Involvement of Lymphocyte Infiltration in the Progression of Mouse Peritoneal Fibrosis Model

Peritoneal fibrosis is a serious complication in patients with severe chronic kidney disease who are undergoing peritoneal dialysis (PD). One of the pathological characteristics of peritoneal fibrosis is the infiltration of macrophages in the thickened submesothelial compact zone. In addition, infiltration of lymphocytes, including T and B lymphocytes, is observed in the fibrotic peritoneum. However, the relationship between lymphocyte infiltration and progression of peritoneal fibrosis remains unclear. In this study, we investigated the role of lymphocytes in the development of peritoneal fibrosis induced by chlorhexidine gluconate (CG) by comparing the histological changes observed in severe combined immunodeficient (SCID) mice (largely lacking functional T and B lymphocytes) with those observed in wild-type (WT) mice. As expected, CG-injected WT mice showed a thickening of the submesothelial compact zone together with massive collagen deposition accompanied by increased numbers of infiltrating macrophages and T and B lymphocytes. In the peritoneum of SCID mice, the submesothelial compact zone was thicker and the number of macrophages and B lymphocytes was significantly higher than that observed in control immunodeficient and WT mice. In contrast, the number of T lymphocytes in the peritoneum of SCID mice was significantly lower than that in the peritoneum of WT mice. These results suggest that T and B lymphocytes modulate the process of peritoneal fibrosis via macrophage infiltration.     

Pleiotrophin triggers inflammation and increased peritoneal permeability leading to peritoneal fibrosis

Long-term peritoneal dialysis induces peritoneal fibrosis with submesothelial fibrotic tissue. Although angiogenesis and inflammatory mediators are involved in peritoneal fibrosis, precise molecular mechanisms are undefined. To study this, we used microarray analysis and compared gene expression profiles of the peritoneum in control and chlorhexidine gluconate (CG)-induced peritoneal fibrosis mice. One of the 43 highly upregulated genes was pleiotrophin, a midkine family member, the expression of which was also upregulated by the solution used to treat mice by peritoneal dialysis. This growth factor was found in fibroblasts and mesothelial cells within the underlying submesothelial compact zones of mice, and in human peritoneal biopsy samples and peritoneal dialysate effluent. Recombinant pleiotrophin stimulated mitogenesis and migration of mouse mesothelial cells in culture. We found that in wild-type mice, CG treatment increased peritoneal permeability (measured by equilibration), increased mRNA expression of TGF-β1, connective tissue growth factor and fibronectin, TNF-α and IL-1β expression, and resulted in infiltration of CD3-positive T cells, and caused a high number of Ki-67-positive proliferating cells. All of these parameters were decreased in peritoneal tissues of CG-treated pleiotrophin-knockout mice. Thus, an upregulation of pleiotrophin appears to play a role in fibrosis and inflammation during peritoneal injury.     

TNP-470, an angiogenesis inhibitor, suppresses the progression of peritoneal fibrosis in mouse experimental model

BACKGROUND: In patients on long-term peritoneal dialysis (PD), angiogenesis and vasculopathy are observed in the peritoneum, and the degree of vascularization correlates with the area of fibrotic tissue, suggesting the involvement of angiogenesis in the progression of peritoneal fibrosis. The aim of the present study was to evaluate the effect of TNP-470, an anti-angiogenic compound, on the development of peritoneal fibrosis induced by chlorhexidine gluconate (CG). METHODS: Peritoneal fibrosis was induced by injection of CG into peritoneal cavity of Institute for Cancer Research (ICR) mice. TNP-470 was injected subcutaneously with CG. Mice were sacrificed, and peritoneal tissues were dissected out at days eight and 16 after CG and TNP-470 injection. The expression patterns of CD31 (as a marker of endothelial cells), vascular endothelial cell growth factor (VEGF), alpha-smooth muscle actin (as a marker of myofibroblasts), heat shock protein 47 (HSP47), type III collagen, F4/80 (as a marker of mice macrophages), proliferating cell nuclear antigen (PCNA), and cyclin-dependent kinase 2 (Cdk2) were examined by immunohistochemistry. RESULTS: CG-injected mice showed thickening of the submesothelial zone and increased number of vessels, myofibroblasts, and infiltrating macrophages. The expression levels of VEGF, type III collagen, and HSP47 were increased, and a large number of PCNA-positive cells and Cdk2-expressing cells were observed in the thickened submesothelial area. Treatment with TNP-470 suppressed the submesothelial zone thickening and reduced collagen III expression as well as angiogenesis. TNP-470 also decreased the number of VEGF-expressing cells, myofibroblasts, macrophages, PCNA-positive cells, and Cdk2-expressing cells. CONCLUSION: Our results indicate the involvement of angiogenesis in the progression of peritoneal fibrosis, and suggest that TNP-470 may be potentially useful for the prevention of peritoneal fibrosis through inhibition of angiogenesis and suppression of myofibroblast proliferation.     

Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease

Macrophage infiltration of the kidney is a prominent feature associated with the severity of renal injury and progressive renal failure. To determine the influence of macrophages in renal disease models in the absence of endogenous T and B cells, we performed adoptive transfer of macrophages into severe combined immunodeficient (SCID) mice. In this study, macrophages were isolated from the spleens of BALB/c mice and stimulated with lipopolysaccharide to induce classically activated M1 macrophages or with interleukin-4 (IL-4) and IL-13 to induce alternatively activated M2 macrophages. These macrophages were then infused into SCID mice with adriamycin nephropathy; an in vivo model of chronic inflammatory renal disease analogous to human focal segmental glomerulosclerosis. Mice infused with M1 macrophages had a more severe histological and functional injury, whereas M2 macrophage-induced transfused mice had reduced histological and functional injury. Both M1 and M2 macrophages localized preferentially to the area of injury and maintained their phenotypes even after 4 weeks. The protective effect of M2 macrophages was associated with reduced accumulation and possibly downregulated chemokine and inflammatory cytokine expression of the host infiltrating macrophages. Our findings demonstrate that macrophages not only act as effectors of immune injury but can be induced to provide protection against immune injury.     

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats

BACKGROUND: Peritoneal fibrosis is a serious complication in patients on continuous ambulatory peritoneal dialysis (CAPD), but the molecular mechanism of this process remains unclear. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules, and is expressed in the tissue of human peritoneal fibrosis. In the present study, we examined the effect of HSP47 antisense oligonucleotides (ODNs) on the development of experimental peritoneal fibrosis induced by daily intraperitoneal injections of chlorhexidine gluconate (CG). METHODS: HSP47 antisense or sense ODNs were injected simultaneously with CG from day 14, after injections of CG alone. Peritoneal tissue was dissected out 28 days after CG injection. The expression patterns of HSP47, type I and type III collagen, alpha-smooth muscle actin (alpha-SMA), as a marker of myofibroblasts, ED-1 (as a marker of macrophages), and factor VIII were examined by immunohistochemistry. RESULTS: In rats treated with CG alone, the submesothelial collagenous compact zone was thickened, where the expression levels of HSP47, type I and type III collagen and alpha-SMA were increased. Marked macrophage infiltration was also noted and the number of vessels positively stained for factor VIII increased in the CG-treated group. Treatment with antisense ODNs, but not sense ODNs, abrogated CG-induced changes in the expression of HSP47, type I and III collagen, alpha-SMA, and the number of infiltrating macrophages and vessels. CONCLUSION: Our results indicate the involvement of HSP47 in the progression of peritoneal fibrosis and that inhibition of HSP47 expression might merit further clinical investigation for the treatment of peritoneal fibrosis in CAPD patients.     

NK cells do not mediate renal injury in murine adriamycin nephropathy

In adriamycin nephropathy (AN), a model of chronic proteinuric renal injury, the absence of functional B and T cells with residual natural killer (NK) cells, and macrophages in severe combined immunodeficient (SCID) mice results in more severe disease than in immunocompetent mice. We have recently shown expression of the stimulatory NK cell molecule NKG2D and its ligand RAE-1 in the adriamycin (ADR) kidney. Therefore, we sought to determine the role of NK cells in AN. We used anti-asialo GM1 NK cell depletion in immunocompetent BALB/c mice with AN, and also compared AN in immunodeficient SCID mice and immunodeficient nonobese diabetic (NOD)-SCID mice (that have impaired NK cell function). The number of NK cells was increased in AN in BALB/c mice compared with normal controls. NK cell depletion or reduction of NK function in NOD-SCID mice did not affect the severity of disease. In both wild type and immunodeficient models, ADR upregulated RAE-1 in the kidney. High levels of Class I major histocompatibility complex molecules were found in both models of AN. In conclusion, NK cells do not play a significant role in AN.     

