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Biomedical subjects

W Pfaller

Publications and source records attributed to W Pfaller.

85 records · Page 5Linked to original sources
PubMedDate not supplied

Interferon alpha2b increases paracellular permeability of renal proximal tubular LLC-PK1 cells via a mitogen activated protein kinase signaling pathway.

The therapeutic administration of Interferon alpha2b (IFNalpha) is often accompanied by impaired renal function, i.e. reduced glomerular filtration rate and sometimes a so-called "capillary leak syndrome". To clarify the mechanism behind the renal dysfunction, confluent monolayers of LLC-PK1 cells were used as a model system to analyze the effects of IFNalpha on renal tubular epithelium. Examination of epithelial barrier function via measurement of transepithelial resistance (TER) revealed a dose dependent increase in paracellular permeability by IFNalpha treatment. The effect was reversible upon removal of IFNalpha at doses up to 5 x 10(3) U/mL. Apical or basolateral application of IFNalpha yielded the same decrease in TER. Tyrphostin A25, an inhibitor of phosphotyrosine kinases, ameliorated the IFNalpha induced decrease of TER. In order to unravel intracellular signal transduction pathways that may mediate IFNalpha induced changes of epithelial barrier function, we inhibited IFNalpha signaling through a mitogen activated protein kinase pathway by the Mek1 inhibitor PD98059. The inhibitor could be shown to prevent IFNalpha induced decrease of transepithelial resistance. Inhibitors of the p38 mitogen activated protein kinase pathway did not affect IFNalpha mediated changes of epithelial barrier function, indicating a highly specific role for the Mek/Erk pathway. Activation of mitogen activated protein kinase pathways by epidermal growth factor or anisomycin could not, per se, imitate the effect of IFNalpha on the paracellular permeability of LLC-PK1 monolayers. These findings provide evidence that IFNalpha can affect barrier function in renal epithelial cells via activation of the Mek/Erk pathway.

Animals↗
PubMedDate not supplied

Quantitative morphology of renal cortical structures during compensatory hypertrophy.

The compensatory hypertrophy in different renal cortical structures was studied in rats 10 and 21 days after unilateral nephrectomy (UNX). Quantitative morphological/stereological analysis revealed significant increases in total renal cortical volume--33% on day 10 and 48% on day 21--after UNX. These changes were paralleled by significant increments in the volumes of proximal convoluted tubule (PCT, 55%), distal convoluted tubule (DCT, 114%), and cortical collecting duct (CCD, 106%) segments on day 10. The corresponding changes on day 21 were 76, 122, and 212%, respectively. These alterations were accompanied by increases in segment length; 3% PCT, 23% DCT, and 50% CCD on day 10 and 9% PCT, 30% DCT, and 142% CCD on day 21 after UNX. The total luminal and basolateral cell membrane surface areas also exhibited a time-dependent increase after UNX. The increments in both luminal and basolateral membrane domains in PCT and DCT after 10 days were not significant, but reached significance after 21 days (PCT: luminal membrane 21%, basolateral membrane 63%; DCT: luminal membrane 98%, basolateral membrane 63%). In contrast, CCD membrane areas had increased substantially already 10 days after UNX (luminal membrane 92%, basolateral membrane 71%). It declined subsequently by day 21 (luminal membrane 57%, basolateral membrane 32%). The cell rubidium concentration after a 30-second rubidium infusion, an index of Na-K-ATPase activity, as well as sodium concentrations were unaltered in cells of all nephron segments investigated. Altogether the stereological analysis shows that the compensatory increase in organ volume can be attributed primarily to an increase in nephron epithelial volume. The PCT responds with 'radial' hypertrophy (thickening of the tubular epithelial wall), while the DCT undergoes 'length' hypertrophy (increase of tubular length without thickening of the tubular wall and without an increase in number of cells). This type of hypertrophy is especially prominent on day 21 after UNX for the CCD which doubles in length. Only on day 10 does the CCD seem to respond with hyperplasia. Adaptive changes in response to UNX develop gradually. Only a few of the morphological parameters studied had completed their change by 10 days, the majority required longer.

