Biomedical subjects
Publications and source records attributed to J D Tange.
The rat kidney has been prepared for microdissection by in vivo perfusion with hyaluronidase and collagenase and subsequent fixation with glutaraldehyde. The procedure ensures preservation of cytoplasmic detail, and the principle of fixation after maceration should provide a foundation for developing a variety of staining and histochemical studies.
The severity of the renal papillary necrosis produced in rats by ethylenimine is dependent both on dose and urinary concentration within the medulla. When this is reduced by diuresis, the severity of the lesion is effectively reduced. Increasing urinary concentration has a reverse effect, but of less magnitude. When the concentrating power of the renal medulla is impaired by a single dose of ethylenimine, insufficient to cause necrosis of the whole papilla, further doses of ethylenimine do not cause progressive damage to the papilla.
Urinary paracetamol metabolites from Sprague-Dawley and Wistar rats were analysed by reversed-phase HPLC. Variations in the metabolic profile were observed as a function of dose, age, sex, species and route of administration. In addition the effect of 3-methylcholanthrene as an inducer of cytochrome P450 mixed function oxidase on paracetamol metabolism was also studied. Increased oxidative metabolism which lead to the formation of 3-thiomethylparacetamol conjugates along with paracetamol mercapturic acid could be correlated with increased susceptibility to hepatic damage. Furthermore it appears that the length of time taken for excretion and the level of free drug excreted may be involved in the aetiology of chronic renal damage.
When isolated rat kidneys are perfused with glucose as the only substrate, there is a progressive diminution in glomerular filtration rate and fractional reabsorption of sodium. This is most marked after 1 h. Renal glutathione content rapidly falls and is less than 30% of control levels after 1 h. Renal concentrating ability is markedly impaired and structural lesions are consistently observed in cells lining the thick ascending limb of Henle's loop. Addition of 20 physiologic amino acids including cysteine to the perfusate prevents the fall in renal glutathione, prevents the anatomical damage to ascending limb cells, permits GFR and fractional sodium reabsorption to remain high and close to their initial levels for as long as 4 h, and improves renal concentrating capacity. If amino acid supplementation is limited to three precursors of glutathione--cysteine, glycine, and glutamic acid--renal glutathione content is preserved and concentrating ability is improved, but GFR and fractional sodium reabsorption are not maintained as well as with comprehensive amino acid supplements. The results suggest that amino acid deficiency and glutathione depletion may contribute to disturbances in renal structure and function.
Deposition of bilirubin at the tip of the renal papilla has been studied in the infant homozygous Gunn rat. The deposits form during the 4th wk of life in ground substance along basement membranes of capillaries and tubules and along the borders of interstitial cells. Deposits also form in capillary lumens. The early ultrastructural appearances indicate that unconjugated bilirubin is not directly cytotoxic. This conclusion has important implications for the pathogenesis of enhanced nephrotoxicity in the homozygous Gunn rat and ultimately for the pathogenesis of analgesic nephropathy.
1. Isolated kidneys from homozygous Gunn rats were perfused with paracetamol in concentrations lower and higher than Km for paracetamol oxidation in the albino rat kidney. 2. Glucuronylation of paracetamol was not detected at either concentration. 3. An increase in oxidative metabolism at the higher concentration, similar to that seen with the Sprague-Dawley rat kidney, did not occur with kidneys from homozygous Gunn rats. 4. This finding does not support the hypothesis that the enhanced nephrotoxicity of paracetamol observed in the homozygous Gunn rat in vivo is due to increased intrarenal formation of reactive metabolites.
The morphologic characteristics of the isolated perfused rat kidney were examined by light and electron microscopy. Following perfusion for 90 min, the cortex appeared normal except for the development of spaces in glomerular mesangial regions and ultrastructural abnormalities in very few proximal tubular cells. In the medulla, descending limbs of the loops of Henle and Collecting tubules appeared normal. During perfusion, however, even under optimal conditions, consistent and reproducible changes rapidly developed in cells of the thick ascending limb of Henle's loop, more specifically, in cells of the straight portion of the distal tubule in the inner stripe of the outer medulla. The changes became progressively more severe as perfusion continued and ranged from swelling of mitochondria at 15 min to complete cellular disruption by 90 min. Horseradish peroxidase added to the perfusion medium was found in and between damaged cells and in the interstitial tissue adjacent to affected areas. The pathogenesis of these selective medullary distal tubular abnormalities may relate to the specialized functional activity of this segment of the nephron. Such abnormalities may contribute to the specific functional defects of the preparation.
Both phenacetin and paracetamol produce acute centrilobular liver necrosis in the homozygous Gunn rat. Paracetamol is more hepatotoxic than phenacetin, and both are more hepatotoxic to the homozygous Gunn rat than to the heterozygous Gunn rat or to the albino rat. These findings have relevance to the role of the compounds in the clinical syndromes of paracetamol induced liver necrosis and analgesic nephropathy.
1. Renal metabolism of paracetamol has been studied in the isolated perfused rat kidney. 2. The major metabolites of paracetamol normally observed in vivo were present in low concentrations in the urine of the isolated perfused rat kidney. 3. Paracetamol was bound covalently to kidney protein in a linear relationship to dose up to 31.9 mM paracetamol in the perfusate. 4. Using Michaelis-Menten kinetics, the apparent Km of 5.5 mM and Vmax of 139 nmol/h/g wet wt. for renal paracetamol oxidation were observed. 5. Concn. of paracetamol greater than 14.2 mM induced immediate diuresis and diminished sodium reabsorption. Lower concn. were without effect on function.
