Mouse Model Of Peritoneal Dialysis Research Articles

Hyperglycolysis in endothelial cells drives endothelial injury and microvascular alterations in peritoneal dialysis.

Endothelial cell (EC) dysfunction leading to microvascular alterations is a hallmark of technique failure in peritoneal dialysis (PD). However, the mechanisms underlying EC dysfunction in PD are poorly defined. We combined RNA sequencing with metabolite set analysis to characterize the metabolic profile of peritoneal ECs from a mouse model of PD. This was combined with EC-selective blockade of glycolysis by genetic or pharmacological inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in vivo and in vitro. We also investigated the association between peritoneal EC glycolysis and microvascular alterations in human peritoneal samples from patients with end-stage kidney disease (ESKD). In a mouse model of PD, peritoneal ECs had a hyperglycolytic metabolism that shunts intermediates into nucleotide synthesis. Hyperglycolytic mouse peritoneal ECs displayed a unique active phenotype with increased proliferation, permeability and inflammation. The active phenotype of mouse peritoneal ECs can be recapitulated in human umbilical venous ECs and primary human peritoneal ECs by vascular endothelial growth factor that was released from high glucose-treated mesothelial cells. Importantly, reduction of peritoneal EC glycolysis, via endothelial deficiency of the glycolytic activator PFKFB3, inhibited PD fluid-induced increases in peritoneal capillary density, vascular permeability and monocyte extravasation, thereby protecting the peritoneum from the development of structural and functional damages. Mechanistically, endothelial PFKFB3 deficiency induced the protective effects in part by inhibiting cell proliferation, VE-cadherin endocytosis and monocyte-adhesion molecule expression. Pharmacological PFKFB3 blockade induced a similar therapeutic benefit in this PD model. Human peritoneal tissue from patients with ESKD also demonstrated evidence of increased EC PFKFB3 expression associated with microvascular alterations and peritoneal dysfunction. These findings reveal a critical role of glycolysis in ECs in mediating the deterioration of peritoneal function and suggest that strategies targeting glycolysis in peritoneal ECs may be of therapeutic benefit for patients undergoing PD.

#3330 INFLAMMATION SECONDARY TO CHRONIC KIDNEY DISEASE AND PERITONEAL DIALYSIS ACCELERATES ATHEROSCLEROSIS IN A NEW MOUSE MODEL

Abstract Background and Aims Inflammation is an established contributor to accelerated atherosclerosis, which is highly prevalent in people with chronic kidney disease (CKD) and even more so in those treated with peritoneal dialysis (PD). We therefore hypothesised that PD-induced systemic inflammation is essential in the development of atherosclerosis. To study this we developed a novel mouse model of CKD and PD accelerated atherosclerosis. Method We performed a 5/6ths nephrectomy in ApoE deficient “black 6” mice, fed these mice a high-cholesterol diet (HCD), and intraperitoneally administered 3.86% Physioneal daily for 67 days. There were three groups of mice: control (HCD-only), CKD+HCD, and CKD+HCD+PD, with relevant shams. Tissues were harvested twelve weeks after the nephrectomy. Assessments of residual kidney function, atherosclerotic plaque pathology and immunology, and systemic immune responses were performed. Results Atherosclerotic plaque size in the aortic arch was larger in the PD group compared to the control, increasing from 83,779 μm2 ±18,430 to 109,973 μm2 ±25,602 (p = 0.0349), whilst plaque size was unchanged in the CKD group. In contrast, both the CKD and PD groups displayed altered plaque stability. There was less collagen in the plaque vs control, decreasing from 43.54% ±10.65 to 29.71% ±9.43 and 29.73% ±10.18 respectively. Additionally, fibrous cap thickness decreased in both groups vs control. The plaques from only the PD group showed infiltration by T-cells compared to control, with no changes in macrophage content in either the CKD or PD group. In the spleen in the CKD and PD groups, naïve CD4 cells increased and effector CD4 cells decreased compared to control, with the latter change also occurring in the circulation. Central memory CD4 cells, terminally differentiated Th1 cells, Th17 cells, and ‘vascular homing’ cells all increased in the PD group compared to control mice. These homing cells also increased in circulation. The PD group showed decreases in the spleen of regulatory T-cells, effector Th1 cells, and effector CD8 cells. Notably, all of these changes were absent from the mesenteric lymph node. There was a large drop in monocytes in the spleen and a loss of immature and mature dendritic cells in the PD group. Conclusion Combining CKD, a high-cholesterol diet, and exposure to PD fluids in ApoE−/-mice successfully generated a new model for cardiovascular disease in human PD patients. This new model is in a widespread and well-understood mouse line representative of human atherosclerosis. Here we demonstrate the pronounced effects of CKD and PD on the innate and adaptive immune response which increases atherosclerotic burden and leads to larger more vulnerable plaques

Restoration of CPT1A-mediated fatty acid oxidation in mesothelial cells protects against peritoneal fibrosis.

