Objective To investigate the molecular mechanism of chemokine released from renal proximal tubular epithelial cells (PTECs) induced by urine protein of minimal change disease (MCD) and focal segmental glomerular sclerosis (FSGS) patients. Methods Patients with MCD or FSGS were diagnosed by clinic and renal biopsy. Cultured PTECs were incubated with urine protein extracted from MCD or FSGS patients. Protein and gene levels of MIF and RANTES were detected by RT-PCR, flow cytometry and ELISA. MARK and extracellular regulated kinase (ERK) activation were measured by Western blotting. Pretreatment of PTECs with specific inhibitor SB203580 or PD98059 was performed to prevent the activation of MARK and ERK. Results Both of the two kinds of urine protein increased RANTES and MIF expression in time- and dose-dependent manner. Additionally, FSGS urine protein mediated a significantly greater secretion of chemokine in PTECs compared to MCD urine protein. Both of the two kinds of urine protein significantly activated p38 MARK and ERK pathway. Pretreatment with SB203580 or PD98059 prevented the effect of FSGS urine protein on RANTES and MIF production, while only SB203580 inhibited the effect of MCD urine protein. Conclusions Urinary protein of FSGS induces a potentially higher expression of chemokines in PTECs as compared to that of MCD through the distinct MAPKs signaling pathway. Component of proteinuria may play a role in determining the severity and progression of tubular inflammation and fibrosis.

Objective To detect the level of serum amyloid A (SAA) and explore its relationship with cardiovascular disease (CVD) in patients with chronic kidney disease(CKD). Methods Clinical data of 178 CKD patients, including 120 non-dialytic (ND), 58 hemodialysis (HD), and 60 controls were collected and analyzed retrospectively. SAA level was detected by enzyme-linked immunosorbent assay（ELISA）. Left ventricular diameter (LVD), left atrial diameter(LAD), left ventricular posterior wall thickness(LVPWT), interventricular septal thickness (IVST) and left ventricular ejection fraction(LVEF) were detected by ultrasonic cardiography. Relationship between SAA level and CVD incidence in CKD patients was analyzed by Logistic regression model. Results The average level of SAA in CKD patients was significantly higher than that in control group (P<0.05), and was significantly higher in HD group as compared with ND group (P<0.05). SAA level increased with the progression of renal dysfunction. In ND group, SAA level was negatively correlated with glomerular filtration rate (GFR) and positively correlated with the proteinuria respectively (P<0.05), while in HD group, it was positively correlated with dialysis duration (P<0.05). However, in both groups, SAA level was positively correlated with C-reactive protein(CRP), interleukin 1β(IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), Tch and Lp(a) respectively(P<0.05). A positive correlation of SAA level with the clinical manifestations such as abnormal echocardiography and cardiovascular disease was found. A negative correlation was also found between SAA level and serum albumin, HDL-C and apoA (P<0.05). Conclusions SAA level elevates markedly in CKD patients which may result from increased synthesis and decreased excretion. SAA may be an isolated risk factor of CVD in CKD patients.

Objective To explore the implication of urine IL-18 in early diagnosis of contrast-induced nephropathy (CIN) after coronary angiography in a prospective study. Methods One hundred and fifty patients undergone coronary angiography were enrolled in this study, and CIN was defined following the traditional criteria. Before and 24 h, 48~72 h after the procedure, the serum creatinine (Scr) was tested by enzymic method. Before and 24 h after the procedure, the clinical data and urine samples were collected. Urine IL-18, NAG and RBP were tested by ELISA method and were compared between CIN and non-CIN groups. Results CIN occured in 13 of 150 enrolled patients, the incidence was 8.7%. Twenty-four hours after the use of contrast medium, the levels of urinary IL-18(ng/L) and NAG (U/L) elevated significantly [15.06(12.21, 21.31) vs. 11.62(9.37, 13.86) and 13.88(7.09, 33.23) vs. 10.09(5.96, 16.62), P<0.05]. The differences of RBP and Scr at this time point were not significant. Compared with the non-CIN group, urine IL-18 (ng/L) level of CIN group increased significantly [18.97(13.64, 48.57) vs. 14.01(11.91, 17.77), P<0.05]. Significant linear correlation was found between the levels of urine IL-18 and Scr (r=0.664, P=0.013), whereas urine NAG and RBP were not significantly correlated with Scr. ROC analysis confirmed the diagnostic accuracy of urine IL-18 in CIN, and the area under the curve was 0.749 (P=0.012). With the cut-off value of IL-18 as 15.8 ng/L, the diagnostic sensitivity and specificity in CIN were 69.2％ and 74.1％, respectively. In cohort study, the cases with significant increment of urine IL-18 after the procedure was more risky to CIN (RR=3.125). The constituent ratio of significant increment of urine IL-18 was the highest among markers tested (P< 0.05). Conclusion Urine IL-18 can diagnose CIN earlier than Scr, which may be a good biomarker for early diagnosis of CIN.

