Expression of heat shock proteins 47 and 70 in the peritoneum of patients on continuous ambulatory peritoneal dialysis

BACKGROUND: Peritoneal sclerosis, characterized by collagen accumulation, is a serious complication in continuous ambulatory peritoneal dialysis (CAPD) therapy. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperon and is closely associated with collagen synthesis. METHODS: We determined the expression of HSP47 and HSP70 (nonspecific for collagen synthesis) by immunohistochemistry in peritoneal tissues of patients on CAPD. The tissue for collagen III, alpha-smooth muscle actin (alpha-SMA), and CD68 (a marker for macrophages) were also stained. Thirty-two peritoneal samples were divided into three groups (group A1, 11 patients who had no ultrafiltration loss; group A2, 9 patients who had ultrafiltration loss; and group B, 12 specimens who had end-stage renal disease prior to induction of CAPD. RESULTS: In group B, staining for HSP47, HSP70, and collagen III in peritoneal tissues was faint, and only a few cells were positive for alpha-SMA and CD68. In contrast, HSP47, HSP70, and collagen III were expressed in areas of thickened connective tissues in fibrotic peritoneal specimens of CAPD patients. The expression level of HSP47, HSP70, collagen III, and alpha-SMA and the number of CD68-positive cells in group A2 were significantly higher than those in groups A1 and B. HSP47/HSP70-positive cells were mesothelial cells, adipocytes, and alpha-SMA-positive myofibroblasts. Furthermore, the expression level of HSP47 was significantly higher in peritoneal specimens from patients with refractory peritonitis than without it and was significantly higher in patients with more than 60 months of CAPD therapy than that in patients with less than 60 months of CAPD. CONCLUSION: Our results indicate that CAPD therapy may induce HSPs in the peritoneal tissue, and that peritonitis in CAPD patients may be associated with the progression of peritoneal sclerosis at least through HSP47 expression and chronic macrophage infiltration. Our data also suggest that the progression of peritoneal sclerosis in such patients is associated with deterioration of peritoneal ultrafiltration function.     

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.     

Interstitial expression of heat shock protein 47 and alpha-smooth muscle actin in renal allograft failure.

BACKGROUND: Tubulointerstitial inflammation and fibrosis are the main pathological features of chronic renal allograft rejection, which is characterized by accumulation of extracellular matrix protein. Heat shock protein 47 (HSP47), known as a collagen-specific stress protein, is thought to be a molecular chaperone during the processing and/or secretion of procollagen. HSP47 is thought to be involved in the progression of fibrosis, but its expression in chronic renal allograft rejection is still unknown. METHODS: We examined the expression of HSP47 together with that of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts, and CD68, a marker of macrophages, by immunohistochemistry in allograft kidney tissues. Uninvolved portions of surgically removed kidneys with tumours served as control tissue. RESULTS: Expression of HSP47 was detected in the interstitium of fibrotic regions of allograft kidneys. Cells positive for HSP47 were also stained for alpha-SMA and type I collagen, and the expression of HSP47 correlated with the degree of interstitial fibrosis. Furthermore, the expression of HSP47 correlated with the number of infiltrating macrophages. In contrast, HSP47 and alpha-SMA were not expressed in the control tissues, sections of 1 h post-transplantation biopsy specimens and acute allograft rejection without fibrosis. CONCLUSION: Our results suggest that HSP47 may contribute to the progression of interstitial fibrosis in allograft renal tissues.     

Diltiazem suppresses collagen synthesis and IL-1beta-induced TGF-beta1 production on human peritoneal mesothelial cells.