Background: Peritoneal dialysis (PD) is limited by gradual fibrotic remodeling in the peritoneum, a process involving profibrotic response of mesothelial cells. However, the role of fatty acid oxidation (FAO) and carnitine palmitoyltransferase 1A (CPT1A) in this process remains unexplored. Methods: FAO and CPT1A expression were characterized in mesothelial cells from patients on long-term PD and from a mouse model of PD using multiple experimental methods, including single-cell sequencing, seahorse assay, real-time quantitative PCR, Western blot, and immunofluorescence staining. Overexpression of CPT1A was achieved in a human mesothelial cell line and in primary mouse mesothelial cells. Finally, genetic and pharmacological manipulations of CPT1A were performed in a mouse model of PD. Results: Herein, FAO and CPT1A expression were reduced in mesothelial cells from patients on long-term PD, which negatively correlated with expression of fibrogenic markers in these cells. This was corroborated in PD mice, as well as in mouse and human mesothelial cells incubated with transforming growth factor (TGF) β1. CPT1A overexpression in mesothelial cells, which prevented TGFβ1-induced suppression of mitochondrial respiration, restored cellular ATP levels and downregulated the expression of fibrogenic markers. Furthermore, restoration of FAO by overexpressing CPT1A in PD mice reversed profibrotic phenotype in mesothelial cells and reduced fibrotic lesions in the peritoneum. Treatment with the CPT1A activator C75 induced similar therapeutic benefit in PD mice. In contrast, inhibition of FAO with a CPT1 inhibitor caused more severe fibrosis in PD mice. Conclusions: A defective FAO is responsible for the profibrotic response of mesothelial cells and thus the peritoneal fibrogenesis. This aberrant metabolic state could be improved by modulating CPT1A in mesothelial cells, suggesting FAO enhancement in mesothelial cells is a potential treatment of peritoneal fibrosis.

Fasting influences aquaporin expression, water transport and adipocyte metabolism in the peritoneal membrane.

The water channels aquaporin-1 (AQP1) and AQP7 are abundantly expressed in the peritoneal membrane. While AQP1 facilitates water transport during peritoneal dialysis (PD), the role of AQP7, which mediates glycerol transport during fasting, remains unknown. We investigated the distribution of AQP7 and AQP1 and used a mouse model of PD to investigate the role of AQP7 in the peritoneal membrane at baseline and after fasting. Single nucleus RNA-sequencing revealed that AQP7 was mostly detected in mature adipocytes, whereas AQP1 was essentially expressed in endothelial cells. Fasting induced significant decreases in whole body fat, plasma glucose, insulin and triglycerides, as well as higher plasma glycerol and corticosterone levels in mice, paralleled by major decreases in adipocyte size and levels of fatty acid synthase and leptin, and increased levels of hormone-sensitive lipase mRNAs in the peritoneum. Mechanistically, fasting upregulated the expression of AQP1 and AQP7 in the peritoneum, with increased ultrafiltration but no change in small solute transport. Studies based on Aqp1 and Aqp7 knockout mice and RU-486 inhibition demonstrated that the glucocorticoid induction of AQP1 mediates the increase in ultrafiltration whereas AQP7 regulates the size of adipocytes in the peritoneum. Fasting induces a coordinated regulation of lipolytic and lipogenic factors and aqua(glycero)porins in the peritoneum, driving structural and functional changes. These data yield novel information on the specific roles of aquaporins in the peritoneal membrane and indicate that fasting improves fluid removal in a mouse model of PD.

