Biomedical subjects
Publications and source records attributed to K Thurau.
A method is described whereby single renal tubules, incubated in varying incubation media can be captured and prepared for electron microprobe analysis for intracellular sodium, potassium, chloride, phosphorus, magnesium amongst others.
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Electron microprobe analysis was used to determine cellular concentrations of potassium and sodium in renal cortical cells of hypertensive rats of the Milan strain (MHS) and spontaneously hypertensive rats of the stroke prone strain (SHRSP) and their respective controls. Potassium concentrations in proximal and distal tubular cells were similar in both strains of hypertensive rats compared with their normotensive controls. In MHS rats proximal tubular cell sodium concentration was lower than in controls by 3.1 mmol/kg ww, whereas in both proximal and distal tubular cells of SHRSP sodium concentrations were higher than in controls by 5.3 and 4.3 mmol/kg ww, respectively. These results indicate that changes in the transport characteristics of the renal tubular epithelium are a feature of both models of hypertension.
Energy dispersive X-ray microanalysis was employed to determine the intracellular electrolyte concentrations in various epithelial tissues. Analyses of the changes in the intracellular electrolyte concentrations during different functional states provided new insights into the mechanisms involved in transepithelial ion transport. Results obtained on the Na transporting frog skin epithelium confirmed the two-barrier concept of transepithelial Na transport involving a passive Na uptake and an active Na extrusion. The Na transport compartment comprises all living epithelial cell layers, only the outer cornified cells, the mitochondria-rich cells and gland cells being exceptions. Na influx across the outer barrier can be inhibited by amiloride or stimulated by novobiocin or vasopressin. In the Cl-secreting frog cornea the electrolyte concentrations in the various epithelial cell layers and cell types were almost identical, indicating that the epithelium forms a functional syncytium with regard to ion transport. Under virtually all experimental conditions the Cl concentration was found to be higher than the expected equilibrium values, favouring a passive exit from the cells towards the tear side. The results are consistent with the existence of a furosemide-sensitive coupled NaCl uptake process at the inner-facing cell membranes. In the rat kidney after K depletion a fall in cellular K by about 20% was observed both in proximal and distal tubular cells. Simultaneously a rise in the intracellular Na concentration was detected which, however, was insufficient to balance the loss in K. No significant differences could be detected between the various epithelial cell types which are thought to be involved in either K reabsorption or secretion. It is concluded that the fall in cellular K is not sufficient to explain the marked reduction in urinary K excretion during K depletion.
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The effect of dietary potassium depletion on nuclear and cytoplasmic element concentrations in cortical renal tubular cells and hepatocytes was investigated using electron microprobe analysis. Significant differences in sodium and potassium concentrations between nucleus and cytoplasm were not detected either under control or under potassium-depleted conditions. Potassium depletion for at least 14 days resulted in a decrease in plasma potassium concentration from 4.4 +/- 0.1 to 2.0 +/- 0.1 mmoles X liter-1. There was a fall in cellular potassium from 151.6 +/- 3.5 to 120.2 +/- 2.1 in distal tubular cells, from 150.1 +/- 2.6 to 117.7 +/- 1.2 in proximal tubular cells, and from 140.6 +/- 1.3 to 128.0 +/- 1.3 mmoles X kg-1 of wet wt in hepatocytes. The cellular chlorine concentrations fell from 19.9 +/- 0.7 to 15.8 +/- 0.3 and from 21.3 +/- 0.4 to 17.2 +/- 0.4 in proximal tubular and liver cells, respectively, but remained unchanged at 11.4 +/- 0.7 and 11.0 +/- 0.4 mmoles X kg-1 of wet wt in distal tubular cells. The intracellular sodium concentrations rose from 10.4 +/- 0.7 to 15.8 +/- 0.8, 19.1 +/- 0.8 to 24.1 +/- 0.7 and 14.1 +/- 0.5 to 16.2 +/- 0.6 mmoles X kg-1 of wet wt in distal tubular, proximal tubular and liver cells, respectively. This rise in cellular sodium was insufficient in any cell type to compensate for the loss of potassium. No significant differences were found in the cellular electrolyte concentrations of the various distal tubular cell types which are thought to be involved in either potassium reabsorption or secretion. The decrease in potassium concentrations in distal tubular cells by about 20% does not seem sufficient to explain the marked fall in urinary potassium excretion.
