| BackgroundAldosterone(Aldo), as a key effector molecule of the renin-angiotensin-aldosterone system (RAAS), plays a critical role in the progression and prognosis of chronic kidney disease (CKD). There are several lines of evidence that aldosterone contributes to the progression of chronic kidney disease with increased inflammation and oxidative stress, vasoconstriction and remodeling, fibrosis, as well as cell proliferation, deformation and differentiation through genomic and non-genomic pathways. Of note, pathological mesangial cell(MC) proliferation and increased mesangial matrix is the most common phenomenon in glomerulonephritis. Therefore, to explore more about its molecular mechanism responsible for aldosterone-induced MC proliferation would be of great contribution in developing a therapeutic target against mesangial proliferative renal disease and in delaying progressive of CKD. With the presence of connexins within the kidney and their functional role in growth regulation, it provides a new angle of view for investigation for the mesangial proliferative renal disease.Connexins(Cxs) are ubiquitous transmembrane proteins that form gap junctions (GJs), allowing the diffusion of ions, second messengers, and other small signaling molecules between the cytoplasm of adjacent cells to achieve gap junctional intercellular communication(GJIC). As now is obvious, connexins and its formed GJs play a crucial role in the regulation of embryonic development, maintenance of tissue/organ homeostasis, cellular metabolism, morphogenesis, and growth control. Among them, Cx43is the most abundant and extensively-studied connexin. Of note, connexins, mainly Cx43, has dual functions in cell growth regulation:first, the well-accepted role in forming a gap junction channel(termed channel-dependent mechanisms) and, second, a direct action on growth(termed connexin-dependent but channel-independent mechanisms). Given that, the purpose of this study is to investigate whether Cx43regulates MC proliferation induced by Aldo, whether this is of functional importance, and whether Aldo regulates Cx43through the same pathways that have been identified to be relative to MC proliferation or some other pathways. Understanding the molecular mechanism of MC proliferation by Aldo aims to provide proper experimental foundation and theoretical basis. Methods1. Effect of different concentrations of Aldo(0.1、1、10、100nM) on MC proliferation and effect of mineralocorticoid receptor (MR) antagonists spironolactone(Spi, lOnM), ERK1/2inhibitor PD98059(10μM), PI3K inhibitor LY294002(10μM) and PKC inhibitor GF109203X(10μM) on Aldo(100nM)-induced MC proliferation were detemined by the incorporation of3H-thymidine (3H-TdR) assay;2. Effect of different concentrations of Aldo on Cx43mRNA and effect of inhibition of MR, ERK1/2, PI3K and PKC pathways on down-regulation of Cx43mRNA response to100nM Aldo treatment were detemined by RT-PCR;3. Effect of different concentrations of Aldo on the protein expression of total CX43and effect of inhibition of MR, ERK1/2, PI3K and PKC pathways on down-regulation of total Cx43protein response to100nM Aldo treatment were detemined by RT-PCR by western blotting;4. Effect of different concentrations of the proteasome inhibition MG132(0.5、1、5、10μM) on the protein expression of total CX43were examined by western blotting and effect of5μM MG132on Aldo (100nM)-induced MC proliferation were detemined by the incorporation of3H-thymidine (3H-TdR) assay;5. Effect of different concentrations of Aldo on GJIC function was detemined by scrape loading and dye transfer (SLDT);6. Effect of different concentrations of Aldo on intracellular calcium concentrations([Ca2+]i) were examined in laser scanning confocal microscopy after loaded by Fura-3/AM.Results1.The incorporation of3H-TdR results showed that:Aldo increased MC proliferation in a dose-dependent manner at concentrations ranging from0.1to100nM(P<0.05). Moreover, Aldo-induced MC proliferation were significantly inhibited by whatever MR antagonist, the ERK1/2blocker, the PI3K blocker or the PKC blocker(P<0.01). However, none of the individual inhibitor has influence on MCs growth compared to the control group (P>0.05).2. RT-PCR results showed that:Taking the mRNA level in the control as1, Aldo decreased Cx43mRNA in a dose-dependent manner at concentrations ranging from0.1nM(0.90±0.02, P<0.05) to100nM(0.32±0.03, P<0.01), and the down-regulation of the Cx43mRNA were significantly reversed by MR antagonist spironolactone, the ERK1/2inhibitor PD98059and the PKC inhibitor GF109203X(p<0.01) other than PI3K inhibitor LY294002(p>0.05) as follows:Spi (0.92±0.02), PD98059(0.78±0.03), LY294002(0.28±0.03), GF109203X (0.67±0.04). However, the inhibitor itself did not exert much change on Cx43mRNA compared to the control group (p>0.05).3. Western blotting results showed that:in parallel the gene level, Aldo decreased the protein expression of total Cx43in a dose-dependent manner at concentrations ranging from0.1nM(0.91±0.02, P<0.05) to100nM(0.43±0.02, P<0.01), and the down-regulation of total Cx43protein were significantly reversed by MR antagonist spironolactone, the ERK1/2inhibitor PD98059and the PKC inhibitor GF109203X (p<0.01) but not PI3K inhibitor LY294002(p>0.05) as follows:Spi(0.88±0.03), PD98059(0.69±0.05), LY294002(0.33±0.04), GF109203X(0.54±0.03). However, the inhibitor itself did not exert much change on total Cx43protein compared to the control group (p>0.05).4. Western blotting results showed that:MG132increased the expression of total Cx43protein in a dose-dependent manner, and MG132at a concentration of5μM successfully caused a elevation in Cx43protein level (P<0.01). Followed by, to further assess the role of Cx43in Aldo-induced MC proliferation, the incorporation of3H-thymidine (3H-TdR) assay was employed to value MC proliferation by treatment with Aldo at different concentrations in the presence or absence of MG132for24h.The results showed that under the condition that5μM MG132elevated Cx43in protein level (P<0.01), Aldo-induced MC proliferation were decreased compared with corresponding concentration.5. None of concentrations of Aldo ranging from0.1nM to100nM had any notable effects on GJIC marked by the diffusion length of Lucifer yellow dye compared to the control group (P>0.05).6. Intracellular calcium concentrations([Ca2+]i) response to Aldo treatment were examined in laser scanning confocal microscopy after loaded by Fura-3/AM. The data showed that Aldo increased [Ca2+]i in a dose-dependent manner ranging from0.1nM to100nM, which was marked by the fluorescence gray value as fowllowing levels: control(21.62±1.90);0.1nMAldo(29.92±1.87,p<0.05);1nMAldo(37.20±2.1,p<0.01);10nMAldo (57.61±1.82,p<0.01);100nMAldo (74.80±1.60,p<0.01). ConclusionAldo promotes MC proliferation, and its possible mechanisms might directly decrease DNA and protein expression of Cx43via its genomic mechanisms, instead of its influence on GJIC function through MR-mediated, the ERK1/2-and PKC-dependent but not PI3K-dependent pathways. |
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