Biomedical subjects

W Pfaller

Publications and source records attributed to W Pfaller.

At least 19 recordsLinked to original sources

PubMed2006

Application of a population balance model to a perfusion in vitro toxicity system.

Perfusion cell culture is different to static cell culture: In perfusion culture, steady state concentrations of nutrients and metabolites can be achieved. When a toxin is added to the medium, it is administered to the cells at a defined constant concentration. Perfusion culture is favourable for time series analysis of experimental data because assays can be performed online in the outflowing medium at a high time resolution. The cytotoxicity exerted by cadmium chloride (CdCl(2)) on LLC-PK(1) cells was investigated in static and in perfusion culture using the EpiFlow system in order to compare both the techniques. For this comparison, cytotoxicity was assessed by the release of the cytosolic enzyme lactate dehydrogenase (LDH), and by the potential of viable cells to reduce resazurin. The results showed a greater sensitivity of LLC-PK(1) monolayers to CdCl(2) with continuous exposure in perfusion culture as compared to a single or repeated dose in static culture, as evidenced by increased LDH release and decreased resazurin reduction. These differences between static and perfusion culture were ascribed to different delivery rates of toxin to the site of absorption. Cadmium accumulation of the cells was measured and found to be higher under perfusion conditions than under static conditions. Time series analysis of the LDH release in perfusion culture was performed by establishing a deterministic population balance model of cell death. An empirical death rate function that accounts for accumulation of the toxin was introduced and could be successfully verified.

Algorithms↗

PubMed2005

Serum free cell culture: the free access online database.

The cultivation of cells in vitro is an important tool for biomedical research and production purposes. The supplementation of animal/human cell culture media with sera (components) of animal origin remains still standard, providing for e.g. necessary nutrition, shear protection, growth factors and cytokines. Because of undefined composition, risk of contaminations, the cost factor and also animal welfare considerations concerning the production of sera, the conversion to serum free alternatives is promoted by regulatory authorities, industry and the research community in general. To support this trend and to help save one of the scientists most valuable resources -- time -- a data bank was compiled of commercially available formulations, searchable for products, applications, cell lines and manufacturers. The database is accessible free of charge in HTML format and as PDF download, the informations are checked and updated twice a year. Problems concerning serum free cell culture are discussed at and comments are welcome.

Animals↗

PubMed2000

Urinary N-acetyl-beta-D-glucosaminidase and neopterin aid in the diagnosis of rejection and acute tubular necrosis in initially nonfunctioning kidney grafts.

AIM: The study aimed at investigating urinary neopterin, a marker of cellular immune response, and urinary N-acetyl-beta-D-glucosaminidase (NAG), a marker of tubular damage, as noninvasive means to differentiate between acute tubular necrosis (ATN) and rejection in initially nonfunctioning (INF) human renal transplants. METHODS: Seventy-two renal transplant patients were studied. Forty-five of them experienced an uncomplicated early posttransplant course, 27 patients suffered from INF. Twenty-two patients experienced ATN, 5 patients had a total of six biopsy-proven rejections. The NAG activity was measured by a colorimetric assay, neopterin by high-performance liquid chromatography. Receiver operating characteristics (ROC) analysis was applied to compute diagnostic performance and an optimal discriminating threshold. RESULTS: Demographic characteristics (age, gender, cold and warm ischemia periods, HLA mismatches) and posttransplant urinary NAG and neopterin excretions did not differ between ATN and rejection groups. Both urinary NAG and neopterin excretions were lower in the control group (NAG 1.8 +/- 1.0 U/mmol urinary creatinine; neopterin 270 +/- 126 nmol/mmol urinary creatinine; mean +/- SD) as compared with the ATN group (NAG 12 +/- 10 U/mmol, p < 0.001 vs. control group; neopterin 303 +/- 195 nmol/mmol, n.s.) and the rejection group (NAG 7 +/- 8 U/mmol, p < 0. 01; neopterin 508 +/- 419 nmol/mmol, p < 0.01). The ratio of urinary neopterin to NAG excretion (uNNR; dimension nmol neopterin/U NAG activity) increased during rejections as compared with ATN (139 +/- 74 vs. 50 +/- 38 nmol/U, p < 0.01). The area under the ROC curve for uNNR was 0.88 +/- 0.07 (p < 0.001). Applying a ROC-estimated optimal discriminator of uNNR (80 nmol/U), 16 patients with ATN and all six rejection episodes were classified correctly. CONCLUSION: The uNNR provides a noninvasive means to aid in the differential diagnosis of rejection and ATN in INF human renal transplants.

