MP051
GENETIC BACKGROUND DETERMINES MURINE PRIMARY MESANGIAL CELL RESPONSE TO TGF-ß Free

INTRODUCTION AND AIMS: C57Bl6/J (B6) mice have been reported as resistant to experimental renal fibrosis. Alb/TGF-beta1 transgenic mice on B6 background develop only mild renal fibrosis compared to CBA mice, despite the elevated circulating TGF-ß levels (Kokeny et al, Clin Kidney J 2011;4(S2):421-429). We therefore aimed to investigate the effects of TGF-ß administration at the cellular level on primary mesangial cells isolated from B6 and CBA mice.

METHODS: Mesangial cells were isolated from a male 6 week-old B6 and a CBA mouse using the magnetic bead glomerular separation method. Glomerular outgrowths were maintained in selective RPMI medium for 21 days, then cells were subcultured and characterized for mesangial markers. Primary cells from P5 to P8 passage number were seeded on 24-well plates and the gene expression profile of B6 and CBA mesangial cells (n=6/group) was analyzed upon PBS or TGF-ß treatment (10 ng/ml) for 48 hours. Data (mean±SD) were analyzed using Kruskal-Wallis test.

RESULTS: Compared to B6 cells, TGF-ß treated CBA cells showed significant overexpression of TGF-ß (B6+PBS: 1.0±0.1; B6+TGFß: 1.1±0.1 vs CBA+PBS: 0.9±0.1; CBA+TGFß: 2.5±0.1, p<0.05) and TIMP-1 (B6+PBS: 1.0±0.2; B6+TGFß: 1.2±0.1 vs CBA+PBS: 0.7±0.1; CBA+TGFß: 3.2±0.5, p<0.01). The fibrosis related transcription factor EGR-1 (early growth response factor-1) was 2-fold elevated in CBA cells (B6+PBS: 1.0±0.2; B6+TGFß: 0.9±0.1 vs CBA+PBS: 1.1±0.1 ; CBA+TGFß: 1.9±0.2, p<0.05), accompanied by marked increase in type-I collagen expression (B6+PBS: 1.0±0.3; B6+TGFß: 0.8±0.1 vs CBA+PBS: 1.1±0.2; CBA+TGFß: 8.7±2.3, p<0.01). The gene expression of Smad molecules was not different in B6 and CBA cells.

CONCLUSIONS: Our in vitro results further confirmed the previous reports on renal fibrosis resistance of C57Bl6/J mice over other inbred mouse strains, which might be related to strain-dependent TIMP-1 response.Funding: Hungarian Scientific Research Fund (OTKA PD 112960).