BACKGROUND: After long-term treatment with continuous ambulatory peritoneal dialysis (CAPD), some patients may develop peritoneal fibrosis. Peritoneal mesothelial cells (PMCs) participate in the inflammatory reactions in the peritoneal cavity, and transforming growth factor-beta1 (TGF-beta1) and interleukin-1beta (IL-1beta) are involved in peritoneal fibrosis. Diltiazem is used frequently in patients with CAPD to treat hypertension. The objectives of this study were to examine the effects of diltiazem on collagen- and IL-1beta-induced TGF-beta1 production on human PMCs and the signalling pathway of diltiazem in this induction. METHODS: Human PMCs were cultured from the enzymatic disaggregation of human omentum. Collagen synthesis was measured by [3H]proline incorporation into pepsin-resistant, salt-precipitated collagen. The expression of collagen I and III, and TGF-beta1 mRNA was evaluated by northern blotting. The production of TGF-beta1 by human PMCs was measured by immunoassay. The changes of intracellular calcium level after adding Fura-2-AM were measured by fluorescence spectrophotometry. Western blotting was used to assess mitogen-activated protein kinase (MAPK) signalling proteins. RESULTS: We found that diltiazem (<0.2 mM) inhibited collagen I and III mRNA expression and collagen syntheses on a dose-dependent basis. Diltiazem (0.2 mM) suppressed IL-1beta- (5 ng/ml) induced TGF-beta1 production on human PMCs at both the protein and mRNA levels. Diltiazem (0.2 mM) also inhibited IL-1beta- (5 ng/ml) induced collagen I and III mRNA expression. Intracellular calcium levels did not change after the treatment with diltiazem, IL-1beta or both. The IL-1beta-treated human PMCs increased phospho-JNK (stress-activated c-Jun N-terminal kinase) and phospho-p38 MAPK expression, while diltiazem could suppress this phenomenon. CONCLUSIONS: Diltiazem suppressed collagen synthesis of human PMCs and inhibited IL-1beta-induced TGF-beta1 production on human PMCs. This signalling transduction may be through p38 MAPK and JNK pathways instead of intracellular calcium. These results suggest diltiazem to be a potential therapeutic regimen in preventing peritoneal fibrosis and support further in vivo studies.     

Role of atrophic changes in proximal tubular cells in the peritubular deposition of type IV collagen in a rat renal ablation model.

BACKGROUND: Tubular atrophy, dilation and interstitial fibrosis are common in tubulointerstitial lesions, but the precise roles and inter-relationships of these components in the development of interstitial lesions have not been determined. This study focused on the origin and roles of atrophic tubules in the peritubular deposition of type IV collagen in a rat renal ablation model. METHODS: Male Wistar rats underwent 5/6 nephrectomy or sham operation, and then were sacrificed at 4, 8 or 12 weeks, their remaining kidneys removed for histological and immuno-histochemical studies as well as in situ hybridization for type IV collagen mRNA. RESULTS: Immuno-histochemistry demonstrated the positive staining of atrophic tubules to vimentin, platelet-derived growth factor-B chain (PDGF) and heat shock protein 47 (HSP47). Cells positive to one or more of PDGF receptor beta, alpha-smooth muscle actin (alpha-SMA), and HSP47 accumulated around atrophic tubules. Type IV collagen was also increased in the proximity of the atrophic tubules. These intimate relationships were more clearly demonstrated in 'mosaic tubules', which are composed of both intact and atrophic proximal tubular epithelial cells, and which had a mixed pattern of staining with vimentin, PDGF and HSP47. The interstitial cells positive to alpha-SMA or HSP47, or both, were in close contact with atrophic but not with intact epithelial cells. Type IV collagen was exclusively deposited between atrophic tubules and HSP47-positive interstitial cells. In situ hybridization of type IV collagen mRNA demonstrated predominant expression in atrophic tubular epithelial cells, but not in surrounding interstitial cells. CONCLUSIONS: These findings suggest that atrophic proximal tubular cells are active in the development of collagen deposition in the peritubular space, i.e. in this model type IV collagen in the interstitial fibrotic area may be produced mainly by atrophic proximal tubules.     

Calcification and osteopontin localization in the peritoneum of patients on long-term continuous ambulatory peritoneal dialysis therapy.

