Objective To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cells (HKC).Methods Cultured HKC were divided into 3 groups: (1) negative control; (2) low dose CTGF treated (rhCTGF 2. 5 ng/ml); (3) high dose CTGF treated (rhCTGF,5. 0 ng/ml). Expression of ?-smooth muscle actin (?-SMA) and fibronectin(FN) mRNA were measured by RT-PCR. Indirect immunofluorescence and flow cytometry methods were used to assess the level of intracellular ?-SMA protein. Concentration of FN secreted into the media was determined by ELBA. Results Upon the stimulations of different concentrations of rhCTGF,expression of ?-SMA and FN mRNA increased markedly(P

To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC), in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5.0 ng/ml). Then the expression of alpha-smooth muscle actin (alpha-SMA) were assessed by indirect immuno-fluorescence, and the percentage of alpha-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of alpha-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of alpha-SMA were markedly stronger than that in negative controls. The percentages of alpha-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9%, 65.5% vs 2.4%, P<0.01). alpha-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F).
Actins/metabolism ; Cell Differentiation/*drug effects ; Cells, Cultured ; Epithelial Cells/*cytology ; Immediate-Early Proteins/*pharmacology ; Insulin-Like Growth Factor Binding Proteins/pharmacology ; Intercellular Signaling Peptides and Proteins/*pharmacology ; Kidney Tubules/*cytology

Objective To study the effects of CD2-associated protein (CD2AP) on podocyte adhesion and extension ability and to explore its possible mechanism. Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. The podocytes were transfected with CD2AP small interfering RNA (siRNA) and serambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagen IV coated plates. The relative cell adhesion and cell area were examined 90 min later. Apoptotic rates of CD2AP siRNA transfected podoeytes and different PAN concentrations incubated podoeytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confoeal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot. Results The relative ceil adhesion of CD2AP siRNA transfected podocytes was apparently lower than that of control group[(41.72±6.07)% vs (64.46±8.53)%, P<0.05]. The cell area analysis had the similar result. The apoptotic rate of CD2AP siRNA transfected podocytes was significantly higher than that of the controls [(5.73±0.61)% vs (3.26±0.45)%, P<0.05]. 100 mg/L PAN could markedly induce podocytes to apoptosis and impair cell adhesion ability (P<0.05). Nevertheless, no significant difference was found in cell body spreading (P>0.05). The distribution of F-actin in CD2AP depletion podocytes was apparently altered. The expression of nephrin protein and its phosphorylation level was conspicuously descended to some degree (P<0.05). Conclusions CD2AP depletion facilitates podocyte apoptosis and impairs cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness caused by CD2AP depletion may he partly responsible for the decline of cell adhesion and spreading.

AIM:To observe the expression of connective tissue growth factor(CTGF)in unilateral ureteral obstruction(UUO)rats,and to explore its pathogenic role in renal tubulointerstitial fibrosis.METHODS:48 Wistar rats were randomly divided into sham-operated and UUO group.The rats were sacrificed at day 1,3,7,and 14.The degree of tubulointerstitial damage was scored according to the Masson staining.The mRNA and protein levels of CTGF,transforming growth factor-?1(TGF-?1),collagen Ⅰ(Col Ⅰ),and plasminogen activator inhibitor-1(PAI-1)were detected by reverse transcriptional-polymerase chain reaction(RT-PCR)and immunohistochemistry,respectively.Expression of CTGF protein in the kidney was also assessed using Western blotting.RESULTS:TGF-?1 mRNA level began to increase as early as 1 day after UUO.This increase was followed by the elevation of CTGF mRNA level,which began to increase at third days after UUO(P

Objective To study the CT findings of dermoid cyst of the orbit.Methods Four cases of dermoid cyst of the orbit proved by operation and pathology were presented. There were three cases of male, one case of female, age 6～21 years. Transverse CT scans(3 mm slice) and coronal CT scans(5 mm slice) were obtained, in which enhancement study was done in two cases.Results Among four cases, lesion was located in the right on three cases, in the left on one case. All lesions were located outside the muscle of upper-lateral guarter. One lesion was showed a multi-lobular shape, with widening of the frontozygomatic fissure and protruding to outside of the orbit. Three lesions were found anoval shape, in which one lesion was showed a bony sclerosis and thickening, another two cases were showed no bony change. On all cases, the edge of the lesions were demonstrated clearly, in which three cases were low-density(-140～-80 HU) similar to fat density, and one case with heterogeneous density. The lesion was showed more clearly on enhancement study on two cases.Conclusion CT findings of dermoid cyst of the orbit is characteristic and the diagnosis can be made before operation.

