Publications by authors named "Hai-chang Huang"

18 Publications

  • Page 1 of 1

[Sirolimus inhibits the expression of type I collagen and fibronectin in cultured renal cortical myofibroblasts].

Beijing Da Xue Xue Bao Yi Xue Ban 2008 Oct;40(5):509-13

Department of Nephrology, Peking University First Hospital; Institute of Nephrology, Peking University, Beijing 100034, China.

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 alpha-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-I mRNA 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 alpha-SMA of myofibroblasts at different time points. (2) The expression of Col-I in 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 < 0.05] compared with control group at each time point, respectively. (3) The levels of procollagen-I mRNA reduced significantly at the end of 1 h and 2 h compared with control group at each time point [(0.38+/-0.05) and (0.55+/-0.16), P < 0.05], but increased to basic level at the end of 4 h. (4) The myofibroblasts had basic expression of Col-I early at the end of 12 h, its expression in supernatant culture medium reduced obviously both at 24 h and 48 h in sirolimus group compared with control group of each time point [(0.59+/-0.25) and (0.52+/-0.21), P < 0.05]. (5) The expression of FN in the whole cell lysates had the same trend as that in supernatant culture medium, which reduced obviously at the end of 24 h in sirolimus group compared with control group at each time point [(0.44+/-0.09) and (0.40+/-0.15), P < 0.05], but the inhibitive effect of sirolimus on FN disappeared at the end of 48 h. (6) The activities of MMP-2 and MMP-9 in the supernatant culture media were not significantly changed along with the experimental time points.

Conclusion: Sirolimus may exert its anti-fibrotic effect through the inhibition of the expression of Col-I and/or FN in cultured renal cortical myofibroblasts.
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October 2008

RhoA required for acid-induced stress fiber formation and trafficking and activation of NHE3.

Am J Physiol Renal Physiol 2007 Oct 8;293(4):F1054-64. Epub 2007 Aug 8.

Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, Texas, USA.

Exposure to an acid load increases apical membrane Na(+)/H(+) antiporter (NHE3) activity, a process that involves exocytic trafficking of the transporter to the apical membrane. We have previously shown that an intact microfilament structure is required for this exocytic process (Yang X, Amemiya M, Peng Y, Moe OW, Preisig PA, Alpern RJ. Am J Physiol Cell Physiol 279: C410-C419, 2000). The present studies demonstrate that acid-induced stress fiber formation is required for stimulation of NHE3 activity. Formation of stress fibers is associated with acid-induced tyrosine phosphorylation and increases in protein abundance of two focal adhesion proteins, p125(FAK) and paxillin. The Rho kinase inhibitor Y27632 completely blocks acid-induced stress fiber formation and the increases in apical membrane NHE3 abundance and activity, but it has no effect on acid-induced tyrosine phosphorylation of p125(FAK) or paxillin. Herbimycin A completely blocks acid-induced tyrosine phosphorylation of p125(FAK) and paxillin but only partially blocks stress fiber formation and NHE3 activation. These studies demonstrate that Rho kinase mediates acid-induced stress fiber formation, which is required for NHE3 exocytosis, and increases in NHE3 activity. Acid-induced tyrosine phosphorylation of the focal adhesion proteins p125(FAK) and paxillin is not Rho kinase dependent. Thus these two acid-mediated effects are associated, yet independent processes.
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http://dx.doi.org/10.1152/ajprenal.00295.2007DOI Listing
October 2007

[The significance of urinary podocytes in patients with active lupus nephritis].

Zhonghua Nei Ke Za Zhi 2007 Feb;46(2):127-30

Renal Division, Peking University First Hospital, Beijing 100034, China.

Objective: To assess the significance of urinary podocyte and its possible implication as a marker of activity of lupus nephritis.

Methods: The presence of podocytes in urinary sediment was detected with immunochemical staining using anti-podocalyxin antibody. The correlation of the number of urinary podocytes with activity index of renal pathological lesions, hematuria, and proteinuria was analyzed respectively. The proliferating podocytes in renal biopsy tissue and urine from patients with class IV lupus nephritis were examined with double immunohistochemical staining.

