0135

EFFECT OF LDL ON THE PROLIFERATION OF RAT GLOMERULAR MESANGIAL CELLS IN VITRO

Wu L, Wang L, Wang YW, et al.

Department of Pediatrics, Changzheng Hospital, Shanghai, China

Objective: To study the effect of low density lipoprotein (LDL)on the expression of transforming growth factor-β(TGF-β) and fibronectin (FN )mRNA in rat glomerular mesangial cells (MsC).

Methods: Mesangial cells were isolated from the glomeruli of male Sprague- Dawley rats (Shanghai experiment animal center) aged 4 to 6 weeks, weighing 100 to 150 gm using a differential sieving technique. Isolated glomeruli were then digested with IV collagenase (Sigma Inc, USA). MsC were cultured in RPMI-1640 medium containing 20% fetal bovine serum (FBS) in CO₂ incubator for 2 to 3 weeks. Cells were subcultured at one to two-week intervals. Cells between passages 3 throngh 4 were used for experiments. Cultured mesangial cells were exposed to medium with LDL prepared from normal human plasma(50, 100, 200ug/ml) for 24 hours and with LDL 150ug/ml for 24, 48, 72 hours. 3H-TdR incorporation was used for the detection of MsC proliferation.Anti-TGF-β antibody 10ug/ml was added to MsC treated with LDL at concentration of 50 to 200 ug /ml for 24 hours, and 150 ug/ml for 24 to 72 hours at the end of incubation ,respectively. According to the standard method ,Northern blot and Dot blot analysis were performed with TGF-β cDNA(2.138 kb) and FN cDNA(1.3 kb) and were used to determine the expression of TGF-βmRNA and FN mRNA. The autoradiographs were scanned with a laser densitometer to determine relative mRNA levels.

Results: (1) LDL had biphasic effect on MsC proliferation. Low concentration stimulated MsC proliferation in concentration-dependent (50 to 150ug/ml) and time-dependent (24 to 72 hours) manner(P<0.05),whereas high concentration (200ug/ml) inhibited MsC proliferation (P<0.05). (2) Northern blot analysis revealed an increase of TGF-βmRNA and FN mRNA levels in concentration-dependent and time-dependent manner in MsC stimulated by LDL.The expression of TGF-β mRNA was 24 hours earlier than that of FN mRNA. (3) Dot blot analysis showed that the expression of FN mRNA in MsC cultured with medium containing anti-TGF-βantibody decreased significantly.

Conclusion: Our results clearly demonstrate that LDL stimulates mesangial cells proliferation and enhances TGF-βmRNA and FN mRNA expression.It is possible that LDL may play an important role in the development and progression of glomerular injury.Our study also suggests that TGF-βhas several potential roles in renal disease by promoting extracellular matrix accumulation in renal cells in vitro.