Adaptation, Physiological↗
PubMedDate not supplied

Lipid peroxidation--an initial event in experimental acute renal failure.

A method was developed to monitor the occurrence of lipid peroxidation (LPO) during ischemia and Na-maleate-induced acute renal failure (ARF) on male rats in vivo by measuring malondialdehyde (MDA) levels in arterial and renal venous blood and in urine. No signs of LPO could be detected under control conditions. In ischemic ARF produced by 45 min of renal artery clamping a steep increase of MDA was found in the renal venous effluent immediately after starting reperfusion. This effect was nearly abolished after 5 min of blood reflow while glomerular filtration remained at 5% of control value during a 90-min postischemic observation period. Intoxication with Na-maleate leads to enhanced LPO in combination with an impaired renal function 2 h after administration. These findings would well explain cellular damage and some aspects of renal dysfunction associated with the initiation phase of ARF.

Acute Kidney Injury↗
PubMedDate not supplied

Quantitative morphologic changes in nephron structures and urinary enzyme activity pattern in sodium-maleate-induced renal injury.

The morphologic changes in sodium-maleate-induced acute renal injury in the rat were quantified by a stereologic analysis. The major changes were confined to an increase in endocytic vacuoles and a decrease in mitochondrial inner membrane surface area. These results were found to be linked to significantly increased urinary activities of the cytosolic of the cytosolic enzymes fructose-1,6-bisphosphatase (FBP) and lactate dehydrogenase, the lysosomal enzyme N-acetyl-beta-glucosaminidase (NAG) and the NAD-dependent mitochondrial isocitrate dehydrogenase (ICDH). The highest increase was found for NAG, followed by FBP and ICDH.

Animals↗
PubMedDate not supplied

Carbonic anhydrase activity in Madin Darby canine kidney cells. Evidence for intercalated cell properties.

Madin Darby canine kidney (MDCK) renal epithelial cell cultures have been investigated with respect to their potency to express carbonic anhydrase activity using histochemical methods. Acetazolamide inhibitable carbonic anhydrase activity could be detected in the cytoplasmic compartment as well as in the apical membrane of cells when grown on solid culture supports. Cells forming domes in MDCK monolayers exhibit the highest histochemically detectable enzyme activity. The attempt to subculture clonal cell lines from MDCK monolayer cultures resulted in the establishment of 5 clones, slightly different with respect to size and shape of cells and their potency to form domes. Scanning electron microscopy ensured the identification of one clone (1A4), which distinctly differed from the others with respect to the apical membrane architecture. Co-localization of peanut agglutinin and carbonic anhydrase activity at the plasma membrane always revealed a combined occurrence of enzyme reactivity and lectin binding in the apical membrane domain. Both, lectin binding and carbonic anhydrase activity were distinctly more intense in plasma membrane regions equipped with microvilli. From the results it is concluded that MDCK cells in tissue culture retained properties of intercalated cells of the nephron collecting duct segment.

Animals↗
PubMedDate not supplied

Volume-regulatory potassium release from isolated perfused rat kidney.

