The Mechanism Of Enhanced ACE2 Regulates Collagen Type Ⅰ Metabolism In Diabetic Nephropathy

Cloning ofACE2 geneGene-specific primers were designed on the basis of published human ACE2 sequence (GenBank no. AF241254) with the primers (sense:5'-CAC CAT GTC AAG CTC TTC CTG-3'; antisense:5'-AAA GGA GGT CTG AAC ATC ATC AGT G-3'). These primers were used to amplify the full-length cDNA from a human fetal heart cDNA library (Gibco, Carlsbad,CA, USA). This was accomplished by using a two-step PCR protocol using PfxUltima DNA polymerase (Invitrogen, Carlsbad, CA, USA).Transfection of recombinant ACE2 gene in MES-13 cellsThe purified PCR product with a full length of 2401 bp was ligated into the pcDNA3.1(-) vector (Invitrogen). The positive recombinant transformants were randomly picked up and then verified by PCR amplification and DNA sequence. Stable transfection of the constructs in MES-13 mouse mesangial cells was performed using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. The cells were selected with G418(Merck,Darmstadt, Germany) at a final concentration of 200μg ml-1.Cell cultureMES-13 cells were grown in a 3:1 mixture of Dulbecco's modified Eagle's and Ham's F-12 media (Invitrogen) supplemented with 5% (v/v) fetal bovine serum containing 50 units ml-1 penicillin G and 50 g ml-1 streptomycin in a humidified atmosphere containing 5% CO2 at 37℃. Cells were first grown up to 70% confluency and synchronized overnight in serum-free medium prior to treatment.For RT-PCR, the quiescent cells were treated with ANGⅡfor various concentrations or durations. In some experiments cells were pretreated with selective inhibitors for indicated durations before ANGⅡtreatment.For western blotting using anti-procollagen type I antibody,MES-13 cells were stimulated with vehicle, ANG II (100 nM) alone, ANG II in combination with losartan (1μM, an AT-1R-specific antagonist),or ANG II in combination with other antagonists including wortmannin(20 nM, a PI3K inhibitor), Akt inhibitor X(10μM), 2,5-DOA(100uμM, an AC inhibitor), RP-cAMP(100μM, an Epac/PKA inhibitor) PP2(10uM, a Src inhibitor), SB431542 (1OμM,a TGFβR I inhibitor) for 24 h.MES-13 mesangial cells were stimulated with vehicle, ANG II (100 nM) alone, ANG II in combination with losartan (1μM, an AT-1R-specific antagonist),or ANG II in combination with other antagonists including wortmannin(20 nM, a PI3K inhibitor), 2,5-DOA(100μM, an AC inhibitor), RP-cAMP(100μM, an Epac/PKA inhibitor), H-89(10μM, a PKA inhibitor), KN-93(5μM, a CaMK inhibitor), PP2(10μM, a Src inhibitor), SB431542 (10μM,a TGFβR I inhibitor) for 2 min. The antagonists were added 1 h before ANG II treatment. Cell lysates were subjected to western blotting using anti-Phospho-Akt and anti-Total-Akt.RT-PCR and Western blottingTotal RNA was isolated from MES-13 cells using TRIzol reagent (Invitrogen). Reverse transcription was carried out on MES-13 cell total RNA followed by PCR. Lysates from cultured MES-13 cells were subjected to western blot analysis as described previously. Protein concentration was determined using the BCA Protein Array Kit (Pierce, Rockford, IL, USA). Protein samples were separated by 8-15% sodium dodecyl sulphatepolyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (Hybond ECL, Amersham, Piscataway,NJ, USA), and then blocked with 1% Blot-Qualified BSA(Promega) for 90 min. The membranes were incubated overnight at 4℃with primary antibodies against procollagen typeⅠ, phospho-Akt,total-Akt andβ-actin (1:250-1:1000) and then incubated with alkaline phosphatase-labelled secondary antibody (Biotechnology, Santa Cruz, CA, USA; 1:4000) for 60 min at room temperature. The positive bands corresponding to aim proteins were developed with the