Acetylglucosaminidase↗

PubMed1999

Effects of interferon alpha-2b on barrier function and junctional complexes of renal proximal tubular LLC-PK1 cells.

BACKGROUND: Interferon alpha-2b (IFNalpha) treatment of diseases can be accompanied by impaired renal function and capillary leak syndrome. To explore potential mechanisms of IFNalpha-induced renal dysfunction, an in vitro cell culture model system was established to investigate the effects of IFNalpha on barrier function and junctional complexes. METHODS: LLC-PK1 cells were cultured on microporous membranes. Transepithelial resistance (TER) was measured, and the dose- and time-dependent effects of IFNalpha were assessed. The expression patterns of junctional proteins were examined by Western blot analysis and by confocal immunofluorescence microscopy. RESULTS: IFNalpha produced a dose- and time-dependent decrease in TER. The effect was reversible on removal of IFNalpha at doses up to 5 x 103 U/ml. Tyrphostin, an inhibitor of phosphotyrosine kinases, ameliorated the IFNalpha-induced decrease in TER. Increased expression of occludin and E-cadherin was detected by Western blot analysis after IFNalpha treatment. Immunofluorescence confocal microscopy revealed a broader staining of occludin and E-cadherin following IFNalpha treatment, with prominent staining at the basal cell pole in addition to localization at the junctional region. A marked increase in phosphotyrosine staining along the apico-lateral cell border was detected after IFNalpha treatment. CONCLUSIONS: These findings provide evidence that IFNalpha can directly affect barrier function in renal epithelial cells. The mechanisms involve enhanced tyrosine phosphorylation and overexpression and possibly displacement or missorting of the junctional proteins occludin and E-cadherin.

Animals↗

PubMed1999

Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures. Are renal cells in tissue culture hypoxic?

When renal proximal tubular cells are brought into tissue culture, they revert from oxidative metabolism and gluconeogenesis to high rates of glycolysis. Among the factors possibly responsible for this metabolic conversion, limited oxygen availability and/or substrate supply are discussed. In order to study the role of these factors on long-term cultures, the impact of growth conditions, culture media volume, and glucose content on carbohydrate metabolism of the continuous renal cell lines LLC-PK(1) (porcine kidney) and OK (opossum kidney) was investigated. The impact of culture media volumes and glucose content, respectively, was determined by overlaying confluent monolayer cultures of LLC-PK(1) and OK cells (i) with increasing volumes of culture medium and thus increasing amounts of glucose, and (ii) with increasing culture medium volumes at constant absolute amounts of glucose by adding glucose-free medium, in order to increase volume at a constant glucose supply. Alternatively, and in order to improve cell oxygenation, LLC-PK(1) cells were also cultured in roller bottles. Cell carbohydrate metabolism was assessed by measuring rates of glucose consumption and lactate production, respectively, and by determination of specific activities of the key glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH). Mitochondrial phosphate-dependent glutaminase (PDG) was assayed as marker enzyme of oxidative metabolism of glutamine. In LLC-PK(1) and OK cells, rates of glucose consumption were independent of the initial glucose concentrations and/or the culture media volumes used. Glucose was quantitatively converted to lactate, which accumulated in a 1:2 molar ratio. Lactate in culture media reached a maximum content after 24 h, and was reutilized by the cell lines thereafter. Interestingly, the rates of lactate reuptake strictly depended on culture medium volume, indicating a volume-induced stimulation of oxidative lactate metabolism. Marked changes were found for the specific activities of glycolytic enzymes. In LLC-PK(1) cells, increased glucose supply caused increases in HK, PFK, PK and LDH activities, which were superimposed to the stimulatory effects of increased media volumes. Enzyme activity showed a biphasic response, indicating that both glucose supply and culture media volumes covering the cell monolayer are factors determining glycolytic rates of LLC-PK(1) renal cells. Conversely, in OK cells glycolytic enzyme activities decreased with increasing culture media volumes at constant glucose levels. As expected, under conditions of enhanced oxygenation of LLC-PK(1) cells in roller bottle culture, glycolytic enzyme activities decreased, whereas PDG activity increased, which was paralleled by increased rates of ammonia generation. Thus, changes in nutrient supply and oxygenation of renal epithelial cell cultures by altered culture media volumes dramatically influence metabolic rates and levels of enzyme activities, respectively.