Results: Thirty-one patients with lupus nephritis undergoing renal biopsy were enrolled into the study. Renal pathological findings of the patients could be classified into WHO class III (25.8%), class IV (64.5%) and class V (9.7%). 90% of the patients had positive urinary podocytes. The number of urinary podocytes was strongly and positively correlated with the severity of hematuria (r=0.639, P=0.000) and glomerular pathological activity index (r=0.487, P=0.014) in patients of class III and class IV. The amount of proteinuria was not correlated with pathological activity index, even though all the patients had proteinuria. Furthermore, the number of urinary podocytes, the severity of hematuria and the amount of proteinuria were all decreased after treatment with methyl prednisone, cyclophosphamide or mycophenolate mofetil. Interestingly, the urinary podocytes could disappear even before the remission of hematuria and proteinuria after treatment. Proliferative podocytes were observed both in biopsied kidney tissue and urinary sediments in patients of class IV.

Conclusion: The presence and the number of urinary podocytes can be used as a valuable marker to grade the activity of lupus nephritis and to evaluate the efficacy of steroid therapy.
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February 2007

[High glucose downregulates the expression of podocalyxin protein in glomerular podocytes of mice].

Beijing Da Xue Xue Bao Yi Xue Ban 2007 Apr;39(2):167-70

Division of Nephrology, Peking University First Hospital, Beijing 100034, China.

Objective: To examine the expression of podocalyxin protein in glomerular podocytes by long-term high glucose exposure in vitro and in vivo.

Methods: Immunohistochemical staining and computer image analysis were applied to detect the expression of podocalyxin protein in glomeruli from db/db mice and Wt mice. The effects of high glucose on the expression of podocalyxin protein were analyzed by Western blotting. The activation of MAPKS signaling pathway (ERK, p38 and JNK) by high glucose was also examined.

Results: The expressions of podocalyxin protein in db/db mice were obviously less than that in Wt mice [(0.18+/-0.07) vs (0.25+/-0.05),P<0.05] assessed by immunostaining and semiquantitative analysis. Basal levels of podocalyxin protein were observed in cultured mouse podocytes. The level of podocalyxin protein declined at each time point by high glucose incubation, reached the lowest level on the 6th day (5.5% of control group, P<0.01), but no significant changes were observed in normal glucose and mannitol glucose incubation groups. High glucose medium induced phosphorylation of ERK1/2 as early as 30 minutes, reached the peak at hour 6; maintained the activation from hour 12 to 24, and declined to the basal level at hour 48. However, activation of ERK1/2 was not detected in normal glucose and mannitol glucose groups. Blockade of activation of ERK1/2 with PD98059, a specific ERK1/2 activation inhibitor, attenuated the high glucose-induced expression of podocalyxin protein on the 6th day.

Conclusion: High ambient glucose decreases the protein level of podocalyxin by podocyte in vitro and in vivo, and the decrease in podocalyxin protein is ERK1/2jdependent in cultured podocytes.
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April 2007

[Hypoxia induces the expression and secretion of connective tissue growth factor and fibronectin by cultured renal cortical myofibroblasts].

Beijing Da Xue Xue Bao Yi Xue Ban 2007 Feb;39(1):67-71

Department of Nephrology, Peking University First Hospital, Institute of Nephrology, Peking University, Beijing 100034, China.

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-1alpha, 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-1alpha 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<0.05 ) , and sustained the high levels by 24 h (143%+/-27%). The protein level of CTGF in supernatant culture medium reached 3.48 times higher than that in normoxic group of cells at h24 (348%+/-99% , P<0.05 ). The levels of secreted FN by myofibroblasts were elevated under hypoxia at h6 (187%+/-42%), h12 (199%+/-51%) and reached the peak level at h24 (210%+/-29%, P<0.05), whereas the levels of cellular FN was declined at the same time points. Furthermore, we found the expression of FN mRNA was increased in cells under hypoxia condition at h3, reached the peak level at h6(135%+/-13%, P<0.05), and then decreased to the comparable level of cells in normoxic group at h12. The activities of MMP-2 and MMP-9 in the supernatant cultured medium were not significantly changed along with the experimental time points.

