Na regulates growth of kidney epithelial cells induced by lowering extracellular K concentration

1984 ◽  
Vol 247 (5) ◽  
pp. C321-C326 ◽  
Author(s):  
M. M. Walsh-Reitz ◽  
H. N. Aithal ◽  
F. G. Toback
Keyword(s):  
Cell Growth ◽  
Epithelial Cells ◽  
Dependent Manner ◽  
Deficient Diet ◽  
Critical Determinant ◽  
Concentration Dependent Manner ◽  
Kidney Epithelial Cells ◽  
K Concentration ◽  
Low K ◽  
Stimulation Of

Accelerated kidney growth and increased tissue Na content have been observed in rats fed a K-deficient diet. These observations suggest that enhanced Na influx could mediate renal growth, a hypothesis that was tested in cultures of kidney epithelial cells of the BSC-1 line. Reduction of the K concentration in the culture medium from 5.4 to 3.2 mM augmented cell growth and induced a transient increase in the cellular content of Na and a decrease in that of K. That low-K-induced growth was Na dependent was shown by decreasing the medium Na concentration from 155 to 150 mM, which abolished the increases in both growth and cell Na content in a concentration-dependent manner. The stimulation of glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity that occurs in cells exposed to low-K medium for 1 h was similarly prevented by decreasing the medium Na concentration. Thus decreased availability of extracellular Na prevented the increase in cell Na content, stimulation of G3PD activity, and accelerated growth induced by low-K medium. The hypothesis was also tested by adding vasopressin to cultures of BSC-1 cells exposed to low-K medium; the hormone prevented the increments in cell Na content, G3PD activity, and growth to the same extent as did decreased availability of extracellular Na. These results are consistent with the interpretation that transient accumulation of Na is a critical determinant of the initiation of kidney epithelial cell growth.

Stimulation of DNA synthesis in kidney epithelial cells in culture by potassium

1984 ◽  
Vol 247 (1) ◽  
pp. C14-C19 ◽  
Author(s):  
F. G. Toback ◽  
K. B. Ekelman ◽  
N. G. Ordonez
Keyword(s):  
Epithelial Cells ◽  
Dna Synthesis ◽  
Calf Serum ◽  
Extracellular Fluid ◽  
Chloride Concentration ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cells In Culture ◽  
Kidney Epithelial Cells ◽  
Number Of Cells

The hypothesis that the K+ concentration of extracellular fluid is a determinant of renal DNA synthesis was examined in quiescent, high-density cultures of monkey kidney epithelial cells of the BSC-1 line. The addition of KCl to the medium increased the number of cells engaged in DNA synthesis in a concentration-dependent manner. The capacity of K+ to stimulate DNA synthesis in a greater number of cells was additive with exogenous NaCl and calf serum and was associated with an increment in the steady-state cell K+ content. Studies with other monovalent cations indicated that the stimulatory effect of K+ on DNA synthesis was not mediated by increments in the chloride concentration or osmotic pressure of the medium. The addition of K+ to confluent cultures was associated with a concentration-dependent increase in cellmultiplication. The commitment of cells to increased multiplication required exposure of the culture to added KCl for longer than 3 but not more than 6 h. Addition of KCl to cultures of mouse fibroblasts did not alter DNA synthesis, multiplication, or cell K+ content. These observations indicate that increased availability of K+ in the extracellular fluid can stimulate DNA synthesis in kidney epithelial cells in culture.

Extracellular potassium modifies the structure of kidney epithelial cells in culture

1985 ◽  
Vol 249 (1) ◽  
pp. C105-C110 ◽  
Author(s):  
S. Waack ◽  
M. M. Walsh-Reitz ◽  
F. G. Toback
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Epithelial Cells ◽  
Unit Surface Area ◽  
Scanning Electron Micrographs ◽  
Membrane Function ◽  
Transmission Electron ◽  
Kidney Epithelial Cells ◽  
K Concentration ◽  
Low K

In animals fed a K-deficient diet, alterations in kidney cell structure and function occur in association with changes in the ionic composition of the extracellular fluid. The hypothesis that the extracellular K concentration mediates these changes in renal tissue was tested in cultures of monkey kidney epithelial cells (BSC-1 line) by reducing the K concentration of the culture medium from the control value of 5.4 to 3.2 mM. Exposure of BSC-1 cells to low-K medium raised the maximal rate of uptake for L-glutamic acid by 39% without a change in apparent Km. To determine whether this alteration in plasma membrane function had a structural correlate, studies of the cell surface were performed using scanning and transmission electron microscopy. Morphometric analysis of scanning electron micrographs revealed that the number of microvilli per cell per unit surface area was 45% greater in cells exposed to low-K medium for 3 min than those exposed to control medium. This observation was confirmed by transmission electron microscopy. The results indicate that an alteration in the extracellular K concentration per se can modify specific structural and functional characteristics of kidney epithelial cells.