1. At a perfusate concn. of 3.5-4.0 mM, 59 plus or minus 9 nmol of paracetamol h per g wet wt. were oxidized by isolated rat kidney. 2. Approx. half the paracetamol undergoing oxidation was converted to a mercapturic acid metabolite and the remainder was covalently bound to kidney protein. 3. Addition of GSH to the perfusate decreased the level of covalent binding. Depletion of cellular GSH, by prior administration of diethyl maleate, significantly decreased formation of the mercapturic acid metabolite. 4. The metabolic pathways of glucoronylation, sulphation and mercapturic acid formation were induced either by 3-methylcholanthrene pretreatment or by prolonged feeding of aspirin or paracetamol; covalent binding of paracetamol to kidney protein was not increased.
p-Aminophenol administration lowered the microsomal cytochrome P-450 and b5 content and decreased the activity of NADPH cytochrome c reductase in kidney, but not in liver. Kidney GSH was depleted to 29% of the control value at 2 h, and only partly restored (50% of control) at 24 h. Liver GSH was transiently decreased, the lowest levels (77% of control) occurring at 30 min. The maximum level of covalently bound radioactivity was at two hours when 16.8% of the total radioactivity in kidney, 1.5% in liver and 3.6% in plasma was protein bound. At this time 81% of the total radioactivity in kidney and 95% of that in the liver was present in the soluble fraction.
Inducers and inhibitors of the microsomal mixed function oxidase system have no consistent effect upon the nephrotoxicity of p-aminophenol, or on binding of the compound in vivo to cell protein. p-[ring-3H]Aminophenol was bound in vitro to kidney microsomal protein and to a lesser extent to liver. The binding was enhanced by preincubation of the p-aminophenol in air and inhibited by ascorbate, GSH, N2 and NADPH. These findings indicate that in contrast to paracetamol hepatoxicity which is dependent upon the mixed function oxidase system, that nephrotoxicity of p-aminophenol is dependent upon oxidation to a toxic metabolite by some other pathway. A similar metabolite may be responsible for the nephrotoxic action of phenacetin.
Increased vascular permeability and visceral oedema without significant hypertension or arterial damage was found in clip-nephrectomized rats dying in the first post-operative week. Increased vascular permeability was demonstrated by intravenous colloidal carbon labelling, and was restricted to capillaries and venules. Rats surviving the first week became hypertensive, and visceral oedema with predominantly venular and capillary labelling continued, although carbon was now present in arteries also, associated with smooth muscle necrosis. These results suggest that the pressor and permeability effects of renal artery constriction are independent.
Constriction of the remaining renal artery in unilaterally nephrectomized rats is associated with a high mortality in the early post-operative phase before hypertension has developed. The high mortality is accompanied by increased vascular permeability, mesenteric and pancreatic oedema. Temporary post-operative water restriction reduces this mortality and also modifies subsequent blood pressure elevation, heart weight and cardiac index in surviving animals.
1. Liver and kidney glutathione are depleted in rats and mice following administration of N-hydroxyparacetamol. 2. Centrilobular hepatic necrosis and necrosis of renal proximal convoluted tubules were also found, the liver lesion predominantly in mice and the renal lesion predominantly in rats. Glutathione depletion was not responsible for this species difference. 3. These results indicate that N-hydroxyparacetamol is the metabolic precursor of the reactive toxic intermediate of paracetamol. They are also relevant to the pathogenesis of the renal damage associated with long term abuse of phenacetin containing compound analgesics.
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1. The metabolic effects of p-aminophenol have been compared with those of paracetamol and other analgesics in studies of rat liver and kidney in vitro. 2. p-Aminophenol injected into rats inhibited gluconeogenesis from lactate in renal cortical tubules, but not in isolated hepatocytes, and reduced kidney ATP content without affecting the ATP content of liver. Perfused kidneys from rats previously injected with p-aminophenol showed a 50% reduction of ATP content, severe inhibition of Na+ reabsorption and reduction of inulin clearance without significant inhibition of gluconeogenesis from lactate. 3. Paracetamol, p-phenetidine, phenazone and aspirin, when given intravenously to rats, had no effect on renal tubular glucose synthesis from lactate or pyruvate. Paracetamol and aspirin both slightly inhibited renal glucose synthesis from several different substrates when added directly to tubules. 4. Paracetamol (4 mmol/l) inhibited glucose synthesis from lactate and other substrates by 50% or more in isolated hepatocytes. Glucose synthesis from lactate was inhibited 30% by concentrations of paracetamol as low as 0.5 mmol/l. 5. These results indicate that p-aminophenol is a potent inhibitor of proximal tubular function, with its main site of action the inhibition of ATP synthesis and energy production, and they confirm the primary hepatotoxic effect of paracetamol.
1. p-Aminophenol, a known nephrotoxin, has been studied as a model for phenacetin-induced renal damage. 2. Respiration, oxidative phosphorylation and ATPase activity were inhibited in mitochondria isolated from the kidneys of treated rats; this could not be reversed by the addition of exogenous loosely bound cofactors and bovine serum albumin to the assay medium. 3. After treatment the mitochondrial levels of sodium and calcium were increased, potassium decreased and magnesium unaltered. 4. Mitochondria isolated from treated rats showed ultrastructural damage. 5. The results are interpreted to indicate that renal tubular cell mitochondrial injury is important in triggering cortical analgesic renal damage.