BACKGROUND: Peritoneal calcification is an uncommon complication of continuous ambulatory peritoneal dialysis (CAPD), which is mainly observed in patients on long-term therapy. Although some asymptomatic patients must have microscopic calcification in their peritoneum, little information on this topic has been published. Recent studies have revealed active participation of adhesive/chemotactic protein osteopontin (OPN) in dystrophic calcification. METHODS: Peritoneal tissue was obtained by biopsy or at autopsy from 18 CAPD patients (median duration, 122 months), 5 control haemodialysis (HD) patients, and 3 pre-CAPD patients. The distribution of calcium deposits and OPN protein was determined by von Kossa staining and immunohistochemistry, respectively. Smooth muscle cells and macrophages were identified with anti-alpha smooth muscle actin (alpha-SMA) and anti-CD68 antibodies. RESULTS: Calcium deposits with various configurations were observed in specimens from 12 of the 18 CAPD patients. They included massive calcification facing the peritoneal cavity, scattered granular or crystalloid deposits in the submesothelial stroma, and oval-shaped deposits formed within hyalinized vasa. Most were present in highly sclerosed areas and accompanied by extracellular OPN precipitation. Cytoplasmic OPN was detected in infiltrating leukocytes, granulation tissue cells, fibroblast-like cells and mast cells. Computerized tomography examination also detected peritoneal calcification in seven of the CAPD patients. No calcium deposits or OPN staining was detected in control specimens. CONCLUSIONS:The results of our study suggest that microscopic peritoneal calcification is frequent in patients on CAPD for more than 10 years. Myofibroblast infiltration, OPN expression, calcium deposition, and associated OPN precipitation seem to be components of the peritoneal changes in such patients.     

Effects of 1,25-dihydroxyvitamin D3 on the expression of connective tissue growth factor and heat shock protein 47 in peritoneum fibrosis rats

Objective To investigate the expression of connective tissue growth factor (CTGF) and heat shock protein 47 (HSP47) in peritoneum fibrosis rats, and the mechanism of 1,25-dihydroxyvitamin D3 [1,25-(OH)2-VitD3] in inhibiting the peritoneum fibrosis. Methods Adult male Sprague-Dawley rats were randomly divided into 3 groups: control group (n=8), model group (n=8) and 1,25-dihydroxyvitamin D3 group (VitD3, n=8). The model of peritoneum fibrosis rats were induced by daily intraperitoneally injection of 15% chlorhexidine gluconate (CHX) 0.2 ml/d with 0.1% glucose for 4 weeks. Rats in VitD3 group were also treated with 1,25-(OH)2-VitD3 [i.p. 6 ng?(100 g)-1?d-1]. Peritoneal transport function, renal function, peritoneum thickness and serum level of 25 hydroxyvitamin D3 were detected. In vitro, primary cultured peritoneal mesothelial cells were divided into control group, high glucose group (HG, 2.5%), CTGF siRNA intervention group (CTGF siRNA+HG), VitD3 intervention group (VitD3+HG) and combined intervention group (CTGF siRNA+VitD3+HG). Real-time PCR, Western blotting and immunofluorescence were applied to measure the expression of CTGF and HSP47, also ELISA was used to detect the protein level of FN in peritoneum and peritoneal mesothelial cells. Results Compared with control group, the peritoneal ultrafiltration in peritoneum fibrosis rats were significantly decreased (P＜0.05), the absorbance level of peritoneal fibrosis, peritoneum thickness, the rate of dialysate urea nitrogen and blood urea nitrogen (DUN/BUN) and the expressions of CTGF and HSP47 were increased (all P＜0.05). After application of 1,25-dihydroxyvitamin D3, peritoneal fibrosis lesion was significantly improved, the peritoneum thickness, the expressions of CTGF and HSP47 were decreased (all P＜0.05). In vitro, 2.5% high glucose induced-peritoneal mesothelial cells were respectively treated by CTGF siRNA, 1,25-(OH)2-VitD3 and combined interventions, the expression of FN, CTGF and HSP47 was significantly lower than that in high glucose group (all P＜ 0.05). Conclusions The expression of CTGF and HSP47 is significantly increased in peritoneal fibrosis rats. 1,25-(OH)2-VitD3 may ameliorate the progression of peritoneal fibrosis via reducing the expression of CTGF, decreasing the expression of HSP47 and FN.     

