Andrew Thewles

Fractionation of livers following diosgenin treatment to elevate biliary cholesterol

The plant saponin, diosgenin, is known to induce a marked increase in biliary cholesterol/phospholipid ratio. We reasoned that putative biliary lipid supply vesicles might be similarly enriched with cholesterol. Seven-day diosgenin feeding to rats resulted in significantly increased biliary cholesterol and cholesterol/phospholipid ratio, but had no effect on total cholesterol or phospholipid content of the liver. Subcellular fractionation of livers showed no selective increase in any fraction (nuclear, mitochondrial, lysosomal, microsomal) of the homogenate. Further subfractionation of microsomal or nuclear (plasma membrane) fractions also showed no difference between control and diosgenin groups. Thus, no intracellular vesicle fraction has been identified with the provision of the enhanced biliary cholesterol and the results are discussed in terms of the possible involvement of cytosolic lipid-binding proteins as putative lipid carriers to the canalicular membrane as an alternative to the presence of the lipid in lipid supply vesicles.

The hypomagnesaemic action of FK506: urinary excretion of magnesium and calcium and the role of parathyroid hormone.

A side-effect of the immunosuppressive drug FK506 (Prograf; tacrolimus) is hypomagnesaemia. We have investigated the effects of short-term (7-day) treatment of rats with FK506, using a protocol designed to indicate whether there are modifications in the renal tubular handling of magnesium and other electrolytes, or in the tissue deposition of magnesium, which may account for the hypomagnesaemia. We have also investigated whether parathyroid hormone has a role in the observed hypomagnesaemia. Two studies have been performed; in the first we administered FK506 (0.5 mg x kg(-1) body weight x day(-1)) or vehicle by intraperitoneal injection for 7 days, and then housed the rats in metabolic cages for the 24 h collection of urine. At the end of the metabolic cage period, the animals were anaesthetized, and blood and tissue samples were taken for analysis. In the second set of experiments the dosage regime was identical, but at the end of the treatment period the animals were anaesthetized for implantation of arterial and venous cannulae, and then received a saline (plus inulin) infusion for 6 h, during which time blood and urine samples were collected. The dose of FK506 employed did not decrease the glomerular filtration rate. FK506 elicited hypomagnesaemia in both sets of experiments, accompanied by inappropriately high fractional excretion of magnesium. There was also evidence of disruption of the normal renal reabsorption of calcium, but this did not result in hypocalcaemia. Plasma parathyroid hormone activity was not significantly different between the two groups, and there was no evidence of altered tissue content of magnesium in kidney, liver, heart, skeletal muscle or bone. The study confirms that hypomagnesaemia is a significant side-effect of FK506, even at a relatively low dose which did not decrease the glomerular filtration rate. The effect is not due to a decrease in parathyroid hormone release, or to translocation of magnesium from plasma to tissues, but does reflect decreased renal tubular magnesium (and calcium) reabsorption.

Vasopressin-induced natriuresis in the conscious rat: role of blood pressure, renal prostaglandin synthesis and the peptide ANF.

1. The response to arginine vasopressin (AVP) at doses of 5 and 10 pmol (100 g body weight)‐1 h‐1 was studied in conscious rats during the infusion of 1% (w/v) dextrose at 11.6 ml h‐1 with and without pre‐treatment with indomethacin. 2. In the absence of indomethacin AVP infusion induced dose‐related increases in sodium output that were positively correlated with increases in mean arterial blood pressure (MAP) and plasma atrial natriuretic factor (ANF) immunoreactivity. Increases in renal prostaglandin E2 (PGE2) synthesis were also associated with AVP infusion. 3. Indomethacin pre‐treatment abolished the AVP‐induced increases in renal PGE2 synthesis and also the dose‐related differences in ANF immunoreactivity. Increases in MAP and sodium output were unaffected at the 10 pmol (100 g body weight)‐1 h‐1 dose of AVP and only slightly attenuated for the 5 pmol (100 g body weight)‐1 h‐1 dose. 4. For both series AVP induced marked falls in glomerular filtration rate (GFR) but only small transient falls in effective renal plasma flow. The observed falls in GFR support the view that the natriuresis is due to changes in tubular handling and not in the filtered load of sodium. 5. It is concluded that the natriuresis elicited by AVP is closely related to the pressor action of the hormone but renal PGE2 synthesis and plasma ANF are not responsible for mediating this response.

Renal excretion of aluminium in the rat: effect of citrate infusion.

