorvastatin and erythropoietin on renal fibrosis induced by partial unilateral ureteral obstruction in rats. Menoufia Med J [serial online] 2014 [cited 2020 Feb 17];27:197-204. Available from: http://www.mmj.eg.net/text.asp?2014/27/1/197/132804


  Introduction Top


Chronic urinary tract obstruction is an important cause of chronic kidney disease and renal failure in all age groups. The decrease in renal function caused by chronic obstruction is associated with structural derangements in the kidney, characterized by tubulointerstitial fibrosis [1]. Permanent renal function loss is accompanied by histological alterations, including tubular dilatation, tubular cell apoptosis, and progressive interstitial compartment fibrosis [2].

Interstitial fibrosis is a complex process involving inflammatory cell infiltration, fibroblast proliferation, excessive extracellular matrix accumulation, and decreased matrix degradations. Shortly after obstruction, inflammatory cells appear in the interstitial space, releasing cytokines and growth factors to stimulate the fibrotic process [3].

In the unilateral ureteral obstruction (UUO) model many readily quantifiable cellular and molecular events occur during the initiation and progression of renal injury, such as apoptosis, inflammation, and fibrosis [2]. Therefore, the UUO model is an appropriate experimental model to study renal disease progression.

Some studies showed that atorvastatin (one member of statins) provided renal protection in experimental models of urinary tract obstruction [4]. Statins have anti-inflammatory and antioxidant potential and are capable of attenuating fibrogenesis [5]. Agents with antioxidant action provide renal protection in cases of partial obstruction. These properties of statins are independent of their cholesterol-lowering effect and are termed as pleiotropic effects [6],[7].

Erythropoietin (EPO) was originally identified as a critical hormone promoting erythrocyte survival and differentiation. In addition to its hematopoietic effect, EPO has multiple paracrine and autocrine functions that coordinate local responses to injury by attenuating primary (apoptosis) and secondary (inflammatory) causes of cell death [8].

EPO attenuates experimental ischemic or inflammatory brain injury and ischemic or infarcted heart injury by inhibiting apoptosis, restoring vascular autoregulation, attenuating the inflammatory response, and augmenting cellular regeneration [9].

As EPO receptors are expressed in the kidney, especially in renal peritubular and mesangial cells [9], the systemic administration of EPO may also protect against renal damage. Indeed, EPO has been found to ameliorate the toxic renal injury caused by cisplatin and ischemia reperfusion renal injury [10].

This work aims at clarification of the effect of atorvastatin and EPO on renal injury induced by partial UUO (PUUO) in rats.


  Materials and methods Top


Fifty adult albino rats of local strains, weighing 150-200 g each, were used. They were divided into five main equal groups as follows:

Group 1 (G1): rats received saline intraperitoneally and methylcellulose orally for 14 days and served as the negative control group.

Group 2 (sham-operated group; G2): rats underwent the same surgical procedure as that in group 3, including removal of the right kidney (right nephrectomy), but the left ureter was not obstructed, and they received saline intraperitoneally for 14 days.

Group 3 (G3): rats underwent right nephrectomy and PUUO of the left ureter, and they received saline intraperitoneally for 14 days.

Group 4 (G4): rats underwent right nephrectomy and PUUO of the left ureter and received atorvastatin at a dose of 50 mg/kg dissolved in methylcellulose solution orally daily for 14 days [11].

Group 5: rats underwent right nephrectomy and PUUO of the left ureter and received EPO at a dose of 3.000 IU/kg daily intraperitoneally for 14 days [12].

Rats were anaesthetized with ketamine (50-100 mg/kg, intraperitoneally). The rats were placed in a supine position. The abdominal skin was cleaned and shaved and laparotomy was performed. For induction of right nephrectomy, the right kidney was exteriorized, decapsulated, mobilized, and the hilum was clamped. This kidney was immediately excised. The renal blood vessels and ureter to this kidney were ligated [13]. For induction of PUUO, the left ureter was ligated alongside a 0.5-mm diameter stainless steel wire with a nylon thread, and the stainless wire was withdrawn [14]. The incision was sutured and each rat was caged individually and allowed to recover.

Systolic blood pressure of the conscious animals was measured at the end of the study using a rat tail blood pressure monitor system (LE5001 System; Harvard Apparatus Inc.) [15]. The average of four to six readings was taken as the value for systolic blood pressure.

