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G Gstraunthaler

Publications and source records attributed to G Gstraunthaler.

At least 19 recordsLinked to original sources
PubMed1992

Ammoniagenesis in LLC-PK1 cultures: role of transamination.

The LLC-PK1 renal epithelial cell line has been used as a model system to study renal ammoniagenesis and its regulation by metabolic acidosis in vitro. Experiments were performed on confluent LLC-PK1 epithelia grown for 10-14 days in conventional monolayer technique. After the medium pH was changed from 7.6 to 7.0 for 24-72 h by lowering the bicarbonate concentration in culture medium, LLC-PK1 cells responded with an adaptive increase in glutamine consumption and ammonia production. The rates of glutamine uptake and ammonia generation displayed a ratio of 1:1, i.e., 1 mol ammonia was produced per mole of glutamine consumed. Glutamine consumption and ammonia formation were paralleled by an equimolar production of L-alanine, indicating that transamination appears to be the main ammoniagenic pathway in LLC-PK1 cells. Analysis of the key enzymes of renal ammoniagenesis, phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GDH), revealed no changes in enzyme activities up to 72 h of adaptation. Alanine aminotransferase (ALT) activity in LLC-PK1 cells also remained unchanged during the adaptation period. Because transamination seems to play a crucial role in channeling the metabolic flux in LLC-PK1 ammoniagenesis, experiments were performed in which transamination was inhibited by (aminooxy)acetate (AOA). After incubation of control and pH 7.0-adapted LLC-PK1 cultures for 24-72 h in 0.2 mM AOA, no alanine production was found, but 2 mol of ammonia were formed per mole of glutamine consumed, again, without adaptive changes in PDG and GDH activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1990

Morphology of the differentiation and maturation of LLC-PK1 epithelia.

In the present study, a stereologic approach was utilized to quantitatively assess morphological changes during the differentiation of LLC-PK1 cells into an epithelial membrane. This renal epithelial cell line has been described to undergo morphological changes during differentiation and maturation from subconfluent culture to a confluent epithelial layer. An increase in the number of apical microvilli, interpreted as an areal increase in this membrane domain was reported. This morphological differentiation was found to be accompanied by an increase in the expression of apical Na(+)-dependent hexose transport and the activities of certain brush border enzymes. Since no data are available that quantify the morphologic changes during LLC-PK1 differentiation, a quantitative morphologic-stereologic-investigation was performed for an early (6 days) and a late (12 days) state of confluence of LLC-PK1 monolayer cultures. The following morphological parameters were determined by light and electron microscopic morphometry: volume fractions (Vv) of nuclei, mitochondria, and lysosomes, and surface densities (Sv) of the apical and basolateral cell membrane domains. For the apical membrane surface, the microvillous fraction has been measured separately. Since the stereologic approach used in the present study allows the determination of absolute cell volumes, the absolute measures of organelle volumes (V) and membrane surfaces (S) per average cell can be calculated from volume and surface densities. Although no changes in cell density were found for 6 and 12 day old LLC-PK1 monolayers, indicating ceased cell proliferation due to contact inhibition, remarkable changes were found concerning the absolute cell volume and apical membrane surface. The observed increase in the apical cell surface was exclusively due to the enlarged microvillous surface fraction. This finding is in good agreement with the increased number of Na(+)-dependent hexose transporters as well as with the increased expression of apical membrane marker enzymes observed during the differentiation of LLC-PK1 monolayers.

Animals↗
PubMed1990

Madin-Darby canine kidney cells. III. Aldosterone stimulates an apical H+/K+ pump.

Functionally and morphologically, Madin-Darby canine kidney (MDCK) cells resemble intercalated cells of urinary epithelia. Experiments were performed on domes of confluent MDCK monolayers to test for apical H+ secretion. Apical application of 10(-3) mol/l amiloride or of Na(+)-free solution significantly reduced the limiting pH gradient across the dome epithelium (delta pHd) consistent with inhibition of apical Na+/H+ exchange. Short-circuit current (SCC) measurements disclosed an acetazolamide-sensitive, (basolateral to apical) positive transepithelial current stimulated by 10(-7) mol/l aldosterone and inhibited by acidification of apical medium to pH = 4.5. Histochemical evaluation of carbonic anhydrase (CA) activity revealed cytoplasmic and apical-membrane-bound CA particularly in dome-forming cells. Apical substitution of Na+ by K+ increased delta pHd, whereas a reduction of K+ concentration to 0.5 mmol/l or addition of barium or omeprazole (10(-5) mol/l) to the apical superfusate reduced delta pHd by at least 75%. Aldosterone-stimulated SCC was completely abolished by the apical application of barium. We conclude that besides Na+/H+ exchange MDCK cells can express an apically located H(+)-K+ pump stimulated by aldosterone and inhibited directly by the anti-ulcer agent omeprazole or indirectly, either by blocking apical K+ recycling or by interfering with the CA-dependent intracellular formation of H+ ions.

