Influences Of ACTH And TGF-β1on Podocytes Phenotype

Objectives:To investigate the effects of synthetic adrenocorticotropic hormone (ACTH1-24and ACTH4-10) and transforming growth factor-beta1(TGF-β1) on slid diaphragm associated protein podocin mRNA level of mouse podocytes, and protein levels of fibronectin(FN) and α-smooth muscle actin (α-SMA), in order to discuss the influences and protecting mechanisms of ACTH1-24and ACTH4-10to mouse podocytes phenotype. And to define whether ACTH4-10has the similar effect to ACTH1-24in preventing the injury TGF-β1to mouse podocytes.Methods:1. Conditional immortalized mouse podocyte clone5(MPC5) culture: according to the references[1], the resurgent MPC5cells were cultured under condition of33℃. More than80%coverage could be achieved after average3～4days. Then the condition was changed to37℃, and the cells would differentiate to mature podocyte cells prior to later experiment after10～14days’culture. These were differentiated podocyte cells, observing the morphology of mouse podocytes. Cells were seeded in6-well cell culture clusters. Prior to the experiment, cells were incubated in RPMI1640withous Fetal Bovine Serum (FBS) to regulate cell growing state, to make podocytes in synchronization.2. Cell groups:cells were divided to ACTH1-24preventing groups and ACTH4-10preventing groups; both ACTH1-24preventing groups and ACTH4-10preventing groups contain A and B groups. A groups:ACTH1-24or ACTH4-10was added,1hour later TGF-β1was added; the podocytes were treated for24hours. B groups:ACTH1-24or ACTH4-10was added,1hour later TGF-β1was added; the podocytes were treated for48hours. According to the references[2,3], podocytes were treated by ACTH1-24three concentrations:low concentration (75ng/ml), middle concentration (150ng/ml), high concentration (300ng/ml); while podocytes were treated by ACTH4-10three concentrations:low concentration (1ng/ml), middle concentration (10ng/ml), high concentration (100ng/ml). According to the references [4], the concentration of TGF-β1treating podocytes was2ng/ml; treated times are24hours and48hours.3. Detection index:mRNA expression levels of slit diaphragm associated protein podocin of mouse podocytes, protein expression levels of FN and a-SMA.Results:ACTH1.24preventing groups:Contrasting with control groups, podocytes treated by TGF-β1, mRNA expression levels of podocin decreased obviously, about54.9%; protein expression levels of FN increased obviously, about2.3times; protein expression levels of a-SMA increased obviously, about2.4times.Contrasting with control groups, podocytes treated by ACTH1-24alone, mRNA expression levels of podocin increased about7.5%; protein expression levels of FN decreased about0.1%; protein expression levels of a-SMA increased about1.7%. Contrasting with control groups, podocytes treated by ACTH1-24three different concentrations and TGF-β1, mRNA expression levels of podocin increased about1.3times、2.4times and1.2times respectively. Contrasting with TGF-β1treated groups, protein expression levels of FN decreased about0.1%、7.3%and40.1%respectively,. For podocytes treated by the low and middle concentrations of ACTH1-24, Protein expression levels of a-SMA increased about4.5%and11.8%respectively, while the high concentration of ACTH1-24and TGF-β1treated groups decreased about22.4%.ACTH4-10preventing groups:Contrasting with control groups, podocytes treated by TGF-β1, mRNA expression levels of podocin reduced obviously, about57.9%; protein expression levels of FN increased obviously, about18.6times; protein expression levels of a-SMA increased obviously, about90.6%. Contrasting with control groups, podocytes treated by ACTH4-10alone, mRNA expression levels of podocin increased about8.3%protein expression levels of FN decreased about4.8%; protein expression levels of a-SMA increased about28.9%. Contrasting with control groups, podocytes treated by three different concentrations of ACTH4-10and TGF-β1, mRNA expression levels of podocin increased about5.5%、64.3%and1.7times respectively. Contrasting with TGF-β1treated groups, for podocytes treated by the low and middle concentrations of ACTH4-10and TGF-β1, protein expression levels of FN increased about8.8%and9.2%respectively; for podocytes treated by the high concentrations of ACTH4-10and TGF-β1, protein expression levels of FN decreased about13.1%. For podocytes treated by the low concentration of ACTH4-10and TGF-β1, protein expression levels of a-SMA increased about3.8%, while for podocytes treated by the middle and high concentrations of ACTH4-10and TGF-β1, protein expression levels of α-SMA decreased about5.6%and35.5%respectively.Conclusions:ACTH1-24and ACTH4-10both exert a direct effect on podocytes, and stabilizes mRNA protein expression of slid diaphragm associated protein podocin, thus maintaining the integrity of slit diaphragm, meanwhile ACTH1-24also could inhibit the increasing of FN and a-SMA, weakening podocytes penotypic conversion TGF-β1to podocytes, thereby protecting podocytes. ACTH4-10has the similar effect to ACTH1-24,in inhibiting podocytes penotypic conversion and preventing the injury TGF-β1to mouse podocytes, however ACTH4-10has some differences from ACTH1-24, in concentrations making effects.