The present study has been performed to test for cell volume regulatory potassium release from the isolated perfused rat kidney exposed to hypotonic perfusate and for its sensitivity to potassium channel blocker barium and calcium channel blocker verapamil. Replacement of 25 mmol/l NaCl with 50 mmol/l mannitol has little effect on effluent potassium activity, whereas subsequent omission of mannitol from the perfusate leads to a transient increase of effluent potassium activity, reflecting volume regulatory potassium release. Barium (1 mmol/l) leads to a marked transient decrease of effluent potassium activity, pointing to net cellular uptake of potassium. Verapamil (1 mumol/l) leads to a slight decrease of effluent potassium activity. Both barium and verapamil virtually abolish the rapid, transient increase of effluent potassium activity upon exposure to hypotonic perfusates. Thus, the substances either block or markedly retard volume regulatory potassium release. The apparent renal vascular resistance is transiently increased by exposure to hypotonic perfusates and by barium, but is reduced by verapamil. Cell volume regulation of isolated perfused mouse straight proximal tubules is retarded but not abolished by verapamil (0.1 mmol/l). In conclusion, cellular potassium release from rat kidney can be determined by continuous measurement of effluent potassium activity. The volume regulatory potassium release and cell volume regulation are impaired by both barium and verapamil. The persisting cell volume regulation could be due either to slow potassium release and/or some mechanism independent of potassium.

Animals↗
PubMedDate not supplied

Modification of membrane protein expression and protein secretion in LLC-PK1 cultures grown on different carbohydrates.

Membrane glycoproteins and glycolipids play an important role in epithelial organization, transport and function. To study the effects of exogenous carbohydrates on the expression of glycoproteins, cells of the renal epithelial line LLC-PK1 were cultured on different nutritive carbohydrate sources and on uridine, which is, despite striking differences, known to substitute all essential nutritive functions of glucose. LLC-PK1 cultures were long-term adapted to growth in culture medium containing 0.5, 5, 10 and 25 mM glucose, and 5 mM fructose, galactose and uridine, respectively, as the sole carbohydrate source. These growth conditions elicited adaptive changes in the expression of enzyme activities of alkaline phosphatase and gamma-glutamyltranspeptidase, integral membrane glycoproteins exclusively localized in the apical membrane of LLC-PK1 cells. SDS-PAGE of membrane preparations of adapted LLC-PK1 cells revealed a strong induction of several protein bands between 13.5 and 47 kD in fructose-grown cells, while in plasma membranes of cells grown in galactose several protein bands between 62 and 70 kD decreased. Changes in the secretion pattern of proteins into the culture medium were most prominent in uridine-grown cells compared to controls grown on 25 mM glucose.

Alkaline Phosphatase↗
PubMedDate not supplied

Morphological and biochemical changes of LLC-PK1 cells during adaptation to glucose-free culture conditions.

The established renal epithelial cell line LLC-PK1 retained in tissue culture several differentiated properties of renal proximal tubular cells. By adapting LLC-PK1 cells to glucose-free culture conditions, we recently succeeded in isolating a gluconeogenic strain of LLC-PK1 cells capable of growing in the absence of hexoses. In contrast to the parental wild type, the isolated strain expressed fructose-1,6-bisphosphatase activity and was, therefore, designated LLC-PK1-FBPase+. Besides the differences in glucose metabolism, the isolated gluconeogenic substrain differs form the parental wild type with respect to morphological appearance and the expression of apical membrane marker enzymes. LLC-PK1-FBPase+ cells display a drastic accumulation of autophagic vacuoles, disappearance of apical membrane alkaline phosphatase activity, and increased gamma-glutamyltranspeptidase activity. In order to find out whether or not a low alkaline phosphatase activity in combination with the enhanced formation of autophagic vacuoles is related to a change in apical membrane surface, we utilized a combined light and electron microscopic morphometric procedure to determine the absolute amount of organelle volumes and membrane surface areas. This stereologic approach shows that LLC-PK1-FBPase+ cells display a tenfold increase in the volume of autophagic vacuoles and the lysosomal compartment. Analysis of lysosomal enzyme activities, however, revealed no changes as compared to wild-type cells. The apical membrane surface of gluconeogenic cells was found to be increased by 80%. Karyotype analysis revealed that LLC-PK1 wild-type cells were diploid, whereas FBPase+ cells exhibited polyploidy with a high percentage of tetraploid nuclei. Culturing LLC-PK1-FBPase+ cells in the presence of 5 mM glucose does not abolish the morphological and biochemical changes described, indicating the stability of the FBPase+ strain.

