Effect of Peripheral Blood Mononuclear Cells Isolated from Children with Minimal Change Nephrotir Syndrome to Glomerular Basement Mernbrane Heparan Sulfate Proteoglycan(GBM HSPG) in Rats Glomerular Epithelial Cell: Including Development of Quantitative RT / 대한신장학회잡지

ABSTRACT

Minimal Change Nephrotic Syndrome(MCNS) reflects a disorder of T-lymphocytes. These T-cells are thought to release a vascular permeability factor (UPF) that injures the glomerular epithelial cells (GECs). Glomerular epithelial cellular damage may lead to proteinuria in MCNS by decreasing the synthesis of polyanions such as heparan sulfate proteoglycan(HSPG) : these polyanions constitute most of the normal charge barrier to glomerular filtration of macromolecules such as albumin. This study evaluates the direct effect of supernatant of culture media of peripheral blood mono- nuclear cells(PBMC) which was isolated from children with MCNS to GBM HSPG mRNA expression in rats GEC. GEC were cultured until confluent. Supernatant of culture media of PBMC from each group of 3 chilren with MCNS, IgA nephropathy or normal healthy were added. Total RNA was extracted at 12, 24 and 72hrs after adding supernatant. RT-PCR using Rat Perlecan Domain-I(RPD- I) specific primers and beta-actin as internal controls was done. Densities and areas of GRM HSPG corresponding bands to beta-actin bands were measured. At 24 hrs, supernatant of culture media of PBMC from 3 children with MCNS caused 62, 70, and 75Vo reductions, respectvely, in GEC's GBM HSPG mRNA expression compared to normal children. However, supernatant of culture media of PRMC from 3 children with IgA nephropathy did not. In addition, reductions of GEC's GBM HSK' mRNA expressions caused by supernatant of culture media of PBMC from 3 children with MCNS were restored upto levels of normal children at 72hrs after adding supernatant. Mutant cDNA was synthesized as primers for competitive PCR to quantify GBM HSPG mRNA expression. Mutant template was 212 base pairs shorter than RPD-I, 497 base pairs. In conclusion, we found that supernatant of culture media of PBMC from children with MCNS reversibly suppressed GBM HSPG mRNA expression in rats GEC. This study suggests cytokines of PBMC from children with MCNS directly injures GEC and leads to decrease in synthesis of GBM HSPG by GEC in the pathogenesis of MCNS.