Inhibition of the interaction of AGE-RAGE prevents hyperglycemia-induced fibrosis of the peritoneal membrane

The peritoneal membrane of long-term peritoneal dialysis patients is characterized by a loss of ultrafiltration capacity, associated morphologically with submesothelial fibrosis and neoangiogenesis. Exposure to high glucose concentrations in peritoneal dialysate and the resultant advanced glycation end-products (AGE) accumulation have been implicated in the development of these changes, but their exact pathophysiological role is unknown. We examined the effect of the interaction of AGE with one of their receptors (i.e., RAGE) on the function and structure of the peritoneum exposed to high ambient glucose concentrations. Streptozotocin-induced diabetic rats and control rats were treated during 6 wk with either neutralizing monoclonal anti-RAGE antibodies or control antibodies. The expression of RAGE was strongly enhanced in the peritoneal membrane of the diabetic animals. The diabetic peritonea were characterized by an elevated transport of small solutes, lower ultrafiltration rates, a higher vascular density, and an upregulation of endothelial nitric oxide synthase expression. These parameters were unaffected by treatment with anti-RAGE antibodies. In contrast, anti-RAGE but not control antibodies prevented upregulation of TGF-beta, development of submesothelial fibrosis, and fibronectin accumulation in the peritoneum of diabetic animals. In conclusion, binding of AGE to RAGE increases the expression of TGF-beta and contributes to the development of submesothelial fibrosis. Neoangiogenesis and the resultant loss of ultrafiltration capacity are mediated by different pathogenetic pathways.     

Advanced glycation end-products and peritoneal sclerosis

Long-term continuous ambulatory peritoneal dialysis (CAPD) often causes peritoneal fibrosis and sclerosis with a loss of function, and some CAPD patients develop sclerosing encapsulating peritonitis. Glucose-based peritoneal dialysis fluids readily produce glucose degradation products by heat sterilization, and glucose degradation products accelerate the formation of advanced glycation end-products (AGE) in the peritoneal cavity. The accumulation of AGE is observed in peritoneal mesothelial and submesothelial layers in CAPD patients, accompanied by enhanced expression of various growth factors and peritoneal thickening. The expression of transforming growth factor-beta1 (TGF-beta1), macrophage-colony stimulating factor, and vascular endothelial growth factor (VEGF) is distributed in the peritoneum similarly to that of AGE. In CAPD patients with low ultrafiltration (UF) capacity, peritoneal membrane is thickened owing to an increase in the number of cells such as fibroblasts and macrophages and collagen in the submesothelial layer. AGE is detected in the fibroblasts and macrophages as well as degenerated collagen. These cells in the submucosal layer are almost positive for the receptor for AGE (RAGE) and uptake AGE. The intensity of AGE accumulation and the expression of growth factors are associated with the severity of UF impairment. In fact, the accumulation of AGE and the expression of growth factors are recognized most markedly in the peritoneum of CAPD patients with low UF and sclerosing encapsulating peritonitis. In conclusion, long-time CAPD with heat-sterilized peritoneal dialysis fluid promotes AGE accumulation in the peritoneal membrane and alteration in peritoneal cell function and dialysis quality, followed by peritoneal sclerosis, and, finally, sclerosing encapsulating peritonitis.     