AqF026 may act as a cancer therapeutic agent via inducing cancer cell oncosis

Cancer is one of the leading causes of death worldwide, and metastasis is the major cause of death in cancer, therefore, treatments to attenuate metastasis are urgently needed. Cell migration is indispensable in metastatic cascade, and aquaporins (AQPs) promote cell migration by facilitating water influx at cell front (lamellipodia). In fact, AQPs overexpressed widely among many cancer types. Accordingly, previous efforts of targeting AQPs as strategies of cancer treatments were based on AQP inhibitors, yet the efficacy of AQP inhibition was limited based on recent surveys. On the contrary, whether AQP agonist has role in cancer treatments has not been explored. AqF026, an AQP1 agonist, was initially applied to a mouse model of peritoneal dialysis. Herein, we aimed to apply AqF026 to magnify the water influx into lamellipodia of migrating cancer cells so as to induce oncosis by causing overloaded cancer cell swelling in advance of metastatic cascade. Cell swelling is a characteristic of oncosis. With impairment or insufficient regulatory volume decrease (RVD), cell swelling can lead to oncosis. Cancer cells with metastatic potentials shared the same population of cancer cells with multidrug resistance (MDR) lineage, and the impairment or insufficient RVD is shown in cancer cells with MDR. Taken together, the author hypothesized that given appropriate concentration or dose of AQP1 agonist AqF026, the AqF026 may induce oncosis of cancer cells preferentially rather than normal cells by causing overloaded water influx via AQP1 and consequent irreversible cell swelling.

Dimethylaminomicheliolide ameliorates peritoneal fibrosis through the activation of autophagy.

Peritoneal fibrosis (PF) is a major cause of ultrafiltration failure in patients receiving long-term peritoneal dialysis (PD), and effective prevention and treatment strategies are urgently needed. The dimethylamino Michael adduct of a natural product-derived micheliolide (MCL), dimethylaminomicheliolide (DMAMCL), is a new lead compound with the advantages of high stability, low toxicity, and sustainable release of MCL. This study aimed to investigate the protective effect of DMAMCL against PD-related PF and the mechanisms involved. In this study, we found that DMAMCL significantly decreased PD-induced extracellular matrix (ECM) deposition in a mouse model of PD, and that delayed DMAMCL administration halted the progression of PF in an established PD model. In addition, rapamycin administration induced autophagy and significantly ameliorated PF. The protective effect of DMAMCL against PF was weakened when co-administered with DMAMCL and 3-methyladenine. Inducing autophagy by rapamycin decreased transforming growth factor-β1-induced ECM accumulation in vitro. MCL promoted autophagy and inhibited ECM deposition. The anti-fibrotic effect of MCL was eliminated when knocking down ATG7 by siRNA. Taken together, DMAMCL might prevent against PF through activating autophagy. The anti-fibrotic effect of DMAMCL may be a new candidate for the treatment in patients with PD-related PF. KEY MESSAGES: Dimethylaminomicheliolide, the pro-drug of micheliolide, protects against peritoneal fibrosis in a mouse peritoneal dialysis model. Micheliolide inhibits TGF-β1-induced extracellular matrix accumulation in vitro. Autophagy plays a protective role against peritoneal fibrosis. The antifibrogenic effect of dimethylaminomicheliolide may be due to the activation of autophagy.

Protein kinase C α inhibition prevents peritoneal damage in a mouse model of chronic peritoneal exposure to high-glucose dialysate

Chronic exposure to commercial glucose-based peritoneal dialysis fluids during peritoneal dialysis induces peritoneal membrane damage leading to ultrafiltration failure. In this study the role of protein kinase C (PKC) α in peritoneal membrane damage was investigated in a mouse model ofperitoneal dialysis. We used 2 different approaches: blockade of biological activity of PKCα by intraperitoneal application of the conventional PKC inhibitor Go6976 in C57BL/6 wild-type mice and PKCα-deficient mice on a 129/Sv genetic background. Daily administration of peritoneal dialysis fluid for 5 weeks induced peritoneal upregulation and activation of PKCα accompanied by epithelial-to-mesenchymal transition of peritoneal mesothelial cells, peritoneal membrane fibrosis, neoangiogenesis, and macrophage andT cell infiltration, paralleled by reduced ultrafiltration capacity. All pathological changes were prevented by PKCα blockade or deficiency. Moreover, treatment with Go6976 and PKCα deficiency resulted in strong reduction of proinflammatory, profibrotic, and proangiogenic mediators. In cell culture experiments, both treatment with Go6976 and PKCα deficiency prevented peritoneal dialysis fluid-induced release of MCP-1 from mouse peritoneal mesothelial cells and ameliorated transforming growth factor-β1-induced epithelial-to-mesenchymal transition and peritoneal dialysisfluid-induced MCP-1 release in human peritoneal mesothelial cells. Thus, PKCα plays a crucial role in the pathophysiology of peritoneal membrane dysfunction induced by peritoneal dialysis fluids, and we suggest that its therapeutic inhibition might be a valuable treatment option for peritoneal dialysis patients.