Animals↗

PubMed1998

Characterization of an ochratoxin-A-dedifferentiated and cloned renal epithelial cell line.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with predominant nephrotoxic action. OTA impairs postproximal renal electrolyte handling and increases the incidence of renal adenoma and carcinoma. Furthermore, it is supposed to be involved in the pathogenesis of different forms of human renal diseases. Previously we have shown that OTA activates extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the C7 clone but not in the C11 clone of renal epithelial MDCK cells. Here we show that nanomolar concentrations of OTA lead to stable and irreversible phenotypical and genotypical alterations, resulting in sustained dedifferentiation of MDCK-C7 cells but not of MDCK-C11 cells. Dedifferentiated MDCK-C7 cells (OTA-C7 cells) display a distinct morphology from the parent cell line (spindle-shape, pleiomorphic, narrow intercellular spaces, increased cell size) and show a reduced proliferation rate and numerical chromosomal aberrations. Functionally, OTA-C7 cells are characterized by a dramatic reduction of transepithelial electrolyte transport and the complete loss of responsiveness to the mineralocorticoid hormone aldosterone. Our data provide further evidence that OTA can lead to cell dedifferentiation and eventually to transformation of cloned quiescent cells. The changes in phenotype due to this dedifferentiation could explain some of the OTA-induced changes in renal function.

Animals↗

PubMed1998

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for organ toxicity.

In the past decade in vitro tests have been developed that represent a range of anatomic structure from perfused whole organs to subcellular fractions. To assess the use of in vitro tests for toxicity testing, we describe and evaluate the current status of organotypic cultures for the major target organs of toxic agents. This includes liver, kidney, neural tissue, the hematopoietic system, the immune system, reproductive organs, and the endocrine system. The second part of this report reviews the application of in vitro culture systems to organ specific toxicity and evaluates the application of these systems both in industry for safety assessment and in government for regulatory purposes. Members of the working group (WG) felt that access to high-quality human material is essential for better use of in vitro organ and tissue cultures in the risk assessment process. Therefore, research should focus on improving culture techniques that will allow better preservation of human material. The WG felt that it is also important to develop and make available relevant reference compounds for toxicity assessment in each organ system, to organize and make available via the Internet complete in vivo toxicity data, including human data, containing dose, end points, and toxicokinetics. The WG also recommended that research should be supported to identify and to validate biological end points for target organ toxicity to be used in alternative toxicity testing strategies.

Animal Testing Alternatives↗

PubMed1998

Nephrotoxicity testing in vitro--what we know and what we need to know.