T cells or active epstein-barr virus infection in the development of lymphoproliferative disease in human B cell-injected severe combined immunodeficient mice

Background: Severe combined immunodeficient (SCID) mice develop Epstein-Barr virus (EBV) containing human lymphoproliferative disease (LPD) tumors when reconstituted with human peripheral blood leukocytes (PBLs) from EBV-seropositive donors, but LPD tumors do not develop in the presence of immunosuppressive agents, such as cyclosporine A or corticosteroids. Methods: Therefore, LPD development in SCID mice was used as a model to explore the relationship among B cells, T cells, and EBV in vivo. SCID mice were engrafted with PBLs isolated by leukapheresis from a single EBV-seropositive donor. Purified populations of CD3⁺ lymphocytes (T cells) or CD19⁺ lymphocytes (B cells) were isolated and engrafted into SCID mice. Results: SCID mice engrafted with purified CD3⁺ lymphocytes (T cells) or CD19⁺ lymphocytes (B cells) did not develop LPD. In contrast, mice engrafted with purified B cells developed LPD if they were co-engrafted with purified T cells or if they were inoculated with infections EBV. Conclusions: This study confirms the requirement of T cells or active EBV infection in the development of LPD in animals engrafted with B cells latently infected with EBV. A greater understanding of the cellular and viral interactions leading to transformation and malignancy may allow the development of specific interventional therapies for malignancies in the immunosuppressed host. Presented at the 46th Annual Cancer Symposium of The Society of Surgical Oncology, Los Angeles, California, March 18–21, 1993.     

Activation of immunocompetent cells in the peritoneum of patients treated with CAPD

Chemiluminescence and flow cytometry, employing a number of monoclonal antibodies, was used to investigate activation of immunocompetent cells in the blood and peritoneum of patients treated with CAPD. Increased expression of HLA-DR and RFD7, both markers of macrophage maturation, was observed on peritoneal macrophages, 78% and 33.5% cells positive, when compared to blood monocytes, which were 46% and 5.3%, respectively (P less than 0.001). Macrophage chemiluminescent response to opsonized zymosan was greater than that of circulating blood monocytes in CAPD patients, whereas the inverse was true for normal controls. Enhanced expression of IL-2 receptor and surface IgG by peritoneal macrophages were 24.9% and 65.3% cells positive compared with monocytes, 5.7% and 12.3% (P less than 0.01), and also suggests their activation. There was a marked increase in the HLA-DR expression by peritoneal lymphocytes from CAPD patients (32% cells positive) compared with those from CAPD blood (13.8%), normal blood (11.3%) and normal peritoneal fluid (14.5%), P less than 0.001. This was partially accounted for by increased numbers of B cells (18%) and activated T cells bearing HLA-DR. TFR and IL-2 receptor expression by CAPD peritoneal lymphocytes was similar to that of blood lymphocytes, implying the lack of an organized immune response within the peritoneum. Taken together, these results suggest that peritoneal macrophages from CAPD treated patients have features of maturation and activation, while changes in the lymphocyte populations are compatable with the actions of IL-1, indicating activity of the cellular immune system within the peritoneum.     

IL-25 induces M2 macrophages and reduces renal injury in proteinuric kidney disease

The kidney contains receptors for the cytokine IL-25, but the effects of IL-25 in CKD are unknown. Here, we induced adriamycin nephropathy in both BALB/c mice and severe combined immunodeficient (SCID) mice, and we injected IL-25 for 7 consecutive days starting at day 5 after adriamycin administration. BALB/c mice treated with IL-25 had less glomerulosclerosis, tubular atrophy, interstitial expansion, and proteinuria than control mice at day 28. IL-25 increased the levels of IL-4 and IL-13 in serum, kidney, renal draining lymph nodes, and CD4+ lymphocytes. IL-25 also directly suppressed effector macrophages in vitro and in vivo and induced alternatively activated (M2) macrophages in vivo. However, in SCID mice and in BALB/c mice treated with IL-4/13-neutralizing antibody, IL-25 failed to protect against renal injury and did not induce M2. In conclusion, IL-25 protects against renal injury in adriamycin nephropathy in mice by, at least in part, inducing Th2 immune responses.     