Dipyridamole inhibits human peritoneal mesothelial cell proliferation in vitro and attenuates rat peritoneal fibrosis in vivo

BACKGROUND: Peritoneal fibrosis (PF) is one of the most serious complications after long-term continuous ambulatory peritoneal dialysis (CAPD). Proliferation of human peritoneal mesothelial cells (HPMC) and matrix over-production are regarded as the main processes predisposing to PF. Dipyridamole (DP) has been reported to have potential as an antiproliferative and antifibrotic agent. We thus investigated the effect of DP in inhibiting proliferation and collagen synthesis of HPMC. A rat model of peritonitis-induced PF was also established to demonstrate the in vivo preventive effect of DP. METHODS: HPMC was cultured from human omentum by an enzyme digestion METHOD: Cell proliferation was measured by the methyltetrazolium assay. Intracellular cAMP was measured using an enzyme immunoassay (EIA) kit. Total collagen synthesis was measured by (3)H-proline incorporation assay. Expression of collagen alpha1 (I) and collagen alpha 1 (III) mRNAs was determined by Northern blotting. The rat model of peritonitis-induced PF was developed by adding dextran microbeads (Cytodex, 8 mg/1 mL volume) to a standardized suspension (3 x 10(9)) of Staphylococcus aureus. DP was administrated via intravenous infusion (4 mg in 1 h) daily for seven days. Macroscopic grading of intraperitoneal adhesions and histological analyses of peritoneal thickness and collagen expression were performed. RESULTS: Addition of DP to HPMC cultures suppressed serum-stimulated cell proliferation and collagen synthesis. The antimitogenic and antifibrotic effects of DP appear to be predominantly mediated through the cAMP pathway, as DP increased intracellular cAMP in a dose-dependent manner. The macroscopic grade of intraperitoneal adhesion and peritoneal thickness were both significantly increased in animals treated with Cytodex plus S. aureus; on the other hand, DP attenuated these fibrotic changes with statistical significance (P < 0.01). Analysis of gene expression of collagen alpha 1 (I) and alpha1 (III) in the peritoneal tissue of experimental animals yielded similar results. CONCLUSIONS: This study suggests that dipyridamole may have therapeutic potential in treating peritoneal fibrosis.     

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.     

Dipyridamole inhibits TGF-beta-induced collagen gene expression in human peritoneal mesothelial cells

BACKGROUND: Peritoneal matrix accumulation is characteristic of peritoneal fibrosis (PF). Continuous ambulatory peritoneal dialysis (CAPD) patients who had persistent transforming growth factor-beta (TGF-beta) in their drained effluent had an increased risk of PF. We previously reported that TGF-beta stimulates the expression of types I and III collagen mRNA in cultured human peritoneal mesangial cells (HPMCs), which may predispose them to develop PF. Pharmacological interventions to attenuate TGF-beta-stimulated matrix accumulation in HPMC may have therapeutic potential for the treatment of PF. The SMAD family and the extracellular signal-regulated protein kinase (ERK1/2, p44/p42) pathways have been shown to participate in TGF-beta signaling. Our current study identified these signal pathways in HPMCs and investigated the molecular mechanisms involved in the inhibitory effects of dipyridamole on TGF-beta-induced collagen gene expression in HPMCs. METHODS: HPMCs were cultured from human omentum by an enzyme digestion METHOD: Expression of collagen alpha1(I) mRNA was determined by Northern blotting. The SMAD proteins and the ERK1/2 activity were determined by Western blotting. RESULTS: TGF-beta-stimulated collagen alpha1(I) mRNA expression of HPMC was inhibited by dipyridamole in a dose-dependent manner. Smad2 and ERK1/2 were activated in response to TGF-beta; however, TGF-beta had little effect on the protein expression of Smad4. The addition of PD98059, which blocked activation of ERK1/2, suppressed TGF-beta-induced collagen alpha1(I) mRNA expression in a dose-dependent manner. At a concentration that inhibited collagen gene expression (17 microg/mL), dipyridamole suppressed ERK1/2 activation by TGF-beta. In contrast, the same concentration of dipyridamole had no effect on TGF-beta-induced activation of Smad2. CONCLUSION: Dipyridamole inhibits TGF-beta-induced collagen gene expression in HPMC through modulation of the ERK pathway. Our study of dipyridamole may provide therapeutic basis for clinical applications in the prevention of PF.     