Syndecan-1 (CD138) deficiency increases Staphylococcus aureus infection but has no effect on pathology in a mouse model of peritoneal dialysis

BackgroundTechnique failure in peritoneal dialysis (PD) due to fibrosis and angiogenesis is complicated by peritonitis. Staphylococcus aureus infection is one of the most common causes of peritonitis in PD. The heparan sulfate proteoglycan, syndecan-1 (CD138), was reported to regulate fibrosis, angiogenesis, inflammation and S. aureus infection. The objectives of this study were to examine the effects of syndecan-1 on S. aureus infection and histopathology in a PD model.ResultsSyndecan-1-/- and wild type mice were dialyzed for 4 weeks and infected intraperitoneally with S. aureus. Tissues were collected after 4 h for histomorphometric analysis. Intravital microscopy was used to observe leukocyte recruitment and to quantify syndecan-1 in the parietal peritoneum microcirculation. The dialyzed syndecan-1-/- mice were more susceptible to S. aureus infection than undialyzed syndecan-1-/- controls and wild type animals. However, peritoneal fibrosis and neovascularization due to PD did not differ between syndecan-1-/- and wild type mice. Intravital microscopy showed that in S. aureus infection, syndecan-1 was removed from the subendothelial layer of peritoneal venules but syndecan-1 deficiency did not affect leukocyte recruitment.ConclusionsThis study indicates that, while syndecan-1 is important for providing a barrier to acute S. aureus infection in PD, it does not affect peritoneal fibrosis and angiogenesis.

A Novel Mouse Model of Peritoneal Dialysis: Combination of Uraemia and Long-Term Exposure to PD Fluid.

Different animal models for peritoneal dialysis (PD) have been used in the past decades to develop PD fluids compatible with patient life and to identify markers of peritoneal fibrosis and inflammation. Only few of those studies have taken into account the importance of uraemia-induced alterations at both systemic and peritoneal levels. Moreover, some animal studies which have reported about PD in a uremic setting did not always entirely succeed in terms of uraemia establishment and animal survival. In the present study we induced uraemia in the recently established mouse PD exposure model in order to obtain a more clinically relevant mouse model for kidney patients. This new designed model reflected both the slight thickening of peritoneal membrane induced by uraemia and the significant extracellular matrix deposition due to daily PD fluid instillation. In addition the model offers the opportunity to perform long-term exposure to PD fluids, as it is observed in the clinical setting, and gives the advantage to knock out candidate markers for driving peritoneal inflammatory mechanisms.

MicroRNA-29b inhibits peritoneal fibrosis in a mouse model of peritoneal dialysis

TGF-β/Smad3 signaling plays a pivotal role in the pathogenesis of peritoneal fibrosis associated with peritoneal dialysis (PD). MicroRNA-29 (miR-29) is known as a potent downstream inhibitor of TGF-β/Smad3 in renal fibrosis. In this study, we examined the therapeutic potential for miR-29b on PD-related peritoneal fibrosis in a mouse model of PD induced by daily infusion of 4.25% dextrose-containing PD fluid (PDF). MiR-29b-expressing plasmid was delivered into the peritoneum via an ultrasound-microbubble-mediated system before and at day 14 after PDF. We found that mice on PD developed peritoneal fibrosis with impaired peritoneal function, which was associated with a loss of miR-29b. In contrast, overexpression of miR-29b before the PDF infusion showed a protective effect on peritoneal fibrosis including EMT and prevented peritoneal dysfunction. Moreover, delayed miR-29b treatment until peritoneal fibrosis was established at day 14 also halted the progression of peritoneal fibrosis at day 28. Further studies identified that blockade of the Sp1-TGF-β/Smad3 pathway may be a mechanism by which miR-29b inhibited peritoneal fibrosis. In conclusion, treatment with miR-29b may represent a novel and effective therapy for PD-associated peritoneal fibrosis.

Quantification of osmotic water transport in vivo using fluorescent albumin.

