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Toxicological studies using isolated glomeruli.

Ahmed, Iftikhar. (1991) Toxicological studies using isolated glomeruli. Doctoral thesis, University of Surrey (United Kingdom)..

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Glomeruli isolated from both rat and pig were demonstrated by enzyme leakage (e.g. LDH leakage 13% of total (350 um/min/mg protein), amino acid incorporation (lysine 0.56 pmol/mg protein, proline 0.36pmol/mg protein, histidine 0.19 pmol/mg protein) thymidine incorporation (0.18 pmol/mg protein), no change in ATP content (8 nmol/mg protein), and pentose phosphate pathway stimulation (ratio of Cl/C6-glucose was 1.56) to be viable for at least 4 hours. The use of enzyme leakage as an indicator of toxicity was found to produce data that was difficult to interpret. This was partially due to the interaction of the compounds directly with the enzymes or the assay procedure (e. g streptomycin inhibited lactate dehydrogenase (LDH) activity and adriamycin (ADR) interfered with N-acetyl-beta-D-glucaminidase (NAG), beta-D-galactosidase (GALACT), glucaminidase (GLUC) and gamma-glutamyltranspeptidase (GGT) assays due to its intense red colour). The release of proteolytic enzymes from the glomerulus may have affected the activity of leaked enzymes. Protein synthesis, measured by the de novo incorporation of amino acids, was a sensitive indicator of toxicity. The inhibition of proline was found to be particularly sensitive. For example, ADR inhibited proline incorporation by 50% at a concentration of 61 uM, but a 50% increase in LDH leakage was only apparent when 600uM of this anthracycline was present. This sensitivity was selective, ADR and its analogue pharmarubicin both inhibited proline incorporation. In contrast the proximal tubular toxin hexachlorobutadiene-cystiene (HCBD-cystiene) had no effect on glomerular proline incorporation at concentrations of 2mM, at this concentration tubular incorporation was inhibited to 34%. ADR inhibited proline incorporation to 50% at a concentration of 61uM (P<0.0005), at this concentration glomerular adenosine triphosphate (ATP) content was unaffected and there was no LDH leakage. Of the total glomerular incorporation of amino acids, 55% of proline, 70% of lysine and 14. 5% of histidine was incorporated into a crude extract of the glomerular basement membrane (GBM). Glucose oxidation and the pentose phosphate pathway (PPP) were shown to be active in isolated rat glomeruli. The PPP was demonstrated to be stimulated equally by the redox cycling drug paraquat and ADR (300% control by 500uM after 4 hours). This stimulation can be partially blocked by desferrioxime suggesting the involvement of free radicals as part of the toxic mechanism of ADR. The addition of free radical scavengers increased the incorporation of proline (150%), reduced thymidine (by 60%) while PPP activity was inhibited. This suggests that damaging free radicals are generated in the isolation of rat glomeruli. The addition of free radical scavengers during isolation may reduce this damage. Thus isolated glomeruli remain viable for at least 4 hours. Selectivity for in vivo glomerular toxins was demonstrated in vitro for some toxins e.g ADR. Proline incorporation appears to be a a sensitive indicator of toxicity and this was not ATP (energy) dependent, this is likely to be free radical mediated although not prevented by the addition of all free radical scavengers.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors :
Ahmed, Iftikhar.
Date : 1991
Contributors :
Additional Information : Thesis (M.Phil.)--University of Surrey (United Kingdom), 1991.
Depositing User : EPrints Services
Date Deposited : 22 Jun 2018 09:50
Last Modified : 06 Nov 2018 16:52

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