A Clinically Relevant Murine Model of Peritoneal Fibrosis by Dialysate and Catheters
Summary
Patients with severe peritoneal fibrosis have high morbidity and mortality. The mechanism of peritoneal fibrosis is unclear. In this study, we describe a simple experimental murine model of peritoneal fibrosis induced by the injection of peritoneal dialysis fluid.
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 hypochlorite6,7,8. 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 angiogenesis10,11. 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
Representative Results
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 macrophages4,14.
<p class=…Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Taiwan University Hospital (NTUH) 111-UN0026.
Materials
| 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 |
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