SP13
THE EFFECTS OF XTRACELLULAR MATRIX COMPONENTS ON
PODOCYTE PHENOTYPE.
Tarunkumar Madne 1 Iain AM MacPhee 2 Mysore K Phanish 1 Mark EC Dockrell 1
1 SWT Institute for Renal Research, London UK 2 St George’s University of London
UK
INTRODUCTION: In addition to providing structural support to cells, the
extracellular matrix (ECM) can also regulate cell proliferation and phenotype as well
creating a specific microenvironment. The glomerular basement membrane (GBM) is
a dynamic structure, the constituents of which change in quality and quantity both
during maturation and disease. Although the GBM is a complex structure where
components may interact to regulate the cells we have started by investigating how
specific proteins influence podocyte behaviour both basally and in the presence of the
powerful pleiotropic growth factor TGFβ1.
OBJECTIVE: We have investigated the role of different matrices on podocyte
phenotype and responses to TGFβ1.
METHODS: Conditionally immortalised human podocytes, developed by Saleem and
colleagues were seeded directly after passage on cell culture dishes or on dishes
coated with: collagen IV (Col IV), cellular fibronectin containing the EDA Exon
(EDA+ Fn), or plasma fibronectin, which lacks the EDA Exon, (EDA –Fn). Cells
were maintained on the respective cell surfaces during proliferation, at the permissive
temperature, and differentiation at the non-permissive temperature. Gross cellular
morphology was studied by phase contrast light microscopy and expression of
EDA+Fn, synaptopodin and alpha-SMA was assessed by RT-PCR and Q-PCR. Cells
were studied in the presence and absence of TGFβ1 (2.5ng/mL).
RESULTS: Marked changes in podocyte morphology were observed by light
microscopy in cells grown on the different matrices. In addition a decrease of ~40%
in synaptopodin expression was observed in cells grown on cellular Fn. The
expression alpha-SMA mRNA did not significantly change at this time point.
Investigating the effects of different cellular matrix protein on TGFβ-mediated
responses demonstrated that the increase in expression of EDA+Fn was completely
abolished in cells grown on collagen IV. The effect of TGFβ on synaptopodin was
also greatly affected by the growth surface, with opposite effects seen on collagen as
compared to fibronectin. Activation of receptor Smads was unaffected by different
matrices.
CONCLUSION: Alteration of the constituents of the GBM is likely to significantly
alter podocyte phenotype and cellular responses to growth factors involved in
podocytopathies.