The kidney is affected by many chemicals. Some of the chemicals may even contribute to end-stage renal disease and thus contribute considerably to health care costs. Because of the large functional reserve of the kidney, which masks signs of dysfunction, early diagnosis of renal disease is often difficult. Although numerous studies aimed at understanding the mechanisms underlying chemicals and drugs that target various renal cell types have delivered enough understanding for a reasonable risk assessment, there is still an urgent need to better understand the mechanisms leading to renal cell injury and organ dysfunction. The increasing use of in vitro techniques using isolated renal cells, nephron fragments, or cell cultures derived from specific renal cell types has improved our insight into the molecular mechanisms involved in nephrotoxicity. A short overview is given on the various in vitro systems currently used to clarify mechanistic aspects leading to sublethal or lethal injury of the functionally most important nephron epithelial cells derived from various species. Whereas freshly isolated cells and nephron fragments appear to represent a sufficient basis to study acute effects (hours) of nephrotoxins, e.g., on cell metabolism, primary cultures of these cells are more appropriate to study long-term effects. In contrast to isolated cells and fragments, however, primary cultures tend to first lose several of their in vivo metabolic properties during culture, and second to have only a limited life span (days to weeks). Moreover, establishing such primary cultures is a time-consuming and laborious procedure. For that reason many studies have been carried out on renal cell lines, which are easy to cultivate in large quantities and which have an unlimited life span. Unfortunately, none of the lines display a state of differentiation comparable to that of freshly isolated cells or their primary cultures. Most often they lack expression of key functions (e.g., gluconeogenesis or organic anion transport) of their in vivo correspondents. Therefore, the use of cell lines for assessment of nephrotoxic mechanisms will be limited to those functions the lines express. Upcoming molecular biology approaches such as the transduction of immortalizing genes into primary cultures and the utilization of cells from transgenic animals may in the near future result in the availability of highly differentiated renal cells with markedly extended life spans and near in vivo characteristics that may facilitate the use of renal cell culture for routine screening of nephrotoxins.

Cell Line↗

PubMed1997

Loss of cytokeratin expression and formation of actin stress fibers in dedifferentiated MDCK-C7 cell lines.

MDCK-C7 cells dedifferentiated either by transient alkaline stress (C7F cells) or by transfection with a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (C7caMEK1 cells) were analyzed by western blot and immunofluorescence microscopy to compare the expression of different cytokeratins, vimentin, and alpha-smooth muscle actin. Expression of all cytokeratins tested, the type II-neutral and basic cytokeratins CK5, CK7, CK8 as well as the type I-acidic keratins CK17 and CK19, was substantially reduced in dedifferentiated cell lines C7F and C7caMEK1 when compared with epithelial wild-type MDCK-C7 cells or mock-transfected MDCK-C7 cells. While vimentin expression was detected in all of the four MDCK-C7 cell lines examined, only the dedifferentiated cell lines C7F and C7caMEK1, which have been reported to express highly active ERK2, exhibited formation of alpha-smooth muscle actin-containing stress fibers. Taken together our results show that, associated with an increase in ERK2 activity, an epithelial to mesenchymal dedifferentiation occurred in both MDCK-C7F cells and caMEK1-transfected MDCK-C7 cells.

Actins↗

PubMed1997

Inhibition of angiotensin-converting enzyme modulates structural and functional adaptation to loop diuretic-induced diuresis.

