A Clinically Relevant Murine Model of Peritoneal Fibrosis by Dialysate and Catheters

Abstract

Peritoneal fibrosis can occur in patients undergoing peritoneal dialysis (PD), and patients with severe peritoneal fibrosis have high morbidity and mortality. Peritonitis, high-glucose peritoneal dialysis fluid, and a long period of PD precipitate the onset of peritoneal fibrosis. An animal study of peritoneal fibrosis is needed due to the limitations of human and in vitro studies. However, most animal models do not mimic clinical conditions. To study peritoneal fibrosis, we developed a clinically relevant murine model by implanting a peritoneal catheter and injecting 2.5% high-glucose PD fluid plus 20 mM methylglyoxal (MGO) into the peritoneal cavity daily for 21 days. Implantation of the peritoneal catheter avoids peritoneal injury by needles and mimics clinical PD patients. Immunofluorescence staining showed that myofibroblasts accumulated in the fibrotic peritoneum. The experimental group had lower ultrafiltration volume and peritoneal membrane transport function (peritoneal equilibration test). In this article, we provide a detailed protocol of the model.

Introduction

Peritoneal dialysis (PD) is one kind of kidney replacement therapy received by about 11% of end-stage renal disease (ESRD) patients¹. Studies have reported that peritoneal fibrosis develops in patients receiving PD². Encapsulating peritoneal sclerosis (EPS), a severe form of peritoneal fibrosis, results in high morbidity and mortality³. The risk factors of peritoneal fibrosis include peritonitis, high-glucose peritoneal dialysis fluid, a long period of PD, and genetic factors⁴. The mechanism of peritoneal fibrosis involves complex changes in the peritoneal microenvironment and crosstalk between different cell types. In vitro experiments and clinical observations are limited to providing mechanistic insights into peritoneal fibrosis, and animal models are needed. Experimental animals such as dogs, rabbits, pigs, rats, and mice are usually used in human disease research⁵, of which mice are the most commonly used due to the advantages of their small size, low cost, and ease of experimentation. Furthermore, specific cells can be studied in disease models of mice using lineage tracing techniques.

Establishing a suitable animal model would be helpful in understanding the pathophysiology of peritoneal fibrosis. Ideally, this model should be inexpensive, be easy to reproduce, and provide the basis of clinical treatment for peritoneal fibrosis.

Currently available animal models of peritoneal fibrosis are established with the intra-peritoneal injection of high-glucose PD fluid daily for 28 days, chlorhexidine gluconate (CG) thrice weekly for 3 weeks, or a single dose of sodium hypochlorite hypochlorite^6,7,8. However, these models have limitations. The injection of high-glucose PD fluid, although highly relevant to clinical practice, induces only mild peritoneal fibrosis and fails to recapitulate the clinical conditions of severe peritoneal fibrosis, such as EPS. CG or hypochlorite can induce severe peritoneal fibrosis mimicking EPS through chemical injury⁹. However, CG and hypochlorite may induce peritoneal injury and fibrosis through different mechanisms from high-glucose PD fluid.

This study describes the protocol for developing a murine model of peritoneal fibrosis. In this model, a PD catheter is inserted first, and then daily injections of high-glucose PD fluid plus methylglyoxal (MGO) are performed for 21 days. MGO is a toxic glucose degradation product in PD fluid, and it promotes the formation of advanced glycation end-products, which induce inflammation and angiogenesis^10,11. The addition of MGO to high-glucose PD fluid induces the accumulation of myofibroblasts and promotes peritoneal fibrosis. This model, thus, mimics clinical conditions.

Protocol

The experiments were approved and conducted according to the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC). All mice were housed under standard care.

1. Animal experiment

1. Use male and female C57BL/6 wild type (WT) mice more than 11 weeks of age.
2. Prepare the surgical materials, including a mouse 4-french silicone port, gloves, drapes, catheters, sutures, and needles.
3. Implant the mouse port as follows:
   1. Anesthetize the mice with ketamine/xylazine (100/10 mg/kg body weight) via subcutaneous injection.
      1. Check the respiratory rate and depth of anesthesia of the mice during surgery to ensure the anesthesia conditions.
         NOTE: If the respiratory rate of the mice becomes rapid and short, add 20% of the previous dose.
   2. Place the mice in the prone position during anesthesia. Shave and clean an area 2 cm x 3 cm in size on the left back for surgery and disinfect the skin with povidone-iodine.
   3. Make a 1.5 cm incision in the skin on the left back.
   4. Dissect carefully between the skin and muscle on the left back to create a space of 1 cmx 2 cm to implant the mouse port.
   5. Perforate a small hole over the left-back muscle.
   6. Insert the total 4-french distal part of the mouse port into the peritoneal cavity
   7. Suture (4-0 nylon) the root of the mouse port to the back muscle.
   8. Put the head of the mouse port between the skin and back muscle.
   9. Close the skin with reflex clips (7 mm).
      NOTE: Reflex clips are better than sutures because mice often bite the wound.
   10. Start the experiment after 7 days of mouse port implantation. Randomly divide the mice into experimental and control groups.
   11. Disinfect the skin at the access site with povidone-iodine before injecting fluid.
   12. Inject the PD fluid (PDF) group with 2.5% PD fluid and 20 mM MGO in a total of 2 mL intra-peritoneally daily for 21 days. Inject the control group with normal saline (NS).

