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Objective To assay the expression chinse of ED1 positive cell in focal segmental glomemlosclerosis in rats,we investigated the relationship between the infiltration of mononuclear phagocytes and the progression of glomendar sclerosis.Methods We used 12 Wistar ratswhichwere dividedintotwo groups.1est group and coutrol group.Themodel offocal segmental glomerursclerosiswas ulade by in jecting PAN 9 mg/100g body weights.The rats ofcontrol group were injected 3ml 0.9％80diuln chloride.The proteinuria,serum creatinine,fipi&and protein of the rats were examined.The rata were killed at the 20th week.All the kidneys were kept and nlade into pathologic slEun-pie.1mmunohistochemical method was applied to detect the protein expression of FIN and the EDI positive cell in renal tissue of all the rats,and the number of Edl positive cell W88 counted.The results were analyzed by SPSS.Results The proteinuria of the mts in FSGS model group was significantly increased,the serum lipid ofthem was also increased.The pathology changes of the rat renal in model group showed that a part of giomemli appeared focal segmental sclerosis or all glomerular sclerosis,and the extracelhlar matrix accumulated.In the renal of model rats,the amount of EDI positive cells was significantly higher than that in normal rats(P<O.05).The expression of FN in the rehal of model rats was significantly higher than that in the normal rats(P<0.05).111e mount ofEDl positive cells WaS significantly positively correlated WitII the expression of FN(P=0.002).The amount of Edl positive cells W88 significantly positively correlated with the pmteinuria of the rats(P=0.014).Conclusion Theinfiltration ofEDI positivecell contributed to the progression of glomerular sclerosis.
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Objective To investigate the inhibitory effect and associated mechanism of rapamycin on proliferation and extracellular matrix (ECM) secretion in myofibroblasts stimulated by connective tissue growth factor (CTGF). Methods Primary cultivated myofibroblasts were divided into 6 groups: control, CTGF (100 μg/L), rapamycin 20 μg/L+CTGF 100 μg/L, rapamycin 40 μg/L +CTGF 100 μg/L, rapamycin 20 μg/L, and rapamycin 40 μg/L alone. 5'-bromodeoxyuridine (BrdU) incorporation assay was used to detect the myofibroblast proliferation.Western blot was used to analysis the secretory FN protein in the supernatant medium of cultured myofibroblasts and the ERK1/2 phosphorylation in myofibroblasts. Results CTGF (100 μg/L)incubation significantly increased the number of Brdu positive myofibroblasts(P<0.01) and the level of FN protein secretory (P<0.05) in cell supernatant medium compared with control group,respectively. The number of Brdu positive myofibroblasts markedly decreased by 62% and 70% (P <0.05) in rapamycin 20 μg/L+CTGF 100 μg/L and rapamycin 40 μg/L+CTGF 100 μg/L groups, respectively. The FN protein levels in supernatant were decreased by 15% and 44% compared with CTGF 100 μg/L group, respectively; but the difference of FN protein levels was significant only in rapamycin 40 μg/L group (P<0.05). CTGF could activate ERK1/2 at 10 minutes; but as myofibroblasts were pretreated with rapamycin 40 μg/L for 30 min, it abolished CTGF-induced ERK1/2 phosphoralation. PD98059, the specific inhibitor of ERK1/2, could block the effect of CTGF-induced proliferation (7%±5% vs 85%±7%, P<0.01) and FN secretion (1.0±0.1 vs 1.6±0.3, P<0.05). Conclusions Rapamycin partially suppresses the proliferation and ECM secretion of myofibroblasts induced by CTGF. Its effect may be through inhibiting CTGF-induced activation of ERKI/2 signaling pathway.