Western Blue stabilized substrate for alkaline phosphatase (ProtoBlotⅡAP System,Promega).ResultsThe cloning, transfection and expression of recombinant ACE2 in cellsThe full-length cDNA of the ACE2 gene was successfully amplified and inserted into the pcDNA3.1(-) vector. The positive recombinant transformants were analysed by PCR amplification with the use of ACE2 genespecific primers in the vector (Figure 1a). RT-PCR and western blot analyses showed that the ACE2 mRNA and protein were overexpressed in ACE2-transfected cells compared with the controls, but not in controls and blank plasmid-transfected MES-13 cells (Figures 1b and c).ACE2 transfer inhibits the typeⅠcollagen synthesis stimulated by AngⅡThe cause of many glomerular diseases is an increase in the synthesis of extracellular matrix components, including collagens. AngⅡhas been shown to increase the synthesis of collagen. We first examine what subtypes of collagen are affected by Ang II treatment, quiescent MES-13 cells were treated with AngⅡunder serum-free condition for various durations (30,60,90,120,240, and 1440 min),and the gene levels of three subtypes of collagen (Coll-1al, Coll-1a2 and Col-4a1) were determined by RT-PCR. MES-13 cells cells expressed only trace amounts of collagen genes. AngⅡinduced a significant increase in Coll-lal and Coll-la2 gene levels 90 min after initiation of treatment, and the effect was still evident after 24 h. In contrast, Coll-4al gene levels was not affected by Ang II.To further determine the most effective in vitro dosage of ANGⅡin the experiment, quiescent MES-13 cells were treated with different concentrations of AngⅡ(10nM, 100nM and 1μM) for 24hours. As illustrated by Figure 2b, Coll-lal mRNA synthesis was most strongly stimulated by 100nM AngⅡ, and this dose was used in all the following experiments.As mentioned above, AngⅡwas assumed to increase the synthesis of the type I collagen. Western blotting results verified a significant increase in the protein levels of procollagen type I in AngⅡ-treated cells, whereas the effect was completely abrogated by ACE2 gene transfer.ACE2 transfer interrupts the typeⅠcollagen synthesis mediated by AngⅡ-AT1R/TGFβRⅠ-PI3K-Akt pathwayTo explore if the AngⅡeffect is mediated by AT-1R, quiescent MES-13 cells were treated with vehicle, AngⅡ, or AngⅡin combination with losartan, an AT-1R-specific antagonist. The synthesis of the typeⅠcollagen protein was completely abrogated by co-treatment with losartan. These results indicated an AT-1R-mediated increase in collagen synthesis.To verify if TGFβRⅠ, PI3K and Akt are downstream signaling molecules induced by AngⅡ, MES-13 cells were treated with AngⅡfor 1,2,5 or 15 min, and phosphorylation of Akt was examined by Western blotting. There was a significant increase in Akt phosphorylation (threonine 308) 1min after AngⅡtreatment. The peak appears at 2 min and then declines. As shown in Figure 5a, the outcome of AngⅡ-induced increases in phospho-Akt was altered by losartan, a selective TGFβR I inhibitor (SB431542,1OμM) or wortmannin, a PI3K inhibitor. The results indicates that acute AngⅡstimulation affectes the activation of TGFβRⅠ-PI3K-Akt signaling pathway in MES-13cells.We have shown that TGFβRⅠ-PI3K-Akt pathway are initiated by AngⅡ. To further detect if Akt, PI3K and TGFβRⅠinhibitor can inhibit the synthesis of the type I collagen, cells were treated with vehicle, AngⅡ(100nM) alone, AngⅡin combination with Akt inhibitor X (10μM), SB431542 or wortmannin. The synthesis of the type I collagen was fully abrogated by Akt inhibitor X,SB431542 and wortmannin. The western blotting results demonstrated that TGFβRI and PI3K-Akt were critical for AT-1R-mediated induction of collagen synthesis.