Osmotic water transport across the peritoneal membrane is applied during peritoneal dialysis to remove the excess water accumulated in patients with end-stage renal disease. The discovery of aquaporin water channels and the generation of transgenic animals have stressed the need for novel and accurate methods to unravel molecular mechanisms of water permeability in vivo. Here, we describe the use of fluorescently labeled albumin as a reliable indicator of osmotic water transport across the peritoneal membrane in a well-established mouse model of peritoneal dialysis. After detailed evaluation of intraperitoneal tracer mass kinetics, the technique was validated against direct volumetry, considered as the gold standard. The pH-insensitive dye Alexa Fluor 555-albumin was applied to quantify osmotic water transport across the mouse peritoneal membrane resulting from modulating dialysate osmolality and genetic silencing of the water channel aquaporin-1 (AQP1). Quantification of osmotic water transport using Alexa Fluor 555-albumin closely correlated with direct volumetry and with estimations based on radioiodinated ((125)I) serum albumin (RISA). The low intraperitoneal pressure probably accounts for the negligible disappearance of the tracer from the peritoneal cavity in this model. Taken together, these data demonstrate the appropriateness of pH-insensitive Alexa Fluor 555-albumin as a practical and reliable intraperitoneal volume tracer to quantify osmotic water transport in vivo.

A pathogenetic role for endothelin-1 in peritoneal dialysis-associated fibrosis.

In patients undergoing peritoneal dialysis (PD), chronic exposure to nonphysiologic PD fluids elicits low-grade peritoneal inflammation, leading to fibrosis and angiogenesis. Phenotype conversion of mesothelial cells into myofibroblasts, the so-called mesothelial-to-mesenchymal transition (MMT), significantly contributes to the peritoneal dysfunction related to PD. A number of factors have been described to induce MMT in vitro and in vivo, of which TGF-β1 is probably the most important. The vasoconstrictor peptide endothelin-1 (ET-1) is a transcriptional target of TGF-β1 and mediates excessive scarring and fibrosis in several tissues. This work studied the contribution of ET-1 to the development of peritoneal damage and failure in a mouse model of PD. ET-1 and its receptors were expressed in the peritoneal membrane and upregulated on PD fluid exposure. Administration of an ET receptor antagonist, either bosentan or macitentan, markedly attenuated PD-induced MMT, fibrosis, angiogenesis, and peritoneal functional decline. Adenovirus-mediated overexpression of ET-1 induced MMT in human mesothelial cells in vitro and promoted the early cellular events associated with peritoneal dysfunction in vivo. Notably, TGF-β1-blocking peptides prevented these actions of ET-1. Furthermore, a positive reciprocal relationship was observed between ET-1 expression and TGF-β1 expression in human mesothelial cells. These results strongly support a role for an ET-1/TGF-β1 axis as an inducer of MMT and subsequent peritoneal damage and fibrosis, and they highlight ET-1 as a potential therapeutic target in the treatment of PD-associated dysfunction.

AqF026 Is a Pharmacologic Agonist of the Water Channel Aquaporin-1

Aquaporin-1 (AQP1) facilitates the osmotic transport of water across the capillary endothelium, among other cell types, and thereby has a substantial role in ultrafiltration during peritoneal dialysis. At present, pharmacologic agents that enhance AQP1-mediated water transport, which would be expected to increase the efficiency of peritoneal dialysis, are not available. Here, we describe AqF026, an aquaporin agonist that is a chemical derivative of the arylsulfonamide compound furosemide. In the Xenopus laevis oocyte system, extracellular AqF026 potentiated the channel activity of human AQP1 by >20% but had no effect on channel activity of AQP4. We found that the intracellular binding site for AQP1 involves loop D, a region associated with channel gating. In a mouse model of peritoneal dialysis, AqF026 enhanced the osmotic transport of water across the peritoneal membrane but did not affect the osmotic gradient, the transport of small solutes, or the localization and expression of AQP1 on the plasma membrane. Furthermore, AqF026 did not potentiate water transport in Aqp1-null mice, suggesting that indirect mechanisms involving other channels or transporters were unlikely. Last, in a mouse gastric antrum preparation, AqF026 did not affect the Na-K-Cl cotransporter NKCC1. In summary, AqF026 directly and specifically potentiates AQP1-mediated water transport, suggesting that it deserves additional investigation for applications such as peritoneal dialysis or clinical situations associated with defective water handling.