During recent years our group has employed the technique of electron microprobe analysis to determine the electrolyte concentrations in various epithelial tissues. The specimen preparation is characterized by shock-freezing of small tissue pieces in liquid propane/isopentane mixtures at 77 K, cryosectioning of 1 micrometer thick serial sections at 170 K and subsequent freeze-drying at 190 K and 10-4 Pa. The analysis of the frozen dried cryosections is performed in a scanning electron microscopy which is equipped with an energy dispersive X-ray detector. The measuring conditions selected are 17-20 kV acceleration voltage and 0.1-0.5 nA probe current. For quantification, the cellular X-ray spectra are compared with those of an internal albumin standard layer. The evaluation of the characteristic X-ray intensities is performed using a computer program. Some critical points of this technique will be discussed.
To investigate the effect of ionic composition of the tubular fluid in the macula densa segment upon the JGA renin activity, the macula densa segment was perfused with different solutions and the renin activity in the attached juxtaglomerular apparatus was measured. Increasing the sodium chloride concentration at the macula densa by perfusion with Ringer's solution or saline increased the JGA renin activity. An activation of JGA renin activity did not occur when amiloride was added to the saline or when the animals were pretreated by a high-salt diet. Perfusing the macula densa segment with a sodium-chloride-free solution, with sodium- or chloride-substituted solutions, or with mock early distal fluid, did not affect the JGA renin activity. These data are in accord with the proposition that in the tubuloglomerular feedback mechanism the renin-angiotensin salt diet. Perfusing the macula densa segment with a sodium-chloride-free solution, with sodium- or chloride-substituted solutions, or with mock early distal fluid, did not affect the JGA renin activity. These data are in accord with the proposition that in the tubuloglomerular feedback mechanism the renin-angiotensin salt diet. Perfusing the macula densa segment with a sodium-chloride-free solution, with sodium- or chloride-substituted solutions, or with mock early distal fluid, did not affect the JGA renin activity. These data are in accord with the proposition that in the tubuloglomerular feedback mechanism the renin-angiotensin system in the juxtaglomerular apparatus acts as a local, humoral mediator between the signal perceived at the macula densa and the hemodynamic response at the glomerulus. The mediation by the renin-angiotensin system, which is influenced by the sodium chloride balance, allows the sensitivity of this control of GFR to be adjusted in a homeostatically appropriate manner.
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The technique of electron microprobe analysis was used to determine the intracellular electrolyte concentrations in proximal or distal tubular cells of the rat kidney during ischemia. When the exposed kidney was maintained in air during ischemia, the composition of the surface cells differed little from control, and the electrolyte disturbances were confined to the deeper lying cells. When maintained in nitrogen, all cells underwent changes in cellular electrolyte concentrations that were uniform, indicating that the surface cells can preserve their composition during ischemia by utilizing oxygen from the air. In the proximal tubular cells, after 20 or 60 min of ischemia in nitrogen, sodium increased from 20 to 93 or 112, chloride rose from 21 to 53 or 66, potassium fell from 141 to 65 or 42, phosphate decreased from 145 to 110 or 95 mmoles.kg-1 of wet wt, and the dry wt dropped from 22.6 to 20.3 or 17.5% of wet wt, respectively. In the distal tubular cells, 20 min of ischemia in nitrogen produced little effect on cellular composition, but after 60 min, sodium increased from 11 to 77, chloride rose from 15 to 48, potassium fell from 134 to 89, phosphate decreased from 168 to 145 mmoles.kg-1 of wet wt, and the dry wt dropped from 20.8 to 18.4% of wet wt. The disturbances in sodium and potassium are caused primarily by an inhibition of the sodium/potassium pump, whereas the changes in chloride, phosphate, and dry weight content result mainly from an influx of extracellular fluid. When blood flow was reintroducing, the electrolyte disturbances were rapidly reversed in all cells, restoration being virtually complete within 60 min, but returned in some proximal cells by 18 hr of reperfusion. Thus, the disturbance in electrolyte composition increases with the duration of ischemia, is less pronounced in the distal than proximal cells and, although initially completely reversible when blood flow is restored, reappeared in the proximal cells 1 days after the initial injury.