Rats were anaesthetized with diethyl ether. Venous blood samples were collected in heparinized capillary tubes from the retro-orbital plexus of rats for biochemical assay of serum urea [16] and creatinine [17] levels. Serum transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-͍ (TNF-͍) levels were determined using ELISA kits (e Bioscience Inc., USA).

Twenty-four-hour urine samples were collected from all rats to determine urine volume/24 h. Fresh urine samples were collected to determine urinary creatinine levels and also for the determination of protein and albumin concentrations.

Creatinine clearance was estimated using the following formula [18]:

Creatinine clearance = U × V/P,

where U is the creatinine concentration in urine; V is the volume of urine/min; and P is the creatinine concentration in plasma.

Urine protein concentrations were measured on a spectrophotometer using commercially available kits, and 24-h urine protein excretion was calculated thereafter and expressed as mg/24 h [19].

Urine albumin concentrations were measured using immunoassay kits on an i-CHROMA Reader (Boditech Med Inc., Korea), and 24-h urine albumin excretion was calculated thereafter and expressed as mg/24 h [20].

After the experimental period, the rats were killed and the left kidney was removed rapidly and cut into two equal halves using a scalpel. One half of the kidney was fixed in 10% neutral buffered formalin for histopathological study, and the other half was rapidly washed in ice-cold saline (0.9%) and homogenized in chilled phosphate buffer (0.1 mol/l, pH 7.4) using a glass tissue homogenizer for determination of malondialdehyde level in renal tissue [21].

Histopathological examination

The dissected kidney from each rat was received at the Pathology Department, Faculty of Medicine, Menoufiya University, preserved in 10% formalin solution, and dehydrated in a graded alcohol series. After xylene treatment, the specimens were embedded in paraffin blocks. Five-micrometer-thick sections were cut and stained with hematoxylin and eosin (H&E) for histopathological study and Masson's trichrome (MT) stain for detection of collagen fiber.

Immuonohistochemical examination of smooth muscle actin

Five-micrometer-thick sections were cut from the paraffin-embedded blocks with subsequent steps of deparaffinization and dehydration in xylene and a graded series of alcohol, respectively. Antigen retrieval was performed by boiling the sections in 10 mmol/l citrate buffer (pH 6.0) for 20 min, followed by cooling at room temperature. The slides were incubated overnight at room temperature with smooth muscle actin (͍-SMA) Clone 1A4 (mouse monoclonal antibody; Dako, Glostrup, Denmark) at a dilution of 1 : 50. The Envision1 (Dako) method was used for detection of ͍-SMA binding. The reaction was visualized using an appropriate substrate/chromogen (diaminobenzidine) reagent with Mayer's hematoxylin as a counter stain.

Statistical analysis

Data were collected, tabulated, and statistically analyzed using the SPSS program for windows (version 13; SPSS Inc., Chicago, Illinois, USA). Results were expressed as mean ± SE. Statistical evaluation was carried out using one-way analysis of variance (ANOVA). All P-values were two-sided. P-values less than 0.05 were considered statistically significant and those less than 0.01 were considered highly significant.


  Results Top


Systolic blood pressure

A statistically significant difference in systolic blood pressure (SBP) was observed between G1 and G3 (P < 0.05). G3 and G4 also showed a statistically significant difference in SBP (P < 0.05; [Table 1]).
Table 1: Effect of atorvastatin and erythropoietin on SBP and serum TGF-β 1, serum TNF-α , and renal MDA levels after 14 days of PUUO in rats

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Serum transforming growth factor-β1 and tumor necrosis factor-͍

As regards serum levels of both TGF-β1 and TNF-͍, G3 showed a significant increase in comparison with G1 (P < 0.05). However, serum TGF-β1 levels in G4 were significantly decreased compared with G3 (P < 0.05). In addition, serum TNF-͍ levels in G4 and G5 showed a significant decrease compared with G3 (P < 0.05; [Table 1]).

Malondialdehyde level in kidney tissue

A statistically significant increase in the level of malondialdehyde was observed in G3 compared with G1 (P < 0.05). In addition, the level of malondialdehyde in G4 and G5 showed a significant decrease compared with G3 (P < 0.05; [Table 1]).