Host defences in continuous ambulatory peritoneal dialysis and the genesis of peritonitis

Continuous ambulatory peritoneal dialysis (CAPD) has come to be extensively used for the treatment of end-stage renal failure in children, and especially infants, such that now more than half of children on dialysis worldwide receive treatment by this means. Peritonitis, however, is commoner in children than in adults receiving treatment, and is a major source of morbidity and treatment failure in children started on CAPD. Only recently has the immunology of the normal peritoneum been studied extensively, with the need to assess the impact of the installation of large volumes of fluid into the peritoneal sac during dialysis. The main phagocytic defences of the peritoneum depend upon a unique set of macrophages which are present free in the peritoneal fluid but also in the submesothelium and in perivascular collections together with B lymphocytes in the submesothelial area. Both the number of macrophages per unit volume and the concentration of opsonic proteins, such as IgG, complement and fibronectin, are reduced to between only 1% and 5% when dialysis fluid is continuously present in the peritoneal sac. In addition, the fluids used for CAPD are toxic to both macrophages and to mesothelial cells. Thus minor degrees of contamination frequently lead to peritonitis and in addition the majority of patients have catheters inserted in their peritoneum which become colonised with organisms capable of producing exopolysaccharide (slime), which promotes adhesion of the organism to the plastic and protects them against phagocytic attack and the penetration of antibiotics. Thus the peritoneum is in a state of continual inflammation, as well as being a markedly more vulnerable site than the normal peritoneum to the entry of organisms. Whether clinical peritonitis appears in this state of chronic contamination probably depends on perturbation in the balance between host defences and the organism. WhilstStaphylococcus epidermidis is the commonest, cause of peritonitis,Staphylococcus aureus and Gram-negative organisms are much more serious and more frequently lead either to temporary catheter removal or discontinuation of dialysis altogether. This review describes, the peritoneal defences in relation to the genesis of peritonitis.Based in part on an address given to the European Society of Paediatric Nephrology in Heidelberg, Germany, October 1993     

Morphologic changes in the peritoneal membrane of patients with renal disease.

This study examined the morphologic features of the parietal peritoneal membranes of 130 patients undergoing peritoneal dialysis (PD) and compared them with the features of the peritoneal membranes of normal individuals, uremic predialysis patients, and patients undergoing hemodialysis. The median thickness of the submesothelial compact collagenous zone was 50 microm for normal subjects, 140 microm for uremic patients, 150 microm for patients undergoing hemodialysis, and 270 microm for patients undergoing PD (P < 0.001 for all versus normal subjects). Compact zone thickness increased significantly with the duration of PD therapy [0 to 24 mo, 180 microm (n = 58); 25 to 48 mo, 240 microm (n = 24); 49 to 72 mo, 300 microm (n = 13); 73 to 96 mo, 750 microm (n = 16); >97 mo, 700 microm (n = 19)]. Vascular changes included progressive subendothelial hyalinization, with luminal narrowing or obliteration. These changes were absent in samples from normal subjects but were present in 28% of samples from uremic patients and 56% of biopsies from patients undergoing PD. In the PD group, the prevalence of vasculopathy increased significantly with therapy duration (P = 0.0001). The density of blood vessels per unit length of peritoneum was significantly higher for patients with membrane failure and was correlated with the degree of fibrosis (P = 0.01). For the first time, a comprehensive cross-sectional analysis of the morphologic changes in the peritoneal membranes of patients undergoing PD is provided. The infrequency of fibrosis in the absence of vasculopathy suggests that vasculopathy may predispose patients to the development of fibrosis. This study provides a sufficiently large cohort of samples to allow structure-function relationships to be established, as well as providing a repository of tissue for further studies.     

Interstitial Fibrosis Restricts Osmotic Water Transport in Encapsulating Peritoneal Sclerosis

Encapsulating peritoneal sclerosis (EPS) is a rare but severe complication of peritoneal dialysis (PD) characterized by extensive fibrosis of the peritoneum. Changes in peritoneal water transport may precede EPS, but the mechanisms and potential predictive value of that transport defect are unknown. Among 234 patients with ESRD who initiated PD at our institution over a 20-year period, 7 subsequently developed EPS. We evaluated changes in peritoneal transport over time on PD in these 7 patients and in 28 matched controls using 3.86% glucose peritoneal equilibration tests. Compared with long-term PD controls, patients with EPS showed early loss of ultrafiltration capacity and sodium sieving before the onset of overt EPS. Multivariate analysis revealed that loss of sodium sieving was the most powerful predictor of EPS. Compared with long-term PD control and uremic peritoneum, EPS peritoneum showed thicker submesothelial fibrosis, with increased collagen density and a greater amount of thick collagen fibers. Reduced osmotic conductance strongly correlated with the degree of peritoneal fibrosis, but not with vasculopathy. Peritoneal fibrosis was paralleled by an excessive upregulation of vascular endothelial growth factor and endothelial nitric oxide synthase, but the expression of endothelial aquaporin-1 water channels was unaltered. Our findings suggest that an early and disproportionate reduction in osmotic conductance during the course of PD is an independent predictor of EPS. This functional change is linked to specific alterations of the collagen matrix in the peritoneal membrane of patients with EPS, thereby validating the serial three-pore membrane/fiber matrix and distributed models of peritoneal transport.     

