Vitamin E and selenium in gentamicin nephrotoxicity.

1. The sequence of renal cellular membrane damage induced by gentamicin was studied in the rat by using the release of alkaline phosphatase, acid phosphatase, muramidase and protein from renal cells as indices of renal damage. 2. The protective effect of a combination of vitamin E and selenium against renal damage was also investigated. 3. Gentamicin (60 mg kg-1 body weight) was nephrotoxic within 12 h of the first dose. 4. The plasma membrane of the renal tubules is damaged before the lysosomal membrane is affected. 5. A combination of vitamin E (1 mg g-1 body weight) and selenium (4 x 10(-3) mg g-1 body weight) attenuates the renal damage induced by gentamicin. Results suggest synergism between vitamin E and selenium in attenuating the renal damage. The possible mechanism of attenuation is discussed. 6. Vitamin E and selenium may have anti-diuretic potential.

Effect of repeated administration of berenil on urinary enzyme excretion with corresponding tissue pattern in rats.

The effect of repeated administration of berenil, a trypanocide, on urinary excretion of some enzyme activities in rat and their corresponding levels in the kidney and serum was investigated. Daily administration of this drug to rats resulted in increased urinary volume, excretion of protein, alkaline phosphatase and lactate dehydrogenase activities. However, the level of acid phosphatase activity was not significantly increased while muramidase activity disappeared completely during the period of drug administration. In the kidney tissue, there was a significant loss of lactate dehydrogenase activity immediately after the first dose and this trend continued until the end of drug administration. In the same tissue, there was an increase in alkaline phosphatase activity while the lysosomal enzymes were not significantly affected. In the serum, except for the increase in alkaline phosphatase activity, all other enzymes were not significantly affected. All these results indicate that there is cellular damage to rat kidney as a result of repeated berenil administration, and that the plasma membrane and the soluble portion of the cytoplasm are the primary site of injury to the cells. They also suggest that urinary enzyme excretion could be useful in determining the site of cellular damage by chemical agents in kidneys.

Studies on correlations between chloroquine-induced tissue damage and serum enzyme changes in the rat.

The administration of chloroquine to rats resulted in a significant elevation of serum enzymes and a corresponding decrease of these enzymes in the tissues. The changes in serum and kidney enzymes were most marked, thus indicating a primary renal dysfunction.

Biochemical changes in the rat during experimentally induced acute ochratoxicosis.

Biochemical changes in rat urine and tissues treated with five consecutive daily doses of ochratoxin A (10 mg/kg body weight) were studied. Urine volume and urinary proteins were moderately raised during the first few days of ochratoxin treatment, and were then highly elevated towards the end of the investigation. Urinary muramidase excretion was significantly raised (p less than 0.01) 24 h after the first insult with the toxin. The urinary output of alkaline and acid phosphatases, lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH) were all elevated but very much later, during the course of injections with ochratoxin A. Kidney alkaline and acid phosphatases, LDH and GDH were correspondingly reduced 7 days from the beginning of ochratoxin A administration. Liver LDH activity was reduced while serum LDH was raised. Liver glycogen level was significantly (p less than 0.0001) increased. Experimental evidence was presented to show that the initial point of interaction of ochratoxin A with the rat renal system may be at the first portion of the proximal convoluted tubular cell region.

Chloroquine-induced inhibition of rat serum muramidase activity in vivo in relation to tissue changes.

Chloroquine was tested for its inhibitory effect in vivo on the activities of rat serum and tissue muramidase. Daily administration of this anti-malarial drug to rats resulted in an appreciable reduction of kidney and liver enzyme activities 1 day after the first dose. No corresponding increases in enzyme activity were detected in the serum of treated animals up to day 9 when muramidase activity was then significantly reduced. Activity observed in the heart tissue was not affected until day 12, when the level was significantly reduced. These results suggest that chronic chloroquine administration to rats may result in an extensive inhibition of muramidase activity probably at the cellular/molecular level.

Protection by selenium against gentamicin-induced acute renal damage in the rat.