Blocking TGF-β1 Protects the Peritoneal Membrane from Dialysate-Induced Damage

During peritoneal dialysis (PD), mesothelial cells undergo mesothelial-to-mesenchymal transition (MMT), a process associated with peritoneal-membrane dysfunction. Because TGF-β1 can induce MMT, we evaluated the efficacy of TGF-β1-blocking peptides in modulating MMT and ameliorating peritoneal damage in a mouse model of PD. Exposure of the peritoneum to PD fluid induced fibrosis, angiogenesis, functional impairment, and the accumulation of fibroblasts. In addition to expressing fibroblast-specific protein-1 (FSP-1), some fibroblasts co-expressed cytokeratin, indicating their mesothelial origin. These intermediate-phenotype (Cyto(+)/FSP-1(+)) fibroblasts had features of myofibroblasts with fibrogenic capacity. PD fluid treatment triggered the appearance of CD31(+)/FSP-1(+) and CD45(+)/FSP-1(+) cells, suggesting that fibroblasts also originate from endothelial cells and from cells recruited from bone marrow. Administration of blocking peptides significantly ameliorated fibrosis and angiogenesis, improved peritoneal function, and reduced the number of FSP-1(+) cells, especially in the Cyto(+)/FSP-1(+) subpopulation. Conversely, overexpression of TGF-β1 in the peritoneum by adenovirus-mediated gene transfer led to a marked accumulation of fibroblasts, most of which derived from the mesothelium. Taken together, these results demonstrate that TGF-β1 drives the peritoneal deterioration induced by dialysis fluid and highlights a role of TGF-β1-mediated MMT in the pathophysiology of peritoneal-membrane dysfunction.

Cyclooxygenase-2 Mediates Dialysate-Induced Alterations of the Peritoneal Membrane

During peritoneal dialysis (PD), exposure of the peritoneal membrane to nonphysiologic solutions causes inflammation, ultimately leading to altered structure and function. Myofibroblasts, one of the cell types that contribute to dysfunction of the peritoneal membrane, can originate from mesothelial cells (MCs) by epithelial-to-mesenchymal transition (EMT), a process that has been associated with an increased rate of peritoneal transport. Because cyclooxygenase-2 (COX-2) is induced by inflammation, we studied the role of COX-2 in the deterioration of the peritoneal membrane. We observed that nonepithelioid MCs found in peritoneal effluent expressed higher levels of COX-2 than epithelioid MCs. The mass transfer coefficient for creatinine correlated with MC phenotype and with COX-2 levels. Although COX-2 was upregulated during EMT of MCs in vitro, COX-2 inhibition did not prevent EMT. In a mouse model of PD, however, COX-2 inhibition with Celecoxib resulted in reduced fibrosis and in partial recovery of ultrafiltration, outcomes that were associated with a reduction of inflammatory cells. Furthermore, PD fluid with a low content of glucose degradation products did not induce EMT or COX-2; the peritoneal membranes of mice treated with this fluid showed less worsening than mice exposed to standard fluid. In conclusion, upregulation of COX-2 during EMT may mediate peritoneal inflammation, suggesting COX-2 inhibition as a potential strategy to ameliorate peritoneal deterioration in PD patients.

Disproportionally low clearance of macromolecules from the plasma to the peritoneal cavity in a mouse model of peritoneal dialysis

This study was performed to establish a model for quantitative measurements of a number of basic peritoneal transport parameters, particularly transperitoneal clearances (Cl) of macromolecules, during mouse peritoneal dialysis. Mice were anaesthetized using 3% isofluorane inhalation anaesthesia. The right jugular vein and the left femoral artery were cannulated for infusion and sampling purposes and for registration of (mean) arterial blood pressure. Access to the peritoneal cavity occurred via a thin abdominal catheter (Ø 0.7 mm). About 2.5 ml of either 4% (n = 9) or 1.5% (n = 5) glucose containing PD-fluid were instilled intraperitoneally (i.p.). Dialysate volume was followed vs time using i.p. RISA ((125)I human serum albumin) as a volume marker, after correcting for RISA mass disappearance from the peritoneum, assessed separately (n = 11). Microsampling (10 microl) of plasma and dialysate was performed for determinations of glucose, haematocrit, radioactivity (RISA and (51)Cr-EDTA) and Ficoll. The i.p. volume vs time curves [V(D)(t)] were, after scaling, similar to those observed in humans (and in rats). Clearance of RISA out of the peritoneal cavity (Cl(out)) was 9.33 +/- 0.83 microl/min and the clearance of RISA to plasma (Cl-->P) and the RISA clearance to the peritoneal cavity (Cl-->D) were 1.49 +/- 0.13 and 0.084 +/- 0.008 microl/min, respectively. The peritoneal transport coefficients for (51)Cr-EDTA and glucose, as well as Cl(out) and Cl-->P, were 13-17% of those previously assessed in 300 g rats, whereas Cl-->D was only approximately 2% of that in rat. All peritoneal transport parameters measured, except Cl-->D, scaled very well to the corresponding human data. The mechanisms of the disproportionally low clearance of macromolecules from the plasma to the peritoneal cavity in mice remain elusive and warrant further study.

