cDNA array analyses of K-ras-induced gene transcription.
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Animals↗
Biomedical subjects
M Knepper
Publications and source records attributed to M Knepper.
Interdiffusion in short-fibre reinforced hydroxyapatite ceramics.
Sintering in air and hot isostatic pressing are production methods regarded as being capable of producing fibre-reinforced hydroxyapatite ceramics for biomedical applications. These composites may have the advantage of improved mechanical properties and be suitable for applications in areas where there are significant levels of load on the material. The use of pure hydroxyapatite is restricted to those free of dynamical load. Obtaining improved mechanical strength is a question of the bond between the matrix phase and the fibre-reinforcement phase. However, a chemical bond between both phases, indicated by large diffusion zones, might lead to the dehydration of the hydroxyapatite leading to undesired tricalcium phosphate in the matrix resulting in a weakening of the mechanical and biological stability of the composites. Composites with three fibre types, alumina, 316L-stainless steel and titanium were prepared and sintered in air or hot isostatically pressed. A reaction zone was noted around the titanium and stainless steel fibres, but not around the alumina fibres. The reaction zone was larger for stainless steel than titanium. Hot isostatic pressing also reduced the reaction zone markedly compared to sintering in air.
Journal Article↗
Concentrating defect in experimental nephrotic syndrone: altered expression of aquaporins and thick ascending limb Na+ transporters.
BACKGROUND: Several pathophysiological states associated with deranged water balance are associated with altered expression and/or intracellular distribution of aquaporin water channels. The possible role of dysregulation of thick ascending limb NaCl transporters, which are responsible for countercurrent multiplication in the kidney, has not been evaluated. METHODS: Semiquantitative immunoblotting and immunocytochemistry were carried out in the kidneys of rat with adriamycin-induced nephrotic syndrome and in vehicle-injected control rats. RESULTS: Preliminary studies confirmed the presence of a severe concentrating defect. Semiquantitative immunoblotting of outer medullary homogenates demonstrated a marked decrease in the abundance of three thick ascending limb Na+ transporters in nephrotic rats, namely the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1), the type 3 Na/H exchanger (NHE-3), and the alpha 1-subunit of the Na-K-ATPase. These results are predictive of a decrease in the NaCl transport capacity of the medullary thick ascending limb and therefore a decrease in countercurrent multiplication. Immunocytochemistry of outer medullary thin sections demonstrated broad (but highly variable) suppression of BSC-1 expression in the outer medullas of adriamycin-nephrotic rats. There was also a large decrease in outer medullary expression of two collecting duct water channels (aquaporin-2 and -3) and the major water channel of the thin descending limb of Henle's loop (aquaporin-1). CONCLUSION: The concentrating defect in adriamycin-induced nephrotic syndrome in rats is a consequence of multiple defects in water and solute transporter expression, which would alter both the generation of medullary interstitial hypertonicity and osmotic equilibration in the collecting duct. Whether a similar widespread defect in transporter expression is present in idiopathic nephrotic syndrome is, at this point, untested.
Animals↗
Acute effects of vasopressin V2-receptor antagonist on kidney AQP2 expression and subcellular distribution.
The acute effect of treatment with the vasopressin V2-receptor antagonist OPC-31260 (OPC) on aquaporin-2 (AQP2) distribution and expression in rat kidney was examined. Immunofluorescence and semi-quantitative immunoelectron microscopy revealed that 15 and 30 min of OPC treatment resulted in significant reduction in apical plasma membrane labeling of AQP2, with a concomitant increase in labeling of vesicles and multivesicular bodies. In parallel, OPC treatment induced a large increase in urine output [0.6 +/- 0.2 vs. 8.3 +/- 1.0 ml/h (n = 4)]. Northern blotting using a 32P-labeled AQP2 cDNA probe and a digoxigenin-labeled AQP2 RNA probe revealed a band of approximately 1.6 kb corresponding to the predicted size of AQP2 mRNA. In control experiments, thirsting increased, whereas water loading decreased AQP2 mRNA levels. Treatment of rats with OPC caused a significant reduction in AQP2 mRNA within 30 min (52 +/- 21%, n = 8, P < 0.025) and 60 min (56 +/- 7%, n = 4, P < 0.001) of treatment compared with intravenous saline-injected controls. Thus a very rapid reduction in AQP2 mRNA was observed in response to vasopressin-receptor antagonist treatment. The reduction in AQP2 mRNA persisted after 24 h (40 +/- 17%, n = 5, P < 0.05) of OPC treatment. There was a parallel increase in diuresis and reduction in urine osmolality. In conclusion, V2-receptor blockade produced a rapid internalization of AQP2 parallel with a rapid increase in urine output. Furthermore, OPC treatment caused a rapid and significant reduction in AQP2 mRNA expression, demonstrating that for rapid regulation of AQP2 expression, modulation of AQP2 mRNA levels is regulated via vasopressin-receptor signaling pathways.
Animals↗
Stability of hydroxyapatite while processing short-fibre reinforced hydroxyapatite ceramics.
