Study on the Mechanism of miR-125b-5p Affecting Melanocyte Biological Behavior and Melanogenesis in Vitiligo through Regulation of MITF.

Objective: The goal was to confirm the mechanism by which miR-125b-5p influences melanocyte biological behavior and melanogenesis in vitiligo by regulating MITF. Methods: oe-MITF, sh-MITF, miR-125b-5p mimic, NC-mimic, NC-inhibitor, and miR-125b-5p inhibitor were transfected into cells by cell transfection. Western blotting was used to detect the related protein expression, qRT-PCR was used to detect miR-125b-5p and MITF expression, immunohistochemistry was used to detect the MITF-positive cells in vitiligo patients tissues, and a dual-luciferase reporter system was used to detect the target of miR-125b-5p and MITF. PIG1 and PIG3V cell proliferation by the CCK-8 method, cell cycle progression and apoptosis by flow cytometry, apoptosis was detected by TUNEL, Tyr activity and melanin content were measured using Tyr and melanin content assay kits. Results: Compared with the healthy control group, the expression of miR-125b-5p in the tissues and serum of vitiligo patients was upregulated, and the expression of MITF was downregulated; compared with PIG1 cells, the expression of miR-125b-5p and MITF in the PIG3V group was consistent with the above. Compared with the NC-minic group, the cell proliferation activity of the miR-125b-5p mimic group decreased, apoptosis increased, and the expression levels of melanogenesis-related proteins Tyr, Tyrp1, Tyrp2, and DCT were downregulated. Compared with the NC-inhibitor group, the above indices in the miR-125b-5p inhibitor group were all opposite to those in the miR-125b-5p mimic group. Transfection of oe-MITF into the miR-125b-5p mimic group reversed the effect of the miR-125b-5p mimic, while transfection of sh-MITF enhanced the effect of the miR-125b-5p mimic. Conclusion: miR-125b-5p affects vitiligo melanocyte biological behavior and melanogenesis by downregulating MITF expression.

The clinical and pathological relevance of waxy casts in urine sediment.

Although casts in urine may imply the underlying pathogenesis and the diagnosis, the waxy cast is poorly understood yet. We aim to investigate the association between waxy casts and clinicopathological indices. Patients undergone renal biopsy and urine sediment examination were enrolled. Waxy casts referred to those presented with a homogeneous melted wax appearance and pre-waxy casts referred to those in which one or more segments demonstrated a waxy-cast appearance. Multivariable logistic regression was used to assess the factors associated with waxy casts. In 1282 patients, the detection rate of waxy casts was 26.3%. If either waxy or pre-waxy cast was considered as a diagnostic marker for renal insufficiency (eGFR < 60 ml/min/1.73 m2), the sensitivity was 0.58 and the specificity was 0.88. If the only waxy cast was considered as the diagnostic marker, the sensitivity was 0.29 and the specificity was 0.97. The patients with waxy or pre-waxy casts had higher blood pressure, more proteinuria, and worse renal function. Waxy or pre-waxy cast was independently associated with eGFR (odds ratio: 0.73 per 10 mL/min/1.73 m2 increase, 95% confidence interval: 0.69-0.77, p < 0.001), proteinuria (odds ratio: 1.07 per 1 g/day increase, 95% confidence interval: 1.03-1.10, p < 0.001) and pathological lesions. Waxy or pre-waxy casts are closely related to impaired renal function. Their presence is a specific indicator of renal insufficiency but is not sensitive enough.

Urinary sediments could differentiate endocapillary proliferative lupus nephritis and endocapillary proliferative IgA nephropathy.

