A stable epithelial-like pig kidney cell strain has been established. This strain has been carried through more than 300 serial passages, has remained free of microbial and viral contaminants, and has retained a near diploid number of chromosomes. Attempts to produce tumors with these cells in immunosuppressed laboratory animals have been uniformly negative. The cells have grown rapidly in monolayer cultures with a split ratio of 1 to 15 at weekly intervals, but have failed to proliferate in suspension cultures. A subline adapted to growth on serum-free medium 199 has been carried through 145 passages on this medium. Several unusual morphologic features have been observed in these cultures including three-dimensional "domelike" structures. These cells have been found susceptible to some viruses and have been especially useful for viruses of domestic animals. LLC-PK1 cells have produced significant levels of plasminogen activator.
"LLC-PK1 cells are kidney proximal tubule epithelial cells ; they were cultured as described . Appropriate concentrations of phenformin, resveratrol or AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) and their effects on AMPK have been determined previously . "
[Show abstract][Hide abstract] ABSTRACT: Phenformin, resveratrol and AICAR stimulate the energy sensor 5'-AMP activated kinase (AMPK) and inhibit the first step of ribosome biogenesis, de novo RNA synthesis in nucleoli. Nucleolar activities are relevant to human health, because ribosome production is crucial to the development of diabetic complications. Although the function of nucleoli relies on their organization, the impact of AMPK activators on nucleolar structures is not known. Here, we addressed this question by examining four nucleolar proteins that are essential for ribosome biogenesis.
Kidney cells were selected as model system, because diabetic nephropathy is one of the complications associated with diabetes mellitus. To determine the impact of pharmacological agents on nucleoli, we focused on the subcellular and subnuclear distribution of B23/nucleophosmin, fibrillarin, nucleolin and RPA194. This was achieved by quantitative confocal microscopy at the single-cell level in combination with cell fractionation and quantitative Western blotting.
AMPK activators induced the re-organization of nucleoli, which was accompanied by changes in cell proliferation. Among the compounds tested, phenformin and resveratrol had the most pronounced impact on nucleolar organization. For B23, fibrillarin, nucleolin and RPA194, both agents (i) altered the nucleocytoplasmic distribution and nucleolar association and (ii) reduced significantly the retention in the nucleus. (iii) Phenformin and resveratrol also increased significantly the total concentration of B23 and nucleolin.
AMPK activators have unique effects on the subcellular localization, nuclear retention and abundance of nucleolar proteins. We propose that the combination of these events inhibits de novo ribosomal RNA synthesis and modulates cell proliferation. Our studies identified nucleolin as a target that is especially sensitive to pharmacological AMPK activators. Because of its response to pharmacological agents, nucleolin represents a potential biomarker for the development of drugs that diminish diabetic renal hypertrophy.
PLoS ONE 01/2014; 9(1):e88087. DOI:10.1371/journal.pone.0088087 · 3.23 Impact Factor
"Since there is no human cell line from distal nephron commercially available, we tried to test whether the renal monkey cell line MA104 was originated from distal nephron. First, we compared the sensitivity of three mammal renal cell lines to urea: MDCK, a canine kidney cell line with characteristics of distal nephron –; LLCPK1, a porcine renal cell line with characteristics of proximal tubules  and MA104, not yet characterized. Cells were incubated for 48 h with various urea concentrations and the cell number and the cellular viability were measured by Trypan Blue staining. "
[Show abstract][Hide abstract] ABSTRACT: Glutathione (GSH) plays an important role in protecting cells against oxidative damage. ABCC1 protein transports GSH. Although this protein is largely studied in cancer, due to multidrug resistance phenotype, its role in the tubular cells of the kidney is unknown. The goal of this study was to find out whether ABCC1 has a role in protecting cells from the distal nephron against the stress caused by high medullar osmolality.
MA104 cells were treated with high concentrations of sodium chloride, urea, or both to raise the osmolality of the culture medium. Cell viability was accessed by MTT and trypan blue assays. ABCC1 expression and extrusion of carboxi-fluorescein (CF), a fluorescent ABCC1 substrate, were measured by flow cytometry.
Incubation of MA104 cells in a high sodium concentration medium resulted in changes in cell granularity and altered expression and activity of ABCC1. Urea did not alter ABCC1 expression or activity, but reversed the observed NaCl effects. High sodium concentrations also had a negative effect on cell viability and urea also protected cells against this effect.
Our findings demonstrate that ABCC1 plays a significant role in the protection of kidney epithelial cells against the stress caused by high sodium environment present in renal medulla.
PLoS ONE 06/2013; 8(6):e68049. DOI:10.1371/journal.pone.0068049 · 3.23 Impact Factor
"LLC-PK1 porcine and HK2 human kidney proximal tubule epithelial cell lines were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD)  . Cells were cultured in a 5% CO 2 incubator in growth medium (GM) consisting of Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/l glucose, 2 mM Lglutamine , 5000 IU/l penicillin, 5 mg/l streptomycin, 125 U/l Fungizone , 2.2 g/l sodium bicarbonate supplemented with 10% fetal calf serum (FCS). "
[Show abstract][Hide abstract] ABSTRACT: Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. In diabetes, renal Na+ K+ ATPase (NKA) activity is downregulated and phosphoinositide metabolism is upregulated. We examined the effects of AGEs on NKA activity in porcine LLC-PK1 and human HK2 proximal tubule epithelial cells. AGE-BSA increased cellular phosphoinositol 4,5 bisphosphate (PIP2) production as determined by immunofluorescence microscopy and thin layer chromatography. AGE-BSA (40 microM) induced 3H-arachidonic acid release and reactive oxygen species (ROS) production via cytosolic phospholipase A2 (cPLA2) activation. Within minutes, AGE-BSA significantly inhibited NKA surface expression and activity in a dose- and time-dependent manner as determined by immunofluorescence staining and [86Rb+] uptake, respectively, suggesting AGEs inhibit NKA by stimulating its endocytosis. The AGE-BSA-induced decrease in cell surface NKA was reversed by a cPLA2alpha inhibitor, neomycin, a PIP2 inhibitor, and PP2, a Src inhibitor. AGE-BSA increased binding of NKA to the alpha-adaptin but not beta2- or mu2-adaptin subunits of the AP-2 clathrin pit adaptor complex. Transfection of HK2 cells with PIP5Kgamma siRNA prevented AGE-BSA inhibition of NKA activity. AGEs may stimulate PIP5Kgamma to increase PIP2 production, which may enhance AP-2 localisation to clathrin pits, increase clathrin pit formation, enhance NKA cargo recognition by AP-2 and/or stimulate cPLA2alpha activity. These results suggest AGEs modulate arachidonic acid and phosphoinositide metabolism to inhibit NKA via clathrin-mediated endocytosis. Elucidation of new intracellular AGE signaling pathways may lead to improved therapies for diabetic nephropathy.
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