Reinforcement by short fibres has been adapted from modern ceramic processing technologies to achieve an improvement of structural properties of hydroxyapatite. However, the influence of the reinforcement fibres on the thermochemical behaviour of the hydroxyapatite has yet to be clarified comprehensively. Titanium, alumina and 316L-stainless steel, all materials with a proven record as implant materials, were chosen as reinforcement materials. Short fibres of these materials were incorporated in a matrix of hydroxyapatite to toughen the hydroxyapatite. Composites were processed by sintering in air, hot isostatic pressing and a method combining sintering in inert gas atmosphere and hot isostatic pressing.
Aluminum Oxide↗
NEUROLAB, a PC-program for the processing of neurobiological data.
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Algorithms↗
The aquaporin family of water channels in kidney: an update on physiology and pathophysiology of aquaporin-2.
The long-standing problem of membrane water transport has been advanced by the recognition of a new family of water transport proteins, the "aquaporins" [1-3]. Not surprisingly, water transport is a major process in kidney physiology, and the biology of aquaporins is most thoroughly understood in that organ. We reviewed in detail the status of aquaporins in the kidney only one year ago [4], but the subsequent progress has dictated the need for an update. This seems especially appropriate in honor of the 100th birthday of Homer Smith, the pioneer whose foresight initiated this field.
Animals↗
Urinary excretion of aquaporin-2 in humans: a potential marker of collecting duct responsiveness to vasopressin.
The vasopressin-sensitive water channel (aquaporin 2; AQP-2) mediates water transport across the apical plasma membrane of the renal collecting ducts and is excreted in human urine. This study presents the hypothesis that measurements of the AQP-2 excretion rate might be used as a marker of collecting-duct responsiveness to vasopressin, and therefore could be useful in the clinical evaluation of various water-balance disorders. This study presents information about the development of an antibody to human AQP-2, and measures the urinary excretion of AQP-2 by quantitative Western analysis. A standard curve of band densities was generated by using known quantities of the modified immunizing peptide to derive the amount of AQP-2 contained in aliquots of urine. AQP-2 urinary excretion changed with short-term alterations in hydration status produced either by water loading (76% decrease, P < 0.01) or by 3% sodium chloride (760% increase, P < 0.01). Steady-state 24-h urinary excretion of AQP-2 was 43 +/- 10 nmol/24 h (or 28.5 +/- 6.9 pmol/mg creatinine), and 20 +/- 6 nmol/24 h (or 18.3 +/- 7.9 pmol/mg creatinine) in men and women, respectively. Therefore, urinary AQP-2 excretion can be quantified by using Western analysis, and may serve as a marker of collecting-duct responsiveness to vasopressin in different physiologic settings.
Adult↗
Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.
Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2 water channels.
Animals↗
Separation of synaptic and spike activity in intracellular recordings for selective analysis.
A software spike filter has been developed which allows the separation of synaptic activity and action potentials in intracellular recordings. The algorithm uses the different velocities of the membrane potential during synaptic and spike activity and a time window to identify action potentials. When spikes are recognized, they are removed and the membrane potential is substituted by interpolated values. The spike filter makes possible a separate quantitative evaluation of postsynaptic potentials and spike activity. Thus a comprehensive characterization of neuron activity can be obtained. The spike filter is part of a modular software package designed for the evaluation of neurobiological data.
Action Potentials↗
Organization of nephron function.
Recent studies of mammalian nephron segments have revealed an unexpected diversity of renal transport functions. Most substances are transported by several segments, and the transport mechanisms differ from segment to segment. In this paper we review some of these findings in order to fit them into an integrated picture of kidney function. The main question we ask is what is the advantage of spatial separation of the various transporters along the nephron. We propose that spatial separation of transport functions allows independent and efficient control of the excretion of substances whose renal handling is interdependent. The organization of sodium and water handling along the nephron is considered in the greatest detail. Sodium and water play central roles in controlling the renal excretion of many other substances. Yet the excretion rates of sodium and water themselves are independently controlled. The intricate anatomical structure of the kidney not only provides spatial separation of transport processes along the nephron but also couples the function of different segments by juxtaposing them in specialized regions within the medulla and cortex. These anatomical arrangements provide a framework for integrating the complex array of renal functions.
Animals↗
NEUROLAB, a comprehensive program for the analysis of neurophysiological and behavioural data.
A comprehensive and versatile computer software for IBM-compatible microcomputers has been developed. It is designed for quantitative off-line analysis of A/D-sampled intracellular or extracellular recordings and behavioural or stimulus data. The program works with single files or file sets. It supports data to be viewed on the monitor and allows sectioning of interesting data for common analysis. It offers 19 filters/operators for data processing and comprehensive possibilities to set and calculate trigger points. Data of trigger points can be exported as ASCII files. Standard neurophysiological histograms like interval-, PST-, phase histograms or auto- and cross-correlograms can be obtained. Time-dependent and phase-dependent averaging is possible for all original and filtered data. All graphical output on the display can directly be copied to a plotter/laser printer or HP-GL file by keyboard commands.
Animals↗