Effects of anaesthesia on fluid and solute transport in a C57BL6 mouse model of peritoneal dialysis

Genetically modified mice show promise as animal models for studying the physiology and pathophysiology of the peritoneum during peritoneal dialysis (PD). Methods for evaluation of the functional characteristics of the mouse peritoneum have not been studied extensively, and the effects of anaesthesia on fluid and solute transport in mouse models of PD are unknown. A single exchange of dialysis solution was performed in C57BL6 mice by injecting fluid into the peritoneal cavity using a 27-gauge needle and allowing fluid to dwell for 30, 60 or 120 min. Experiments evaluated the effect of ketamine (plus xylazine) anaesthesia on fluid and solute transport; these effects were examined in separate experiments using glucose and mannitol as the osmotic agent added to the injected dialysis solution. After euthanasia, blood was collected, the remaining dialysis solution was drained and their contents analysed for concentrations of the osmotic solute (glucose or mannitol), urea nitrogen (UN), sodium (Na) and a volume marker (fluorescein-labelled albumin) added to the initial, injected dialysis solution. Determined parameters included final volume of dialysis solution (drained plus residual fluid volume), dialysate concentration (D/D0) of glucose (or D/D0 mannitol), dialysate-to-plasma concentration ratio for (D/P) UN and D/P Na and the apparent dialysis solution volume by indicator dilution. Peritoneal permeability-area (PA) values or mass transfer-area coefficients were also calculated for the osmotic solutes. Final volumes of dialysis solution were higher when mice were anaesthetized with ketamine than in unanaesthetized mice, independent of whether glucose or mannitol was used as the osmotic agent. The increases in final volume were paralleled by higher dialysate concentrations (D/D0 values) and lower calculated PA values for both glucose and mannitol. When using either osmotic agent, anaesthesia also increased plasma glucose concentrations, suggesting that ketamine altered glucose metabolism. Ketamine anaesthesia in the mouse decreases PA values for glucose and mannitol when used as osmotic agents in PD solutions. The decrease in transperitoneal transport for these osmotic agents increases the final volume of fluid which can be obtained from the peritoneal cavity.

Mice that lack endothelial nitric oxide synthase are protected against functional and structural modifications induced by acute peritonitis.

Pharmacologic studies suggest that the release of nitric oxide (NO) by endothelial NO synthase (eNOS) contributes to functional alterations of the peritoneal membrane (PM) induced by acute peritonitis. In this study, peritoneal permeability parameters in a mouse model of peritoneal dialysis were characterized, and the effects of eNOS deletion on the PM structure and permeability at baseline and after catheter-induced bacterial peritonitis were examined. Exposure of C57BL/6 mice to standard dialysate yielded a transport of urea and glucose, a sodium sieving, and a net ultrafiltration that were remarkably similar to the values obtained in rats. In comparison with controls, mice with catheter-induced peritonitis were characterized by structural changes in the PM (mononuclear cells infiltrate, vascular proliferation), upregulation of endothelial and inducible NOS, increased permeability for urea and glucose, decreased ultrafiltration, and increased protein loss in the dialysate. Comparison of eNOS wild-type and knockout mice revealed that the permeability modifications and structural changes induced by acute peritonitis were significantly reversed in eNOS knockout mice, resulting in a net increase in ultrafiltration. In contrast, the deletion of eNOS in mouse peritoneum was not reflected by permeability modifications or structural changes at baseline. These results are the first to take advantage of a knockout mouse model to demonstrate directly the crucial importance of eNOS in the permeability and structural modifications caused by acute peritonitis. The characterization of this mouse model suggests that genetically modified mice represent useful tools to investigate the molecular bases of the peritoneal changes during peritoneal dialysis.