Ichiro Hirahara

Methylglyoxal induces peritoneal thickening by mesenchymal-like mesothelial cells in rats

BACKGROUND The epithelial-to-mesenchymal transition (EMT) of mesothelial cells was observed in patients on peritoneal dialysis and may be involved in peritoneal thickening. Conventional peritoneal dialysis fluids (PDFs) that contain glucose degradation products (GDPs), such as methylglyoxal (MGO) and formaldehyde (FA), are bioincompatible. The aim of this study is to analyse the participation of EMT in peritoneal thickening induced by GDPs in rats. METHODS Rat mesothelial cells were cultured with various GDPs, and the gene expression of Snail was analysed by polymerase chain reaction (PCR). Sprague-Dawley rats were administered intraperitoneally 20 mM MGO/PDFs, 20 mM FA/PDFs or 0.1% chlorhexidine gluconate (CHX)/15% ethanol/saline every day for 21 days. On Day 22, the expression of transforming growth factor-beta (TGF-beta), collagen 1, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), Snail and receptor for advanced glycation end-products (RAGE) was analysed by PCR, enzyme-linked immunoassay or immunohistological staining. RESULTS In cell-culture experiments, the expression of Snail was enhanced by MGO, but not FA. In rats treated with 20 mM MGO, peritoneal fibrous thickening with the proliferation of mesenchymal-like mesothelial cells was observed. The expression of TGF-beta, collagen 1, MMP-2, VEGF, Snail and RAGE increased significantly (P < 0.01). In FA- or CHX-treated rats, the peritoneum was thickened with sparse collagen fibres, but mesenchymal-like mesothelial cells were not observed. CONCLUSIONS MGO induced peritoneal fibrous thickening with the proliferation of mesenchymal-like mesothelial cells in vivo. These cells may be transdifferentiated from mesothelial cells by EMT via Snail and play an important role in peritoneal fibrous thickening.

Serial analysis of matrix metalloproteinase-2 in dialysate of rat sclerosing peritonitis models

AbstractBackground. Sclerosing peritonitis (SP) is a serious complication of continuous ambulatory peritoneal dialysis (CAPD). In order to carry out CAPD safely, it is important to analyze the development of SP. Methods. We prepared animal models of SP by the intraperitoneal administration of chlorhexidine gluconate (CHX) or talc, using male Sprague-Dawley rats. The peritoneal equilibration test and histological examinations were performed in the model rats, and dialysate drained from them was analyzed by gelatin zymography. Results. In the two types of SP animal models, matrix metalloproteinase-2 (MMP-2) level in dialysate correlated with the changes of inflammation, thickness of peritoneum, D/D0 glucose level (glucose level of drained dialysate obtained 90 min after the injection of 2.5% glucose containing peritoneal dialysis fluid divided by that obtained immediately after the injection), and net ultrafiltration. Conclusions. From these results, MMP-2 level in drained dialysate was considered to change with the development and progression of SP in rat models. Thus, MMP-2 has potential as a diagnostic marker for SP.

Captopril directly inhibits matrix metalloproteinase-2 activity in continuous ambulatory peritoneal dialysis therapy

BACKGROUND Matrix metalloproteinase (MMP)-2 plays an important role in tissue remodeling related to inflammation during continuous ambulatory peritoneal dialysis (CAPD) therapy. But its inhibitors were not applied clinically. We determined whether an angiotensin-converting enzyme (ACE) inhibitor, captopril, inhibits MMP-2 activity in peritoneal effluents from patients on CAPD, and simulated molecular models of the MMP-2-captopril complex. METHODS The inhibitory effect of captopril on MMP-2 activity was measured in peritoneal effluents from 17 patients on CAPD. Molecular models of the MMP-2-captopril complex were simulated by 1000 iterations of random docking and energy minimization. RESULTS Captopril directly inhibited MMP-2 activity in peritoneal effluents from patients on CAPD (IC50; 48 micromol/l), and that captopril binding to the MMP-2 active site could be formed in each complex model without molecular distortion. CONCLUSION ACE inhibitors, such as captopril, may be applied as important compounds for MMP-2 inhibition in inflammation caused by CAPD.

Increase of matrix metalloproteinase‐2 in dialysate of rat sclerosing encapsulating peritonitis model

SUMMARY: Sclerosing peritonitis (SP) and sclerosing encapsulating peritonitis (SEP) are serious complications of continuous ambulatory peritoneal dialysis (CAPD). the mortality rate of SP/SEP is extremely high. It is important to clarify the mechanism of progression of SP/SEP, and to prevent this complication. We prepared an animal model of SEP by intraperitoneal administration of chlorhexidine gluconate (CHX) using male Sprague‐Dawley rats. Dialysate drained from these animals was analysed by gelatin zymography. In this animal model of SEP, fibrous peritoneal thickening accompanied by cellular infiltration and peritoneal adhesion, were observed. Four of six rats presented with a so‐called abdominal cocoon. an increase of peritoneal absorption of glucose was also confirmed. Zymographic analysis revealed that the matrix metalloproteinase‐2 (MMP‐2) level was high in the dialysate from the animal model, although MMP‐9 was hardly detected. From these results, the MMP‐2 level in drained dialysate was considered to increase in SP/SEP. Matrix metalloproteinase‐2 might be associated with the progression of SP/SEP.

