Int Urol Nephrol (2015) 47:2047–2051 DOI 10.1007/s11255-015-1116-8
NEPHROLOGY - ORIGINAL PAPER
Effect of bevacizumab, a vascular endothelial growth factor inhibitor, on a rat model of peritoneal sclerosis Sibel Ada1 · Sibel Ersan1 · Aykut Sifil1 · Mehtat Unlu2 · Efsun Kolatan3 · Mehmet Sert1 · Sulen Sarioglu2 · Osman Yilmaz3 · Taner Camsari1
Received: 20 May 2015 / Accepted: 18 September 2015 / Published online: 3 October 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Purpose Peritoneal fibrosis is almost uniform feature encountered in peritoneal dialysis patients. The transition of epithelial cells to mesenchymal phenotype, neovascularization, and consequently development of peritoneal fibrosis occur due to the involvement of peritoneal membrane by various insults such as uremia itself, peritonitis attacks, and exposure to bio-incompatible peritoneal dialysis fluids. Bevacizumab is a monoclonal antihuman antibody developed against vascular endothelial growth factor and can reduce fibrosis by preventing neovascularization. There has been no study so far that demonstrates the effect of bevacizumab on peritoneal fibrosis in a rat model. Methods A total of 41 female Wistar albino rats were divided into six groups. The control group (C) received 0.9 % isotonic saline (2 ml/day) intraperitoneally (i.p) for 21 days. Chlorhexidine group (CH) received 15 % ethyl alcohol and 0.1 % chlorhexidine gluconate (CG) in saline (2 ml/day) i.p for 21 days. The resting group (R) received CG 2 ml/day i.p for 21 days. The bevacizumab-1 group (B1) received CG 2 ml/day i.p for 21 days and bevacizumab 2.5 mg/kg i.p as a single dose on day 21. The bevacizumab-2 group (B2) received CG 2 ml/day for 21 days and bevacizumab 2.5 mg/kg i.p on day 0 and day 21. The bevacizumab-3 group (B3) received bevacizumab 2.5 mg/
* Sibel Ersan [email protected] 1
Division of Nephrology, Department of Internal Medicine, Dokuz Eylul University Hospital, Izmir, Turkey
2
Department of Pathology, Dokuz Eylul University Hospital, Izmir, Turkey
3
Department of Laboratory Animals Science, Dokuz Eylul University Hospital, Izmir, Turkey
kg i.p on day 0 and day 21. Peritoneal samples were taken from the left anterior abdominal wall. The thickness, vascularization, and fibrosis scores in the peritoneal samples were assessed using a light microscope. Results On histopathological evaluations, peritoneum thicknesses, vascularization scores, and fibrosis significantly decreased in bevacizumab groups B1 and B2. Conclusion Histopathologically, bevacizumab was proven to attenuate fibrotic process in experimental peritoneal sclerosis model. Keywords Fibrosis · Growth factors · Peritoneal dialysis · Peritoneal membrane
Introduction Peritoneal dialysis (PD) is a treatment modality that can be performed both at home and in center, seems to be associated with 48 % lower mortality rate than hemodialysis over the first 2 years of dialysis therapy, and improves quality of life [1]. Preservation of residual renal function and of individual membrane performance improves survival in PD patients. However, ongoing background peritoneal inflammatory reaction per se is a major concern that results in oxidative stress with generation of free radicals and local production of several cytokines. This inflammatory milieu unfavorably effects peritoneal remodeling promoting peritoneal fibrosis and culminates in decreased technical and patient survival which is characterized by impaired solute and fluid clearances [2–4]. Simple fibrosis develops in all patients 1–3 years after PD, but some of them develop encapsulating peritoneal sclerosis (EPS), which is a rare but serious complication that occurs in long-term peritoneal dialysis patients and
13
2048
