We investigated composition and content of glycosphingolipids (GSLs) of rat renal glomerular epithelial cells (SGE1) in vitro, and showed GSLs change during growth and differentiation of the cells. To analyze GSLs, we used a high sensitivity fluorescent labelling method on thin-layer chromatogram. The method could detect GSLs from as little as 10⁷ cells. The GSLs of SGE1 were composed of CMH, CDH, CTH, globoside, paragloboside, Forssman antigen and other minors for neutral GSLs, and GM3, GM2, GM1, GD1a and other minors for ganglioside. We used 2 culture ways; one was monolayer culture and the other was collagen-embedding 3-dimentional (3D) culture that induced cell differentiation. In the case of monolayer culture, GM3 content increased at 6 days after seeding, with increase of γ-GTP activity and with dome formation, the markers for SGE1 cell differentiation. In the case of collagen-embedding 3D culture, content of GD1a of ganglioside, CMH and globoside of neutral GSL as well as GM3 increased, with increase of γ-GTP activity and 3D cyst formation. These results showed that GSLs change of SGE1 cells quantitatively correlated with cell growth and differentiation of renal epithelial cell.

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Long-term culture by extended serial passage of renal glomerular epithelial cells has not yet been described in detail, although it has been known that the cells exhibit outgrowth from isolated glomeruli in primary culture. In the present review article, establishment and characterization of an epithelial cell line, SGE1, from isolated rat glomeruli are described. In the serum-free condition, SGE1 cells required collagen type 1 or IV-coated substratum for cell adhesion and growth. Essential medium supplements for the adhesion and growth were epidermal growth factor and transferrin, respectively. Morphological observations found that in a monolayer culture, SGE1 cells formed typical domes, and in a collagen-embedding culture, they formed cystic spheres having features of a simple cuboidal epithelium. We also found that retinyl acetate regulated morphology, functions, and growth of cells. In addition, SGE1 cells expressed Fx1A antigens, which are nephritogenic antigens localized on their microvilli.

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In this article, pathological studies of glomerulopathy using cultured glomerular visceral epithelial cells (GEC) are described. The mechanism of puromycin aminonucleoside (PAN)-inducedn ephrosis, which is similar to minimalc hange disease in humans, has not yet been well defined. We investigated roles of active oxygen species in GEC injury in vitro induced by PAN. Concomitant addition of either hydrogen peroxide scavenger or iron chelating agent to the culture medium with PAN caused a striking reduction of cellular injury. The release of hydrogen peroxide into the medium by cultured GEC exposed to PAN was shown to increase dose-dependently. These results strongly suggested that active oxygen species might play an important role in PANinduced GEC injury in vitro as well as in vivo. Moreover, we also found that injection of antibodies raised against membrane fractions purified from cultured GEC to rats induced rapid and severe proteinuria associated with GEC damage. Thus, these experimental models hopefully will lead to a greater understanding of the mechanism by which proteinuria is induced and developed.

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In this review, we describe two kinds of substances, liver tumor promoters [phenobarbital and barbital] and a glutathione-related tripeptide [S- (1,2dicarboxyethyl)glutathione (DCE-GS)], which induce DNA synthesis in primary cultured rat hepatocytes in the presence of epidermal growth factor (EGF). The two promoters, phenobarbital and barbital, enhanced DNA synthesis in suckling rat hepatocytes which seems to be stimulated by autocrine growth factor(s). On the other hand, phenobarbital suppressed DNA synthesis in adult rat hepatocytes in the absence of growth factors such as insulin and EGF, but enhanced DNA synthesis in the cells stimulated by these growth factors. The tripeptide DCE-GS, which is mainly synthesized in the liver, showed no effect on DNA synthesis in adult rat hepatocytes by itself, but significantly enhanced DNA synthesis in the cells when added simultaneously with EGF. The DCE-GS level was increased in the regenerating rat liver, reaching a maximal level (4.7-fold) on day 2, and reverted to the normal level within 1 week. The time courses of the increase in DCE-GS level and DNA synthesis in the regenerating rat liver closely correlated to each other. These findings probably indicate that the intrinsic tripeptide DCE-GS is closely related to liver regeneration.

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Serine dehydratase (SDH), which is involved in gluconeogenesis, is induced in adult liver under diabetic conditions and by high protein diet. Its expression is inhibited in rapidly growing, immature liver such as fetal or regenerating liver. The molecular mechanisms of regulation of its expression in various conditions were investigated. Results indicated that in fetal rat liver, transcription of the SDH gene is repressed by a negative trans-acting factor(s), which may decrease or be inactivated during terminal differentiation of hepatocytes. On the oontnury, the decrease in its expression in regenerating liver seemed to be caused by decrease of positive trans-acting factors. The rat SDH gene contains glucocorticoid- and cAMP- responsive e|ements, but the mechanism of the synergistic effects of glucocorticoid and glucagon (cAMP) on enzyme induction is unknown.

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Hepatocyte growth factor (HGF) was originally identified as a potent growthpromoting factor for rat hepatocytes in primary culture. Recent progress, however, has revealed that HGF is mitogenic to some epithelial cells other than hepatocytes and to endothelial cells. It is also a multifunctional cytokine with mitogenic, motogenic, morphogenic and tumor-suppressing activities. Despite its broad-spectrum of biological activities, HGF is most likely the physiological hepatotrophic factor that triggers or modulates liver regeneration. HGF levels in plasma and liver of experimental animals, partially hepatectomized or treated with hepatotoxins, increase prior to induction of hepatic DNA synthesis. In this review, we focus upon regulation of gene expression and production of HGF by cultured fibroblasts and other cells. Human HGF (hHGF) mRNA expression is upregulated by interleukin-1, probably via the nuclear factor-interleukin-6binding element located near the transcription initiation site of the hHGF gene. Tumor necrosis factor-a, and activators of protein kinase C stimulate HGF gene expression and production of HGF. Injurin, a heat-stable protein found in sera of rats that have undergone injury of the liver or kidney, also induces HGF mRNA expression, although its physicochemical and biological properties are only partially characterized. Dexamethasone and transforming growth factor-fl inhibit gene expression and production of HGF. The effects of the latter are more potent than those of the former. The physiological significance of the stimulators and inhibitors of HGF production is discussed.

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Since the angiotensin-converting enzyme inhibitor has a potent suppressive effect upon the development of glomerular sclerosis, angiotensin II is believed to be involved in the disease progress. Angiotensin II causes glomerular hypertension, a proposed predominant factor leading to glomerular sclerosis, through more prominent contraction of the efferent than the afferent arterioles as well as through elevating systemic blood pressure. In vitro studies using cultured mesangial cells have revealed that angiotensin II also causes cell proliferation and matrix production, a hallmark of various glomerular diseases, as well as cell contraction which is important in the regulation of the glomerular filtration process. Furthermore, the contraction and proliferation of mesangial cells are biologically linked to each other and may play a synergistic role in the progression of glomerular sclerosis. The direct in vivo role of angiotensin II in physiological and pathophysiological regulation of mesangial cell functions should be further studied in future.

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