Role of mast cells and myofibroblasts in human peritoneal adhesion formation

OBJECTIVE: To study fibroblasts and mast cells in human peritoneal adhesions and to evaluate whether their interaction plays a role in adhesion development. SUMMARY BACKGROUND DATA: Myofibroblasts play a critical role in wound repair/fibrosis. Mast cells influence the formation of peritoneal adhesions in a rat model, and they are modulators of fibroblast functions. METHODS: Peritoneal adhesion biopsies were processed for either histology (H&E, toluidine blue) or immunohistochemistry (tryptase, laminin, collagen type IV and VIII, and alpha-SMA) or grown as explants for obtention of fibroblasts. The effects of mast cell (HMC-1) sonicate and selected mast cell mediators and cytokines on fibroblast proliferation ([ (3)H]thymidine) and collagen synthesis ([ (3)H]proline) and on fibroblast contractile activity (tridimensional collagen lattice) were evaluated. Mast cell mediators influencing fibroblast proliferation were partially characterized by enzymatic susceptibility and FPLC gel filtration column chromatography. RESULTS: Most of the fibroblasts in peritoneal adhesions were identified as alpha-SMA-positive myofibroblasts. Mast cell hyperplasia was observed and more than one third of the mast cells were degranulated. Few mast cells showed a faint staining for laminin or collagen type IV and VIII. Mast cell sonicate increased fibroblast proliferation and contractile activity while decreasing collagen synthesis. Mast cell sonicate proliferating activities were found to be proteinase-sensitive with a molecular weight of more than 158 kd, of approximately 40 kd, and of less than 10 kd. TGF-beta and tryptase enhanced collagen synthesis; TNF-alpha and chymase decreased it. None of the selected mediators increased fibroblast proliferation. CONCLUSIONS: Myofibroblasts are the main connective tissue cells present in human peritoneal adhesions, and mast cells play a direct role in peritoneal adhesion formation.     

Endostatin peptide, an inhibitor of angiogenesis, prevents the progression of peritoneal sclerosis in a mouse experimental model

Peritoneal sclerosis is a major and serious complication in patients on long-term continuous ambulatory peritoneal dialysis (PD). The involvement of angiogenesis and proangiogenic factors such as vascular endothelial growth factor (VEGF)-A in progressing peritoneal sclerosis has been reported. We previously reported the therapeutic efficacy of endostatin peptide, a potent inhibitor of angiogenesis derived from type XVIII collagen, in a mouse diabetic nephropathy model. Here, we examined the therapeutic effect of endostatin peptide in preventing progression in a mouse peritoneal sclerosis model. Male ICR mice received intraperitoneal injections of chlorhexidine gluconate (CG) every other day to induce peritoneal sclerosis. Endostatin peptide (1 or 4 mg/kg/day) was administered via subcutaneously implanted osmotic minipumps. Peritoneal sclerosis (day 24) was significantly suppressed by endostatin peptide in a dose-dependent manner. Peritoneal accumulation of type III collagen was significantly suppressed by endostatin peptide. Increase in the number of CD31(+) blood vessels, F4/80(+) monocyte/macrophage accumulation, and 5-bromodeoxyuridine(+) proliferating cells was significantly inhibited by endostatin peptide. Increase in peritoneal expression of VEGF-A, profibrotic transforming growth factor-beta1, and alpha-smooth muscle actin was suppressed by endostatin peptide. Immunoreactivity for endogenous endostatin (whole molecule) and endostatin receptor alpha5beta1-integrin was increased and colocalized to CD31(+) blood vessels in the thickened peritonea of CG-injected mice. These results demonstrate the potential use of antiangiogenic endostatin peptide as a novel therapeutic agent in preventing peritoneal sclerosis, a severe complication in patients undergoing long-term PD.     