Aberrant responses to growth-regulatory signals by variant kidney epithelial cells

1988 ◽  
Vol 254 (5) ◽  
pp. F747-F753
Author(s):  
M. M. Walsh-Reitz ◽  
R. I. Feldman ◽  
F. G. Toback
Keyword(s):  
Epithelial Cells ◽  
Growth Regulation ◽  
Growth Inhibitor ◽  
Soft Agar ◽  
Isoenzyme Analysis ◽  
Variant Cell ◽  
Kidney Epithelial Cells ◽  
Potent Growth ◽  
Potent Growth Inhibitor ◽  
Low K

Cultures that achieved a higher cell density than expected were noted during study of growth regulation in monkey kidney epithelial cells of the BSC-1 line. Multiplication of the variant cells was accelerated, compared with parental cells, as the cultures approached confluence. Cytogenetic analysis, immunofluorescence antibody reactions with specific monkey serum, isoenzyme analysis, microbiological studies, and lack of growth in soft agar indicated that the variant cells were not a contaminating cell type, lacked new isoenzymes, were free of microbial contamination, and were not transformed. Confluent variant cultures did not respond to a purified growth inhibitor protein produced by BSC-1 cells that inhibits multiplication and reduces cell Na content in subconfluent variant and parental cells. Vasopressin, which is a mitogen for parental cells, was a potent growth inhibitor for confluent cultures of variant cells. Low-K or high-Na media, which stimulate proliferation of parental cells, had no effect on growth of the variant cell line. These results suggest that enhanced multiplication of the variant cells is mediated by altered signal transduction pathways and/or receptors for growth-regulatory molecules.

Cellular and molecular basis of increased ammoniagenesis in potassium deprivation

2011 ◽  
Vol 301 (5) ◽  
pp. F969-F978 ◽  
Author(s):  
Shaikh Abu Hossain ◽  
Farrukh A. Chaudhry ◽  
Kamyar Zahedi ◽  
Faraaz Siddiqui ◽  
Hassane Amlal
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Molecular Mechanisms ◽  
Intracellular Acidosis ◽  
Deficient Diet ◽  
Proximal Tubule Cells ◽  
Glutamine Transporter ◽  
Additive Increase ◽  
Acid Base Status ◽  
Low K ◽  
Stimulation Of

Hypokalemia is associated with increased ammoniagenesis and stimulation of net acid excretion by the kidney in both humans and experimental animals. The molecular mechanisms underlying these effects remain unknown. Toward this end, rats were placed in metabolic cages and fed a control or K+-deficient diet (KD) for up to 6 days. Rats subjected to KD showed normal acid-base status and serum electrolytes composition. Interestingly, urinary NH4+ excretion increased significantly and correlated with a parallel decrease in urine K+ excretion in KD vs. control animals. Molecular studies showed a specific upregulation of the glutamine transporter SN1, which correlated with the upregulation of glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase. These effects occurred as early as day 2 of KD. Rats subjected to a combined KD and 280 mM NH4Cl loading (to induce metabolic acidosis) for 2 days showed an additive increase in NH4+ excretion along with an additive increment in the expression levels of ammoniagenic enzymes GA and GDH compared with KD or NH4Cl loading alone. The incubation of cultured proximal tubule cells NRK 52E or LLC-PK1 in low-K+ medium did not affect NH4+ production and did not alter the expression of SN1, GA, or GDH in NRK cells. These results demonstrate that K+ deprivation stimulates ammoniagenesis through a coordinated upregulation of glutamine transporter SN1 and ammoniagenesis enzymes. This effect is developed before the onset of hypokalemia. The signaling pathway mediating these events is likely independent of KD-induced intracellular acidosis. Finally, the correlation between increased NH4+ production and decreased K+ excretion indicate that NH4+ synthesis and transport likely play an important role in renal K+ conservation during hypokalemia.

scholarly journals CP30, a Cysteine Proteinase Involved inTrichomonas vaginalis Cytoadherence

2000 ◽  
Vol 68 (9) ◽  
pp. 4907-4912 ◽  
Author(s):  
M. Remedios Mendoza-López ◽  
Cecilia Becerril-Garcia ◽  
Loriz V. Fattel-Facenda ◽  
Leticia Avila-Gonzalez ◽  
Martha E. Ruíz-Tachiquín ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Hela Cell ◽  
Trichomonas Vaginalis ◽  
Cysteine Proteinase ◽  
Collagen Iv ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Monolayers ◽  
Thiol Proteinase

ABSTRACT We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.