The roles of elevated cell sodium concentrations and the angiotensin-aldosterone system (AAS) in the structural and functional adaptation of the distal tubule and collecting duct system to a chronic increase of sodium delivery were examined using electron microprobe and quantitative morphologic/stereologic analyses. Studies were performed on rats given the loop diuretic torasemide acutely (20 min) or chronically (12 days), either alone or in combination with the angiotensin-converting enzyme (ACE) inhibitor, enalapril. In the sodium-absorbing cells of the distal tubule and cortical collecting duct-that is, in distal convoluted tubule (DCT), connecting tubule (CNT) and principal cells-an acute increase in sodium delivery caused a significant rise in intracellular sodium concentration and rubidium uptake, the latter an index of in vivo Na,K(Rb)-ATPase activity. The elevated cell sodium concentrations returned to, or close to, control values during chronic torasemide treatment. Intracellular rubidium concentrations, measured after a 30-second rubidium exposure, were not different from controls in DCT and CNT cells but were still higher in principal cells. Since, however, the distribution space for rubidium was significantly increased in chronic torasemide animals, rubidium uptake, and hence Na,K-ATPase activity, must have increased in proportion to cell volume in DCT and CNT cells, but more than proportionately in principal cells. When ACE was inhibited during chronic torasemide, the epithelial volume of DCT and cortical collecting duct (CCD) was increased mainly by lengthening and not, as was the case in rats given torasemide alone, by thickening of the tubule wall. Adaptation of the proximal tubule exclusively by lengthening was not affected by inhibition of the ACE. These data indicate that changes in cell ion composition may participate in initiating cell processes leading to adaptation of distal nephron segments to chronically increased salt delivery. Inhibition of the ACE reverses the torasemide-induced increase in apparent Na pump density in principal cells and seems to shift the relationship between hypertrophy and hyperplasia noted in DCT and CCD after chronic torasemide in favor of hyperplasia.

Aldosterone↗

PubMed1997

Differential expression and activation of MAP kinases in dedifferentiated MDCK-focus cells.

Mitogen-activated protein kinases (MAPK) play a key role in the regulation of cellular processes such as cell growth, cell differentiation, and apoptosis. However, the specific function of single isoforms of the MAPK family in renal epithelial cell differentiation and/or proliferation has not been investigated so far. We now report stable reduction of extracellular signal-regulated kinase 1 (ERK1) protein expression and lack of serum-induced ERK1 activation in alkali-dedifferentiated Madin-Darby canine kidney-C7 focus (MDCK-C7F) cells compared with their parental epithelial MDCK-C7 cells. The changes in ERK1 protein expression and activation were accompanied by a small rise in c-jun NH2-terminal kinase 1 (JNK1) protein expression but slightly decreased basal and anisomycin-stimulated JNK1 activity. In contrast, ERK2 phosphorylation, as assessed by using an antibody which detects phosphorylated tyrosine 204 of both ERK1 and ERK2, as well as enzymatic ERK2 activity, was substantially increased in untreated and fetal calf serum-treated MDCK-C7F cells, although ERK2 protein expression remained unchanged. Differential expression and activation of ERK1, ERK2, and JNK1 were accompanied by an inhibition of serum-induced MDCK-C7F cell proliferation. Together, our results demonstrate an association between changes in the activation of certain MAPK and alkali-induced stable MDCK-C7 cell dedifferentiation. Moreover, these data provide evidence for distinct signaling functions of ERK1 and ERK2 in these cells.

Alkalies↗

PubMed1997

Interactions of ultrapure bovine hemoglobin with renal epithelial cells in vivo and in vitro.

Using an ultrapurified hemoglobin (Hb) solution, we investigated the physiological effects and cellular processing of Hb in rat kidneys and in cultured opossum kidney (OK) cells. Rats infused with < 5.0 g/kg Hb showed no change in baseline serum creatinine (SCr) values (0.58 +/- 0.05 mg/dl) over 48 h, whereas transient acute renal failure followed infusion of 7.5 g/kg Hb (SCr 3.4 +/- 1.02 mg/dl, P = 0.02). Histology of Hb-infused kidneys demonstrated tubular epithelial cell injury. Renal injury was not caused by volume or oncotic load, cardiovascular effect, or ATP depletion. After Hb infusion, heme oxygenase, the rate-limiting enzyme in Hb catabolism, was induced in an organ-specific fashion. Inhibiting heme oxygenase activity with cimetidine did not alter Hb renal injury. Using OK cells, we determined that renal epithelia process Hb by fluid-phase endocytosis. Proton permeability of fluorescein Hb endosomes was unaltered compared with fluorescein dextran controls, demonstrating that Hb does not alter endosomal membrane integrity. These data suggest that Hb renal injury in rats occurs following large doses of ultrapure Hb, does not alter early steps in Hb endosomal processing by renal epithelia, and involves a mechanism that is not heme oxygenase dependent.