Progressive renal fibrosis in murine polycystic kidney disease: an immunohistochemical observation

BACKGROUND: The appearance of interstitial fibrosis in polycystic kidneys is emblematic of progressive disease. Matrix forming this scar tissue is derived from local renal cells in response to cystogenesis. We investigated the phenotype of collagen-producing cells in the cystic kidneys of DBA/2-pcy mice to better characterize the spectrum of interstitial cells associated with renal fibrogenesis. METHODS: The extent of interstitial fibrosis and the number of fibroblasts in cystic kidneys were first quantitated over time using computer-assisted image analysis. Subsequently, antisera to four cell protein markers were studied by coexpression immunohistochemistry during progression of fibrosis using confocal microscopy. The antisera included fibroblast-specific protein 1 (FSP1) for fibroblast phenotype, alpha-smooth muscle actin (alpha-SMA) for contractile phenotype, vimentin (VIM) for mesenchymal phenotype, and heat shock protein 47 (HSP47) for interstitial collagen-producing phenotype. RESULTS: Interstitial fibrosis in cystic kidneys gradually increased throughout the 30-week observation period of our study. With progression of cystogenesis, most of the tubules in pcy mice either dilated or disappeared with time. FSP1+ fibroblasts were distributed sparsely throughout the renal interstitium of young pcy and wild-type mice. Their number increased in the widening fibrotic septa by 18 weeks of age and persisted through 30 weeks of the study interval. Some epithelia among remnant tubules trapped within fibrotic septa around adjacent cysts also acquired the phenotype of FSP1+, HSP47+ collagen-producing fibroblasts, suggesting a possible role for epithelial-mesenchymal transformation (EMT) in this process. Most FSP1+ fibroblasts were alpha-SMA-, but HSP47+, suggesting they were producing collagen proteins for the extracellular matrix. alpha-SMA+, FSP1-, HSP47+ or HSP47- cells were also observed, and the latter tended to distribute independently in a linear pattern, reminiscent of vasculature adjacent to forming cysts. VIM+ expression was not observed in alpha-SMA+ cells. CONCLUSIONS: Many nonoverlapping as well as fewer overlapping populations of FSP1+ and alpha-SMA+ cells shared in the collagen expression associated with progressive fibrogenesis in pcy mice undergoing cystogenesis. Some FSP1+ fibroblasts are likely derived from tubular epithelium undergoing EMT, while alphaSMA+, VIM- cells probably represent vascular smooth muscle cells or pericytes surviving vessel attenuation during the chaos of fibrogenesis. Importantly, not all interstitial cells producing collagens are alpha-SMA+.     

Interstitial expression of heat-shock protein 47 correlates with capillary deposition of complement split product C4d in chronic allograft nephropathy

BACKGROUND: Chronic allograft nephropathy (CAN), associated with late-allograft dysfunction is caused by alloantigen-dependent and -independent mechanisms, and eventually leads to interstitial fibrosis (ci). Activation of complement cascade is considered to be a poor prognostic marker of graft survival. This study was designed to examine the relationship between the expression of C4d and heat-shock protein 47 (HSP47, a collagen-specific chaperone) in the development of interstitial fibroproliferative lesions in CAN. METHODS: Sixty-three renal allograft biopsy specimens, obtained from 48 patients, were examined for the expression of C4d, HSP47, CD68 and alpha-smooth muscle actin (alpha-SMA) by immunohistochemistry. Double-staining was performed to determine the colocalization of C4d and HSP47. The relationship of between the expression of C4d, HSP47, CD68 and alpha-SMA and the clinical and histopathological parameters were statistically analysed. RESULTS: No expression of C4d was noted in the tubulointerstitium including peritubular capillary (PTC) of the control kidney. C4d was expressed in PTC in one-third of allograft renal tissues with morphological evidences of CAN. The interstitial cells around the fibrotic areas of the PTC of CAN were positive for the expression of HSP47. The deposition of C4d in PTC correlated with interstitial expression of HSP47 around the PTC. Most HSP47 expressing cells were phenotypically altered myofibroblasts, as determined by the dual staining of alpha-SMA. CONCLUSIONS: The increased expression of HSP47 positively correlated with the expression of C4d in PTC, and might contribute to the progression of interstitial ci in CAN.     

