Geoffrey G. Duggin

Differential distribution of glutathione and glutathione-related enzymes in rabbit kidney: possible implications in analgesic nephropathy

Whole tissue reduced glutathione (GSH) concentration was found to be lowest in rabbit renal inner medulla and progressively higher in outer medulla and cortex. Activities of cytosolic glutathione reductase in inner medulla and outer medulla were similar, and each was only approximately 50% of that of cortex. Whole tissue and microsomal gamma-glutamyl transpeptidase activities were high in cortex and outer medulla but were low in inner medulla. Cytosolic activity of selenium-dependent glutathione peroxidase ( GPx -I) was similar in both outer medulla and inner medulla but was only 50% of that of cortex. Activity of cytosolic selenium-independent glutathione peroxidase ( GPx -II) was highest in cortex and lowest in inner medulla (approximately 15% of cortex and approximately 50% of outer medulla). Cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate was high in all three regions of kidney. With 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-(4-nitrophenoxy)propane as substrates, cytosolic glutathione S-transferase activities were very low in cortex, outer medulla, and inner medulla. Microsomal activities of glutathione reductase, GPx -I, GPx -II and glutathione S-transferases were much lower than activities of corresponding cytosolic enzymes. Activities of the glutathione peroxidases in renal inner medulla would hence be expected to cause little interference to prostaglandin endoperoxide synthetase mediated cooxidative activation of paracetamol. It has been demonstrated that the paracetamol metabolite can react rapidly with GSH, forming not only glutathione conjugate but also paracetamol itself and oxidized glutathione. Low GSH concentrations, as well as low activities of glutathione reductase, GPx -I, GPx -II, and gamma-glutamyl transpeptidase, may therefore render the inner medullary region of kidney particularly vulnerable to paracetamol-related analgesic nephropathy.

Metabolic oxidation of acetaminophen (Paracetamol) mediated by cytochrome P-450 mixed-function oxidase and prostaglandin endoperoxide synthetase in rabbit kidney☆

Abstract Long-term abuse of acetaminophen, in combination with other antipyretic analgesics, is thought to be responsible for papillary necrosis in analgesic nephropathy. Cytochrome P -450-mediated oxidative metabolism requiring NADPH and O 2 has been believed, to date, to be the sole pathway for the toxic metabolic activation of acetaminophen. Using microsomes from different regions of rabbit kidney, protein covalent binding of acetaminophen by NADPH-dependent metabolism was highest in cortex, less in outer medulla, and minimal in inner medulla. In vivo studies have shown that the highest binding of acetaminophen occurred in renal inner medulla compared to liver and renal cortex. However, cytochrome P -450 could not be detected in rabbit renal inner medulla. Hence, another metabolic pathway for the activation of acetaminophen was presumed to be operative in renal inner medulla. This alternate pathway was recognized as cooxidation of acetaminophen mediated by prostaglandin endoperoxide synthetase, requiring arachidonic acid as well as O 2 , and was found to be active predominantly in renal inner medulla. Glutathione, ascorbic acid, and ethoxyquin inhibited protein covalent binding of acetaminophen arising from both pathways. Indomethacin and aspirin inhibited only the arachidonic acid-dependent cooxidation of acetaminophen. Butylated hydroxyanisole inhibited both NADPH- and arachidonic acid-dependent metabolism, the latter more effectively. Arachidonic acid-dependent metabolism of acetaminophen is probably mediated by the hydroperoxidase activity of prostaglandin endoperoxide synthetase. This alternate pathway could be a significant contributing factor for the genesis of papillary necrosis, as manifested in analgesic nephropathy.

Circulatory adaptation to pregnancy--serial studies of haemodynamics, blood volume, renin and aldosterone in the baboon (Papio hamadryas).

To test the hypothesis that haemodynamic changes in pregnancy precede any significant increase in circulating blood volume, serial haemodynamic studies were performed in eight baboon pregnancies using Swan-Ganz catheterization and arterial cannulation. Simultaneous measurements were made of red cell and plasma volumes, and of plasma renin activity and aldosterone concentration. Haemodynamic changes identified by 4 weeks gestation included significant (P less than 0.01) reductions in right atrial pressure, systemic and pulmonary arterial pressures, and systemic and pulmonary vascular resistance. Stroke volume increased in early pregnancy (P less than 0.01), with a consequent increase in cardiac output. Plasma renin activity and aldosterone concentration were elevated by 4 weeks (P less than 0.01), but plasma volume did not expand until 12 weeks. At no stage in middle or late pregnancy was cardiac filling pressure increased. These results provide the first haemodynamic evidence that pregnancy is a state of reduced effective blood volume associated with vasodilatation from the early weeks.