Conclusion: Hypoxia could potentiate renal interstitial fibrosis through stimulating the expression and secretion of CTGF and FN in cultured cortical myofibroblasts.
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February 2007

[High glucose regulates the expression of connective tissue growth factor and its receptor (low density lipoprotein receptor-related protein) in cultured podocytes].

Beijing Da Xue Xue Bao Yi Xue Ban 2006 Jun;38(3):262-5

Division of Nephrology, Peking University First Hospital, Beijing 100034, China.

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 (P< 0.05), began to decline on the 6th day, returned to the basal level on the 8th day (P>0.05). The levels of CTGF expression in normal glucose and mannitol glucose groups did not change markly. High glucose medium induced phosphorylation of ERK1/2 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 ERK1/2 was not detected in normal glucose and mannitol glucose groups. Blockade of phosphorylation of ERK1/2 with PD98059, a specific ERK1/2 activation inhibitor, 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 ERK1/2-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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June 2006

[Regulation of heat shock protein 27/activating transcription factor-5 complex and its implication in podocyte apoptosis induced by high glucose].

Zhonghua Yi Xue Za Zhi 2006 Feb;86(6):394-8

Division of Nephrology, First Hospital & Institute of Nephrology, Peking University, Beijing 100034, China.

Objective: To investigate the regulation of heat shock protein (HSP)27/activating transcription factor (ATF)-5 complex in podocytes induced by high glucose and relevant mechanisms.

Methods: Mice kidney podocytes were cultured in culture fluid with D-glucose at normal concentration (5.5 mol/L) (Group NG) or with D-glucose at high concentration (30 mmol/L) (Group HG) cells of these 2 groups were collected at different time points after glucose stimulation to detect the cell apoptosis by Hoechst 33342 staining and fluorescence microscopy and flow cytometry. Western blotting was used to analyze the activation of extracellular signal-regulated kinase (ERK = MAPK) and p38 signaling pathway. The HSP27/ATF5 complex was assessed by co-immunoprecipitation. ERK pathway blocker PD98059 and p38 signal pathway blocker SB203580 were added into the culture fluid of Group HG and Group NG respectively, and then the podocytes were collected at different time points to detect the high glucose-induced HSP27/ATF5 complex and cell apoptosis.

Results: The apoptotic rate of the podocytes of Group HG 24 hours after high glucose incubation was 14.3% +/- 6.2%, and that 48 h after was 27.2% +/- 8.9%, significantly higher than that of Group NG (10.6% +/- 2.7%, P < 0.05). HSP27/ATF5 complex could detected in the cells of Group NG too, however, the level of HSP27/ATF5 complex in Group HG 12 hours after incubation was 195% +/- 36% that of Group NG (P < 0.05). Both the ERK signal pathway and p38 signal pathway of Group HG began to be activated 10 min after incubation, peaked 30 min after, remained at the highest level till 1 hour after, and returned almost to the baseline level 2 hours after. No activation of these 2 pathways was observed in Group NG. The HSP27/ATF5 complex level of the PD98059 + high glucose group was 109% +/- 19% that of Group NG, significantly lower than that of Group HG (211% +/- 46% that of Group NG, P < 0.05). The apoptotic rate of the PD98059 + high glucose group was 51% +/- 4%, significantly higher than that of PD98059 + normal glucose group (16% +/- 3%, P < 0.05) and that of Group HG (27% +/- 9%, P < 0.05). The apoptotic rate of the SB203580 + HG group was 16% +/- 6%, significantly lower than that of Group HG (27% +/- 9%, P < 0.05). The HSP27/ATF5 complex level of the SB230580 + HG group was 290% +/- 43% that of Group NG, not significantly different from that of Group HG (231% +/- 20% that of Group NG, P > 0.05).

Conclusion: High glucose stimulates the formation of HSP27/ATF5 complex in podocytes through ERK signaling pathway but not P38 signaling pathway, and the HSP27/ATF5 complex may have a regulatory effect in podocyte apoptosis induced by high glucose.
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February 2006

[Tissue transglutaminase and renal fibrosis].