Transient overexpression of TGF-{beta}1 induces epithelial mesenchymal transition in the rodent peritoneum

Epithelial mesenchymal transition (EMT), a process involved in many growth and repair functions, has been identified in the peritoneal tissues of patients who undergo peritoneal dialysis. The sequence of changes in gene regulation and cellular events associated with EMT after TGF-beta1-induced peritoneal fibrosis is reported. Sprague-Dawley rats received an intraperitoneal injection of an adenovirus vector that transfers active TGF-beta1 (AdTGF-beta1) or control adenovirus, AdDL. Animals were killed 0 to 21 days after infection. Peritoneal effluent and tissue were analyzed for markers of EMT. In the animals that were treated with AdTGF-beta1, an increase in expression of genes associated with EMT and fibrosis, such as type I collagen A2, alpha-smooth muscle actin, and the zinc finger regulatory protein Snail, was identified. Transition of mesothelial cells 4 to 7 d after infection, with appearance of epithelial cells in the submesothelial zone 7 to 14 d after exposure to AdTGF-beta1, was demonstrated. This phase was associated with disruption of the basement membrane and increased expression of matrix metalloproteinase 2. By 14 to 21 d after infection, there was evidence of restoration of normal submesothelial architecture. These findings suggest that EMT occurs in vivo after TGF-beta1 overexpression in the peritoneum. Cellular changes and gene regulation associated with EMT are evident throughout the fibrogenic process and are not limited to early time points. This further supports the central role of TGF-beta1 in peritoneal fibrosis and provides an important model to study the sequence of events involved in TGF-beta1-induced EMT.     

Evidence that specific cellular immunity cannot account for death of Mus caroli embryos transferred to Mus musculus with severe combined immune deficiency disease

Mus caroli blastocysts transferred to pseudopregnant Mus musculus implant but die about midgestation. Death of the M caroli embryos is associated with infiltration of the fetoplacental units by M musculus lymphocytes, including functional cytotoxic T lymphocytes (CTL). To determine if specific maternal antifetal immune responses are responsible for the initiation of M caroli failure, survival of M caroli embryos transferred to M musculus with severe combined immune deficiency disease (SCID) was studied. C.B.-17 scid mice, homozygous for the scid gene, are reported to be unresponsive to T cell or B cell mitogens and to alloantigens. We have evaluated responses of C.B.-17 scid mice to M caroli xenoantigens in vivo and in vitro. C.B.-17 scid fail to reject M caroli skin grafts, and their spleen cells do not proliferate when mixed with M caroli splenocytes in a 1-way mixed leukocyte culture. Further, neither primary nor secondary challenge of C.B.-17 scid footpads with M caroli splenocytes elicits a delayed-type hypersensitivity (DTH) response. These results suggest that C.B.-17 scid mice are unable to recognize or respond specifically to M caroli xenoantigens. M caroli embryos transferred to C.B.-17 scid mice implant but fail to survive while cotransferred M musculus embryos are normal. Histologic examination of resorbing embryos revealed granulocytes, macrophages, large granular lymphocytes, but very few small lymphocytes at the implantation sites. Thus, it appears that specific maternal antifetal immune responses are not responsible for initiation of M caroli embryo failure in the M musculus uterus. These studies also indicate that efficient resorption of embryos does not require the function of T cells or B cells.     