Adaptation, Physiological↗
PubMedDate not supplied

Endothelin-3 modulates glomerular filtration rate in the isolated perfused rat kidney.

The present study has been performed to evaluate hemodynamic and tubular effects of various endothelin-3 (ET-3) concentrations on the isolated perfused rat kidney. Using this experimental system we observed a profound reduction of renal perfusate flow at all ET-3 concentrations tested (50, 250 and 500 pmol/l), suggesting that the vasoconstrictive potency of ET-3 in the kidney is comparable to that described for endothelin-1 (ET-1). The effects on glomerular filtration rate (GFR) differed depending on the ET-3 dose applied. While 250 pmol/l ET-3 increased GFR by 30%, 500 pmol/l ET-3 markedly reduced GFR. 50 pmol/l ET-3 did not alter GFR although renal vascular resistance significantly increased. Infusion of 1 mumol/l N-nitro-L-arginine, a specific and potent inhibitor of nitric oxide synthesis in endothelial cells, abolished the GFR elevation induced by 250 pmol/l ET-3. In parallel with the changes of GFR we observed an increase in sodium reabsorption at 250 pmol/l and a decrease of this parameter at 500 pmol/l ET-3. Moreover, an ET-3 concentration (500 pmol/l), which induced a dramatic fall in tubular sodium load, led to an increase of fractional sodium excretion and to a decrease of renal oxygen consumption. We conclude that ET-3 is a potent vasoconstrictor in the isolated perfused rat kidney. Furthermore, it modulates GFR in a differentiated mode, depending on the concentration used. The GFR increase at 250 pmol/l ET-3 seems to be mediated by endothelium-derived nitric oxide. In addition to its glomerular action, ET-3 might also affect tubular sodium transport.

Animals↗
PubMedDate not supplied

Ultrapure polymerized bovine hemoglobin improves structural and functional integrity of the isolated perfused rat kidney.

Since it became evident that organ dysfunctions after acute hemolysis are not induced by hemoglobin per se, but by stroma-contaminated hemoglobin, solutions of ultrapure stroma-free hemoglobins were regarded to be possible substitutes for blood in transfusion medicine. We tested one of the recently developed modified bovine hemoglobins (Ultrapure polymerized bovine hemoglobin 1; UPPBHb1) in the isolated perfused rat kidney (IPRK) model, using a recirculating system. Control kidneys were perfused with a substrate-enriched Ringer solution containing hydroxyethyl starch (HES) to produce isoncotic conditions. In the experimental group HES was substituted in part by UPPBHb1 (34 g/l). For determination of functional parameters, the kidneys were perfused for 180 min. A separate set of kidneys of both groups was perfusion fixed after 80 min of perfusion which is the period of optimal function. Light and electron microscopic analysis revealed major alterations only for the outer medulla of HES kidneys. Only these suffered from a considerable extent of proximal tubular S3 damage, exhibiting condensed tubular epithelia. In the inner stripe of the outer medulla, which is the zone of greatest sensitivity to damage in the isolated perfused kidney, severe hydropic degeneration, cell detachment, and necrotic destruction of the medullary thick ascending limb were seen in the HES-perfused group, too. In the UPPBHb1 group, the medullary thick ascending limb was well preserved, and S3 showed only a minor degree of damage. UPPBHB1 kidneys were further characterized by the occurrence of intracapillary and interstitial precipitates of UPPBHb1 in inner stripe of the outer medulla and inner medulla. The glomerular filtration rate was significantly higher in UPPBHb1-perfused kidneys (870 +/- 80 vs. 630 +/- 55 microliters/min/g kidney weight for HES). Absolute reabsorption of sodium paralleled the behavior of the glomerular filtration rate. The values for renal perfusate flow and urinary flow rate did not differ significantly between both groups. Renal autoregulation was better preserved in UPPBHb1-perfused kidneys (74 +/- 6% of full autoregulatory response) than in HES-perfused controls (42 +/- 4%). Our results suggest that perfusion of isolated rat kidneys with UPPBHb1 improves kidney function and morphology, providing better oxygenation than in control kidneys. UPPBHb1 does not exert additional nephrotoxic effects on the IPRK that will exceed the noxious potential of the method itself. Thus, it must be concluded that UPPBHb1 may be an oxyphoretic blood substitute with nephroprotective characteristics when compared with nonoxyphoretic substitutes. At least, UPPBHb1 seems to be a promising candidate as oxyphoretic additive to perfusates for the IPRK model.