Sheng Li Ke Xue Jin Zhan 2005 Oct;36(4):314-8

Renal Division, Department of Medicine, First Hospital and Institute of Nephrology, Peking University, Beijing.

Tissue transglutaminase (tTG), an enzyme which can catalyze the posttranslational modification of proteins, is widely distributed and plays an important role in both physiological and pathological processes. More recently, tissue transglutaminase has been believed to participate in tissue fibrosis. Cross-linked extracellular matrix proteins by tissue transglutaminase are stable and highly resistant to proteolytic degradation, thus lead to accumulation of the ECM proteins in tissue fibrosis. This review summarizes the important features of this multifunctional enzyme and particularly introduces the correlation between tTG and renal fibrosis.
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October 2005

[Significance of detecting urinary podocytes in patients with active glomerulonephritis].

Beijing Da Xue Xue Bao Yi Xue Ban 2005 Oct;37(5):463-6

Renal Division, Peking University First Hospital, Beijing 100034, China.

Objective: To establish a reliable method for detecting urinary podocytes, as a non-traumatic marker to evaluate glomerular injury in patients with glomerulonephritis.

Methods: Sixty patients with renal diseases in our renal wards were diagnosed based on the pathological findings in their kidney biopsy tissues, which was examined by light microscopy, immunofluorescence and electron microscopy. Sediments of morning urinary samples were collected and centrifuged onto glass slides before kidney biopsy. Thirty healthy volunteers were enrolled as controls. The podocytes were identified by immunofluorescence staining by using monoclonal antibody against human podocalyxin (PCX) presenting on the surface of podocytes. The patients were divided into active inflammation group and chronic injury group according to their glomerular lesions.

Results: (1)The anti-human PCX antibody we used could specifically recognize the antigen expressed on podocytes in urine sediments examined by indirect immunofluorescence staining. (2) The PCX-positive staining cells in the urine were observed in various glomerulonephritis, and were absent in the healthy controls. (3) The rate of appearance of urinary podocytes was significantly higher in active inflammation group compared with that in chronic injury group (72% vs 22.7%, P<0.05). (4) The glomerular injury index in the patients with PCX-positive staining cells in the urine was markedly increased than that in the patients with PCX-negative staining cells (154+/-60 vs 82+/-46, P<0.05).

Conclusion: The urinary podocytes could be detected in urine sediments from patients with glomerulonephritis by using anti-human PCX antibody, and this method may find further application in the markers to predict the activity of glomerular lesions.
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October 2005

[Hypoxia stimulates expression of connective tissue growth factor through p38 signaling pathway in human renal interstitial fibroblasts].

Beijing Da Xue Xue Bao Yi Xue Ban 2005 Aug;37(4):378-81

Department of Nephrology, Peking University First Hospital, Beijing 100034, China.

Objective: To assess the expression of connective tissue growth factor (CTGF), and relevant mechanism for the regulation of CTGF expression by hypoxia in human renal interstitial fibroblast.

Methods: A human renal interstitial fibroblast cell line TK173 was treated under hypoxia (1% O(2)) or nomoxia (21% O(2)) condition. The expressions of HIF1-alpha, hypoxia marker protein, and CTGF protein were analyzed by Western blotting. RT-PCR was carried out to measure the levels of CTGF mRNA. The activations of MAPKs (ERK, JNK, p38) signaling pathways were assessed at different time points (30 min, 1 h, 6 h, and 12 h ), and the changes of CTGF expression were detected after the inhibitors of activation of MAPKs were applied, respectively.

