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OBJECTIVE To investigate the effect ofγ-secretase inhibitor N-[N-(3,5-difluorophen?acetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on phenotypic transformation and matrix accu?mulation induced by aristolochic acid(AA) in renal tubular epithelial cells(NRK-52E)and explore the mechanism. METHODS NRK-52E cells were divided stochastically into normal cell control group,AA 10 mg·L-1 group and AA 10 mg·L-1+DAPT 1 and 10μmol·L-1 group. After 24 h,the mRNA expressions of Notch1,Jagged1,Numb,E-cadherin,transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),bone morphogenic protein 7 (Bmp7),typeⅠ a1 (Col1a1) and Ⅲ collagens a1 (Col3a1)were quantified by quantitative real-time RT-PCR. The protein expressions of Notch1,Jagged1,α-SMA,and Col3a1 in NRK-52E cel s were detected by immunofluorescence staining. RESULTS In NRK-52E cells,AA enhanced the expression of TGF-β1,α-SMA and Col3a1 mRNA(P<0.05),reduced the expression of E-cadherin mRNA(P<0.05),up-regulated the mRNA expression of Notch1 mRNA(P<0.01)and Jagged1(P<0.05),and down-regulated the mRNA expression of Numb mRNA(P<0.05) compared with normal cell control group,indicating that phenotypic transformation and matrix accumu?lation occurred in AA-treated NRK-52E cells,accompanied by activated Notch signaling. Treatment with DAPT inhibited Notch signaling by decreasing the expression of Notch1 and Jagged1 (P<0.05),and increasing the expression of Numb mRNA(P<0.05). Furthermore, DAPT also down-regulated the expression levels of TGF-β1,α-SMA,Col1a1 and Col3a1 mRNA(P<0.05), and up-regulated the expression level of Bmp7 and E-cadherin mRNA(P<0.05) compared with AA group,suggesting that DAPT inhibited phenotypic transformation and matrix accumulation in AA-treated NRK-52E cells. CONCLUSION AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells,which is inhibited by DAPT treatment. The possible mechanism is that DAPT suppresses the activation of Notch signaling,resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.
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AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .
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OBJECTlVE To investigate the association between CYP3A5 genotypes and the early efficacy of tacrolimus ( Tac) and cyclosporin A ( CsA) in renal transplantation recipients, and provide a basis for individualized treatment. METHODS Seventy-four kidney transplantation recipients were en-rolled in this study between August 2012 and April 2013. Thirty-one patients were treated with the combi-nation of CsA, MMF and methylprednisolone while the rest were treated with Tac, MMF and methylpred-nisolone. The genotype CYP3A5 was detected by sequence specific primer-polymerase chain reaction ( SSP-PCR) before transplantation. The levels of Tac and CsA were detected by ELlSA and chemilumi-nescence, respectively, to monitor the blood concentration/dose of drugs ( c/D) at 2 weeks, 1 month, 2 months, 3 months and 6 months after transplantation. Simultaneously, the concentrations of blood glu-cose, creatinine, urea nitrogen and uric acid were determined with hexokinase method, creatininase method, urease method and uricolase method, respectively. RESULTS Among the 74 recipients, 9.5%carried CYP3A5?1/?1, 48.6%carried CYP3A5?1/?3 and 41.9%carried CYP3A5?3/?3. According to the phenotype of CYP3A5, the patients were divided into CYP3A5 expression group ( including CYP3A5?1/?1 and CYP3A5?1/?3) and non-expression group ( including CYP3A5?3/?3) , which accounted for 58.1%and 41.9%of the cases, respectively. Among the patients taking Tac, the median value of c/D at 2 weeks, 1 month, 2 months, 3 months and 6 months was 25.49, 49.64, 53.72, 51.9 and 44.5 in CYP3A5 expression group, and 65.48,100.84,99.54,123.01 and 133.21 in non-expression group. The c/D ratio of CYP3A5 non-expressers was higher than among CYP3A5 expressers at each time point ( P<0.05) . The initial dose of Tac 0.1 mg·kg-1 was high for CYP3A5 non-expressers, and the kidney function recovered more slowly than among CYP3A5 expressers and kidney damage occurred. However, there was no association between CYP3A5 genotype and the early efficacy of CsA. The levels of blood glucose, creatinine, urea nitrogen and uric acid were not significantly different between CYP3A5 expression and non-expression groups. CONCLUSlON CYP3A5 non-expression recipients whose starting amount of Tac was 0.1 mg·kg-1 have drug overdoses. CYP3A5 genotype is one of the factors affecting the efficacy of Tac. CYP3A5 genotype has no association with the efficacy of CsA in renal transplantation recipients.