Animals↗
PubMedDate not supplied

In vitro experiments on catheter-related infections due to gram-negative rods.

Although many complications may arise with the use of central venous catheters, catheter-related bacteremia is considered to be the most serious complication. Microflora on the patient's skin (coagulase-negative staphylococci, Staphylococcus aureus) is thought to represent the major source of microorganisms causing catheter-related infections. Gram-negative rods are increasingly observed as causative agents. We therefore performed an in vitro study to elucidate the pathogenesis of polymer-associated infections caused by enterobacteriaceae and nonfermenters and to study the resistance of polymer surface-grown gram-negative rods against antibiotics. Using a modification of the semiquantitative method for culturing vascular cannulas on solid media described by Maki et al., we studied the adherence of various gram-negative rods to 1-cm segments of silastic catheters, a material used for Port-A-Cath-systems. Organisms were obtained from the American Type Culture Collection (Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, Enterobacter cloacae). All organisms but Klebsiella showed extensive irreversible adherence. Production of an extracellular slime substance was observed only with antimicrobially treated Pseudomonas. After 'infection', the silastic catheter segments were treated with various antimicrobial agents. The following antibiotics were used either individually or in combination: cefotaxime, ciprofloxacin, imipenem, fosfomycin and gentamicin. Despite the fact that the antibiotic concentrations used were many times greater than the respective MICs of the various antibiotics, none of the antibiotics tested succeeded in eliminating the organisms. E. coli was found to be the most susceptible organism. These in vitro data substantiate our clinical impression that it is hardly possible to successfully eliminate the colonization of central venous catheters with gram-negative rods by using the antimicrobial agents employed here.

Anti-Bacterial Agents↗
PubMedDate not supplied

Ammoniagenesis in renal cell culture. Lack of extracellular ammoniagenesis at the apical surface of LLC-PK1 epithelia.

In renal ammoniagenesis, two major pathways of glutamine metabolism have been described: (i) intracellular metabolism by phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase and (ii) extracellular metabolism by phosphate-independent glutaminase. The latter has been identified as the hydrolytic activity of the apically membrane-bound gamma-glutamyl transpeptidase (gamma-GT). The growth properties of cultured renal epithelia enable the study of in vitro extracellular metabolic properties occurring at the apical epithelial surface in the culture dish. Therefore, confluent epithelia of the LLC-PK1 renal epithelial cell line were used to elucidate the role of extracellular (apical) hydrolysis of glutamine by gamma-GT in LLC-PK1 ammonia production. To distinguish between intra- and extracellular metabolism of glutamine, confluent LLC-PK1 epithelia were incubated with either D-glutamine as substrate, which cannot be metabolized intracellularly by PDG, or with L-glutamine and hippurate to stimulate, and AT-125 (acivicin) to inhibit gamma-GT activity, respectively. In addition, cellular uptake of the glutamate, extracellularly formed by gamma-GT, was inhibited by D-aspartate. D-Glutamine (2 mM) did not increase ammonia formation above endogenous production levels, indicating the negligible role of extracellular hydrolysis of glutamine by gamma-GT. After modulating gamma-GT activity by hippurate or AT-125, almost identical ammonia production rates were found within the various experimental protocols, further confirming that extracellular metabolism of glutamine does not significantly contribute to LLC-PK1 ammoniagenesis.

Amino Acids↗