Aldosterone↗
PubMed1990

Morphology of renal tubular damage from nephrotoxins.

The proximal renal tubular cells' vulnerability to the direct toxic action of chemicals is largely due to the role played by this nephron portion in absorption and secretion. This is an energy-demanding function so that these cells must have a high rate of oxidative metabolism and thus contain many mitochondria supplying the Na+/K+ pumps at the basolateral plasma membrane domain, thereby driving the carrier systems for entry of water and solutes across the luminal membrane. Thus toxic mechanisms leading directly or indirectly to disturbances of the renal cells' energy metabolism will result in cell injury and acute renal insufficiency. Quantitative morphological-stereological-analysis of at least two, the mercuric chloride- and the maleate-induced experimental models of toxic acute renal failure, show a very early substantial loss of ATP-generating mitochondrial inner membrane surface as well as substantial decrease in those functions protecting cells against oxidative or auto-oxidative processes, i.e. glutathione content, activities of the free-radical-scavenging systems superoxide dismutase, glutathione peroxidase and glutathione reductase and catalase. The cellular dysfunction following these early events may be considered as causative of the subsequent development of most of the morphological alterations described, which are fairly similar in appearance regardless of the toxic principle acting upon the kidney.

Acute Kidney Injury↗
PubMed1990

Xanthine oxidase: evidence against a causative role in renal reperfusion injury.

The conversion rates of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) were compared with the time course of in vivo lipid peroxidation (LPO) in an ischemia-reperfusion model of acute renal failure in the rat. LPO, measured as the renal release of malondialdehyde (MDA), was found to be markedly elevated only during the first 5 min of blood reflow following a 45-min interval of ischemia (arteriovenous MDA difference -277.3 +/- 53.5 vs. 3.7 +/- 5.7 nmol/l in controls, n = 14). After 30 min of reperfusion, arteriovenous MDA differences nearly reached control values (9.7 +/- 31.8 nmol/l, n = 7). In contrast to enhanced LPO, no significant conversion of XDH to XO was found (XO activity in controls: 23 +/- 1% of XO plus XDH activity vs. 26 +/- 3% after 45 min of ischemia, n = 7). Therefore XO-derived superoxide anion radicals cannot be considered causative for LPO in the reperfusion interval of experimental ischemic acute renal failure.

Animals↗
PubMed1989

Differential inhibitory action of the fungal toxin orellanine on alkaline phosphatase isoenzymes.

The inhibitory action of orellanine (3,3',4,4'-tetrahydroxy-2,2'-dipyridyl-1,1'-dioxide), a fungal toxin of Cortinarius orellanus Fr. and C. orellanoides R. Hry., on alkaline phosphatase isoenzymes was studied. Orellanine specifically inhibited alkaline phosphatase activity in LLC-PK1 renal epithelial cell cultures and in the colon carcinoma cell line Caco-2 without affecting gamma-glutamyl transpeptidase activity. Kinetic studies revealed that orellanine acts on renal alkaline phosphatase as a noncompetitive inhibitor, whereas the intestinal and placental isoforms are inhibited competitively.

2,2'-Dipyridyl↗
PubMed1989

Fusion of cultured dog kidney (MDCK) cells: I. Technique, fate of plasma membranes and of cell nuclei.

The evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line. Madin Darby canine kidney cells, originally derived from dog kidney, to "giant" cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical "giant" cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.

Animals↗
PubMed1987

Isolation, growth, and characterization of a gluconeogenic strain of renal cells.

LLC-PK1 cells, derived from pig kidney, retain several properties of the proximal tubule, but are incapable of gluconeogenesis, due to the lack of fructose-1,6-bisphosphatase (FBPase) [Am. J. Physiol. 248 (Cell Physiol. 17): C181-185, 1985]. Cells incapable of gluconeogenesis require a hexose, pentose, or nucleoside to provide ribose-5-phosphate for RNA biosynthesis. To induce or select cells that express FBPase activity, we cultured LLC-PK1 cells in glucose-free medium. We obtained cells (designated LLC-PK1-FBPase+) that express FBPase activity and are capable of growing in the complete absence of sugars or nucleosides. The cells have apical membrane enzyme activities that differ from those of wildtype cells. Tests of metabolic flow through the gluconeogenic pathway, using 3-mercaptopicolinic acid, a specific inhibitor of phosphoenolpyruvate carboxykinase, confirmed that the cells are gluconeogenic. LLC-PK1-FBPase+ cells grown in medium containing 5 mM glucose for five weekly passages continued to express FBPase activity and apical membrane enzyme activities characteristic of the FBPase+ strain. When switched back to glucose-free medium, they proliferated well. The strain appears to be stable. It should provide a model for studying the relationship between gluconeogenesis and other proximal tubule functions. An incidental finding is that in both strains, the activity of lactate dehydrogenase varied directly with the concentration of glucose in the growth medium, indicating that the expression of lactate dehydrogenase may be regulated by glucose or a metabolite of glucose.