characterized by malnutrition and intestinal obstruction, may cause fatal outcome in approximately 20 % of patients [2, 5]. The exposure to bio-incompatible solutions with high glucose content, recurrent peritonitis, and long-lasting PD treatment are the main contributing factors in the development of EPS. The denudation of mesothelial cells, on the other hand, is an observation in the first weeks of PD, and neovascularization and fibrosis progress as a consequence of high amount of advanced glycosylation end products (AGE), low-pH dialysate, and recurrent peritonitis attacks [6–8]. Mesothelial cells in the normal peritoneal membrane have the characteristics of epithelial cell and synthesize many substances to enable peritoneal homeostasis; however, when exposed to bio-incompatible dialysis solutions, they lose their epithelial phenotype and gain myofibroblast character and transform into mesenchymal cells, which are more migratory, invasive, and fibrinogenic. This change in mesothelial cells is called as epithelial mesenchymal transformation (EMT) [9, 10]. Although EMT has given a lot of attention in the mechanism of peritoneal fibrosis, the source of myofibroblasts in the peritoneal cavity in EPS patients is still under debate. Different population of cells such as mesenchymal-derived cells (like a pericyte), cells from bone marrow, cells derived from the epithelium (EMT), as well as cells from the endothelium have been postulated as potential sources. Recent studies point to the pericytes of vessels as the source of myofibroblasts. Furthermore, pericytes are involved in angiogenesis [11]. The neovascularization is also caused by hypoxic stimulus derived from thickened peritoneum, which induces vascular endothelial growth factor (VEGF) [12]. Increased number of capillary vessels (neoangiogenesis) due to secretion of angiogenic cytokines (VEGF) has resulted in decrease in UF in PD patients [13, 14]. VEGF has been shown in the peritoneal dialysis fluids of PD patients and is synthesized by mesothelial cells. It is a proangiogenetic factor that enhances endothelial cell proliferation and generation of new vessels [15–17]. It has been demonstrated that VEGF and angiopoietin play a role in neoangiogenesis in a rat model of EPS [17]. The main features of pathophysiology of EPS include inflammation, fibrosis, and angiogenesis, and effective treatment is debatable [18–20]. There has been no reported study so far on the effect of bevacizumab on experimental peritoneal sclerosis model in rats.
Methods This study was approved by laboratory animals ethical committee of the university where the study was conducted. The study comprised a total of 41 female nonuremic Wistar albino rats weighing between 230 and 260 g, which were fed with laboratory pellet and kept in the cages. All animals
13
Int Urol Nephrol (2015) 47:2047–2051
were kept at 24 °C room temperature under 12-h dark/light cycle. They were sheltered under most appropriate conditions and fed with standard pellet. At the end of study, toxic-dose ether anesthesia was used to kill rats. Any infection and systemic event other than peritoneal sclerosis encountered during study period, or the animal’s being in a condition unable to live any longer for another reason was considered as the exclusion criterion. Experimental design The rats enrolled in the study were weighed prior to the study and were then divided into six groups each including seven rats except for the control group, which included six rats. Study period was specified to be 21 days [3 weeks] for the control and chlorhexidine groups and 42 days [6 weeks] for the other groups. In order to create peritoneal fibrosis, the mixture of 0.1 % CG plus 15 % ethanol dissolved in saline was injected i.p for 21 days [13]. Bevacizumab [Altuzan vial 100 mg/4 ml (Roche)] was administered at a dose of 2.5 mg/kg. C group (n; 6) were administered 0.9 % isotonic saline i.p at a dose of 2 ml/day for 3 weeks. The rats were killed at the end of 21st day. CH group (n; 7) were given a solution of 15 % ethyl alcohol and 0.1 % chlorhexidine in saline i.p at a dose of 2 ml/ day for 21 days. The rats were killed at the end of day 21. R group (n; 7) were administered a solution of 15 % ethyl alcohol and 0.1 % chlorhexidine in saline i.p at a dose of 2 ml/day for 21 days and the peritoneum rested until the 42nd day. The rats were killed on day 42. B1 (n; 7) group received a solution of 15 % ethyl alcohol and 0.1 % chlorhexidine in