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Objective: To address the significance of urinary podocytes in the diagnosis of human focal segmental glomerulosclerosis(FSGS). Methods: Twelve patients with FSGS and 20 patients with minimal change disease (MCD) were diagnosed by routine renal biopsy, and 8 healthy persons as controls. Morning urinary sediments was collected and centrifuged onto glass slides. Urinary podocytes were identified by immunofluorescent staining of podocyte specific protein Podocalyxin(PCX). The state of podocytes in glomeruli was observed using immunofluorescence. Results: Urinary podocytes were found in 8 out of 12 FSGS patients(66.67%), whereas none of 20 patients with MCD and control had podocytes in their urine. FSGS patients with positives urinary podocytes had prominent manifestation of nephropathy syndrome, whereas no nephrotic syndrome in patients with negative urinary podocytes. Focal absence of the expression of PCX, a marker protein of podocytes in glomeruli was found in FSGS patients, and the locations of absence were consistent with the lesions of focal sclerosis in glomeruli. In contrast, PCX was expressed integrally in MCD patients. Conclusion: Appearances of podocytes in urine of patients with nephropathy may be used as one of the reliable, convenient and unharmful accessorial methods for distinguished diagnosis of FSGS and MCD.
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Objective: To investigate whether hypoxia can affect the expression and secretion of connective tissue growth factor(CTGF) and fibronectin(FN) in primary cultured rat renal cortical myofibroblasts . Methods: The primary cultured rat renal cortical myofibroblasts were subjected to hypoxic (1%O_2) or normoxic (21% O_2) conditions for a variety of times. The protein levels of HIF - 1?, CTGF and FN protein were analyzed by Western blotting in both the whole cell lysates and supernatant culture medium 6 h,12 h and 24 h after incubation, respectively. RT-PCR was carried out to measure the levels of FN mRNA at different time points (2 h,3 h,6 h and 12 h). The activity of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell medium was assayed by gelatin zymography. Results: The expression of HIF - 1?was induced at h6 in cells under hypoxia incubation. The levels of cellular CTGF protein were increased in hypoxia treated myofibroblasts at h6 (175%?52%),significantly elevated at h12 (347%?67%,P
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Objective:To observe the expression of connective tissue growth factor(CTGF) and its receptor-low density lipoprotein receptor-related protein (LRP), and the relevant signaling pathway for the regulation by long-term high glucose exposure in cultured podocytes. Methods:The effects of high glucose on the expression of CTGF and its receptor LRP were analyzed by western blotting. The activation of mitogen activated protein kinase ( MAPKS )signaling pathway by high glucose was also examined. Results: Basal levels of CTGF were observed in cultured mouse podocytes, the levels of CTGF protein were increased by high glucose medium groups on the 2nd day, reached the peak on the 4th day(P0.05).The levels of CTGF expression in normal glucose and mannitol glucose groups did not change markly. High glucose medium induced phosphorylation of ERK_ 1/2 at as early as minute 30, reached the peak at hour 6; maintained the activity at hours 12 and 24, and declined to the basal level at hour 48. However, phosphorylation of ERK_ 1/2 was not detected in normal glucose and mannitol glucose groups. Blockade of phosphorylation of ERK_ 1/2 with PD98059, a specific ERK_ 1/2 activation inhibitior, did decrease the high glucose-triggered expression of CTGF protein in 4 days. High glucose had no effect on the expression of LRP protein at each time point. Conclusion: Acute high glucose (2-4 days)stimulated the expression of CTGF protein via ERK_ 1/2-dependent signaling pathway in cultured podocytes, while cultured in high glucose for 6-8 days, the podocytes did not increase its CTGF level. Long-term high glucose had no effect on the expression of LRP in podocytes.