Results: The expression of HIF-1alpha protein appeared in cells under hypoxia for 6 h. The expressions of CTGF protein were up-regulated in TK173 cells under hypoxia for 12 h, reached the peak levels in 2 folds of normoxia group cells for 24 h, and return to the levels of control cells by 48 h. The levels of CTGF mRNA were elevated in cells under hypoxia for 1 h, significantly increased at 6 h (6.6+/-1.0, P=0.000 2), and returned to the levels of normoxia group cells by 24 h. Activations of ERK1/2, JNK and p38 were seen in hypoxic cells. Activation of ERK1/2 and JNK were occurred as early as at 10 min, and reached the peak levels at 1 h, while the peak levels of activated JNK were seen at 30 min, then the levels of activated ERK1/2, p38, and JNK were all declined at 6 h, back to the baseline levels at 12 h. Blockade of ERK activation with PD98059, and blockade of JNK activation with SP600125 did not suppress hypoxia-induced expression of CTGF protein, whereas blockade of p38 MARK activation with SB203580 abolished hypoxia-induced expressions of CTGF protein and CTGF mRNA.

Conclusion: Hypoxia could stimulate the expression of CTGF in human renal interstitial fibroblast through the activation of p38 MARK signaling pathway.
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August 2005

[High glucose regulates the production of MMP-9 in podocyte through ERK1/2 signal pathway].

Zhonghua Yi Xue Za Zhi 2005 Jun;85(21):1451-5

Renal Division, Peking University First Hospital & Institute of Nephrology, Peking University, Beijing 100034, China.

Objective: To assess the effect of high glucose on the production of gelatinase and collagen alpha (IV) protein in podocytes and its possible signal pathway.

Methods: Mouse podocytes of an immortalized cell line were cultured and divided into 3 groups: NG group, treated with normal concentration of D-glucose (100 mg/dl), HG group, treated with high concentration of D-glucose (450 mg/dl), and MN group, treated with mannitol (350 mg/dl) plus D-glucose (100 mg/dl). The culture medium supernatants were collected every day. The activity of MMP-9 and MMP-2 was detected by gelatin zymography, the level of collagen alpha5 (IV) protein and the activation of MAPKs (Erk, p38, and JNK) signaling pathway in podocytes were detected by Western blot analysis, and the level of MMP-9 mRNA was detected by RT-PCR. Another podocytes were pretreated by PD9805, a specific inhibitor of MEK1 activation, and then divided into 3 groups as mentioned above so as to detect the effects of high glucose on the MMP-9 activation, and expression of MMP-9 mRNA and collagen alpha5 (IV) protein.

Results: The MMP-2 and MMP-9 activity in the medium supernatants of the NG and MN groups remained constant during the 10 days' incubation. High glucose incubation also did not affect the activity of MMP-2. The MMP-9 activity in the supernatant of the HG group began to increase in the 2nd day, reached the maximum in the 3rd day (144.2 +/- 18.1% that of the NG group, P = 0.006), then began to decline since the 5th day, back to the basal level in the 7th day (76.6 +/- 16.4% that of the NG group, P = 0.218), and remained at the basal level until the 10 th day. The basal level of collagen alpha5 (IV) protein in the supernatant of the NG group was quite high. The collagen alpha5 (IV) protein level in the supernatant of the HG group began to decrease since the 2nd day, reached the minimum in the 3rd day (41.9 +/- 25.5% that of the NG group, P = 0.047), then backed to the basal levels in the 5th day, and retained at that level to the 7th days. The MMP9 activity in the supernatant of the HG group had a strongly negative correlation with the levels of collagen alpha5 (IV) protein (r = -0.577, P < 0.006). The levels of collagen alpha5 (IV) protein in the supernatant of NG and MN groups showed no significant change during the 7 days' incubation. The level of MMP-9 mRNA of the HG group was 199.8 +/- 40.2% that of the NG group (P = 0.003) 2 days after stimulation, and was 90.9 +/- 8.8% that of the NG group 5 days after incubation (P = 0.411). Phosphorylation of ERK1/2 occurred as early as 30 min after simulation by high glucose, reached the peak level 6 hours later, remained at this level for 24 hours, then backed to the basal level 48 hours later, whereas the activation of p38 and JNK remained undetectable. Pretreatment with PD98059, for 30 min abolished the HG-stimulated increase of MMP-9 activity and MMP-9 mRNA, as well as the decrease of collagen alpha5 (IV) protein.

