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Peritonitis remains the most important factor in patient morbidity and technical failure associated with continuous ambulatory peritoneal dialysis (CAPD). In vitro examination of bacterial infection of cultured human peritoneal mesothelial cells (HPMC) is an attractive approach to the study of peritonitis in CAPD, yet there are few reports on this subject. Previous studies have shown two limitations: (i) cell cultures of HPMC lasted for days only when incubated in culture medium and (ii) short‐term studies of <30min were done in HPMC when incubated with peritoneal dialysis fluid (PDF). Human peritoneal mesothelial cells, maintained in a conventional single chamber culture system with PDF alone, were unable to survive more than 40min. The present study was designed to prolong the viability of HPMC cultured in PDF, with the object of using cells under different conditions, such as that of simulating CAPD. HPMC were cultured using plastic microtiter plates, where they were grown to confluence and growth was arrested. PDF containing different concentrations of NaHCO3and human serum albumin was added. Cell viability after exposure for up to 24h was measured by trypan blue, Cell Death Detection ELISA and Annex‐V flow cytometry. The data confirmed the ‘toxic’ effect of PDF, with cell viability being <40% after 2h incubation in 4.25% glucose in PDF. However, the survival time of HPMC increased significantly in 4.25% glucose PDF at a physiological pH and even further after the addition of human albumin. These experimental conditions simulating CAPD may allow future in vitro studies of mesothelial physiology and peritonitis related to CAPD treatment.
Peritonitis remains the most important factor in patient morbidity and technical failure associated with continuous ambulatory peritoneal dialysis (CAPD). In vitro examination of bacterial infection of cultured human peritoneal mesothelial cells (HPMC) is an attractive approach to the study of peritonitis in CAPD, yet there are few reports on this subject. Previous studies have shown two limitations: (i) cell cultures of HPMC lasted for days only when incubated in culture medium and (ii) short‐term studies of <30min were done in HPMC when incubated with peritoneal dialysis fluid (PDF). Human peritoneal mesothelial cells, maintained in a conventional single chamber culture system with PDF alone, were unable to survive more than 40min. The present study was designed to prolong the viability of HPMC cultured in PDF, with the object of using cells under different conditions, such as that of simulating CAPD. HPMC were cultured using plastic microtiter plates, where they were grown to confluence and growth was arrested. PDF containing different concentrations of NaHCO3and human serum albumin was added. Cell viability after exposure for up to 24h was measured by trypan blue, Cell Death Detection ELISA and Annex‐V flow cytometry. The data confirmed the ‘toxic’ effect of PDF, with cell viability being <40% after 2h incubation in 4.25% glucose in PDF. However, the survival time of HPMC increased significantly in 4.25% glucose PDF at a physiological pH and even further after the addition of human albumin. These experimental conditions simulating CAPD may allow future in vitro studies of mesothelial physiology and peritonitis related to CAPD treatment.
The development of animal models in peritoneal dialysis has led to some breakthroughs in the application of this dialysis modality in clinical practice. These breakthroughs are (1) a better understanding of the physiology and pathophysiology of solute transport and ultrafiltration mechanisms, (2) the observation and integration of the long-term structural and functional alterations of the membrane, (3) a better understanding of the biocompatibility issues involved in PD, leading to the clinical introduction of more biocompatible dialysis solutions and finally, (4) the development of colloid osmotic solutions containing polyglucose polymers for application in the long dwells. Intravital miscroscopy provides information in live animals about diverse functional parameters, such as blood flow rate, vessel diameter, permeability to macromolecules, leukocyte-endothelium interaction, capillary recruitment, and lymph vessel kinetics. Also evaluation of different parameters in a living experimental animal, allowing integration of function and structure is possible. A variety of chronic PD models have been developed, mainly to study effects of long-term peritoneal dialysate exposure on peritoneal membrane function and structure. The implementation of different blocking agents of biochemical substances in these models has elucidated many molecular biological mechanisms involved in these processes. The important roles of aquaporins, vascular endothelial growth factor, nitric oxide, advanced glycation end product formation and their receptor (RAGE) upregulation and the integrated roles of all these factors in the fibrotic alterations of the membrane as observed in patients on long-term PD have been investigated. More recently, genetically modified mice have been used as an important tool to investigate the molecular basis of peritoneal changes during dialysis and during acute peritonitis.
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