Gentamicin has been shown to induce renal tubular damage in man and laboratory animals and to result in elevated urinary excretion of some enzymes associated with specific cell regions in the kidney. In the present investigation, the possible protective effect of selenium against gentamicin-induced renal damage was tested by measuring the urinary excretion of some enzymes in the presence and absence of selenium. Our results show that a prior subcutaneous injection of selenium to rats for two days followed by a simultaneous S.C. injection of gentamicin and selenium resulted in a marked reduction in the excretion of such biochemical systems as the urine volume, urinary proteins, alkaline and acid phosphatases, beta-glucuronidase, muramidase, and glutamate dehydrogenase. Renal functional studies revealed that selenium-treated rats suffered less adverse effects compared to rats treated with gentamicin alone. Urinary acid phosphatase, beta-glucuronidase and muramidase, the three lysosomal enzymes tested, appeared to respond most readily to protection by selenium.

Studies on gentamicin-induced labilization of rat kidney lysosomes in vitro. Possible protection by selenium.

The labilizing effect of gentamicin, an aminoglycoside antibiotic, on isolated rat kidney lysosomes was investigated. The light-scattering behavior of lysosomal suspensions and the release of lysosomal acid hydrolase enzymes (acid phosphatase, beta-glucuronidase and muramidase) from incubated lysosomal suspensions, in the presence of gentamicin, were used as indices of lysosomal membrane labilization. Gentamicin was found to cause a decrease in light absorbance and a release of lysosomal acid hydrolases, which indicate lysosomal membrane swelling. In the presence of selenium, in the form of potassium selenate, the decrease in light absorbance of lysosomal suspensions and the release of lysosomal acid hydrolases from isolated lysosome particles were reduced markedly. This suggests that selenium protects against gentamicin-induced lysosomal membrane labilization. The possible mechanisms of protection by selenium are discussed.

Renal effects of an aminoglycoside antibiotic in the rat.

The renal effect of the daily administration of gentamicin to male albino rats (20 mg/kg body weight) for 6 consecutive days on some biochemical systems of the kidney was examined. Urine volume and urinary protein levels were found to be progressively raised following gentamicin. Urinary alkaline phosphatase, acid phosphatase, muramidase and glutamate dehydrogenase (GDH) activities were found to be markedly elevated. Acid phosphatase and GDH activities in urine were raised 24 h and 48 h, respectively, from the onset of gentamicin administration. The sequence in which some regions of the renal cells were involved in gentamicin nephrotoxicity was determined and the probable mechanism of interaction of gentamicin antibiotic with the renal tubular cells is proposed.

Further studies on the in vivo effect of cephaloridine on the stability of rat kidney lysosomes.

The stabilizing effect of cephaloridine, an antibiotic, on rat kidney lysosomal membranes was tested by a single subcutaneous injection. The release of two lyososomal enzymes, acid phosphatase and muramidase, was used as an index of lysosome membrane integrity. The levels of these enzymes in the kidney extracts as well as in the isolated kidney lysosome fractions were found to be raised considerably, compared to the controls. In rats treated with cephaloridine, the supernatant fraction obtained from the kidney homogenates, after centrifugation at 15,000 gav, contained lower enzyme activities than were found in the control animals. It is suggested that cephaloridine may inhibit the release of acid phosphatase and muramidase from rat kidney lysosomes and, therefore, may exert a stabilizing effect on the lysosomal membrane system. The possible mechanism of interaction of this antibiotic with rat kidney lysosomal membranes is proposed.

Some biochemical changes in the rat during repeated chloroquine administration.

The effect of chronic administration of chloroquine phosphate on the urinary excretion of proteins and selected enzymes was investigated in male rats. Urinary volume and protein levels were both increased. Alkaline phosphatase activity was markedly raised after 12 h, while lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH) activities were moderately elevated. These observations suggest that chronic chloroquine administration to rats might impair renal reabsorptive functions, interfere with the properties of some membrane-bound enzymes and alter the membrane permeability of the various organelles in renal cells.

Effect of chloroquine on the stability of rat kidney lysosomes in vivo and in vitro.

1. Chronic administration of chloroquine to rats results in increased urinary excretion of lysosomal acid phosphatase, muramidase and cathepsin D. 2. Various concentrations of chloroquine caused lysosomal membrane swelling as shown by decrease of light absorbance in lysosomal suspensions. 3. Incubating lysosomal suspensions in the presence of chloroquine resulted in a marked lysosomal acid phosphatase release. 4. Addition of acetylsalicylic acid, a lysosomal membrane stabilizer, into a lysosomal suspension containing chloroquine, reduced the degree of lysosomal membrane swelling and acid phosphatase release. 5. The results suggest a labilizing effect of chloroquine on rat kidney lysosomes.

The monitoring of lysosomal integrity by pH-stat and light scattering measurements.