saline i.p at a dose of 2 ml/day for 21 days. Bevacizumab 2 ml was administered at a single dose of 2.5 mg/kg i.p on day 21. The rats were killed at the end of day 42. B2 group (n; 7) were given a solution of 15 % ethyl alcohol and 0.1 % chlorhexidine in saline i.p for 21 days. Bevacizumab was administered i.p at two doses of 2.5 mg/kg on day 0 and day 21. The rats were killed on the 42nd day. B3 group (n; 7) were given bevacizumab i.p at two doses of 2.5 mg/kg (2 ml) on day 0 and day 21. Rats in groups C, CH, and B3 were killed on day 21, and the rats in groups R, B1, and B2 were killed on day 42 under ether anesthesia. After killing, peritoneal samples were taken from the left anterior abdominal wall. The specimens were immediately put into 10 % formaldehyde for pathological examination. Histopathological examination Peritoneal thickness was measured as a mean thickness of submesothelial compact zone between basal border of surface mesothelial cells and upper border of peritoneal adipose
Int Urol Nephrol (2015) 47:2047–2051
tissue (SMC) from ten different sites of HE-stained specimens by an image analysis microscope (Olympus BX50, Olympus Optical Co., Tokyo, Japan) linked to a compatible computer, and expressed as micrometers (µm). The sections were embedded into paraffin and stained with HE and MT. The sections were examined under light microscope for peritoneal thickness, fibrosis, and neovascularization. Peritoneal fibrosis was measured on MT-stained samples by the same image analysis microscope as percentage of green-stained areas that reflect fibrosis by MT [21]. Vascular density was measured by counting vessels within SMC area of more 0.3 mm2. The number was divided by total SMC (mm2) and mesothelial surface length (mm). Statistical analysis Nonparametric methods were used for statistical analysis. Mann–Whitney U test was used to compare the data between groups. The dependent variables in each group were compared by Wilcoxon signed-rank test. The level of significance was considered to be p
NEPHROLOGY - ORIGINAL PAPER
Effect of bevacizumab, a vascular endothelial growth factor inhibitor, on a rat model of peritoneal sclerosis Sibel Ada1 · Sibel Ersan1 · Aykut Sifil1 · Mehtat Unlu2 · Efsun Kolatan3 · Mehmet Sert1 · Sulen Sarioglu2 · Osman Yilmaz3 · Taner Camsari1
Received: 20 May 2015 / Accepted: 18 September 2015 / Published online: 3 October 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Purpose Peritoneal fibrosis is almost uniform feature encountered in peritoneal dialysis patients. The transition of epithelial cells to mesenchymal phenotype, neovascularization, and consequently development of peritoneal fibrosis occur due to the involvement of peritoneal membrane by various insults such as uremia itself, peritonitis attacks, and exposure to bio-incompatible peritoneal dialysis fluids. Bevacizumab is a monoclonal antihuman antibody developed against vascular endothelial growth factor and can reduce fibrosis by preventing neovascularization. There has been no study so far that demonstrates the effect of bevacizumab on peritoneal fibrosis in a rat model. Methods A total of 41 female Wistar albino rats were divided into six groups. The control group (C) received 0.9 % isotonic saline (2 ml/day) intraperitoneally (i.p) for 21 days. Chlorhexidine group (CH) received 15 % ethyl alcohol and 0.1 % chlorhexidine gluconate (CG) in saline (2 ml/day) i.p for 21 days. The resting group (R) received CG 2 ml/day i.p for 21 days. The bevacizumab-1 group (B1) received CG 2 ml/day i.p for 21 days and bevacizumab 2.5 mg/kg i.p as a single dose on day 21. The bevacizumab-2 group (B2) received CG 2 ml/day for 21 days and bevacizumab 2.5 mg/kg i.p on day 0 and day 21. The bevacizumab-3 group (B3) received bevacizumab 2.5 mg/
* Sibel Ersan [email protected] 1
Division of Nephrology, Department of Internal Medicine, Dokuz Eylul University Hospital, Izmir, Turkey
2
Department of Pathology, Dokuz Eylul University Hospital, Izmir, Turkey
3
Department of Laboratory Animals Science, Dokuz Eylul University Hospital, Izmir, Turkey