OBJECTIVE: This study investigated the differences in urinary sediment findings between patients with endocapillary proliferative lupus nephritis (LN) and patients with endocapillary proliferative IgA nephropathy (IgAN) and further evaluated the associations of leukocyturia with disease activity, pathological features and prognosis. METHODS: The urinary sediments of 126 patients, including 92 with LN and 34 with IgAN, with renal-biopsy-proven endocapillary proliferative glomerulonephritis (EPGN) were examined by a standardized method. The urinary elements investigated included various cells, casts and crystals. The associations of leukocyturia with disease activity, pathological features and prognosis were further analyzed. RESULTS: In the patients with EPGN, normal to mild leukocyturia (≤12/HPF) and moderate to severe leukocyturia (>12/HPF) were found in 52 (41.27%) and 74 (58.73%) patients, respectively. The proportion of moderate to severe leukocyturia and the frequencies of urinary white blood cell casts and waxy casts were significantly higher in endocapillary proliferative LN than those in endocapillary proliferative IgAN (P < 0.001, P = 0.020, P = 0.010, respectively). In the endocapillary proliferative LN group, the levels of leukocyturia were significantly correlated with serum creatinine (r = 0.288, P = 0.005), eGFR (r = -0.284, P = 0.006), serum C3 (r = -0.275, P = 0.009), SLEDAI scores (r = 0.383, P ≤ 0.001) and glomerular leukocyte infiltration (r = 0.285, P = 0.002). A multivariate analysis showed that leukocyturia was an independent risk factor for renal outcomes in endocapillary proliferative LN (HR: 1.456, 95% CI: 1.083-1.957, P = 0.013) but not in IgAN. CONCLUSIONS: Urinary sediments of LN with EPGN and IgAN with EPGN differed in many aspects. Leukocyturia could reflect the disease activity and prognosis of EPGN, especially in endocapillary proliferative LN.

[Clinical significance of urine sediment spectrum analyses in crescentic glomerulonephritis].

OBJECTIVE: To explore whether the analyses of urine sediment spectrum contribute to the diagnosis of crescentic nephritis and whether special cells in urine could be a biomarker for the early stage crescentic nephritis. METHODS: Thirty-five patients diagnosed as crescentic nephritis with renal biopsy were recruited. The phase-contrast microscope was used to observe the early morning urine and offer comprehensive descriptions of urine sediment spectrum. And podocalyxin antibody was utilized to detect podocytes in urine and renal specimens by immunohistochemistry. RESULTS: Marked hematuria and casts were present in the urine of crescentic nephritis and "special cells" appeared in over 50% subjects. The detection rates of "special cells" were 75%, 41% and 0 respectively in early, middle and later stages of crescentic nephritis. Podocytes were identified in the urine of 8/9 subjects. CONCLUSIONS: The urine sediment spectrum contributes to the diagnosis of crescentic nephritis. And special cells in urine are helpful to gauge the stage of crescentic nephritis.

[Urine sediment combined with urine protein as a biomarker for renal injury].

OBJECTIVE: To investigate whether combination of urine sediment and urine protein can predict the renal pathological changes. METHODS: We prepared 146 specimens of routine fresh fasting morning urine. Sediment analysis was performed with phase-contrast microscopy and 24-hour urine protein was measured. Both urine protein and sediment data were integrated to form three urine analysis groups. Urine group I: proteinuria, hematuira, urine white blood cells, red/white cell casts. Urine group II: proteinuria, few cell hyaline/fine granular casts. Urine group III: minor proteinuira, epithelial cells of tubule, granular/cell casts. The renal pathological lesions were predicted before and then confirmed by renal biopsy. Statistical analyses were performed using kappa test, chi-square test, and significance was accepted at P<0.05. RESULTS: After renal biopsy, we identified 95 cases of glomerular lesion with proliferation, 46 cases of glomerular disease without obvious proliferation and 5 cases of tubular interstitial lesion. According to the sediment analysis, only 67 cases (46%) could be attributed to urine group I. When combined with urine protein, we could pick out another 75 cases from urine groups I and II, and 8 cases from urine group III. The combined urine analysis could predict glomerular disease (77.7%). CONCLUSION: Clinically we can take advantage of the combined urine analysis to predict the pathological lesion of kidney disease, which is especially suitable for primary care doctor, who can not perform renal biopsy.

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

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.

Connective tissue growth factor stimulates renal cortical myofibroblast-like cell proliferation and matrix protein production.

Myofibroblasts primarily contribute to the pathogenesis of renal interstitial fibrosis by unregulated cell proliferation and synthesis of excessive amounts of extracellular matrix (ECM) proteins. We used cultured myofibroblast-like cells obtained by outgrowth from explants of rat kidney cortex to study the effects and relevant signaling pathway of connective tissue growth factor (CTGF) on cell proliferation and ECM production. Exogenous CTGF stimulated proliferation of myofibroblast-like cells in a dose- and time-dependent manner. CTGF also increased the secretion of fibronectin and collagen I protein in the supernatant medium. Nevertheless, CTGF did not affect matrix-degrading metalloproteinases-2 and -9 activities in supernatant medium measured by gelatin zymography. CTGF induced activation of extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinase pathway as early as 5 minutes. Inhibition of ERK1/2 activation with PD98059 completely blocked CTGF-induced cell proliferation as well as secretion of fibronectin and collagen I protein. The above results indicate that CTGF triggers cell proliferation and production of ECM proteins in cultured myofibroblast-like cells through the ERK1/2 mitogen-activated protein kinase pathway.