A potential pathological role of angiopoietins (Ang) in glomeruli following podocyte injury-induced progressive glomerulosclerosis was explored. Eighty male Wistar rats were randomly allocated into sham operation group (Sham, n = 25), Uninephrectomy group (UPHT, n = 25) and Uninephrectomy+Daunorubicin group (DRB, n = 30). In DRB group, daunorubicin (5 mg/kg) was injected via tail vein on the 7th and 14th day after uninephrectomy. At week 1, 2, 4, 6 and 8 respectively following establishment of the animal model, 5 rats in Sham group and UPHT group, and 6 in DRB group were taken respectively for determining 24-h urinary protein excretion rate (24hUPER), blood urea nitrogen (BUN) and serum creatinine (Scr). The sections of kidneys were examined by an electric microscope, PAS staining, immunohistochemical staining and in situ hybridization histochemistry. The results showed that 24hUPER, BUN and Scr in DRB group were more than those in Sham group and UPHT group at the same time points, and there was a trend towards an increase on level of GSI in DRB group from week 2 to week 8. Electric microscopy revealed that podocyte injury presented in DRB group. The expression of Ang1 mRNA and protein in glomeruli of DRB group was decreased, while the expression of Ang2 protein in glomeruli of DRB group increased. Meanwhile, the expression of Ang1 mRNA had a negative correlation with the expression of Ang2 mRNA, and the expression of Ang1 protein had a positive correlation with the expression of Ang1 mRNA, and had a negative correlation with 24hUPER, BUN, Scr, glomerular sclerotic index (GSI), the expression of Ang2 protein and CoIV protein. The expression of Ang2 protein had a positive correlation with the expression of Ang2 mRNA, and had a positive correlation with 24hUPER, BUN, Scr, GSI, the expression of CoIV protein. It was concluded that podocyte injury might lead to an alteration in the expression of Ang1 and Ang2 within glomeruli. Ang2 may get rid of inhibition from Ang1 for downregulation of the Ang1 expression, which facilitate upregulation of the Ang2 expression in glomeruli to promote progressive glomerulosclerosis in the rats.

To investigate the role and mechanisms of apoptosis and apoptosis signaling pathway in 5/6 nephrectomy rat model (SN(x)), the mRNA and protein levels of caspase-3, -8, -9 and apoptosis were detected by in situ end labeling (TUNEL), immunohistochemistry, RT-PCR, Western-blotting 1, 2, 4, 8, 12, 16, 26 and 40 weeks after 5/6 nephrectomy rat model was made respectively. The rats in the model group developed glomerular sclerosis and renal interstitial fibrosis. The number of the apoptototic cells in glomeruli, renal tubule and renal interstitium was remarkably higher in the model group than that in the control group (P < 0.05, P < 0.01). Changes of mRNA and protein level of caspase-3, -8, -9 had the same tendency and was up-regulated wavily in the rat model compared with the control group (P < 0.05). Peaks in model appeared on the 4th and the 40th week respectively. The growth amplitude of caspase-9 was remarkably higher than that of caspase-8. It is concluded that the development of 5/6 nephrectomy rat model was correlated with the apoptosis of glomeruli, renal tubule and renal interstitium. Both of death receptor and mitochondria signaling pathways are involved in the process and the latter might play a primary role.