EVIDENCE THAT ALTERATIONS IN RENAL METABOLISM AND LIPID PEROXIDATION MAY CONTRIBUTE TO CYCLOSPORINE NEPHROTOXICITY

This study was designed to investigate aspects of renal xenobiotic metabolism and the renal cellular response to drug-induced injury, in mediating cyclosporine nephrotoxicity. The relation between CsA and renal enzyme activity has not previously been investigated. In this study, CsA induced alterations in rat renal cortical microsomal NADPH cytochrome P-450 reductase activity, microsomal and mitochondrial lipid peroxidation, and renal cortical glutathione leveis were investigated. CsA, in vivo (50 mg/kg/day for 4 days), increased in vitro lipid peroxidation in microsomes and mitochondria. CsA produced a significant uncompetitive inhibition of renal NADPH eytochrome P-450 reductase activity. The low activity and maximal enzyme velocity (Vmax) suggest that the amount of renal enzyme available for metabolism may be a rate-limiting step and could contribute to the development of toxicity, CsA in vivo reduced the renal cortical glutathione ratio (GSH/GSSG), which may also reduce the renal cellular response to CsA injury. This study has demonstrated that CsA nephrotoxicity may, in part, be mediated by CsA-induced alterations in renal xenobiotic metabolism.

Renal function and lithium treatment: Initial and follow-up tests in manic-depressive patients

Assessment of renal function was carried out in an unselected sample of patients with bipolar manic-depressive disorder receiving lithium for an average period of 4.5 years. Overall, glomerular filtration rate (GFR) fell within the established normal range based on sex and age, whereas measures of urinary concentrating ability were generally impaired. There was no relationship between duration of lithium treatment and either GFR or impairment of urinary concentrating ability. Moreover, there was no evidence of a progressive impairment of glomerular or tubular function in patients re-tested after 2 years. The results of this study confirm the safety of lithium administration in the majority of patients and emphasize the importance of careful clinical monitoring to avoid lithium intoxication.

Fetotoxicity of angiotensin-converting enzyme inhibition in primate pregnancy: A prospective, placebo-controlled study in baboons (Papio hamadryas)

OBJECTIVES Serious concerns have been raised about angiotensin-converting enzyme inhibition in pregnancy. The central question remains: does toxicity of angiotensin-converting enzyme inhibition pertain to pregnant humans? STUDY DESIGN A prospective, placebo-controlled study was performed to investigate the effect of angiotensin-converting enzyme inhibition on pregnancy outcome in the baboon. Subjects (N = 12) received active and placebo treatments sequentially in a crossover protocol. Data were analyzed with two-sample t tests, analysis of variance, Fishers exact test, or Kaplan-Meier survival analysis, where appropriate. RESULTS Chronic administration of enalapril (7.5 mg per day) from before conception achieved moderate but sustained angiotensin-converting enzyme inhibition as determined by repeated measures of renin-angiotensin system parameters (serum angiotensin-converting enzyme activity, plasma renin activity and plasma angiotensin I, angiotensin II, and aldosterone concentrations). Serum angiotensin-converting enzyme activity was significantly reduced throughout (< 10 nmol.ml-1.min-1, p < 0.01), with significant increases in plasma renin activity and angiotensin I (p < 0.01). Angiotensin II and aldosterone were maintained unchanged compared with placebo. There was a significant incidence of fetal death or intrauterine growth retardation in fetuses exposed to enalapril (eight of 13, zero on placebo, p < 0.01). When the definition of adverse pregnancy outcome was restricted to fetal death alone (four of 13) the difference remained significant (p < 0.05). Maternal arterial pressure was unchanged before conception, but a small and significant fall (10 to 15 mm Hg, p < 0.01) was detected throughout pregnancy. There was no fetal malformations. CONCLUSION The study provides definitive evidence for serious consequences of angiotensin-converting enzyme inhibition in pregnancy of high-order primates.

Aspirin, protein transacetylation and inhibition of prostaglandin synthetase in the kidney.