Connective tissue growth factor increases matrix metalloproteinase-2 and suppresses tissue inhibitor of matrix metalloproteinase-2 production by cultured renal interstitial fibroblasts.

The involvement of gelatinase (matrix metalloproteinase-2 [MMP-2] and MMP-9) in the matrix remodeling and development of tubulointerstitial fibrosis has been studied recently, but relatively little is known about the regulators and the mechanisms controlling the activation and expression of gelatinase in renal fibroblasts. In these studies, the production and underlying signaling pathway for gelatinase by exogenous connective tissue growth factor (CTGF) treatment were investigated. Here, we show that CTGF acts as a potent promoter of the activation and expression of MMP-2, but not MMP-9 in normal rat kidney fibroblasts cell line (NRK-49F). We found that CTGF significantly increased the activity of MMP-2, as well as MMP-2 protein in conditioned medium and MMP-2 mRNA levels in cells. In studies to address the mechanisms involved in the regulation of MMP-2 activity, we found that the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), the inhibitor of MMP-2, decreased significantly when cells were treated with CTGF. Further studies showed that extracellular signal-regulated kinase (ERK) signaling is responsible for most of the CTGF-induced MMP-2 expression and TIMP-2 suppression. When NRK-49F fibroblasts were incubated with CTGF, activation of ERK1/2 signaling was observed. Suppression of ERK1/2 activation with nontoxic concentrations of PD98059, a specific inhibitor of ERK activation, was associated with a reduction of CTGF-stimulated MMP-2 activity and protein expression. In addition, the CTGF-mediated reduction of TIMP-2 activity and protein expression was prevented when ERK1/2 activation was inhibited by PD98059. These results provide evidence that CTGF augments activation of MMP-2 through an effect on MMP-2 protein expression and TIMP-2 suppression, and that these effects are dependent on the activation of the ERK1/2 pathway.

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

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.

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

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.

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

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.

High ambient glucose levels modulates the production of MMP-9 and alpha5(IV) collagen by cultured podocytes.

Recent evidences have demonstrated an important role for glomerular visceral epithelial cell (podocyte) in the development and progression of diabetic nephropathy. We investigated the high-glucose (HG)-triggered signaling pathway and its role in matrix metalloproteinase (MMP) production in murine podocytes. The activity of 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2), in an HG medium significantly increased during incubation of 2 to 3 days and decreased during incubation of more than 5 days revealed by Gelatin zymography. Opposite to the increases in MMP-9 activity, HG medium produced significant decreases in the protein levels of alpha5(IV) collagen. Changes in MMP-9 activity were associated with the same pattern as MMP-9 mRNA levels in podocytes exposed to HG media. HG medium rapidly activated ERK1/2 MAPK in podocytes. Moreover, ERK1/2 activation was required for HG-induced enhancement of MMP-9 activity and a decrease in the level of alpha5(IV) collagen. HG incubation rapidly induced an increase in the nuclear accumulation of Ets-1 protein. Blocking the ERK pathway suppressed HG-induced expression and nuclear accumulation of transcriptional factor Ets-1, and MMP-9 mRNA expression. We suggest that short- or long-term exposure to HG concentrations increases or decreases MMP-9 production and alpha5(IV) collagen expression in podocytes, this may contribute to the GBM abnormality caused by an imbalance in extracellular matrix (ECM) synthesis and degradation, and may play a critical role in the pathogenesis of proteinuria in diabetic nephropathy.

ERK-dependent signaling pathway and transcriptional factor Ets-1 regulate matrix metalloproteinase-9 production in transforming growth factor-beta1 stimulated glomerular podocytes.