Acute Kidney Injury↗

PubMed1997

Differential activities of H+ extrusion systems in MDCK cells due to extracellular osmolality and pH.

The aim of the present study was to obtain detailed information on MDCK cell proton secretion characteristics under various growth conditions. Confluent monolayers cultured on glass coverslips were adapted over 48 h to media with different osmolality and pH (200 mosmol/kgH2O, pH 7.4; 300 mosmol/kgH2O, pH 7.4; and 600 mosmol/kgH2O, pH 6.8) corresponding to the luminal fluid composition of the collecting duct segments found in the in renal cortex, the outer stripe of outer medulla and inner medulla. Proton fluxes were determined from the recovery of intracellular pH following an acid load induced by an NH4Cl pulse times the corresponding intrinsic buffering power (beta(i)). The intracellular buffering power was found to change only with culture medium osmolality but not with culture medium pH. In addition to an amiloride and Hoe-694-sensitive Na+/H+ exchange, Madin-Darby canine kidney (MDCK) cells possess a Sch-28080-sensitive, K+-dependent H+ extrusion mechanism that is increased upon adaptation of monolayers to hyperosmotic-acidic culture conditions. A significant contribution of the bafilomycin A1-sensitive vacuolar H+-ATPase could be found only in cells adapted to hyposmotic culture conditions. Exposure of MDCK cells to 10(-5) or 10(-7) M aldosterone for either 1 or 18 h did not alter the H+ extrusion characteristics significantly. The results obtained show that different extracellular osmolality and pH induce different MDCK phenotypes with respect to their H+-secreting systems.

Adaptation, Physiological↗

PubMed1997

Graft ischemia correlates with urinary excretion of the proximal marker enzyme fructose-1,6-bisphosphatase in human kidney transplantation.

This study was undertaken to test the hypothesis that ischemia prior to transplantation causes tubular damage without clinical evidence of graft dysfunction. The urinary excretion of fructose-1,6-bisphosphatase (EC 3.1.3.11, FBPase), a cytosolic enzyme located exclusively in the proximal tubules, and the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) were measured daily between postoperative days 1 and 4 in 25 renal cadaveric graft recipients who enjoyed an entirely uncomplicated first postoperative month. During the first 4 posttransplant days urinary FBPase excretion was 0.9 +/- 0.5 U/g (0.1 +/- 0.06 U/mmol) urinary creatinine [+/-SD; range 0.2-2.1 U/g (0.02-0.24 U/mmol)]. Cold ischemia time was 20.6 +/- 8.4 h (median 22 h, range: 3-32 h). Multiple regression revealed a significant correlation between cold ischemia time and posttransplant urinary FBPase excretion (multiple R = 0.65, p < 0.001). There were no confounding effects of recipient's age and gender, number of previous transplants, cyclosporin A levels, warm ischemia time, anastomosis time, donor age and gender. Urinary FBPase excretion was significantly lower in grafts stored for a shorter time than the median cold ischemia time of 22 hours (0.69 +/- 0.42 U/g, n = 13) as compared to those stored for a longer period of time (1.13 +/- 0.56 U/g; n = 12; p = 0.035). These results indicate that graft injuries occur even in the absence of graft dysfunction and that the duration of cold ischemia itself correlates with a degree of tubular cell damage as defined by urinary FBPase excretion.

Adult↗

PubMed1996

Reduced renal allograft survival is related to low urinary N-acetyl-beta-D-glucosaminidase excretion during the first posttransplant month.