Accumulating evidence suggests that the small G protein Rho and its downstream effector Rho kinase may play important roles in kidney biology. The present study examined the effects of a Rho-kinase inhibitor, fasudil, on daunorubicin-induced progressive glomerulosclerosis and explored the underlying mechanism by which fasudil ameliorates glomerulosclerosis. Thirty-six male SD rats were randomly allocated into sham-operation group (sham group, n=12), unilateral nephrectomy (UNX)+daunorubicin (DRB) group (model group, n=12), UNX+DRB+Fasudil group (treatment group, n=12). Two to four weeks after the establishment of the animal model, 6 rats in each group were taken randomly for the detection of 24-h urine protein excretion. Kidney sections were examined by HE and PAS staining, immunohistochemistry and transmission electric microscopy (TEM). The expression of Rho-kinase mRNA and P27 mRNA in kidney were detected by RT-PCR. It was found that the 24-h urine protein excretion in model group was increased significantly as compared with sham group (P<0.01). But this increase was significantly suppressed by fasudil (P<0.05). At 4 week, the foot process effacement in podocytes, mesangial proliferation and ECM accumulation were observed in model group, presenting as focal segmental glomerulosclerosis. But in the treatment group, the fasudil alleviated glomerular injury, with proliferating cell nuclear antigen (PCNA)-positive cell infiltration ameliorated and the expression of P27 increased. The expression of Rho-kinase mRNA was significantly enhanced in model group and was suppressed in treatment group. Moreover, fasudil up-regulated the mRNA expression of P27. Our study demonstrated that the glomerulosclerosis was substantially ameliorated by inhibiting the expression of Rho-kinase. It is suggested that Rho-kinase pathway is involved in the renal injury and the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury.

To investigate the effects of mycophenolate mofetil (MMF) on the process of renal interstitial fibrosis, unilateral ureteral obstruction (UUO) model was established in rats. Twenty Sprague-Dawley rats underwent UUO and received vehicle (n = 10) or MMF (20 mg.kg-1.d-1, by daily gastric gavage, n = 10) during a period of 5 days following surgery, and the additional 10 rats were served as sham-operated group. The rats were killed 5 days after surgery. Immunohistochemistry was performed on renal tissue for proliferating cell nuclear antigen (PCNA), alpha-smooth muscle actin (alpha-SMA) and type I and III collagen (col I, col III). Histological studies were also done by MASSON staining. Five days after surgery, proliferating cells in tubules, interstitium as well as interstitial myofibroblast (MyoF) infiltration and interstitial col I, col III deposition were all significantly reduced by MMF treatment. MMF also alleviated the histological changes of UUO rats. These results suggested that the reduction of interstitial MyoF infiltration may be an important event by which MMF prevents renal injury caused by UUO and MMF could be used to limit the progression of renal fibrosis.
Fibrosis ; Kidney/*pathology ; Kidney Diseases/etiology ; Kidney Diseases/pathology ; Kidney Diseases/*prevention & control ; Mycophenolic Acid/*analogs & derivatives ; Mycophenolic Acid/*pharmacology ; Random Allocation ; Rats, Sprague-Dawley ; Ureteral Obstruction/*complications

[Abstract ] Objective To understand the identification and in vitro antifungal susceptibility of Penicillium marneffei(PM)in yeast phase,and guide clinic antifungal application.Methods Strains isolated from blood and bone marrow of 23 patients infected with PM in a hospital between 2009 and 2016 were collected,colony morpholo-gy of PM in yeast phase was observed,susceptibility to itraconazole,voriconazole,amphotericin B,and fluconazole were detected with E-test method.Results Colony morphology of PM were as follows:direct microscopic examina-tion of Wright’s staining of tissue specimens found visible oval or round spore with apparent septum,and mainly lo-cated in macrophage;Gram staining of blood culture specimens found that strains were with bulbous and slightly curved ends,occasionally branched and with septum. PM was dimorphic fungi,presented mycelium at 28° C,pro-duced red pigment and diffused into medium;PM presented yeast form at 35° C,there were typical colony morpholo-gy. Minimum inhibitory concentrations (MICs)of itraconazole,voriconazole,amphotericin B,and fluconazole to PM in yeast phase were 0.002-0.016,0.012-0.125,0.002-0.500,and 0.500-16.000μg/mL respectively. Conclusion Typical colony morphology and fungal spore of PM in bone marrow and peripheral blood are important features for identification. PM is most susceptible to itraconazole,followed by voriconazole and amphotericin B, while fluconazole is less susceptible.