Objective To study the effect of CD2-associated protein (CD2AP) on podocyte proliferation and division. Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃ permissive condition. After labeling with red fluorescent dye PKH-26, podocytes were transfected with CD2AP small interfering RNA (siRNA) using transfection reagent metafectene. Transfection efficiency was measured by flow cytometer 24 hours later and inhibitory effect of CD2AP siRNA was determined by RT-PCR and Western blot at 48 hours after transfection. Proliferation index and cell cycle of podocytes were examined by flow cytometer 72 hours later. Microbule of podocyte was detected by Oregon Green○Ｒ488-conjugated paclitaxel. The ratio of binuclear and polynuclear podocytes was counted under laser scanning confocal microscope (LSCM). Results The transfection efficiency of CD2AP siRNA was 66.27%. Forty-eight hours after transfection with specific siRNA, the expression levels of CD2AP mRNA and protein were down-regulated by 57％ and 39％ respectively. The podocyte proliferation index apparently descended (P<0.05) and the cells in the phase of G2/M accumulated significantly when detected at 72 hours (P<0.05). Under LSCM, podocytes that could not separate after M-period were easily found. The ratio of binuclear and polynuclear podocytes in CD2AP siRNA transfected group was markedly higher than that in control group (P<0.05). Conclusion Knocking-down CD2AP gene expression can interfer cell separation after mitosis and inhibit the podocyte proliferation.

Objective To explore the effect of overexpression of Smad7 on epithelial-mesenchymal transition (EMT) of mesothelial cells in rat peritoneal fibrosis model. Methods Thirty rats were randomly allocated into four groups: normal control group (n=6), peritoneal fibrosis model group (n=12), empty vector group (n=6) and Smad7 gene transfer group (n=6). pTRE-m2 Smad7 plus pEFpurop-Tet-on plasmids plus sonovue or substitution of pTRE-m2 Smad7 with empty vector were injected simultaneously with 4.25% peritoneal dialysate (PDS) and lipopolysaccharide (LPS) on day 15, after intraperitoneal injection of PDS and LPS alone. Peritoneal tissue was dissected out at days 14 and 28 after intraperitoneal injections of PDS and LPS, and the expression patterns of TGF-β1, p-Smad2/3, α-SMA, and E-cadherin were examined by RT-PCR, Western blot or immunofluorescence. Results Comparing with the rats in normal control group, the expression levels of TGF-β1, p-Smad2/3, and α-SMA were increased in model group and vector group (P<0.01), but E-cadherin was reduced (P<0.01). Smad7 gene transfer group abrogated PDS- and LPS-induced changes in the expression of p-Smad2/3, α-SMA, and E-cadherin (P<0.01). Conclusion Blockade of Smad2/3 activation is a central mechanism by which overexpression of Smad7 prevents EMT of mesothelial cells and progressive peritoneal fibrosis in rat peritoneal fibrosis model induced by PDS and LPS.