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Objective: To investigate peroxisome proliferator activated receptor ? (PPAR ?) expressions in patients with actively proliferating glomerulonephritis such as type Ⅳ lupus nephritis (LN) and cellular crescentic glomerulonephritis (RPGN). Methods: All patients were divided into 4 groups as follows: RPGN (17 cases); LN type Ⅳ (15 cases); mild mesangial proliferative IgA nephropathy (IgAN, 7 cases) and minimal change disease (MCD, 10 cases). Clinical parameters, immunohisto chemistry stain and in situ hybridization of renal biopsy specimens were performed. Results: Clinically, proteinuria and hematouria were increased and Ccr were decreased in LN and RPGN patients, and increased ESR and decreased complement C3 were found in group LN. Active index of renal specimens were significantly higher in LN and RPGN groups than in IgAN and MCD groups. Renal specimens of MCD patients showed no positive PPAR ? staining in all sections; little immunoreactivity was detected in sections of glomerular, tubular and interstitial cells from IgAN patients. Glomerular positive staining of PPAR ? in renal sections in LN and RPGN patients[(3.3?1.8) and (2.8?1.2) cells per section of glomerulus, respectively] were significantly increased compared with that in IgAN patients [(0.7?0.5) cells per section of glomerulus]. Similarly, tubular positive staining of PPAR ? in LN and RPGN patients (27.38? 12.46, 23.36?10.55) were also elevated compared with that in IgAN and MCD patients(6.51?3.49, 1.72?0.31). The relevance assay results showed positive relationship between active index and glomerular or tubular PPAR ? immunohisto chemistry staining cell numbers (0.478, P
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Objective:To investigate the anti-fibrotic effect of sirolimus(rapamycin)at the cell level.Methods:The primary cultured rat renal cortical myofibroblasts were divided into two groups,control group and sirolimus 40 mg/L group at each time point.The protein levels of ?-SMA,Col-Ⅰ,fibronectin(FN)were analyzed by Western blot in both the whole cell lysates and supernatant culture media 12 h,24 h and 48 h after incubation,respectively.Real-time quantitative PCR was carried out to measure the levels of procollagen-ⅠmRNA 1 h,2 h,4 h,and 6 h after cell incubation.The activities of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell media were assayed by gelatin zymography.Results:(1)Sirolimus had no effect on the expression of ?-SMA of myofibroblasts at differnet time points.(2)The expression of Col-Ⅰin the whole cell lysates both reduced at the end of 24 h and 48 h in sirolimus group significantly [(0.58?0.05)and(0.63?0.18),P
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AIM: To investigate the effect of hypoxia on the myofibroblast transdifferentiation from fibroblasts,and associated signaling of hypoxia on the production of collagen Ⅰ in cultured rat renal cortical myofibroblasts.METHODS: The study is composed of two relevant parts.In the first part,a normal rat renal interstitial fibroblast cell line NRK-49F was treated with hypoxia(1% O2) or normoxia(21% O2) for 6 h,12 h and 24 h.The expression of hypoxia inducible factor-1?(HIF-1?) was examined by Western blotting in order to make sure the hypoxic condition is reliable.The myofibroblast transformation from fibroblasts induced by hypoxia was assayed by detecting the protein levels of ?-smooth muscle actin(?-SMA).In the second part,the object was done on the primary cultured rat renal cortical myofibroblasts.Myofibroblasts were subjected to hypoxic or normoxic conditions for variety of times.The levels of HIF-1? in cell lysates and collagen I protein in supernatant culture medium and the activation of extracellular signal-regulated kinase(ERK)1/2 MAPK pathway were analyzed by Western blotting.RT-PCR was carried out to measure the levels of collagen I mRNA at different time points(2 h,4 h and 6 h).The distribution of HIF-1? in myofibroblasts was demonstrated by immunocytochemistry.The changes of collagen I production were detected after PD98059,a specific inhibitor of ERK1/2 activation pretreatment and during the hypoxia incubation.The activity of gelatinase matrix metalloproteinase-2(MMP-2) and MMP-9 in the supernatant medium from the cultured cells were assayed by gelatin zymography.RESULTS: Significant increased levels of HIF-1? protein appeared in cell lysates under hypoxia for 6 h.Furthermore,HIF-1? was translocated into nuclei of myofibroblasts after 6 h exposure of myofibroblasts to hypoxia.The levels of ?-SMA protein increased in NRK-49F under hypoxia for 12 h(187%?32%,P
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AIM: To understand the expression of low density lipoprotein receptor-related protein (LRP) in tubulointerstitial fibrosis of unilateral ureter obstruction (UUO) rats. METHODS: The localization of LRP within kidneys were assessed by immunohistochemical staining, the protein level of LRP and connective tissue growth factor (CTGF) in kidney were analysised by Western blot. RESULTS: The location of LRP positive-staining and the protein level of LRP were in the same tendency with CTGF, the level of LRP had strong correlationship with the level of CTGF (r=0.786, P
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As constructive cells of the glomerular filtration barrier, podocytes plays an essential role in the maintenance of the glomerular blood filtration, and also in the prevention of protein loose from blood. Many factors cause podocyte injury, and consequently contribute to the onset and progression of glomerular lesions. This review focuses on relationship between podocyte injury and glomerular disease.