Light scattering measurements were used to monitor the integrity of isolated rat kidney lysosomes during prolonged incubation at 37 degrees C or following the addition of lysolecithin. The fall in extinction at 520 nm (E520) was shown to correlate very well with the fall in the particulate enzyme activity and the corresponding rise in the soluble enzyme activity. Measurements were also made of the release of H+ from the lysosomes into the suspending medium following treatment with lysolecithin. A good relationship was obtained between acidification of the medium and changes in the light scattering (E520) of the lysosomal suspension. The value of these techniques in following rapid changes in the integrity of lysosomes is discussed.

The effect of cephaloridine on the stability of rat kidney lysosomes.

The administration of cephaloridine to rats caused a decrease in the excretion of acid phosphatase into the urine. The antibiotic itself had no effect on urinary acid phosphatase and inhibitors or proteolytic enzymes were not present in the urine from treated rats. Cephaloridine may therefore be stabilizing the lysosomal membrane in vivo and experiments with isolated lysosomes confirm this hypothesis. The lysosomal integrity was followed by measuring the acid phosphatase activity and the light scattering properties of the particles. A good correlation was obtained between these parameters in the case of thermal disruption and progesterone induced lysis of the lysosomes and low concentrations of cephaloridine (0.1-1.0 mmol/1) protected the lysosomes against this form of damage.

The sensitivity of urinary enzyme measurements for detecting renal injury.

The relative sensitivity of urinary enzyme measurements for detecting renal damage was determined for two nephrotoxins. Injection of a single dose of sodium phosphate (10 mmoles/kg) caused damage to the proximal tubules and led to a 15 fold increase in lactate dehydrogenase (LDH) activity excreted into the urine. In contrast to this change the serum LDH remained normal. Similar results were obtained following the injection of cephaloridine (2 g/kg) with an 18 fold increase in urinary LDH and a marginal increase in urinary glutamate dehydrogenase (GDH). By contrast the serum LDH was unchanged. Urinary enzymes are therefore more sensitive for detecting renal injury than enzymes. The four enzymes investigated are located in specific regions of the cell so that the involvement of the organelles and regions of the cell can be followed. Damage to the organelles does not appear to occur as the excretion of the lysosomal enzymes remained normal and only in the case of cephaloridine were marginal changes in the mitochondrial GDH excretion seen. The average alkaline phosphatase was also normal suggesting no gross damage to the plasma membrane although a few individual rats excreted abnormal activities of alkaline phosphatase. These rats however, also excreted high activities of LDH. This suggests that damage to the membrane causes leakage of LDH and in severe cases release of the plasma membrane enzyme alkaline phosphatase. The administration of cephaloridine at various doses showed that urinary enzyme measurements were as sensitive as histology for demonstrating renal damage and that of these enzymes, LDH was by far the most useful.

Cephaloridine and the rat kidney lysosomes stability in vitro.

Two methods employing lysosomal enzyme release and light scattering measurements have shown that Cephaloridine Stabilises rat kidney lysosomes in vitro against beta-Progesterone and lysolecithin, the two well known agents to labilise lysosomal membranes. Treatment of the isolated lysosomes suspended in 0.25 M-sucrose-EDTA (0.34 mM) pH 7.4 with cephaloridine (100mcM) at 37 degrees C for 10 min. and subsequent incubation of the mixture with progesterone (0.25mM) at the same temperature for varying periods of time up to 50 min. resulted in no significant changes from the normal values in the release of acid phosphatase activity obtained from the supernatant fraction after centrifugation of the incubated mixture at 33,000g. for 5 min. at 4 degrees C. When lysosomes were mixed with cephaloridine before treatment with progesterone or lysolecithin, and then subjected to light scattering measurements, lysosomal swelling was considerably decreased, indicating that cephaloridine could exert a stabilising effect on rat kidney lysosomal membrane and thereby limit the release of lysosomal acid hydrolases.

Partial purification and characterisation of bat liver ribonuclease. Hydrolysis of purine and pyrimidine polynucleotides by bat liver RNase.

A ribonuclease has been extracted from bat liver and purified. From polyacrylamide gel electrophoresis, it was found that there is only one molecular weight unit in this enzyme and this has a molecular weight of about 28,000. This enzyme was found to hydrolyse both purine and pyrimidine polynucleotides (e.g. polyadenylic acid and polycytidylic acid respectively). Hydrolysis of polyadenylic acid was found to be faster than that of polycytidylic acid.