When aluminium is administered intravenously to rats, the speciation of the aluminium has a major effect on its renal excretion. Aluminium administered as citrate is much more effectively excreted than that administered as chloride or sulphate. This suggests that citrate could be therapeutically useful in patients who have been exposed to aluminium. Accordingly, we have performed two series of experi ments in rats, in which a citrate infusion (intravenous), was begun either immediately after, or one hour after, the administration of an intravenous aluminium sulphate bolus. Both protocols led to markedly enhanced alumini um excretion compared to controls in which only 0.7% NaCl was infused. The enhancement of aluminium excre tion was 783% if citrate infusion was begun immediately after aluminium administration, and 335% if the citrate infusion began after an hour delay. The increased excre tion was due to an increase in the freely filterable fraction of aluminium. In the control experiments, in which aluminium sul phate administration was followed by 0.7% NaCl infusion, aluminium was found to be deposited in the liver. Administration of citrate one hour after the aluminium bolus did not reduce this liver deposition. The results indicate that a fraction of the plasma alu minium is accessible to the citrate infused and can thereby be converted into a filterable form which can be excreted. It appears that, for maximum therapeutic effect, citrate should be infused as rapidly as possible after an alumini um load, to limit aluminium binding to ligands which allow it to enter cells.

Renal aluminium handling in the rat: a micropuncture assessment

Uncertainties exist over the glomerular filtration of aluminium and virtually nothing is known about its segmental handling along the nephron. The present study has used micropuncture, combined with electrothermal atomic absorption spectroscopy, to determine directly the aluminium content of glomerular filtrate and of late PCTs (proximal convoluted tubules) and early distal tubules in anaesthetized Munich-Wistar rats infused with three different doses of aluminium citrate (plasma aluminium concentrations, 2.9+/-0.1, 5.2+/-0.4 and 10.0+/-0.9 microg.ml(-1) respectively). Aluminium filtration into Bowmans space was found to be considerably greater than that predicted by an in vitro filtration system: in all three groups it was essentially filtered freely. No significant aluminium reabsorption took place along the PCT, but with every dose the FD(Al) (fractional delivery of aluminium; tubular fluid:plasma aluminium/inulin concentration ratio) was lower at the early distal site than at the late PCT (P<0.001 in each case), indicating net aluminium reabsorption in the loop of Henle. This reabsorption amounted to 19-26% of the filtered aluminium load. In the low- and medium-dose groups, there was no significant difference between FD(Al) at the early distal site and that in the final urine; however, in the high-dose group, FD(Al) in the urine (1.02+/-0.06) exceeded that at the early distal tubule (0.75+/-0.04; P<0.001), suggesting aluminium secretion in the distal nephron. The results indicate that aluminium loads, when complexed with citrate, are excreted efficiently owing to a combination of glomerular filtration and minimal reabsorption.

Renal filtration and excretion of aluminium in the rat: dose-response relationships and effects of aluminium speciation

The known toxicity of aluminium, and the toxicity of agents (such as desferrioxamine) used to remove alumini um from the body, has prompted us to investigate whether there may be ways of enhancing aluminium excretion by exploiting the normal renal handling of aluminium. Aluminium (as sulphate or citrate) was administered intravenously to conscious rats at doses ranging from 25 μg (0.93 μmol) to 800 μg (29.6 μmol) aluminium, and alu minium excretion was monitored over the following 2 h. Measurements of the filterability of aluminium from the rat plasma, and the glomerular filtration rate (inulin clearance), enabled us to calculate the filtered load of alu minium, and hence determine aluminium reabsorption. At all doses of administered aluminium, that adminis tered as sulphate was excreted less effectively than that administered as citrate. This difference was attributable to the much greater filterability of aluminium administered as citrate. However, for any given filtered load, the excre tion of aluminium administered as citrate was not signifi cantly different (in either fractional or absolute terms) from the excretion of aluminium administered as sulphate. It seems likely that, following aluminium sulphate administration, the filtered aluminium may be an alumini um citrate form which is then reabsorbed in the same way as aluminium administered as citrate. It is thus apparent that aluminium removal from the body could be further enhanced if it were possible to pre vent the tubular reabsorption of the aluminium species which is so effectively filtered following aluminium citrate administration.

Role of nitric oxide in a toxin-induced model of haemolytic uraemic syndrome

Abstract The role of nitric oxide in the pathogenesis of glomerular thrombotic microangiopathy was explored using an established rat model in which ricin with or without lipopolysaccharide induced glomerular thrombosis. Ricin alone caused a small rise in the plasma concentration of nitric oxide (control 9.2±0.7 µM, ricin 23.3±6.3 µM at 7 h). This increase occurred after the development of glomerular thrombosis. Nitric oxide synthase (NOS) activity in the kidney showed no significant change from control values (control 5.66±2.7 pmol/min per ml homogenate, ricin 7.52±1.8 pmol/min per ml homogenate, total activity). When ricin and lipopolysaccharide were administered together, calcium-independent NOS activity increased whereas calcium-dependent activity decreased (1.22±2.6 pmol/min per ml homogenate). The increase in calcium-independent NOS activity correlated with a high plasma concentration of interleukin-1β in the ricin plus lipopolysaccharide group (4,036.83±1,001.5 pg/ml). These data indicate that thrombus formation in a rat model of haemolytic uraemic syndrome is independent of the effects of nitric oxide.