Notch通路在高浓度葡萄糖腹膜透析液所致大鼠腹膜纤维化模型中的活化

目的 观察Notch通路在高浓度葡萄糖透析液所致大鼠腹膜纤维化模型中的变化并探讨其可能机制。 方法 给予SD雄性大鼠每日腹腔注射高浓度葡萄糖腹膜透析液，于实验后2周和4周杀检。取壁层腹膜组织行光镜检查；免疫印迹检测转化生长因子β1 （TGF-β1）、 E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA) 和I型胶原蛋白（Col I）的表达；RT-PCR检测Notch通路的下游靶基因Hes-1的表达；免疫印迹和RT-PCR检测Notch配体Jagged-1和Notch通路的负性调节因子Numb的表达。 结果 HE染色显示模型组腹膜明显增厚，间皮细胞减少；Masson染色显示壁层腹膜中可见明显的胶原沉积。与健康对照组相比，模型组的TGF-β1、α-SMA 和 Col I的表达增加而E-cadherin的表达下降（均P < 0.01）。4周模型组与对照组相比Jagged-1表达明显增加（P < 0.05），同时Hes-1的表达亦明显增加（P < 0.01），而Notch通路的负性调节因子Numb的表达下降（P < 0.01）。 结论 在高浓度葡萄糖腹膜透析液所致的大鼠腹膜纤维化模型中有Notch通路的活化，而该通路的活化可能与Notch通路的负性调节因子Numb表达的下调有关。高表达Notch通路的负性调节因子，如Numb，可能是治疗腹膜透析患者腹膜纤维化的新途径。     

Smad7 transgene attenuates peritoneal fibrosis in uremic rats treated with peritoneal dialysis

Transforming growth factor beta (TGF-beta) plays a critical role in the pathogenesis of the peritoneal fibrosis that complicates long-term peritoneal dialysis (PD). We studied the TGF-beta/Smad signaling pathway in peritoneal fibrosis induced in uremic rats treated with PD and explored the therapeutic potential of Smad7 to prevent fibrogenesis. After subtotal nephrectomy, uremic rats were treated with peritoneal dialysis using 4.25% dextrose-containing fluid. The peritoneum of uremic rats treated with PD demonstrated fibrosis, increased TGF-beta expression, increased Smad2/3 activation, decreased Smad7 expression, and increased expression of fibrogenic and angiogenic factors. In addition, peritoneal function was impaired and its structure was altered, including a thickened submesothelial layer. In rats transfected with a Smad7 transgene using an ultrasound-microbubble-mediated system, peritoneal fibrosis was attenuated, peritoneal function was improved, and Smad2/3 activation was inhibited. We suggest that administration of Smad7 inhibits peritoneal fibrogenesis in uremic rats treated with PD by correcting the imbalance between downregulated Smad7 and activated Smad2/3. Blockade of the TGF-beta/Smad signaling pathway may represent a novel therapeutic approach to prevent peritoneal fibrosis in patients treated with PD.     

The effect of cyclosporine on macrophage oxidative burst potential during graft-versus-host reactions in mice

The development of graft-versus-host reactions in mice was characterized by an increase in activated macrophage populations in the peritoneum and spleen of the animals. In the present study we tested the effect of the immunosuppressive agent CsA on the appearance and activity of such macrophages. Parental spleen lymphocytes were injected intraperitoneally into F1 hybrids (BALB/c x C57Bl/6), and 13 days following injection we monitored the number of peritoneal exudate cells (PEC), spleen enlargement, and the oxidative burst (OB) of adherent peritoneal macrophages (APM). Macrophage OB was assessed by measuring O2- and H2O2 production, following stimulation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In GVHR mice a 72% increase in their spleen to body weight ratio was observed, whereas in CsA-treated GVHR mice only a 32% increase was evident. Assessment of the effect of CsA on the number and function of peritoneal cells revealed that CsA caused a 73% reduction in the number of infiltrating PEC in GVHR mice. Furthermore, measurements of H2O2 and O2- production by APM revealed that the overall OB capacity of APM from CsA-treated mice was significantly reduced compared to APM from nontreated GVHR mice. CsA had no effect on the number and OB activity of paraffin oil elicited or resident PEC. These results indicate that CsA may prevent recall and activation of macrophages via its effect on T cell lymphokine release and thus may lessen the contribution of macrophage-derived toxic reagents to the damage inflicted by GVHR.