Objective To investigate the effect of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanism. Methods Glomerular mesangial cell line (HMCL) cells were cultured and divided into control group, IL-1β group and different concentration rapamycin groups. Intracellular cholesterol accumulation was observed and measured by oil O staining and HPLC. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression of LDLR, PPARγ, LXRα, ABCA1 in HMCL after the treatment with IL-1β and rapamycin. Results Rapamycin had no significant influence on intracellular cholesterol concentration under normal condition. IL-1β significantly increased the intracellular cholesterol concentration by 143％ of control (P<0.05), and 10, 50, 100 ?滋g/L rapamycin could significantly inhibit the effect of IL-1β (87％, 116％, 96％ of control respectively, all P<0.05). Rapamycin could suppress the increased expression of LDLR caused by IL-1β on both mRNA and protein level in a dose-dependent manner（P<0.01）. Rapamycin dose-dependently up-regulated the reduced mRNA and protein expression of ABCA1, the decreased mRNA expression of PPARγ and LXRα induced by IL-1β as well (P<0.01）. Conclusion Rapamycin may contribute to the maintenance of intracellular cholesterol homeostasis in glomerular mesangial cells under inflammatory state by both reducing cholesterol uptake and promoting cholesterol efflux.

Objective To investigate the effects of bradykinin (BK) on proliferation and collagen secretion in cultured renal mesangial cells (MCs) and to elucidate the mechanism associated with PDGF-BB and ERK signaling pathway. Methods Cultured MCs in vitro were stimulated by BK alone or following incubation of PDGF-BB. Proliferation of MCs was measured by MTT, secretion of collagen by ELISA and protein expression by Western blotting. After incubation with HOE-140 (BK receptor specific inhibitor), OV (tyrosine phosphatase inhibitor) and U0126 (ERK pathway inhibitor), MCs were co-stimulated by BK and PDGF-BB, then protein expression of ERK was examined by Western blotting. Results BK (10~1000 μg/L) alone increased the proliferation, secretion of ColⅠ, Col Ⅳ and induced phosphorylation of ERK1/2 in MCs. Similar results were found in MCs stimulated by PDGF-BB, but such effects were inhibited by BK in a dose-dependent manner. Meanwhile above inhibition of BK against PDGF-BB was blocked by 1 μmol/L HOE-140 and 0.5 mmol/L OV. In other hand, the effects of HOE-140 and OV were inhibited by U0126. Conclusions Stimulation of BK on MCs is mainly mediated through ERK signaling pathway. BK can inhibit the similar stimulation of PDGF-BB and possesses a double effect in which B2 receptor and tyrosine phosphatase may be involved.

Objective To investigate whether Rho/Rho kinase is involved in the pathogenesis of aldosterone/salt-induced renal injury. Methods Twenty-six uninephrectomized rats which were limited to drink only 1% sodium chloride were divided randomly into three groups: control[2% ethanol, subcutaneous(SC)], aldosterone(0.75 μg/h aldosterone, SC) and aldosterone(0.75 μg/h aldosterone, SC) plus fasudil(10 mg&#8226;kg-1&#8226;d-1, SC). After treatment for 5 weeks, systolic blood pressure, proteinuria, renal histological examination, angiotensin Ⅱ level, expression of phospho-RhoA, phospho-MYPT1(represent Rho kinase activity) and ACE by Western blot and mRNA expression of TGF-β1, collagen I and collagen Ⅲ by real-time PCR were all tested. Results Aldosterone plus salt induced severe hypertension[（193±3） mm Hg vs. (130±4) mm Hg, P<0.05], mass proteinuria[(362±93) mg/24 h vs. (22±3) mg/24 h, P<0.05] and severe glomerular proliferative lesion and tubular interstitial fibrosis, accompanied by augmented angiotensin Ⅱ level[(154±16) pmol/g vs. (81±8) pmol/g, P<0.05）] and increased expression of phospho-RhoA and phospho-MYPT1, as well as TGF-β1, collagen I and collagen Ⅲ mRNA in the cortex. But fasudil, a kind of Rho-kinase inhibitor, significantly ameliorated the above effects without changes of angiotensin Ⅱ level, ACE and phospho-RhoA expression and systolic blood pressure. Conclusions Increased cortical Rho/Rho-kinase is involved in aldosterone-induced renal injury. The renoprotection of fasudil is not carried out via suppressing increased cortical angiotensin Ⅱ level and ACE overexpression.