Biochemical parameters

Serum urea, creatinine, urinary protein, and albumin levels in G3 were significantly increased compared with those in G1 (P < 0.05). However, creatinine clearance showed a significant decrease in G3 compared with G1 (P < 0.05; [Table 2]). Moreover, serum urea, creatinine, urinary protein, and albumin levels were significantly decrease in both G4 and G5 compared with their levels in G3 (P < 0.05). However, creatinine clearance showed a significant increase in both G4 and G5 compared with G3 (P < 0.05; [Table 2]).
Table 2: Effect of atorvastatin and erythropoietin on serum urea, creatinine, urinary protein, and albumin levels, as well as creatinine clearance, after 14 days of PUUO in rats

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Histopathological changes

Assessment of H&E sections of renal tissue from both the control group (G1) and the sham-operated group (G2) showed unremarkable architecture or cytological changes. MT-stained sections showed minimal collagen fibers in the connective tissue interstitium [Figure 1].
Figure 1:

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Sections of renal tissue from the PUUO group (G3) stained with H&E showed marked histological changes [Figure 2]a-c. MT-stained sections showed extensive tubulointerstitial fibrosis, as illustrated in [Figure 2]d.
Figure 2:

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Sections of renal tissue from both the atorvastatin-treated group (G4) and the EPO-treated group (G5), stained with both H&E and MT, showed mild improvement in renal architecture with significant reduction of tubular atrophy and interstitial fibrosis [Figure 3] and [Figure 4].
Figure 3:

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Figure 4:

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Expression of smooth muscle actin

Kidney sections from both the control group (G1) and the sham-operated group (G2) showed negligible ͍-SMA expression [Figure 5]. Expression of ͍-SMA was significantly increased in G3 compared with G1 [Figure 6]. The intensity and distribution of immunostaining was decreased in both G4 and G5 compared with G3 [Figure 7].
Figure 5:

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Figure 6:

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Figure 7:

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  Discussion Top


Renal interstitial fibrosis occurs in virtually every type of chronic kidney disease. The severity of tubulointerstitial fibrosis has long been considered a crucial determinant of progressive renal injury in both human and experimental glomerulonephritis [22]. PUUO is one of the most commonly applied rodent models to study the pathophysiology of renal fibrosis. This model reflects important aspects of inflammation and fibrosis that are prominent in human kidney diseases [23].

In the current study, a significant increase in rat tail SBP and serum levels of urea, creatinine, TGF-β1, and TNF-͍ were detected in PUUO rats compared with the control group. In addition, malondialdehyde levels in renal tissue, urinary protein levels, and albumin concentration were all significantly elevated, and creatinine clearance was significantly decreased, indicating deterioration in renal functions and renal fibrosis in this group compared with the control group, which was further confirmed by histopathological and immuonohistochemical findings.

These results were in agreement with those of Garamaleki and colleagues, who demonstrated that obstructive nephropathy leads to permanent and progressive changes in renal function, as well as fibrosis of renal tissue, tubular atrophy, tissue inflammation, and renal cell apoptosis. Various other factors such as nuclear factor-kappa B (NF-κB), TGF-β, intracellular adhesive molecule, and TNF-͍ were involved in the pathogenesis of this clinical condition [24].

TGF-β has long emerged as a prominent master regulator of renal fibrogenesis in general, and specifically in obstructive nephropathy [25]. This cytokine has pleiotropic actions, which are cell-type dependent. Among its diverse biological activities are growth arrest and apoptosis, as well as contributions to mesenchymal transition in epithelial cells. TGF-β is also an important regulator of fibroblasts [26].

Circulating serum TGF-β1 may also contribute to the development and progression of renal scarring. Experimental studies by Sanderson et al. [27] in transgenic mice revealed that an increased serum TGF-β1 level due to its increased hepatic production resulted in renal extracellular matrix accumulation, interstitial fibrosis, and mesangial cell proliferation.

Meldrum et al. [28] demonstrated that renal cortical TNF-͍ levels increase early after UUO, whereas TNF-͍ neutralization with a pegylated form of soluble TNF receptor type 1 significantly reduced obstruction-induced TNF-͍ production. In addition, NF-κB activation, angiotensinogen expression, and renal tubular cell apoptosis were significantly reduced, thus suggesting a major role for TNF-͍ in activating NF-κB.