Conclusion: The production of MMP-9 and the levels of collagen alpha5 (IV) protein can be regulated by high glucose, and the ERK1/2 transduction pathway mediate such regulation.
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June 2005

[Connective tissue growth factor promotes the proliferation of myofibroblast through Erk-1/2 signaling pathway].

Zhonghua Yi Xue Za Zhi 2005 May;85(19):1322-6

Division of Nephrology, Peking University First Hospital & Institute of Nephrology, Peking University, Beijing 100034, China.

Objective: To investigate the mechanism by which connective tissue growth factor (CTGF) enhances transforming growth factor-beta(1) (TGF-beta(1))-mediated myofibroblastic activation in renal interstitial fibroblast NRK-49F.

Methods: NRK-49F cells were pretreated by TGF-beta(1) so that some cells transform into myofibroblasts, and then the cells were devided into vechile, CTGF treated group, TGF-beta(1) treated group, and PD98059 intervene group. The hallmark of myofibroblast, alpha-SMA immunostaining and marker of cellular proliferation, BrdU incorporation were determined by immunocytochemistry doublestaining. The protein level of alpha-SMA was determined by Western blot analysis.

Results: CTGF induced a proliferative response in myofibroblast initiated by TGF-beta(1), whereas TGF-beta(1) had no action on proliferation. Although CTGF could not induce myofibroblastic activation in renal interstitial fibroblast, it upregulated the protein level of alpha-smooth muscle actin significantly in cells pretreated by TGF-beta1 (P < 0.05). Significant phosphorylation of Erk-1/2 was detected after incubation with CTGF for 30 min in the cells pretreated by TGF-beta(1), while TGF-beta(1) did not have this ability. Inhibition of Erk-1/2 activation by Mek kinase inhibitor PD98059 suppressed CTGF-mediated myofibroblasts proliferation and significantly down-regulated expression of alpha-SMA protein in cells pretreated by TGF-beta(1) (P < 0.05).

Conclusion: CTGF induced a proliferative response in TGF-beta(1)-initiated myofibroblasts, and this action is likely dependent on the activation of Erk-1/2 signaling pathway.
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May 2005

[Transforming growth factor-beta1 stimulates matrix metalloproteinase-9 production through ERK activation pathway and upregulation of Ets-1 protein].

Zhonghua Yi Xue Za Zhi 2005 Feb;85(5):328-31

Division of Nephrology, Peking University First Hospital & Institute of Nephrology, Beijing 100034, China.

Objective: To investigate the molecular mechanism of transforming growth factor-beta1 (TGF-beta1) in regulating the production of matrix metalloproteinase-9 (MMP-9) protein.

Methods: Mouse immortal podocyte cells were cultured. Different concentrations (1, 2, and 5 ng/l) of TGF-beta1 were added into the culture medium. Cell culture without TGF-beta1 stimulation was used as control group. The activity of MMP-9 in the supernatant of the culture medion was assayed by gelatin zymography, expression of MMP-9 mRNA was assessed by RT-PCR; the activation of ERK pathway and the level of a transcriptional factor Ets-1 protein was analyzed by Western blotting. PD98059, a specific inhibitor of ERK1/2 activation, was added into the culture fluid of the podocytes for 30 minutes, than 2 ng/ml TGF-beta1 was added. The above mentioned tested were conducted to observe the influence of the inhibitor of ERK1/2 activation.

Results: The MMP-9 activity was very week in the supernatant of culture fluid of the control group and was increased in the TGF-beta1 groups dose-dependently. After the podocytes were co-incubated with 1 ng/ml TGF-beta1 for 24 hours, the MMP-9 activity was 26.86 times that of the control group (P < 0.01). Since the 12th hour after co-incubation with 2 ng/ml TGF-beta1 the MMP-9 activity in the supernatant of culture fluid began to be significantly increased and remained at high level till the 48 th hour. RT-PCR showed that low-level MMP-9 mRNA expression in the control group. After stimulation of 2 ng/ml TGF-beta1 for 6 hours the MMP-9 mRNA expression was 2.71 times that of the control group (P < 0.01) and the high-level expression lasted 24 hours. Western blotting showed low-level Ets-1 protein in the control group. At the time point of 12 th hour after stimulation of TGF-beta1 the Ets-1 protein expression was increased in all the three TGF-beta1 groups. After stimulation with 2 ng/ml TGF-beta1 for 4 hours the Ets-1 protein expression was 2.71 times that of the control group (P <0.01). After pretreatment of the podocyte with PD98059 for 30 minutes, the added 2 ng/ml TGF-beta1 failed to increase the MMP-9 activity and up-regulate the MMP-9 mRNA expression.

