|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1994 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1993 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Three lines of human mesangial cells (HMC) were cultured from isolated glomeruli, which were obtained from the normal renal cortices of three patient with renal tumor by sieving techniques. Fcalpha-receptor (FcalphaR) mRNA expression in these cultured HMC was examined by RT-PCR and northern blot analysis (NB). The FcalphaR mRNA expression was detected on an only one line of cultured HMC by RT-PCR but not NB.Modulation of the FcalphaR mRNA expresson by stimulation with cytokines was examined by RT-PCR and ribonuclease protection assay. HMC were incubated in the presence or absence of IL-1, IL-6, IL-8, TNF-alpha, PDGF,TGF-beta and heat aggregated IgA for 12 or 24 hours. Although IL-1, IL-6 and TNF-alpha enhanced the FcalphaR mRNA expression on HMC,these changes were not significant. Doses of each cytokine and incubated times did not affect the leve lsof FcalphaR mRNA on HMC.
The cytoplasmic polyeptide of FcalphaR was expressed as a fusion protein with glutathione S-transferase (GST) in E.
coli. The fusion protein with GST was purified by absorption with glutathione sepharose column. A polyclonal antibody to the cytoplasmic polypeptde of FcalphaR was made by menae of immunazed the recombinant protein into a white rabbit. The 60 kDa band was detected by immunoprecipitation of HMC's lysates and this antibody. This result may suggest that the precipitates were the FcalphaR proteins on HMC.While human renal biopsy specimens were performed indirect immunofluorescent staining using this antibody, the FcalphaR protein was not distinctly stained in mesangium.
A silent one point mutation (C*T) was found on sty I site (670 base) of FcalphaR cDNA fragments, which amplified from cultured HMC by RT-PCR.Therefore, restriction fragment length polymorphism (RFLP) of FcalphaR were examined by southern blot analysis in 45 patients with IgA nephropathy and 30 healthy volunteers. However, RFLP of FcalphaR with Sty I digestion were not recognized.
In this study, the expression of FcalphaR gene and protein were determined in cultured HMC.The expression of FcalphaR on HMC was scanty and unstable, and cytokines did not change the FcalphaR mRNA levels on HMC.It is suggested that HMC express constitutively low levels of FcalphaR mRNA,and FcalphaR on HMC may play more important roles of the IgA-mediated signaling pathways than the catabolism of IgA immune complexes in mesangium. Less