OBJECTIVE: To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). METHODS: A monoclonal antibody against AM developed by ...OBJECTIVE: To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). METHODS: A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index. RESULTS: A specific monoclonal antibody against AM was succesfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect. CONCLUSION: AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.展开更多
Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms. Methods Intracellular cholesterol accumulation was measured by Oil Red O stain...Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms. Methods Intracellular cholesterol accumulation was measured by Oil Red O staining and high performance liquid chromatography. The effects of rapamycin on interleukin-1β（1L-1β）-induced mRNA and protein changes of low-density lipoprotein receptor （LDLR） and ATP-binding cassette transporter Al （ABCAl） were assayed by quantitative real-time PCR and Western blot. Transient expressions of 3 types of mammalian target of rapamycin （mTOR）, including mTOR-WT （wild type）, mTOR-RR （rapamycin resistant, with kinase activity）, and mTOR-RR-KD （rapamycin resistant, without kinase activity）, were obtained by plasmid transfection. Results Rapamycin had no significant influence on intracellular cholesterol concentration trader normal condition, but it significantly decreased the intracellular cholesterol concentration in the presence of IL-1β. Rapamycin dose-dependently suppressed the increased expression of LDLR induced by IL-1β and up-regulated the suppressed expression of ABCAl caused by IL-1β Transient expression of 3 types of mTOR all reduced ABCAl mRNA expression significantly, which all could be overroded by rapamycin. Conclusions Rapamycin may contribute to the maintaining of glomerular mesangial cell intracellular cholesterol homeostasis under inflammatory state by both reducing cholesterol uptake and increasing cholesterol effiux. And the effect may be not completely mediiated by mTOR.展开更多
文摘OBJECTIVE: To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). METHODS: A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index. RESULTS: A specific monoclonal antibody against AM was succesfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect. CONCLUSION: AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.
文摘Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms. Methods Intracellular cholesterol accumulation was measured by Oil Red O staining and high performance liquid chromatography. The effects of rapamycin on interleukin-1β（1L-1β）-induced mRNA and protein changes of low-density lipoprotein receptor （LDLR） and ATP-binding cassette transporter Al （ABCAl） were assayed by quantitative real-time PCR and Western blot. Transient expressions of 3 types of mammalian target of rapamycin （mTOR）, including mTOR-WT （wild type）, mTOR-RR （rapamycin resistant, with kinase activity）, and mTOR-RR-KD （rapamycin resistant, without kinase activity）, were obtained by plasmid transfection. Results Rapamycin had no significant influence on intracellular cholesterol concentration trader normal condition, but it significantly decreased the intracellular cholesterol concentration in the presence of IL-1β. Rapamycin dose-dependently suppressed the increased expression of LDLR induced by IL-1β and up-regulated the suppressed expression of ABCAl caused by IL-1β Transient expression of 3 types of mTOR all reduced ABCAl mRNA expression significantly, which all could be overroded by rapamycin. Conclusions Rapamycin may contribute to the maintaining of glomerular mesangial cell intracellular cholesterol homeostasis under inflammatory state by both reducing cholesterol uptake and increasing cholesterol effiux. And the effect may be not completely mediiated by mTOR.