Conclusion: TGF-beta1 stimulates the production of MMP-9 by activation of cytoplasmic ERK signaling pathway and upregulation of Ets-1 expression.
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February 2005

[Expression and implication of tissue transglutaminase and connective tissue growth factor at fibrotic tubulointerstitium in kidneys from UUO rats].

Beijing Da Xue Xue Bao Yi Xue Ban 2005 Apr;37(2):143-6

Department of Nephrology, Peking University First Hospital, Beijing 100034, China.

Objective: To observe the expression and co-locolization of tissue transglutaminase (tTG) and connective tissue growth factor (CTGF) in kidneys from rats with tubulointerstitial fibrosis.

Methods: The animal models of unilateral ureteral obstruction (UUO) were used. The rats were randomly divided into the sham-operation group (n=5), the UUO group (n=6), and the Enalapril-treated UUO group (n=6). All the rats were sacrificed on day 9, the kidneys were collected, and renal interstitial fibrosis was examined by periodic acid-Schiff (PAS) staining. The expression and localization of tTG, CTGF, and Fibronectin( FN) in the obstructed kidneys were detected by immunohistochemistry staining. The protein levels of tTG and CTGF of the whole kidney homogenates were determined by Western blot analysis.

Results: Weak signals of tTG, CTGF, and FN-positive immunostaining were observed in renal tubular cells and tubulointerstitium, and very rare positive signals were seen in the glomeruli in the sham-operation group. However, much intensive signals of tTG, CTGF, and FN-positive immunostaining were observed in renal tubular cells and tubulointerstitium in the UUO group, and the intensity of signals were decreased in the Enalapril-treated UUO group. Co-localization of tTG and CTGF immunoreactivity were observed in tubulointerstitium in the UUO group. The levels of tTG and CTGF protein analyzed by Western blotting were increased remarkably in the UUO group compared with the sham-operation group, and a significant reduction of tTG and CTGF protein levels were presented in the Enalapril-treated UUO group compared with the UUO group. The levels of tTG protein had positive correlation with the levels of CTGF protein (r=0.683, P<0.05), and with the semi-quantitative levels of FN expression (r=0.737, P<0.05).

Conclusion: Both tTG and CTGF may play a concordant role in the pathogenesis of renal tubulointerstitial fibrosis,and Enalapril may suppress the development of fibrosis partially via down-regulation of tTG and CTGF expression.
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April 2005

[Transforming growth factor beta 1 modulates connective tissue growth factor expression via Smad2 signaling pathway in podocyte in vitro].

Zhonghua Yi Xue Za Zhi 2004 Apr;84(7):574-7

Department of Nephrology, Peking University First Hospital, Beijing 100034, China.

Objective: To assess the expression of connective tissue growth factor (CTGF), and the signaling pathway for the regulation of CTGF by transforming growth factor beta1 (TGF beta(1)) in podocytes.

Methods: In this study, we observed the effects of three potent profibrotic growth factors-TGF beta(1), Platelet-derived growth factor (PDGF), and Angiotensin II (AngII) on the expression of CTGF protein by Western blot analysis in cultured mouse podocytes, which is one of the most important cell construction of glomerular filter barrier, and we also investigated the underlying ERK and Smads signaling pathway through which TGF beta(1) regulates CTGF expression. The levels of CTGF mRNA were assayed by RT-PCR.