Endothelium and mechanical responses of isolated monkey pulmonary veins to histamine

1990 ◽  
Vol 259 (4) ◽  
pp. H1032-H1037 ◽  
Author(s):  
T. Matsuki ◽  
T. Ohhashi
Keyword(s):  
Pulmonary Veins ◽  
Dependent Manner ◽  
High Concentration ◽  
Concentration Dependent Manner ◽  
Mechanical Responses ◽  
Relaxing Factor ◽  
Prostaglandin F2 Alpha ◽  
H2 Receptors ◽  
Endothelium Derived Relaxing Factor ◽  
Stimulation Of

Ring strips of monkey pulmonary veins precontracted with a high concentration of prostaglandin F2 alpha (PGF2 alpha) relaxed in a concentration-dependent manner in response to histamine. Treatment with mepyramine and/or famotidine attenuated the relaxation. 2-Pyridylethylamine (2PEA) and dimaprit caused relaxations in the precontracted preparations, which were inhibited by pretreatment with mepyramine and famotidine, respectively. Removal of endothelium reversed the histamine- and 2PEA-induced relaxations to dose-related contractions. On the other hand, the removal had no effect on the dimaprit-induced relaxations, which were significantly reduced by pretreatment with famotidine. Histamine-induced relaxations in the precontracted strips with endothelium in the presence and absence of famotidine were suppressed or abolished by treatment with methylene blue or hemoglobin but were unaffected by aspirin. It may be concluded that histamine-induced relaxation in monkey pulmonary veins precontracted with PGF2 alpha is mediated by H2-receptors in smooth muscle and H1-receptors in endothelium. Also, stimulation of the endothelial H1-receptors liberates an endothelium-derived relaxing factor.

Adhesion of uric acid crystals to the surface of renal epithelial cells

2000 ◽  
Vol 278 (6) ◽  
pp. F989-F998 ◽  
Author(s):  
Rima M. Koka ◽  
Erick Huang ◽  
John C. Lieske
Keyword(s):  
Uric Acid ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Hydrophobic Interactions ◽  
Calcium Phosphates ◽  
Apical Cell ◽  
Apical Surface ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Renal Tubular Cells

Adhesion of microcrystals that nucleate in tubular fluid to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones, 12% of which contain uric acid (UA) either alone or admixed with calcium oxalates or calcium phosphates. UA crystals bind rapidly to monolayer cultures of monkey kidney epithelial cells (BSC-1 line), used to model the surface of the nephron, in a concentration-dependent manner. The urinary glycoproteins osteopontin, nephrocalcin, and Tamm-Horsfall glycoprotein had no effect on binding of UA crystals to the cell surface, whereas other polyanions including specific glycosaminoglycans blocked UA crystal adhesion. Specific polycations also inhibited adhesion of UA crystals and appeared to exert their inhibitory effect by coating cells. However, removal of anionic cell surface molecules with neuraminidase, heparitinase I, or chondroitinase ABC each increased UA crystal binding, and sialic acid-binding lectins had no effect. These observations suggest that hydrogen bonding and hydrophobic interactions play a major role in adhesion of electrostatically neutral UA crystals to renal cells, unlike the interaction of calcium-containing crystals with negatively charged molecules on the apical cell surface via ionic forces. After adhesion to the plasma membrane, subsequent cellular events could contribute to UA crystal retention in the kidney and the development of UA or mixed calcium and UA calculi.

scholarly journals 1289. Pravibismane is a Potent, Broad Spectrum Anti-Infective Small Molecule that Rapidly Disrupts Bacterial Bioenergetics and Halts Bacterial Growth

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S659-S660
Author(s):  
Brett Baker
Keyword(s):  
Membrane Potential ◽  
Cell Growth ◽  
Protein Expression ◽  
Broad Spectrum ◽  
Time Lapse ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Time Lapse Microscopy ◽  
Atp Levels