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[ABSTRACT]AIM:ToinvestigatetheeffectofimmunosuppressantFK506onserumglucoseinratsandtoex-plore its mechanism .METHODS: Sprague-Dawley rats ( n =12 ) were randomly divided into drug group and normal group.The rats in drug group were intraperitoneally injected with FK 506 at dose of 1 mg· kg-1 · d-1 and the rats in nor-mal group received saline (1 mL· kg-1 · d-1 , ip) for 14 d.The fasting weight and fasting glucose were regularly meas-ured every 2 d.Visceral fat was isolated from the rats at the end of experiment .The mRNA expression of adiponectin , lep-tin, visfatin, resistin, retinol-binding protein 4 ( RBP4) and peroxisome proliferator-activated receptors γ( PPAR-γ) was determined by real-time fluorescence quantitative PCR .The protein expression of PPAR-γand adiponectin was measured by Western blotting .RESULTS:Compared with normal group , the concentration of fasting blood glucose in model group was significantly increased from the 10th day (P<0.05).At day 14, the fasting blood glucose of the model group increased from (5.10 ±0.62) mmol/L to (7.73 ±0.73) mmol/L.No significant change of blood glucose in normal group between the 10th day and the 14th day [from (4.66 ±0.32) mmol/L to (5.80 ±0.10) mmol/L] was observed.Compared with normal group , the mRNA expression of PPAR-γ, adiponectin and leptin in the adipose tissue of model group was signifi-cantly decreased ( P <0.01 ) , whereas the expression of visfatin , resistin and RBP4 was significantly increased ( P <0.05).Compared with normal group, the expression of PPAR-γand adiponectin in model group was decreased (P <0.01).CONCLUSION:FK506 may decrease the expression of PPAR-γto change the expression of adipocytokines and induce hyperglycemia in rats .
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OBJECTIVE To investigate the molecular mechanisms of resveratrol( Res)in renal interstitial fibrosis(RlF)in rats with unilateral ureteral obstruction(UUO). METHODS Forty-eight Spra-gur-Dawley rats were randomly divided into UUO( normal saline,n = 16),UUO with Res treatment (Res,20 mg·kg-1 ,n=16),and sham-operation(sham,n=16)models. The kidneys were excised on the 7th and 14th day. The deposition of collagen fiber in the kidney was detected with HE and Masson staining. The levels of sonic hedgehog(SHH,an inducer of SHH pathway)in kidney tissues were deter-mined by ELlSA. lmmunohistochemical analysis was performed to evaluate the protein expression of SHH signaling-related molecules,including SHH,smoothened(Smo),patched-1(Ptch1),and Gli1, proliferating cell nuclear antigen(PCNA)and matrix component typeⅢ collagen. The mRNA expression levels of Smo,Ptch1 and Gli1 were detected by real-time RT-PCR. RESULTS The degree of RlF observed with HE and Masson staining was obviously increased in UUO kidneys,but decreased in Res-treated kidneys. Enhanced expression levels of typeⅢ collagen and PCNA in UUO rats were suppressed by Res treatment(P﹤0.05). Res administration decreased the expression levels of SHH,Smo,and Gli1 (P﹤0.05),but increased the expression of Ptch1(P﹤0.05),suggesting that Res inhibit the obstruction-induced activation of SHH signaling. CONCLUSION Res can attenuate RlF in UUO rats,and the possi-ble mechanism is that Res down-regulates the activity of SHH signaling and inhibits cellular proliferation, resulting in inhibition of matrix accumulation in renal interstitium of UUO rats.