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.     

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.     

Transforming growth factor-beta 1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction

BACKGROUND: Interstitial expression of transforming growth factor-beta1 (TGF-beta1) is important in tubulointerstitial fibrosis, a common process in most progressive renal diseases. However, no effective therapy for progressive interstitial fibrosis is known. Recently, we developed an artificial viral envelope (AVE)-type hemagglutinating virus of Japan (HVJ) liposome-mediated retrograde ureteral gene transfer method, which allowed us to introduce the genetic material selectively into renal interstitial fibroblasts. METHOD: We introduced antisense or scrambled oligodeoxynucleotides (ODNs) for TGF-beta 1 into interstitial fibroblasts in rats with unilateral ureteral obstruction, a model of interstitial fibrosis, to block interstitial fibrosis by retrograde ureteral injection of AVE-type HVJ liposomes. RESULTS: TGF-beta 1 and type I collagen mRNA increased markedly in the interstitium of untreated obstructed kidneys, and those were not affected by scrambled ODN transfection. Northern analysis and in situ hybridization revealed that the levels of TGF-beta 1 and type I collagen mRNA were dramatically decreased in antisense ODN-transfected obstructed kidneys. Consequently, the interstitial fibrotic area of the obstructed kidneys treated with antisense ODN was significantly less than that of the obstructed kidneys untreated or treated with scrambled ODN. CONCLUSION: The introduction of TGF-beta 1 antisense ODN into interstitial fibroblasts may be a potential therapeutic maneuver for interstitial fibrosis.     

Localization of HSP47 in cisplatin-treated rat kidney: a possible role in tubulointerstitial damage

Background. A 47-kDa heat-shock protein (HSP47) is a major collagen-binding stress protein and is assumed to play an important role in the fibrotic process, but its role in cisplatin-induced tubulointerstitial fibrosis is not yet clear. To explore the possible role(s) of collagen-binding stress protein HSP47 in cisplatin-induced tubulointerstitial fibrosis, the expression of HSP47 was examined in cisplatin-treated rat kidneys. Methods. Eighteen male Wistar rats were divided into two groups; group I were age-matched controls and group II, animals were injected intraperitoneally with a single dose of cisplatin (6 mg/kg body weight). One hour after cisplatin injection, three rats from group II were killed along with control rats. The remaining rats in both groups were killed on the 7th, 14th, and 28th days of the experiment and the kidneys were collected for morphological and immunohistochemical study. The expression of collagen-binding HSP47 with various proteins implicated in phenotypic modulation (α-smooth muscle actin and vimentin) and fibrosis (type I and type III collagens) was examined in control and cisplatin-treated kidneys. Results. Cisplatin induced marked tubulointerstitial damage, including interstitial fibrosis, which was characterized by increased deposition of type I and type III collagens. Increased expression of HSP47 was also noted in and around the expanded interstitium in cisplatin-treated rats; by double-staining, HSP47-expressing cells were found to be α-smooth muscle actin-positive myofibroblasts and vimentin-positive tubular epithelial cells. In addition, colocalization of HSP47 and collagens was seen in and around the interstitial fibrosis in cisplatin-treated kidneys. Conclusion. From the results, we concluded that overexpression of HSP47 by phenotypically altered renal cells may play an important role in the excessive assembly of collagens and could thereby contribute significantly to the development of the tubulointerstitial fibrosis found in cisplatin-treated rat kidneys. Received: October 5, 1998 / Accepted: March 5, 1999