Erk5 is a mediator to TGFβ1-induced loss of phenotype and function in human podocytes

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.