Abstract Background The rise in resistance to existing antimicrobials has prompted a need for the development of novel antibiotics. Microbion has identified a novel compound, pravibismane, with potent broad spectrum anti-infective and anti-biofilm activity. Methods Here we used a variety of assays, including Bacterial Cytological Profiling (BCP), to analyze pravibismane in E.coli to gain insight into its likely mechanism of action (MOA). The BCP profile of pravibismane suggested it rapidly shut down cell growth, potentially by turning off cellular gene or protein expression. This was confirmed using a plasmid based GFP induction assay in E.coli tolC that showed pravibismane strongly reduced expression of GFP. The kinetics, reversibility and MOA of pravibismane was further characterized by using time-lapse microscopy, wash out experiments and measurements of both membrane potential and relative intracellular ATP levels. Results We found that pravibismane acts rapidly (within 30 mins) to completely halt cell growth rather than causing immediate cell lysis such as that observed with non-specific cell damaging agents bleach or detergent. Inhibitor wash out experiments in which cells were exposed to pravibismane for 2 hours, washed to remove the compound, and then observed using time-lapse microscopy revealed that the effect of pravibismane is reversible and that cells recovered 8-12 hrs after removing the compound. Wash out experiments with an E.coli tolC strain carrying a plasmid with an IPTG inducible GFP demonstrated that transcription and translation ultimately resumed in most cells after washout. The bioenergetics of the membrane was measured using DiBAC 4(5), a membrane potential sensitive dye which can enter depolarized cells, which revealed that pravibismane caused depolarization of the membrane within 30 mins of exposure in a concentration dependent manner. Finally, a luciferase assay determined pravibismane reduced ATP levels (resulting in decreased luminescence) within 15 mins of exposure in a concentration dependent manner unlike antibiotic controls that had modest or no effect on luminescence. Conclusion Our results suggest that pravibismane acts rapidly to disrupt cellular bioenergetics, resulting in the immediate cessation of cell growth and protein expression. Disclosures Brett Baker, M.Sc., D.C., Microbion Corporation (Board Member, Employee)

scholarly journals Anticancer effects of mifepristone on human uveal melanoma cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Prisca Bustamante Alvarez ◽  
Alexander Laskaris ◽  
Alicia A. Goyeneche ◽  
Yunxi Chen ◽  
Carlos M. Telleria ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Progesterone Receptor ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Caspase 3 ◽  
Annexin V ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free Dna

Abstract Background Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. Methods The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. Results MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

Adhesion of hydroxyapatite crystals to anionic sites on the surface of renal epithelial cells

1997 ◽  
Vol 273 (2) ◽  
pp. F224-F233 ◽  
Author(s):  
J. C. Lieske ◽  
R. Norris ◽  
F. G. Toback
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Alcian Blue ◽  
Crystal Surface ◽  
Cetylpyridinium Chloride ◽  
Apical Surface ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Subsequent Formation ◽  
Renal Tubular Cells

Adhesion of microcrystals that nucleate in tubular fluid to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones, 20% of which contain hydroxyapatite (HA). HA crystals bound rapidly to monolayer cultures of monkey kidney epithelial cells (BSC-1 line), used to model the surface of the nephron, in a concentration-dependent manner. Adhesion was blocked by diverse polyanions including heparin, pentosan polysulfate, polyaspartate, and polyglutamate, as well as many found in tubular fluid such as chondroitin sulfates A and B, heparan sulfate, citrate, nephrocalcin, and osteopontin. The polycations cetylpyridinium chloride and cationized ferritin, as well as the cationic dyes alcian blue, polyethylenimine, and brilliant blue R, also inhibited adhesion of HA crystals, as did specific lectins including Triticum vulgaris (wheat germ agglutinin). Anions that inhibited adhesion of crystals appeared to act on the crystal surface, whereas cations and lectins exerted their effect on the cell. Treatment of cells with neuraminidase inhibited binding of crystals, suggesting that anionic cell surface sialic acid residues function as HA crystal receptor sites that can be blocked by specific cations or lectins. Adherence of HA crystals to cells of another renal line (MDCK) and, to 3T3 fibroblasts was also inhibited by heparin, polyaspartate, alcian blue, and T vulgaris lectin, suggesting that these crystals bind to analogous molecules on the surface of different types of cells. These results suggests that the structure, quantity, and/or function of soluble anions in tubular fluid, as well as those anchored to the cell surface, could be critical determinants of HA crystal retention in the nephron and the subsequent formation of a renal stone.

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