A case of encapsulating peritoneal sclerosis at the clinical early stage with high concentration of matrix metalloproteinase-2 in peritoneal effluent.

In encapsulating peritoneal sclerosis (EPS), matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases are involved in the remodeling of peritoneal tissue. We measured the MMP-2 concentration in the peritoneal effluents of a patient with EPS who discontinued continuous ambulatory peritoneal dialysis (CAPD) therapy because of ultrafiltration failure and/or underdialysis. First, we report a 58-year-old female patient who discontinued CAPD therapy because of underdialysis. Several months after cessation of CAPD, she complained of slightly blood-colored ascites and had an elevated level of C-reactive protein (CRP) in plasma. She was diagnosed as having clinical early-stage EPS. Peritoneal effluents drained from this case, and from 11 patients who discontinued CAPD therapy because of ultrafiltration failure and/or underdialysis, and who underwent peritoneal lavage with 1.5% dextrose peritoneal dialysis fluid for several months, were analyzed by gelatin zymography. MMP-2 concentration was also measured by enzyme-linked immunosorbent assay (ELISA). MMP-2 concentration in peritoneal effluent of this patient was highest compared with that of the other patients. There was some tendency of a positive correlation between MMP-2 concentration per 1 g protein and D/Pcr, and was negative correlation between MMP-2 concentration per 1 g of protein and D/D0 glucose. We concluded that MMP-2 is involved in the peritoneal remodeling of long-term CAPD therapy and the progression of EPS.

Matrix metalloproteinase 2 induces epithelial-mesenchymal transition in proximal tubules from the luminal side and progresses fibrosis in mineralocorticoid/salt-induced hypertensive rats

Objectives Excess mineralocorticoids such as deoxycorticosterone acetate (DOCA) together with salt are known to cause tubulointerstitial fibrosis, but the mechanisms underlying fibrosis progression are unclear. Therefore, we investigated the role of matrix metalloproteinase 2 (MMP2) in the epithelial-mesenchymal transition and fibrosis progression. Methods Uninephrectomized rats drank 0.9% NaCl and 0.3% KCl solution and were treated with DOCA alone, DOCA + spironolactone, or vehicle for 1, 4, or 8 weeks. SBP, kidney function and morphology, and kidney and urine MMP2 activity were compared among the groups. Results At week 4, the DOCA-treated group exhibited hypertension, tubulointerstitial fibrosis, increased MMP2 activity in the kidney and urine, and overexpression of MMP2 in proximal tubule cells and MMP14 in apical membranes; these results were more pronounced at week 8. At week 8, the proximal tubule cell apicolateral surface proteins villin, claudin 2, and E-cadherin were downregulated, and the mesenchymal marker &agr;-smooth muscle actin was upregulated in the tubulointerstitium of DOCA-treated rats. These DOCA/salt-induced changes (except for hypertension) and fibrosis progression observed at week 8 were reversed by TISAM (a selective MMP2 inhibitor), which was administered from week 4 to week 8. All of the effects of DOCA/salt at week 8 were attenuated by spironolactone. Conclusion Eight weeks of treatment with DOCA/salt activated MMP2, primarily on the apical surface of proximal tubule cells, which induced epithelial-mesenchymal transition from the luminal side and promoted tubulointerstitial fibrosis progression. These MMP2-induced changes occurred via downstream processes regulated by mineralocorticoid receptors.

Neutral-pH peritoneal dialysis solution improves peritoneal function and decreases matrix metalloproteinase-2 (MMP-2) in patients undergoing continuous ambulatory peritoneal dialysis (CAPD)

BackgroundConventional lactate-buffered peritoneal dialysis (PD) solutions have several bioincompatible characteristics, including acidic pH, lactate buffer, and the presence of glucose degradation products (GDPs), and these characteristics contribute to membrane dysfunction in PD patients. The formation of GDPs can be reduced by separating the glucose component of the solution from the lactate component during sterilization. This study was carried out to evaluate the clinical effect of dual-chambered neutral-pH PD solution in patients on continuous ambulatory peritoneal dialysis (CAPD).MethodsThirteen CAPD patients using conventional PD solution were enrolled in this study. The fast peritoneal equilibration test (fast PET) was performed periodically before and after treatment with neutral PD solution. The concentration of matrix metalloproteinase-2 (MMP-2) in dialysate effluent was measured using 4-h dwelling 2.5% glucose dialysis solution. The patients were categorized into two groups, according to the value of the initial dialysate/plasma (D/P) creatinine ratio: i.e., lower transporters (group L, D/PCr < 0.65) and higher transporters (group H, D/PCr ≥ 0.65).ResultsThe mean D/P creatinine ratio measured by fast PET, was significantly decreased (0.72 ± 0.09 to 0.60 ± 0.06; P < 0.03) after treatment with neutral PD solution in group H. The mean glucose level in 4-h dwelling dialysate effluent was elevated (824.6 ± 195.9 mg/dl to 942.6 ± 147.8 mg/dl; P < 0.022) in all subjects. In group H, a significant decrease of MMP-2 in the dialysate effluent was recognized from 15 months after the beginning of treatment with the neutral PD solution (141.4 ± 52.5 ng/ml to 91.3 ± 15.1 ng/ml; P < 0.05), with the lowest value being shown at 21 months (80.0 ± 31.8 ng/ml; P < 0.03).ConclusionsNeutral-pH peritoneal dialysis solution decreased the MMP-2 level in dialysate and improved peritoneal function in high-transporter patients with CAPD treatment.

