South West Thames Institute for Renal Research Surrey, UK ; St. George's, University of London London, UK.
St. George's, University of London London, UK.
Front Pharmacol. 2014 Apr 21;5:71. doi: 10.3389/fphar.2014.00071. eCollection 2014.
Podocytes are highly specialized cells integral to the normal functioning kidney, however, in diabetic nephropathy injury occurs leading to a compromised phenotype and podocyte dysfunction which critically produces podocyte loss with subsequent renal impairment. TGFβ1 holds a major role in the development of diabetic nephropathy. Erk5 is an atypical mitogen-activated protein (MAP) kinase involved in pathways modulating cell survival, proliferation, differentiation, and motility. Accordingly, the role of Erk5 in mediating TGFβ1-induced podocyte damage was investigated.
Conditionally immortalized human podocytes were stimulated with TGFβ1 (2.5 ng/ml); inhibition of Erk5 activation was conducted with the chemical inhibitor BIX02188 (10 μM) directed to the upstream Mek5; inhibition of Alk5 was performed with SB431542 (10 μM); Ras signaling was inhibited with farnesylthiosalicylic acid (10 μM). Intracellular signaling proteins were investigated by western blotting; phenotype was explored by immunofluorescence; proliferation was assessed with a MTS assay; motility was examined with a scratch assay; barrier function was studied using electric cell-substrate impedance sensing; apoptosis was studied with annexin V-FITC flow cytometry.
Podocytes expressed Erk5 which was phosphorylated by TGFβ1 via Mek5, whilst not involving Ras. TGFβ1 altered podocyte phenotype by decreasing P-cadherin staining and increasing α-SMA, as well as reducing podocyte barrier function; both were prevented by inhibiting Erk5 phosphorylation with BIX02188. TGFβ1-induced podocyte proliferation was prevented by BIX02188, whereas the induced apoptosis was not. Podocyte motility was reduced by BIX02188 alone and further diminished with TGFβ1 co-incubation.
These results describe for the first time the expression of Erk5 in podocytes and identify it as a potential target for the treatment of diabetic renal disease.
足细胞是肾脏正常功能所必需的高度特化的细胞，然而，在糖尿病肾病中，损伤发生导致表型受损和足细胞功能障碍，这会导致足细胞大量丢失，随后发生肾脏损害。TGFβ1 在糖尿病肾病的发生发展中起着重要作用。Erk5 是一种非典型的丝裂原激活蛋白（MAP）激酶，参与调节细胞存活、增殖、分化和迁移的途径。因此，研究了 Erk5 在介导 TGFβ1 诱导的足细胞损伤中的作用。
用 TGFβ1（2.5ng/ml）刺激条件永生化人足细胞；用化学抑制剂 BIX02188（10μM）靶向上游 Mek5 抑制 Erk5 激活；用 SB431542（10μM）抑制 Alk5；用法呢基硫代水杨酸（10μM）抑制 Ras 信号。用 Western blot 检测细胞内信号蛋白；用免疫荧光法检测表型；用 MTS 法检测增殖；用划痕实验检测迁移；用电细胞-底物阻抗传感法研究屏障功能；用 Annexin V-FITC 流式细胞术检测凋亡。
足细胞表达 Erk5，TGFβ1 通过 Mek5 磷酸化 Erk5，但不涉及 Ras。TGFβ1 通过减少 P-钙黏蛋白染色和增加α-SMA，以及降低足细胞屏障功能来改变足细胞表型；这两种作用均被 BIX02188 抑制 Erk5 磷酸化所阻止。BIX02188 可阻止 TGFβ1 诱导的足细胞增殖，但不能阻止其诱导的凋亡。BIX02188 单独可降低足细胞迁移，与 TGFβ1 共孵育时进一步降低。
这些结果首次描述了 Erk5 在足细胞中的表达，并确定其为治疗糖尿病肾病的潜在靶点。
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