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AIM: To investigates the role of phosphatidic acid (PA) in the expression of several inflammatory mediators produced by glomerural macrophages (GM?). METHODS: The study was performed on a rat model of accelerated anti-glomerural basement membrane (anti-GBM) glomerulonephritis (GN). GN-GM? were isolated and identified. Peritoneal M? (P-M?) of both normal and GN rats were used as controls. Block and reverse test were investigated with rhIL-1? stimulated, lisofylline (LSF) and phosphatidic acid (PA). Macrophage expression of ICAM-1 and TGF-? 1 were assessed at the level of protein and gene by immunocytochemistry, northern blot and RT-PCR. RESULTS: (1) After stimulated with rhIL-1?, GN-GM? produced much more ICAM-1, MCP-1 and TGF-? 1 than P-M?, and it's gene expression was similar as protein product. (2) mRNA expression of these factors was up-regulated again after the GN-GM? were pretreated with LSF then PA was added. CONCLUSIONS: Since GN-GM? plays an important role for PA in the mediation of glomerular injury, inhibiting of PA production is the keypoint of blocking M? mediated inflammatory effects. LSF may be an effective medicine in therapy for acute inflammatory forms of GN.
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AIM: To study the expression of connective tissue growth factor (CTGF) induced by hypoxia, and the role and mechanism of hypoxia on promoting renal interstitial fibrosis. METHODS: Renal interstitial fibrosis was induced by unilateral ureteral obstruction (UUO) in rat animal model for 9 days as in vivo studies; marker of hypoxia-HIF-1? mRNA and protein, the expression of CTGF in the obstructed kidneys were assessed by RT-PCR, immunohistochemistry and Western blotting respectively. In vitro , normal rat kidney interstitial fibroblast cells (NRK-49F) were exposed to hypoxia (1%O 2) for up to 6 hours, hypoxia was confirmed by detecting the expression of HIF-1? protein in cells, cellular level of CTGF mRNA and protein were assessed by RT-PCR and Western blotting respectively. RESULTS: Neither HIF-1? mRNA nor HIF-1? protein was expressed in the kidney from sham-operated group of rats. High level of HIF-1? mRNA were occurred, and strongly HIF-1? positive immunostaining were seen in the tubular and interstitial cells in kidney from UUO rats. Expression and location of CTGF protein were paralleled and relevant with the expression of HIF-1? protein in kidney of UUO rats. In cultured NRK-49F cell line, subjected to hypoxia even for 6 hours stimulated the expression of CTGF mRNA and protein. CONCLUSION: Our results indicated that hypoxia could stimulate the expression of CTGF mRNA and protein in kidney from UUO rats, which may in turn contribute to renal interstitial fibrosis.
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Connective tissue growth factor (CTGF) has recently received much attention as a possible key determinant of progressive fibrosis. It promotes tissue fibrosis through different pathways, such as cell proliferation, extracellular matrix accumulation and cell transdifferentiation. A number of regulators of CTGF expression have been identified, including transforming growth factor ?, vascular endothelial growth factor, tumor necrosis factor ?, etc. The mechanism of profibrotic effect by CTGF was reviewed. [