2. Peritoneal function test (peritoneal equilibration test)

1. Prepare 2.5% PD fluid (2 mL) and measure the glucose concentration of the PD fluid, defined as initial glucose concentration (D0).
2. Inject the PD fluid into the mouse port.
3. After 30 min, sacrifice the mice via isoflurane (100%) overdose.
4. Collect the intra-abdominal fluid with a syringe, then measure the fluid volume, defined as the ultrafiltration volume.
5. Measure the glucose concentration of the intra-abdominal fluid, defined as the final glucose concentration (D).
6. Calculate the peritoneal equilibration or peritoneal function¹².
   ​NOTE: Peritoneal equilibration test¹² = Final PD fluid glucose concentration (D)/ Initial PD fluid glucose concentration (D0). The glucose concentration is detected by the hexokinase method with a biochemical analyzer¹³.

3. Tissue preparation and histological analysis

1. Collect the peritoneal tissues: right upper abdominal wall (1 cm x 1 cm) and liver. Fix the peritoneal tissue with 4% paraformaldehyde for 2 h, then overnight in 18% sucrose solution⁷.
2. Prepare 4 µm thick frozen sections of peritoneal tissue and perform histological analysis as previously published⁷.
3. Perform immunofluorescence staining.
   1. Use primary antibodies against the following proteins for immunolabeling: α-smooth muscle actin (SMA; 2 h, 1:200) for detecting myofibroblasts, cytokeratin (2 h, 1:200) for mesothelial cells detection, and 4′,6-diamidino-2-phenylindole (DAPI) (5 min, 1:1000) for detecting cell nuclei.
4. Express the data obtained in an appropriate format.
   NOTE: In this study, data were expressed as mean ± SEM. Statistical analyses were carried out using appropriate statistical analysis software (Table of Materials), and the statistical significance was evaluated by one-way ANOVA or t-test.

Representative Results

The histology of the peritoneal tissue was examined, which showed that peritoneal fibrosis was induced successfully in the PD fluid plus MGO (PDF) group. Immunofluorescence staining of the visceral peritoneum of the liver surface showed more myofibroblast accumulation in the injured peritoneum of the PDF group than in the control group (Figure 1). As shown in Figure 2A, the PDF group had a lower ultrafiltration volume than the control group. As shown in Figure 2B, there was a lower peritoneal equilibration test in the PDF group compared with the control group. These results show that daily injection of 2.5% PD fluid plus MGO for 21 days induced successfully peritoneal inflammation and fibrosis.

Figure 1: Myofibroblasts accumulated in injured peritoneum. (A) Immunofluorescence staining showed that cytokeratin-FITC mesothelial cells were on the visceral peritoneum of the liver surface. (B) PD fluid plus methylglyoxal induced αSMA-RFP + myofibroblast accumulation in the injured peritoneum, and mesothelial cells were lost. Scale bar: 20 µm. Original magnification: 630x. Please click here to view a larger version of this figure.

Figure 2: PD fluid plus methylglyoxal damaged peritoneal function. The PD fluid (PDF) group had lower (A) ultrafiltration volume and (B) peritoneal equilibration than the control group (NS). Data were expressed as mean ± SEM, P < 0.005. Please click here to view a larger version of this figure.

Discussion

The peritoneum has a visceral and parietal peritoneum, covering the abdominal organs and abdominal walls. It is composed of a monolayer of mesothelial cells, a submesothelial layer, serosa, fibroblasts, lymphocytes, and secreted proteins¹⁴. Peritoneal fibrosis is the loss and denudation of mesothelial cells, submesothelial thickening, and the accumulation of many inflammatory cells, such as myofibroblasts and macrophages^4,14.

Animal models of peritoneal fibrosis use peritoneal dialysis to induce acute and chronic peritoneal inflammation. Previous acute peritoneal inflammation models have been induced by Staphylococcus epidermidis cell-free supernatant, bacterial peritonitis, and PD fluid with lipopolysaccharide^6,15,16. The addition of lipopolysaccharide to PD fluid has been shown to induce inflammatory cells and increase peritoneal cytokine production and solute transport¹⁵. Previous chronic peritoneal inflammation models have involved peritoneal fibrosis induced by high-glucose PD fluid with/without MGO, the over-expression of transforming growth factor-β1, or chemical injury with a low pH solution, sodium hypochlorite, and chlorhexidine gluconate (CG)^{6,7,8,9,17,18,19}. Chemical peritoneal injury causes the severe type of fibrosis, EPS⁷.

The insertion of a PD catheter can mimic clinical end-stage renal disease patients undergoing PD and avoid peritoneal injury due to repeated intraperitoneal injection with a needle. In this model, the mouse port is implanted into the back, making it easy to hold the mice. This model is easy to reproduce and can be used for studies on the peritoneum.

The limitation of this model is that the experimental mice have normal renal function, not uremic status. Uremic models probably more closely mimic clinical ESRD patients. However, uremic models involve complications, such as hyperkalemia and metabolic acidosis, and the success rate of five out of six for nephrectomy is dependent on the operator.

Materials

Name	Company	Catalog Number	Comments
anti-wide spectrum Cytokeratin antibody	abcam	ab9377
Beckman Coulter	Beckman Coulter	AU5800	Biochemical analyzer
DAPI, 4′,6-diamidino-2-phenylindole antibody	Sigma-Aldrich	98718-90-3
Drapes			any
FITC goat anti-rabbit	Jackson ImmunoResearch Secondary Antibody	111-095-144
Gloves			any
GraphPad Prizm	GraphPad Software	GraphPad Software 9.0
Kellys			any
Methylglyoxal (MGO)	Sigma-Aldrich
Mini-UTE Mouse Port	Access Technologies	MMP-4S 061108B	Mouse 4-French silicone port
Monoclonal anti-actin, α-smooth muscle-Cy3 antibody	Sigma-Aldrich	C6198
Needles			any
Reflex clips 7 mm			any
Suture 4-0 Nylon			any