The unregulated synthesis of glomerular basement membrane (GBM) components, extracelluar matrix (ECM) proteins, or the secretion of ECM-degradation enzymes, matrix metalloproteinases (MMPs), by podocytes under pathological conditions might be major factors in GBM damage. The present study examined the effects and the underlying molecular mechanism of transforming growth factor beta1 (TGFbeta1) on the production of gelatinase in cultured murine podocytes. Our results showed that TGFbeta1 is the most potent inducer of MMP-9 secretion in both a dose- and time-dependent manner, but has very little effect on MMP-2 secretion. TGFbeta1 upregulated MMP-9 mRNA levels, but did not affect the expression of matrix mettaloproteinases TIMP-1 mRNA. TGFbeta1 induced activation of both Smad2 and extracellular signal-regulated kinases (ERK1/2). However, blockade of Smad2 signaling pathway by Staurosporine did not affect the TGFbeta1-stimulated secretion of MMP-9, whereas inhibition of activation of ERK1/2 by PD98059 abolished TGFbeta1-stimulated secretion of MMP-9 and expression of MMP-9 mRNA. Protein levels of the transcriptional factor Ets-1 increased and were sustained for 12 h by TGFbeta1-stimulation. Our data also showed that blockage of ERK1/2 activation by PD98059 led to a reduction in the level of Ets-1 protein and to a consequent decrease in MMP-9 mRNA levels. These results demonstrate that TGFbeta1 can induce the production of MMP-9 in podocytes through the ERK1/2 MAPK pathway, and suggested that an increase in MMP-9 enzymatic activities may be involved in the damage of the GBM in response to inflammatory factors, ultimately leading to glomerulosclerosis.

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

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.

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

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.

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

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.

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

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.

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

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.

Anti-inflammatory effect of PPARgamma in cultured human mesangial cells.

Our aim is to investigate whether peroxisome proliferator-activator receptor-gamma (PPARgamma) expression was altered in human mesangial cells under inflammatory stress and whether PPARgamma could retard the inflammatory responses. Based on cultured human mesangial cell lines (HMCLs), PPARgamma expressions at protein and mRNA levels were observed by Western blot analysis and reverse transcriptase polymerase chain reaction. Informatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay. Our results demonstrated that PPARgamma protein expression was dramatically increased in HMCLs stimulated by IL-1beta (10 ng/mL). The levels of IL-6 and TNF-alpha in HMCL supernatants, protein, and mRNA expressions of PPARgamma in IL-1beta challenge cells were significantly increased more than those in untreated cells. Importantly, PPARgamma agonists troglitazone, rosiglitazone, and 15-deoxy-delta(12, 14)-prosglandin J2 significantly decreased the up expression of TNF-alpha and IL-6 in HMCL supernatants stimulated by IL-1beta. Furthermore, troglitazone downregulated TNF-alpha and IL-6 mRNA expression from IL-1beta challenge HMCLs. Our data suggest that PPARgamma plays an important role in mesangial cells responding to inflammatory stress. PPARgamma may prove to be a pharmacological target in glomerulonephritis.

Tyrosine phosphorylation of the LDL receptor-related protein (LRP) and activation of the ERK pathway are required for connective tissue growth factor to potentiate myofibroblast differentiation.

Renal myofibroblasts play a crucial role in the accumulation of excess extracellular matrix during renal fibrosis. Both transforming growth factor-beta1 (TGFbeta1) and connective tissue growth factor (CTGF) are important profibrotic growth factors, which interact in the pathogenesis of fibrosis. In this study, we demonstrate that CTGF alone has no influence on myofibroblast transformation and fibronectin secretion in kidney interstitial fibroblasts, whereas incubation of CTGF in combination with TGFbeta1 enhanced TGFbeta1 responses, including myofibroblast activation, de novo expression of alpha-SMA, and extracellular accumulation of fibronectin. CTGF induced tryrosine phosphorylation of the cytoplasmic domain of the low-density lipoprotein receptor-associated protein (LRP) in fibroblasts, and the LRP-antagonist, receptor-associated protein (RAP) inhibited CTGF-induced tryrosine phosphorylation of LRP. Inhibition of LRP signaling reduced CTGF-mediated synergistic induction of alpha-SMA protein. Furthermore, the potentiating action of CTGF was neither dependent on modulation of TGFbeta1-induced Smad2 phosphorylation and its association with Smad4, nor did it result from nuclear accumulation of activated Smad2. When TGFbeta1-pretreated fibroblasts were incubated with CTGF, activation of ERK1/2 MAPK signaling was observed. Inhibition of ERK activation by the MEK1 inhibitor PD98059 was associated with a reduction of CTGF-promoted alpha-SMA protein expression. Our in vitro studies provide evidence that CTGF potentiates TGFbeta1-mediated myofibroblast differentiation and activates differentiated myofibroblasts.