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The thrombogenicity of five suture materials--polypropylene (Prolene), polyester (Mersilene), polyglactin 910 (Vicryl), nylon (Tthilon), and silk--was examined in vivo by scanning electron microscopy. The most blood-compatible material appeared to be Prolene. A rather satisfactory antithrombogenciity was shown by Mersilene, in comparison with which Ethilon and Vicryl provided less thromboresistance. The possible connection between the chemical changes which occur during the hydrolysis of Vicryl and its blood compatibility is discussed. Of the tested materials, silk proved to be the most thrombogenic. This study shows that the thrombogenicity of a suture material could be easily and conveniently tested in an experimental model by scanning electron microscopy, thus enabling the first choice of a suitable suture for the cardiovascular surgery, before one continues with other more sophisticated methods for the evaluation of thromboresistance.
The effect of saline adaptation on the intracellular Na, K, Cl, P concentrations and dry weight content of the toad skin epithelium (Bufo viridis) was studied using the technique of electron microprobe analysis. The measurements were performed on isolated abdominal skins either directly after dissection or after additional incubation in Ussing-type chambers. Adaptations of the toads to increasing NaCl concentrations for 7 days resulted in increased blood plasma osmolarity and a parallel increase in the cellular electrolyte, P and dry weight concentrations of the epithelium, the K increase representing the most significant fraction of the intracellular osmolarity increase. No evidence was obtained to show that the nucleus and cytoplasm reacted differently from each other and all living epithelial cell types basically showed the same response. Incubation of the isolated skins under control conditions showed a drastic inhibition of the transepithelial Na transport after adaptation to high salinities. In spite of the large variations in the transport rate almost identical intracellular electrolyte concentrations were observed. In tap water adapted toads the average cellular concentrations were 8.8 mmole/kg wet weight for Na, 109.6 for K, 41.5 for Cl, and 135.3 for P, respectively. Incubation of the skin with Ringer's solution of different osmolarities demonstrated that the epithelial cells are in osmotic equilibrium with the inner bathing solution. The results are consistent with the view that the osmotic adaptation is mainly accomplished by the movement of water.
The concentrations of intracellular elements were determined by electron microprobe analysis in the nucleus and cytoplasm of freeze-dried cryosections of superficial proximal and distal tubules of the rat kidney. For the nucleus of the proximal tubular cell, the concentrations of sodium and chloride were 20 and 23 mmoles/kg of wet wt, and those of potassium and phosphorus were 144 and 150 mmoles/kg wet wt. For the nucleus of the distal tubular cell, the concentrations of sodium and chloride were significanlty lower (11 and 13 mmoles/kg wet wt), that of potassium was unchanged (143 mmoles), and that of phosphorus was significantly higher (175 mmoles). Towards the basolateral infoldings of the proximal and distal tubules and the brush border of the proximal tubules, the concentrations of sodium and chloride were higher and those of potassium and phosphorus were lower than those obtained in the nucleus, indicating the presence of extracellular compartments in these regions. Measurements performed in the centrally located cytoplasm of proximal and distal tubular cells, close to the nucleus, showed sodium and potassium concentrations to the indistinguishable from those in the nucleus, whereas chloride and phosphorus concentrations were considerably higher. These data demonstrate differences in the intracellular concentrations of phosphorus, sodium, and chloride between proximal and distal tubular cells. In neither the proximal nor the distal tubular cells, however, could a concentration difference for sodium and potassium between nucleus and cytoplasm be determined.
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In order to be able to examine the processes involved in transepithelial transport in tissues, which are not composed of a single cell type, methods are required, which permit analysis at a cellular level. The technique of electron microprobe analysis permits the intracellular concentrations of many elements to be determined simultaneously in various portions of the cell. The application of this method to renal cortical tissue has shown that the best estimates of the cytoplasmic concentrations are to be obtained in regions close to the nucleus, farthest from the basolateral infoldings and microvilli, which separate the intracellular environment from the extracellular space. The nuclear concentrations of Na and K do not differ from those in the surrounding cytoplasm, although those of P and Cl are somewhat higher in cytoplasm. The intracellular element concentrations in the different cell types vary somewhat, proximal tubular cells contain higher concentrations of Na and Cl and lower ones of P than distal tubular cells. Following ischaemia, a manoeuvre know to result in a disturbance of intracellular electrolytes, Na was observed to rise and K to fall only in the non-surface cells of kidneys exposed to the air, but in all cells, if the kidneys were kept air-free in an atmosphere of N2. The proximal and distal tubular cells showed a variable resistance to ischaemia, the distal tubular cells being much more resistant. Despite the severity of the electrolyte disturbance following ischaemia, the intracellular composition was completely restored one hour after re-introducing renal blood flow.