1 The effect of aspirin on the kidney has been investigated in mice and rabbits. [Acetyl‐14C]‐aspirin was administered intraperitoneally in doses ranging from subtherapeutic to toxic. The degree of acetylation of protein was determined by the radioactivity remaining on protein precipitates of renal cortex and medulla after sequential washing designed to remove non‐covalently bound material. Controls were established, by the use of [carboxyl‐14C]‐aspirin. 2 The acetyl‐14C residue was bound to renal proteins in a linear manner in increasing amounts with increasing dosage up to 100 mg/kg. The [carboxyl‐14C]‐aspirin was not bound and thus the salicylate portion of the molecule was not bound covalently to the renal protein. The time course of the acetylation was rapid, consistent with the rate of aspirin absorption. The disappearance of acetylated protein was slow, with a Tl/2 of 112.5 h in the renal cortex, and 129.5 h in the renal medulla. 3 Differential centrifugation, Sephadex chromatography and gel electrophoresis were carried out on tissue homogenates to determine the site of acetylation. The acetylation was greatest in the microsomal fraction, although all protein fractions showed some degree of acetylation. 4 The prostaglandin synthetase activity of a particulate preparation from rabbit kidney was determined by a spectrophotometric assay of malondialdehyde formation. Aspirin (10 mg/kg, i.v.) significantly inhibited prostaglandin synthetase in the renal cortex and medulla. 5 Aspirin and renal proteins undergo a transacetylation reaction resulting in stable acetylated protein, with acetylation being greatest in the microsomal fraction. Aspirin has been shown to inhibit prostaglandin synthetase and this could lead to functional impairment of the tissue.

Survival After a Massive Hydrofluoric Acid Ingestion

BACKGROUND Hydrofluoric acid ingestion is known to have a very high mortality rate secondary to the rapid development of hypocalcemia and fatal arrhythmias. CASE REPORT A 33-year-old man ingested an estimated dose of hydrofluoric acid 6 times that considered to be lethal. The patient survived with minimal morbidity despite having multiple ventricular fibrillation arrests. His survival is attributed to early, high dose calcium therapy given via the nasogastric and intravenous routes.

Characterisation and uptake of paraquat by rat renal proximal tubular cells in primary culture

1 Uptake of the herbicide paraquat (PQ), by rat proximal tubular cells (PTC) in primary culture grown on a collagen coated support was investigated. 2 The uptake of PQ by PTC was predominantly from the basolateral side. The basolateral uptake of PQ was saturable with time and increasing concentrations, energy dependent and could be inhibited by certain organic cations. Using Michaelis Menten kinetics, the apparent Km was 778 ± 241 μM and Vmax was 0.97 ± 0.24 pmol/μg protein/15 min for the basolateral uptake of PQ. Cimetidine (5.7 ± 0.4 pg/μg protein/ 30 min, P < 0.001) was the most potent inhibitor of PQ uptake, followed by quinine (6.5 ± 0.4 pg/μg pro tein/30 min, P < 0.01) and then tetraethylammonium (8.2 ± 0.5 pg/μg protein/30 min, P < 0.05) when com pared with control (11 ± 1 pg/μg protein/30 min). N- methylnicotinamide, p-aminohippurate and putres cine did not inhibit the basolateral uptake of PQ. The sodium hydrogen exchange inhibitors, amiloride and its analogue, 5-(N,N hexamethylene) amiloride (HMA) inhibited both the apical and basolateral uptake of PQ. 3 The apical uptake of PQ was not saturable with increasing concentrations and was not inhibited by 2,4-dinitrophenol, but it was reduced by cimetidine (P < 0.01), quinine (P < 0.05) and a sodium potassium ATPase inhibitor, ouabain (P<0.01). 4 It is concluded that PQ was taken up from the basolateral side of primary cultured rat PTC by an energy dependent transport system.

Structure-activity relationships of cyclosporines: toxicity in cultured renal tubular epithelial cells

Proximal (LLC-PK1) and distal (MDCK) renal epithelial cell cultures were used to investigate early biochemical changes in cellular metabolism following exposure to cyclosporine A (CsA). 3H-thymidine and 3H-leucine incorporation into the cells were used as indices of DNA and protein synthesis. The cells were exposed to concentrations of CsA ranging from 0.2 microgram/ml to 20 micrograms/ml. By 20 hr there was a decrease in the total cell count at concentrations of 10 and 20 micrograms/ml that was more pronounced by 5 days of exposure. At 5 days there was also a reduction in cell count at the lower concentrations of CsA. There was an initial increase in DNA and protein synthesis at 2 hr with inhibition of DNA synthesis evident by 20 hr. Protein synthesis was increased in the LLC-PK1 cells and decreased in the MDCK cells. At 5 days there was evidence of increased DNA and protein synthesis, most marked in the remaining viable cells exposed to the higher concentrations of CsA. Similar alterations in cellular metabolism were evident when the cells were exposed to the immunologically inert cyclosporine H (D-N-MeVal11-Cs). These studies demonstrate that cyclosporine produces alterations in cellular function as early as 2 hr after exposure to the drug. At the lower concentrations there is evidence of sublethal cellular toxicity and cellular regeneration. The toxicity appears to be related to the molecular structure of cyclosporine and its incorporation into cell membranes. We postulate that cyclosporine nephrotoxicity is the summation of several subtoxic alterations in cellular function the final expression of which is modified by other factors affecting renal function.