The excretion of urinary N-acetyl-beta-D-glucosaminidase (NAG) was measured daily between day 7 and day 28 in 33 renal allograft recipients enjoying an entirely uncomplicated first postoperative month. Graft status was evaluated after 4 and 6 years and related to NAG excretion. After 4 years, 6 patients had experienced graft loss due to chronic rejection. Posttransplant urinary NAG excretion in the group of patients with failing grafts was significantly lower (9.4 +/- 6.3 vs. 17.2 +/- 8.5 U/g urinary creatinine, P = 0.036). Univariant analysis of recipient and donor characteristics revealed urinary NAG excretion to be the only parameter significantly differing between the groups. After 6 years, a total of 8 patients had lost their grafts. The posttransplant urinary NAG excretion in this group was 10.8 +/- 6.2 U/g; in the 25 patients with functioning grafts NAG excretion was 17.4 +/- 8.8 U/g (P = 0.064). A very low urinary NAG excretion ( < 7 U/g) was seen in 5 patients and associated with poor graft survival after 4 and 6 years (odds ratios 12.5 (1.9-82.1) and 6.9 (1.1-44.8), respectively. Kaplan-Meier analysis showed a reduced graft survival in this subgroup (P = 0.031). Receiver operating characteristics (ROC) analysis demonstrated an association between low NAG excretion and graft survival rates both at 4 and 6 years (area under the ROC curve 0.799 +/- 0.115, P, 0.05, and 0.747 +/- 0.104, P < 0.05, respectively). Cox proportional hazards analysis identified a low urinary NAG excretion as an independent prognostic risk factor. Urinary NAG excretion was expressed as unit per gram of urinary creatinine; as the amount of NAG excreted depends on the graft mass, and the amount of urinary creatinine depends on the recipient body mass, a low NAG excretion (in terms of U/g urinary creatinine) could be a surrogate marker of an unfavorable low graft to body weight ratio, which, in turn, might be associated with a reduced graft survival.

Acetylglucosaminidase↗

PubMed1996

Sustained ERK-2 activation in rat glomerular mesangial cells: differential regulation by protein phosphatases.

The duration of extracellular signal-regulated protein kinase (ERK) activation is critical for cell signaling decisions and probably determines whether a stimulus elicits proliferation or differentiation. We studied the intracellular signals regulating sustained ERK-2 activity in glomerular mesangial cells (GMC), utilizing combination of GMC mitogens of different potency. Incubation of GMC with both endothelin-1 (ET-1) and platelet-derived growth factor BB (PDGF-BB) led to a long-lasting, monophasic increase in ERK-2 activity. In contrast, when ET-1 was administered together with epidermal growth factor (EGF), a less pronounced and shorter activation occurred. Long-term stimulation of ERK-2 was accompanied by an increase in p45 MEK activity, which again was more pronounced when ET-1 was administered together with PDGF-BB compared with EGF. In the presence of actinomycin D (Act D), an inhibitor of RNA synthesis, ERK-2 activity induced by ET-1 and PDGF-BB but not by ET-1 and EGF remained elevated more than sixfold throughout the whole incubation period of 6 h. The effect of Act D on ET-1- and PDGF-BB-induced ERK-2 activation was mimicked by the protein phosphatase inhibitor sodium orthovanadate. In addition, vanadate also unmarked an ET-1- and EGF-induced ERK-2 activity after 6 h. The serine/threonine phosphatase inhibitor okadaic acid (OA) did neither alter agonist-induced ERK-2 activity after 6 h (0.5 nM OA) nor after 10 min or 1 h (250 nM). Together these results suggest that, in GMC, long-term activation of the mitogen-activated protein kinase ERK-2 is differentially regulated, depending on the combination of agonists administered. ET-1- and PDGF-BB-induced long-term activation of ERK-2 is regulated by a vanadate-sensitive protein phosphatase(s) and by a transcriptionally regulated protein(s). In contrast, ET-1- and EGF-induced sustained ERK-2 stimulation is regulated by a vanadate-sensitive protein phosphatase(s) but not by a transcriptionally regulated protein. Agonist-specific and time-dependent stimulation of ERK-2-regulating protein phosphatases may be critical for the length of ERK-2 activation in GMC and could thus be of pathophysiological significance in glomerular diseases associated with alterations in cell proliferation or cell differentiation.