kg i.p on day 0 and day 21. Peritoneal samples were taken from the left anterior abdominal wall. The thickness, vascularization, and fibrosis scores in the peritoneal samples were assessed using a light microscope. Results On histopathological evaluations, peritoneum thicknesses, vascularization scores, and fibrosis significantly decreased in bevacizumab groups B1 and B2. Conclusion Histopathologically, bevacizumab was proven to attenuate fibrotic process in experimental peritoneal sclerosis model. Keywords Fibrosis · Growth factors · Peritoneal dialysis · Peritoneal membrane
Introduction Peritoneal dialysis (PD) is a treatment modality that can be performed both at home and in center, seems to be associated with 48 % lower mortality rate than hemodialysis over the first 2 years of dialysis therapy, and improves quality of life [1]. Preservation of residual renal function and of individual membrane performance improves survival in PD patients. However, ongoing background peritoneal inflammatory reaction per se is a major concern that results in oxidative stress with generation of free radicals and local production of several cytokines. This inflammatory milieu unfavorably effects peritoneal remodeling promoting peritoneal fibrosis and culminates in decreased technical and patient survival which is characterized by impaired solute and fluid clearances [2–4]. Simple fibrosis develops in all patients 1–3 years after PD, but some of them develop encapsulating peritoneal sclerosis (EPS), which is a rare but serious complication that occurs in long-term peritoneal dialysis patients and
13
2048
characterized by malnutrition and intestinal obstruction, may cause fatal outcome in approximately 20 % of patients [2, 5]. The exposure to bio-incompatible solutions with high glucose content, recurrent peritonitis, and long-lasting PD treatment are the main contributing factors in the development of EPS. The denudation of mesothelial cells, on the other hand, is an observation in the first weeks of PD, and neovascularization and fibrosis progress as a consequence of high amount of advanced glycosylation end products (AGE), low-pH dialysate, and recurrent peritonitis attacks [6–8]. Mesothelial cells in the normal peritoneal membrane have the characteristics of epithelial cell and synthesize many substances to enable peritoneal homeostasis; however, when exposed to bio-incompatible dialysis solutions, they lose their epithelial phenotype and gain myofibroblast character and transform into mesenchymal cells, which are more migratory, invasive, and fibrinogenic. This change in mesothelial cells is called as epithelial mesenchymal transformation (EMT) [9, 10]. Although EMT has given a lot of attention in the mechanism of peritoneal fibrosis, the source of myofibroblasts in the peritoneal cavity in EPS patients is still under debate. Different population of cells such as mesenchymal-derived cells (like a pericyte), cells from bone marrow, cells derived from the epithelium (EMT), as well as cells from the endothelium have been postulated as potential sources. Recent studies point to the pericytes of vessels as the source of myofibroblasts. Furthermore, pericytes are involved in angiogenesis [11]. The neovascularization is also caused by hypoxic stimulus derived from thickened peritoneum, which induces vascular endothelial growth factor (VEGF) [12]. Increased number of capillary vessels (neoangiogenesis) due to secretion of angiogenic cytokines (VEGF) has resulted in decrease in UF in PD patients [13, 14]. VEGF has been shown in the peritoneal dialysis fluids of PD patients and is synthesized by mesothelial cells. It is a proangiogenetic factor that enhances endothelial cell proliferation and generation of new vessels [15–17]. It has been demonstrated that VEGF and angiopoietin play a role in neoangiogenesis in a rat model of EPS [17]. The main features of pathophysiology of EPS include inflammation, fibrosis, and angiogenesis, and effective treatment is debatable [18–20]. There has been no reported study so far on the effect of bevacizumab on experimental peritoneal sclerosis model in rats.