Animals↗
PubMed1987

Precursors of ribose 5-phosphate suppress expression of glucose-regulated proteins in LLC-PK1 cells.

Withdrawal of glucose from the medium bathing mammalian cells in culture results in cessation of growth and induces the synthesis of two stress proteins (Mr approximately 94-100 kDa and 78-80 kDa) that have been termed glucose-regulated proteins (GRPs). In LLC-PK1 cells, proteins of the same molecular weights, assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography, are fully induced by 24 h in glucose-free medium. The GRPs from LLC-PK1 cells cross-react with antibodies to GRPs of nonpolar cells, confirming their identification. Since glucose is not essential for energy production in cultured cells, but is essential for ribose 5-phosphate and nucleotide biosynthesis (Wice et al. J. Biol. Chem. 256: 7812-7819, 1981), we tested the effect of several precursors of ribose 5-phosphate on the induction of GRPs. The addition of 25 mM fructose or galactose to glucose-free medium suppressed the induction of GRPs. The pyrimidine ribonucleosides uridine and cytidine also suppressed GRP synthesis, ribose and the purine ribonucleosides guanosine and adenosine suppressed partially. The results, coupled with indirect evidence in the literature, lead to the suggestion that cell ribose 5-phosphate or a related metabolite regulates the expression of GRPs.

Adenosine↗
PubMed1986

Urinary enzyme analysis in renal allograft transplantation.

The urinary excretion of four enzymes (fructose-1,6-bisphosphatase, glutathione S-transferase, N-acetyl-beta-D-glucosaminidase and pyruvate kinase) was assayed daily in 59 patients following renal cadaveric allografting. 51 patients were given cyclosporin A (CyA group) as an immunosuppressive, 8 patients were treated conventionally with azathioprine and prednisolone (CON-group). Urinary enzyme output was evaluated by two different mathematical models. Model A follows single enzyme excretion, whereas model B also analyzes enzyme patterns. The best results were obtained by a combined analysis of all four enzymes with model B. In the CON-group the sensitivity was 1.00, the specificity 0.85, the predictive values of positive test 0.45 and all 12 graft rejections were diagnosed correctly. In the CyA group the sensitivity was 0.40, the specificity 0.99, the predictive value of positive test 0.33, and 6 out of 9 rejections were recognized. The evaluation of the single enzymes did not produce similarly good results with either model.

Acetylglucosaminidase↗
PubMed1985

Electrical properties of Madin-Darby canine kidney cells. Effects of extracellular potassium and bicarbonate.

To gain some insight into electrogenic transport processes across the plasma membrane of Madin-Darby canine kidney (MDCK)-cells, continuous measurements of the potential difference across the plasma measurements of the potential difference across the plasma membrane (PD) were made during step changes of extracellular ion composition as well as application of barium or valinomycin. During control conditions mimicking in vivo extracellular fluid, PD approaches -51.5 +/- 0.8 mV (n = 62). Step increase of extracellular potassium concentration from 5.4 to 10, to 20 or to 35 mmol/l, depolarizes PD by +5.5 +/- 0.8 mV (n = 7), by +15.8 +/- 0.5 mV (n = 64) and by +23.8 +/- 1.2 mV (n = 12), respectively. 1 mmol/l barium depolarizes PD by +19.8 +/- 0.6 mV (n = 38) and abolishes the effect of increasing extracellular potassium from 5.4 to 10 mmol/l but not to 35 mmol/l. Ten mumol/l valinomycin hyperpolarizes PD to -69.3 +/- 2.9 mV (n = 7). In the presence of valinomycin, increase of extracellular potassium from 5.4 to 20 mmol/l depolarizes PD by +31.0 +/- 1.0 mV (n = 7). Ouabain depolarizes PD and reduces the sensitivity of PD to extracellular potassium concentration. Omission of extracellular bicarbonate and carbon dioxide as well as increase of extracellular bicarbonate at constant carbon dioxide lead to a hyperpolarization and enhanced sensitivity of PD to extracellular potassium. In the presence of barium, the effects of omitted bicarbonate and carbon dioxide of MDCK-cells is highly conductive to potassium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Biochemical characterization of renal epithelial cell cultures (LLC-PK1 and MDCK).