Animals↗

PubMed1995

The effect of sodium fluoride at prophylactic and toxic doses on renal structure and function in the isolated perfused rat kidney.

To assess the renal effects of fluoride, isolated rat kidneys were perfused in single pass mode for 120 min. Five, 15 and 50, as well as 150, 500 and 1500 mumol NaF were administered 60, 80 and 100 min after starting the perfusion, respectively. Kidneys were perfused with constant pressure (100 mmHg). The perfusate consisted of a substrate supplemented Ringer solution containing hydroxy ethyl starch (HES) to produce isoncotic conditions. Concentrations below 500 mumol/l NaF did not induce major changes in the main parameters of renal function. Only upon admixture of the highest concentration of 1500 mumol/l NaF severe changes in renal function could be observed, resulting in complete anuria and a drastic reduction of renal perfusion to 5% of control, associated with a cessation of glomerular filtration. Due to the lack in tubular load, tubular reabsorptive processes inevitably declined to zero. The morphological analysis of kidneys exhibited to 500 mumol/l NaF revealed the occurrence of vesicular material within the urinary space. These vesicles could electron microscopically be identified as membrane enclosed material of podocytic origin. The interstitium was widened. Upon admixture of 1500 mumol/l NaF, kidneys responded with a decrease of the interstitial space. Moreover, epithelial cell swelling, hydropic degeneration of all proximal and distal tubular segments, bleb formation and intraluminal casts were observed frequently. Glomerular capillaries were filled with fine precipitates and their endothelium was severely damaged. The results of our studies in the isolated perfused rat kidney (IPRK) model clearly demonstrate a direct dose dependent acute nephrotoxic effect of NaF only for extremely high doses, which, however, may be reached in human cases of severe fluoride intoxication. On the contrary, for low fluoride doses, especially for those concentrations occurring in human plasma upon cariesprophylactic intake of fluorides, no signs of direct acute nephrotoxic action could be observed in the IPRK model.

Animals↗

PubMed1995

LLC-PK1 epithelia as a model for in vitro assessment of proximal tubular nephrotoxicity.

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were used as a model system for assessment of nephrotoxic side effects of three cephalosporin antibiotics: cephaloridine, ceftazidime, and cefotaxime. Toxic effects of these xenobiotics were monitored on confluent monolayers by light and electron microscopy and by the release of cellular marker enzyme activities into the culture medium. In addition, LLC-PK1 cells were grown on microporous supports, and cephalosporin-induced alteration of epithelial functional integrity was monitored by a novel electrophysiologic approach. For this purpose, an Ussing chamberlike experimental setup was used. The dose-dependent effects on transepithelial ionic permselectivity were monitored under conditions in which defined fractions of the apical culture medium NaCl contents were replaced iso-osmotically by mannitol. This method of determining the functional intactness of the epithelial barrier by measuring dilution potentials was found to be far more sensitive than monitoring cell injury by means of morphology or measurement of enzyme release. As expected from animal experimental data, a dose-dependent disruption of monolayer integrity was detected with all three methodologies applied. Cephaloridine was found the most toxic compound followed by ceftazidime, where a 3-fold, and cefotaxime, where a 10-fold dose of that of cephaloridine was needed to produce cell injury. Measurement of transepithelial dilution potentials was more sensitive as compared to the release of the apical plasma membrane marker enzyme activities alkaline phosphatase and gamma-glutamyltranspeptidase, the cytosolic lactate dehydrogenase, or the mitochondrial glutamate dehydrogenase. The data were compared to the effects of the aminoglycoside antibiotic gentamicin, which at least with respect to its effects on LLC-PK1 morphology and enzyme release, but not transepithelial electrical properties, was already investigated.

Acetylglucosaminidase↗