Peritoneal Fibrosis Induced by Intraperitoneal Methylglyoxal Injection: The Role of Concurrent Renal Dysfunction

Background: Peritoneal fibrosis (PF) is a serious pathophysiology of peritoneal dialysis (PD). An ongoing focus of research is the potential fibrogenic nature of methylglyoxal (MGO) in conventional PD fluid (PDF). The aim of the current study was to explore the effects of the uremic milieu on the promotion of PF by MGO using rats with adenine-induced renal failure (RF). Methods: Adenine-treated Sprague-Dawley rats were randomly assigned to receive continuous peritoneal injections of PDF with or without MGO for three weeks or were left untreated for the same duration. Rats without RF were also assigned to three groups. The peritoneal histology and expression levels of type I collagen, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (αSMA), Snail, matrix metalloproteinase-2 (MMP-2), advanced glycation end-products (AGEs) and the receptor for AGE (RAGE) were then analyzed. Results: Peritoneal treatment with 5 mM MGO accelerated the fibrous peritoneal thickening progression promoted by exposure to standard PDF in the rats with RF, but not in the rats with a normal renal function. Treatment with MGO significantly augmented the proliferation of mesenchymal-like mesothelial cells, accumulation of AGE, de novo expression of αSMA and RAGE and gene expression of type I collagen, TGF-β1, Snail and MMP-2, whereas both MGO and RF alone had, at most, marginal effects on the changes in these biological parameters. Conclusions: In the present study, the adverse effects of MGO on the peritoneum became more prominent under conditions of a uremic milieu. These findings imply that MGO and uremia act cooperatively to induce PF.

Attenuation of methylglyoxal-induced peritoneal fibrosis: immunomodulation by interleukin-10.

Peritoneal fibrosis (PF), a serious pathophysiology of peritoneal dialysis (PD), is implicated in various types of chronic inflammation. In the present study, we examined the benefits of interleukin (IL)-10, which exerts anti-inflammatory effects, in an experimental rat model of methylglyoxal (MGO)-induced PF. We injected an adeno-associated virus (AAV) vector encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male Sprague-Dawley rats at 6 weeks of age. Four weeks later, the rats received continuous peritoneal injections of conventional PD fluid (PDF) with MGO for 3 weeks. Then, the peritoneal histology and the expression levels of fibrogenic mediators and proinflammatory cytokines were analyzed. The rats demonstrating persistent IL-10 expression showed significantly reduced fibrous peritoneal thickening compared with those with GFP expression. The infiltration of macrophages, the expression of tumor necrosis factor-α, IL-1β, IL-6, transforming growth factor-β1, Snail, and matrix metalloproteinase 2 genes as well as the proliferation of mesenchymal-like mesothelial cells augmented by MGO were all significantly suppressed by IL-10 expression. IL-10 also abrogated the extent of MGO-induced bowel adhesions mimicking a cocoon-like mass. Our findings provide valuable insight into the potential benefit of immunomodulation with IL-10 as one potentially effective therapeutic strategy for preventing the onset of peritoneal injury resulting in PF.

Methylglyoxal Induced Basophilic Spindle Cells with Podoplanin at the Surface of Peritoneum in Rat Peritoneal Dialysis Model

Peritoneal dialysis (PD) is a common treatment for patients with reduced or absent renal function. Long-term PD leads to peritoneal injury with structural changes and functional decline. At worst, peritoneal injury leads to encapsulating peritoneal sclerosis (EPS), which is a serious complication of PD. In order to carry out PD safely, it is important to define the mechanism of progression of peritoneal injury and EPS. We prepared rat models of peritoneal injury by intraperitoneal administration of glucose degradation products, such as methylglyoxal (MGO) or formaldehyde (FA), chlorhexidine gluconate (CG), and talc. In rats treated with MGO, peritoneal fibrous thickening with the appearance of basophilic spindle cells with podoplanin, cytokeratin, and α-smooth muscle actin at the surface of the peritoneum was observed. These cells may have been derived from mesothelial cells by epithelial-to-mesenchymal transition. In FA- or CG-treated rats, the peritoneum was thickened, and mesothelial cells were absent at the surface of the peritoneum. The CG- or MGO-treated rats presented with a so-called abdominal cocoon. In the talc-treated rats, extensive peritoneal adhesion and peritoneal thickening were observed. MGO-induced peritoneal injury model may reflect human histopathology and be suitable to analyze the mechanism of progression of peritoneal injury and EPS.