The expression of enzymes in LLC-PK1 and MDCK cells was used to study the retention of differentiated properties of the renal epithelial cell lines by a biochemical approach. Activities of marker enzymes, for which intracellular and intranephron localization is known, were determined from crude cell homogenates of LLC-PK1 and MDCK monolayer cultures. The activity patterns of the particular enzymes found were then compared with the in vivo distribution of the enzymes along the rat nephron. LLC-PK1 cells exhibit high activities of apical membrane enzymes when compared with MDCK cells, whereas in the latter high activity of Na-K-ATPase could be detected. The activities of lysosomal enzymes, mitochondrial enzymes, and transaminases were higher in LLC-PK1 than in MDCK cells. Glycolytic enzymes, however, displayed identical activity levels in both the LLC-PK1 and MDCK cells, which may be due to the fact that these are continuous cell lines and to the culture conditions used, since glucose is a major energy source in the culture media.

Animals↗
PubMed1985

Interrelation between oxygen consumption and Na-K-ATPase activity in rat renal proximal tubule suspension.

The portion of Na-K-ATPase activity on oxygen consumption was determined in suspensions of rat proximal tubules by monitoring oxygen consumption (QO2) under different metabolic states: in the presence or absence of succinate or lactate (10 mmol/l) and increasing concentrations of ouabain (0.4; 0.8; 1.2 and 1.6 mmol/l). In the metabolic states tested, the ouabain induced decrease of QO2 was identical, which implies a fixed rate between Na-K-ATPase activity and QO2. On the basis of these results, Ki and maximal inhibition rate were determined by Lineweaver-Burk and Eadie-Hofstee plots Ki was 0.67 mmol/l ouabain and the maximal inhibition of QO2 was 77%. This corresponds to an absolute decrease of QO2 of 1,630 mumol O2 . h-1 . g-1 protein. Since this value represents the real portion of Na-K-ATPase on QO2, the activity of Na-K-ATPase can be calculated, yielding an activity of 163 mumol ATP . min-1 . g-1 tubule protein.

Animals↗
PubMed1984

[Urinary enzymes in the non-invasive diagnosis of kidney epithelial lesions in acute kidney failure].

The present investigation describes the urinary output of four different enzymes localized within nephron cells in two models of experimental acute renal failure. The activities of fructose-1,6-diphosphatase (FDP), glutathione-S-transferase (GST), N-acetyl-beta-D-glucosaminidase (NAG) and pyruvate kinase (PK) were determined in the urine of rats after maleate or HgCl2 intoxication. 2 hours after maleate intoxication the urinary output of FDP, GST and NAG was significantly increased above control values. 6 hours after HgCl2 poisoning FDP, GST and NAG showed increased urinary enzyme activities. The urinary activity of each enzyme was significantly increased 24 hours after intoxication. These results are in good accordance with the damage observed on light and electron microscopic investigations carried out with both experimental models. Furthermore, general problems of urinary enzyme measurements are discussed in this paper.

Acetylglucosaminidase↗
PubMed1984

Immunocytochemical localization of gamma-glutamyl-transferase on isolated renal cortical tubular fragments.

The localization of gamma-Glutamyltransferase (gamma-GT, E.C.2.3.2.2) was studied on isolated tubular fragments from rat kidney cortex immunocytochemically. Monospecific antibodies raised in the goat against rat kidney gamma-GT were used. Antigoat immunoglobulin from the rabbit conjugated with ferritin was used for visualisation of the antibody binding sites. The enzyme was found to be localized at the brush border membrane of proximal tubules, the luminal membrane of distal tubules and collecting duct segments. The enzyme could further be localized on the antiluminal or basolateral cell membranes of proximal and distal tubular fragments, whereas no such localization was verified for collecting duct segments. The role of this basolateral gamma-GT localization in context with the kidney's ability to extract over 83% of the renal arterial glutathione (GSH) input during a single passage is discussed.

Animals↗
PubMed1983

Glutathione depletion and in vitro lipid peroxidation in mercury or maleate induced acute renal failure.

Nephrotoxic acute renal failure was experimentally induced in male rats by s.c. application of mercuric chloride and i.p. administration of maleate, respectively. Mercuric chloride and maleate are known to enhance the formation of free radicals and peroxides, which presumably overload the cell's natural elimination mechanisms for these highly reactive intermediates. In addition, a reduction in activities of superoxide dismutase, catalase and glutathione-peroxidase, enzymes responsible for the protection of cells against peroxidative action of superoxide anions and hyperperoxides was found. In both models of acute renal failure, enhanced lipid peroxidation in kidney homogenates in vitro, monitored as malondialdehyde production, was observed. Furthermore, HgCl2 and maleate may react with free SH-groups and thus lead to a depletion of glutathione in tubular cells. Indeed, renal cortical contents of reduced and oxidized glutathione were drastically diminished. These results suggest that alterations in membrane integrity, possibly caused by peroxidative processes, can be considered the cause underlying the well-known disturbances in renal function commonly observed during the initiation phase of HgCl2 and maleate induced acute renal failure.

Acute Kidney Injury↗