Results: Basal levels of CTGF protein were observed in cultured podocytes, treatment with 20 ng/ml PDGF and 10(-6) mol/L Ang. II for 24 h did not stimulate the expression of CTGF protein compared with control (P > 0.05), but significantly increase in levels of CTGF protein were seen in 1 ng/ml TGF beta(1) treated cells compared with with control (P < 0.05), and the levels of CTGF were up-regulated in a TGF beta(1) dose-dependent manner; The level of CTGF mRNA was also stimulated by 1 ng/ml TGF beta(1) at 12 h. 1 ng/ml TGF beta(1) induced phosphorylation of Smad(2) and ERK(1/2), and both reached the peak at 30 min; suppression of phosphorylation of Smad(2) with Staurosporine, a Serine/Threonine kinase inhibitor, diminished TGF beta(1)-triggered expression of CTGF protein, while blockade of phosphorylation of ERK(1/2) with PD98059, a specific ERK(1/2) activation inhibitor, did not decrease the TGF beta(1)-triggered expression of CTGF protein.

Conclusion: TGF beta(1) stimulated the expression of CTGF protein via Smad(2)-dependent and ERK(1/2)-independent signaling pathway in podocyte in vitro.
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April 2004

[Connective tissue growth factor synergistically with transforming growth factor beta 1 to promote renal fibrosis].

Zhonghua Yi Xue Za Zhi 2004 Apr;84(7):569-73

Institute of Nephrology & Renal Division, Peking University First Hospital, Beijing 100034, China.

Objective: To investigate the influence of CTGF and TGF-beta(1) on the synthesis and secretion of matrix metalloproteinase-2 (MMP-2) and myofibrotic activation in renal fibroblasts.

Methods: Equal numbers of renal fibroblasts (NRK-49F) were planted and divided into vechile, CTGF treated alone, TGF-beta(1) treated alone, and CTGF plus TGF-beta(1) treated groups. Gelatin zymography and Western-blot analysis were used for assay of the MMP-2 activity and protein level in the supernatant cultured medium, respectively. The levels of MMP-2 mRNA were assessed by real time-PCR. Western-blot analysis was carried out to measure the expression of alpha-smooth muscle actin (alpha-SMA), a maker protein of myofibroblast in cells, and the levels of extracellular matrix (ECM) component Fibronectin in supernantant medium.

Results: The activity and protein level of MMP-2 were no significant difference between the groups when cells were stimulated for 24 hours. While cells were stimulated for 48 hours, 100 ng/ml CTGF and 5 ng/ml TGF- beta(1) induce a increase in MMP-2 activity and protein levels compared with vechile, respectively (P < 0.05); different dose of CTGF plus TGF-beta(1) had the tendency to suppress MMP-2 activity and protein level, and a significant decrease was seen in 50 ng/ml CTGF plus 5 ng/ml TGF-beta(1) group, 100 ng/ml CTGF plus 5 ng/ml TGF-beta(1) group compared with CTGF group and TGF-beta(1) group, respectively (P < 0.05). When cells were stimulated for 12 hours, the levels of MMP-2 mRNA were increased significantly in 100 ng/ml CTGF group and 5 ng/ml TGF-beta(1) group compared with vechile respectively beta(1.72), 1.68 vs 1.29, (P < 0.01), decreased significantly in CTGF plus TGF-beta(1) group compared with CTGF group and TGF-beta(1) group, respectively (0.67 vs 1.72, 1.68, P < 0.01). 100 ng/ml CTGF had no prominent effect on the expression of alpha-SMA in cells and FN in supernatant medium (P > 0.05), whereas 5 ng/ml TGF-beta(1) significantly stimulated both the expression of alpha-SMA and FN (P < 0.05), and CTGF plus TGF-beta(1) induced more alpha-SMA and FN compared with TGF-beta(1) (P < 0.05).

Conclusion: CTGF synergistically with TGF-beta(1) to induce the formation of myofibroblasts and down-regulate the production of MMP-2 in renal fibroblast.
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April 2004

[The clinic significance of urinary podocytes in patients with focal segmental glomerulosclerosis].

Beijing Da Xue Xue Bao Yi Xue Ban 2004 Apr;36(2):135-8

Department of Nephrology, Peking University First Hospital Peking University Institute of Nephrology, Beijing 100034, China.

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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April 2004