Further, Asanuma and colleagues demonstrated that renal cortical TNF-͍ mRNA expression was significantly increased in response to 1 and 4 weeks of obstruction compared with that in sham-treated animals and that TNF-͍ is produced predominantly by tubular epithelial cells in response to renal obstruction and has a significant role in obstruction-induced renal injury [29],[30]. TNF-͍ directly stimulates obstruction-induced renal tubular cell apoptosis, ͍-SMA expression and deposition, collagen deposition, and a deterioration in renal function [31].

The present study showed that all serological and histopathological parameters were significantly improved after 14 days of atorvastatin and EPO administration in G4 and G5, respectively, compared with the PUUO group, except for SBP in the EPO-treated group, in which there was a nonsignificant change compared with the PUUO group.

Our results were in accordance with those of Kamdar et al. [32], who demonstrated that atorvastatin treatment affords protection of renal function in acute UUO and reduces urinary microalbumin levels without lowering cholesterol levels in a so-called pleiotropic effect. This pleiotropic effect of atorvastatin on preservation of renal hemodynamics may be important in attenuating subsequent renal structural injury in chronic UUO.

In a recent study, statins were found to lower blood pressure, attenuate proteinuria and glomerulosclerosis due to anti-oxidative effects (increased eNOS and NOS protein expression), and decrease MCP-1 mRNA expression. Using the animal model of the spontaneously hypertensive rat (SHR), Ito et al. [33] confirmed that atorvastatin upregulates NO synthases in the kidney by Rho-kinase inhibition and Akt activation. In an animal model of obesity and hypertension, simvastatin reduced oxidative stress, thereby protecting against endothelial dysfunction and renal injury [34].

The current results are in accordance with those of Chuang et al. [35], who demonstrated that administration of atorvastatin might ameliorate the tissue damage occurring in the obstructed ureters, through the inhibition of TGF-β1 expression and by diminishing the effects of proinflammatory cytokines. Atorvastatin significantly decreased the expression of TGF-β1 and TNF-͍ in the ligated ureters of rats of the treated group as compared with the untreated groups, especially on day 21 after ligation.

As regards SBP, our results are in agreement with those of Kitamura and colleagues, who examined the effect EPO treatment on blood pressure in the UUO model and found that one of three rats treated with EPO showed markedly elevated blood pressure; they explained this effect as being caused by EPO being prothrombotic in a dose-dependent manner, which is partly mediated by augmented expression of P-selectins and E-selectins [36],[37]. In addition, EPO induces the production of young, hyper-reactive platelets [38], which is particularly problematic for chronic administration of EPO and for the high doses that would be required for renoprotection [39].

Many clinical and experimental studies explained cytoprotective, antiapoptotic, and antioxidant effects of EPO. Toba et al. [40] demonstrated the direct renoprotective effects, which include attenuation of proteinuria and inhibition of renal dysfunction, ameliorating changes in creatinine clearance, serum creatinine and BUN levels, and fibrosis after chronic treatment with the low dose of EPO in the streptozotocin-induced diabetic rat model.

It was proposed that the protective effect of EPO on the proximal tubular epithelial cells is mediated through activation of EPO receptors. Activation of EPO receptors in turn leads to upregulation of Janus-activated kinase 2 signaling. Janus-activated kinase 2 activation was shown to enhance PI3K and Akt (a serine/threonine protein kinase B) phosphorylation in the endothelial and neuronal cells [41]. Activated Akt has several functions along with antiapoptotic effects, including phosphorylation of caspase-9, maintenance of mitochondrial membrane potential, and preservation of glycolysis and ATP synthesis [42],[43].


  Conclusion Top


Our study indicated that atorvastatin and EPO had renoprotective and antifibrotic potential in an experimental model of obstructive nephropathy.


  Acknowledgements Top


Conflicts of interest

None declared.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]


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1 Effects of High Salt-Low Potassium Diet on Blood Pressure and Vascular Reactivity in Male Sprague Dawley Rats
Lebogang Mokotedi,Frederic S. Michel,Angela J. Woodiwiss,Gavin R. Norton,Aletta M. E. Millen
Journal of Cardiovascular Pharmacology. 2018; 71(6): 340
[Pubmed] | [DOI]



 

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