Chen Cheng, Liang Wei, Jia Junya, van Goor Harry, Singhal Pravin C, Ding Guohua
Division of Nephrology, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Nephron Exp Nephrol. 2009;113(1):e26-34. doi: 10.1159/000228080. Epub 2009 Jul 9.
Podocytes play a critical role in the pathogenesis of glomerulosclerosis. Increasing evidence suggests that aldosterone (ALD) is involved in the initiation and progression of glomerular damage. It is, however, unknown whether there is a direct injurious effect of ALD on podocytes. Therefore, in the present study, we evaluated the effect of ALD on podocyte apoptosis and studied the role of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways in this process.
Podocytes were incubated in media containing either buffer or increasing concentrations of ALD (10(-9) approximately 10(-5)M) for variable time periods. The cells were also treated with either wortmannin (inhibitor of PI3-K, 100 nM), SB202190 (SB20, inhibitor of p38MAPK, 10 microM) or buffer. All treatments were performed with or without ALD (10(-7)M) for 24 h. At the end of the incubation period, apoptosis was evaluated by cell nucleus staining and flow cytometric analyses. Activation of PI3-K/Akt and p38MAPK phosphorylation of cultured rat podocytes was evaluated by performing Akt kinase assay and Western blot, respectively.
Apoptosis of cultured rat podocytes was induced by ALD in a dose- and time-dependent manner. ALD inhibited the activity of PI3-K/Akt and increased the activation of p38MAPK. PI3-K/Akt activity was further inhibited by the addition of wortmannin to the cells in the presence of ALD. This was accompanied by a significant increase in apoptosis. ALD-induced p38MAPK phosphorylation and apoptosis were inhibited when the cells were pretreated with SB20. Furthermore, treatment with spironolactone not only attenuated the proapoptotic effect of ALD, but also significantly reversed its effects on PI3-K/Akt and p38MAPK signaling pathways.
ALD induces apoptosis in rat podocytes through inhibition of PI3-K/Akt and stimulation of p38 MAPK signaling pathways. Spironolactone attenuates ALD-induced podocyte apoptosis, thereby positioning this compound as a potential promising target of intervention in human renal damage.
足细胞在肾小球硬化的发病机制中起关键作用。越来越多的证据表明，醛固酮（ALD）参与肾小球损伤的起始和进展。然而，ALD对足细胞是否有直接损伤作用尚不清楚。因此，在本研究中，我们评估了ALD对足细胞凋亡的影响，并研究了磷脂酰肌醇3激酶/蛋白激酶B（PI3-K/Akt）和p38丝裂原活化蛋白激酶（p38MAPK）信号通路在此过程中的作用。
将足细胞在含有缓冲液或浓度递增的ALD（10⁻⁹至10⁻⁵M）的培养基中孵育不同时间段。细胞还分别用渥曼青霉素（PI3-K抑制剂，100 nM）、SB202190（SB20，p38MAPK抑制剂，10 μM）或缓冲液处理。所有处理均在有或无ALD（10⁻⁷M）的情况下进行24小时。孵育期结束时，通过细胞核染色和流式细胞术分析评估凋亡情况。分别通过进行Akt激酶测定和蛋白质印迹法评估培养的大鼠足细胞中PI3-K/Akt的激活和p38MAPK磷酸化情况。
培养的大鼠足细胞凋亡由ALD以剂量和时间依赖性方式诱导。ALD抑制PI3-K/Akt的活性并增加p38MAPK的激活。在存在ALD的情况下向细胞中添加渥曼青霉素进一步抑制了PI3-K/Akt活性。这伴随着凋亡的显著增加。当细胞用SB20预处理时，ALD诱导的p38MAPK磷酸化和凋亡受到抑制。此外，螺内酯处理不仅减弱了ALD的促凋亡作用，而且显著逆转了其对PI3-K/Akt和p38MAPK信号通路的影响。
ALD通过抑制PI3-K/Akt和刺激p38 MAPK信号通路诱导大鼠足细胞凋亡。螺内酯减弱ALD诱导的足细胞凋亡，从而使该化合物成为人类肾损伤潜在的有前景的干预靶点。
