title: Erk5 is a mediator to TGFβ1-induced loss of phenotype and function in human podocytes.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: South West Thames Institute for Renal Research Surrey, UK ; St. George's, University of London London, UK.
authors.forename: Irbaz I
authors.initials: II
authors.lastname: Badshah
authors.affiliation: St. George's, University of London London, UK.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Baines
authors.affiliation: South West Thames Institute for Renal Research Surrey, UK.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Dockrell
keywords: Erk5; TGF-beta; apoptosis; barrier function; diabetic nephropathies; migration; podocytes