Methods This study was approved by laboratory animals ethical committee of the university where the study was conducted. The study comprised a total of 41 female nonuremic Wistar albino rats weighing between 230 and 260 g, which were fed with laboratory pellet and kept in the cages. All animals
13
Int Urol Nephrol (2015) 47:2047–2051
were kept at 24 °C room temperature under 12-h dark/light cycle. They were sheltered under most appropriate conditions and fed with standard pellet. At the end of study, toxic-dose ether anesthesia was used to kill rats. Any infection and systemic event other than peritoneal sclerosis encountered during study period, or the animal’s being in a condition unable to live any longer for another reason was considered as the exclusion criterion. Experimental design The rats enrolled in the study were weighed prior to the study and were then divided into six groups each including seven rats except for the control group, which included six rats. Study period was specified to be 21 days [3 weeks] for the control and chlorhexidine groups and 42 days [6 weeks] for the other groups. In order to create peritoneal fibrosis, the mixture of 0.1 % CG plus 15 % ethanol dissolved in saline was injected i.p for 21 days [13]. Bevacizumab [Altuzan vial 100 mg/4 ml (Roche)] was administered at a dose of 2.5 mg/kg. C group (n; 6) were administered 0.9 % isotonic saline i.p at a dose of 2 ml/day for 3 weeks. The rats were killed at the end of 21st day. CH group (n; 7) were given a solution of 15 % ethyl alcohol and 0.1 % chlorhexidine in saline i.p at a dose of 2 ml/ day for 21 days. The rats were killed at the end of day 21. R group (n; 7) were administered a solution of 15 % ethyl alcohol and 0.1 % chlorhexidine in saline i.p at a dose of 2 ml/day for 21 days and the peritoneum rested until the 42nd day. The rats were killed on day 42. B1 (n; 7) group received a solution of 15 % ethyl alcohol and 0.1 % chlorhexidine in saline i.p at a dose of 2 ml/day for 21 days. Bevacizumab 2 ml was administered at a single dose of 2.5 mg/kg i.p on day 21. The rats were killed at the end of day 42. B2 group (n; 7) were given a solution of 15 % ethyl alcohol and 0.1 % chlorhexidine in saline i.p for 21 days. Bevacizumab was administered i.p at two doses of 2.5 mg/kg on day 0 and day 21. The rats were killed on the 42nd day. B3 group (n; 7) were given bevacizumab i.p at two doses of 2.5 mg/kg (2 ml) on day 0 and day 21. Rats in groups C, CH, and B3 were killed on day 21, and the rats in groups R, B1, and B2 were killed on day 42 under ether anesthesia. After killing, peritoneal samples were taken from the left anterior abdominal wall. The specimens were immediately put into 10 % formaldehyde for pathological examination. Histopathological examination Peritoneal thickness was measured as a mean thickness of submesothelial compact zone between basal border of surface mesothelial cells and upper border of peritoneal adipose
Int Urol Nephrol (2015) 47:2047–2051
tissue (SMC) from ten different sites of HE-stained specimens by an image analysis microscope (Olympus BX50, Olympus Optical Co., Tokyo, Japan) linked to a compatible computer, and expressed as micrometers (µm). The sections were embedded into paraffin and stained with HE and MT. The sections were examined under light microscope for peritoneal thickness, fibrosis, and neovascularization. Peritoneal fibrosis was measured on MT-stained samples by the same image analysis microscope as percentage of green-stained areas that reflect fibrosis by MT [21]. Vascular density was measured by counting vessels within SMC area of more 0.3 mm2. The number was divided by total SMC (mm2) and mesothelial surface length (mm). Statistical analysis Nonparametric methods were used for statistical analysis. Mann–Whitney U test was used to compare the data between groups. The dependent variables in each group were compared by Wilcoxon signed-rank test. The level of significance was considered to be p
