Vasopressin stimulates urinary kallikrein excretion in the isolated erythrocyte-perfused rat kidney

We have found that arginine vasopressin (AVP) (10 pg/ml) stimulates urinary kallikrein in the isolated erythrocyte perfused rat kidney. (In this model, perfusate flow rate approximates blood flow rates in vivo and morphology is normal.) Urinary kallikrein excretion rose from 6.9 +/- 0.8 to 14.9 +/- 2.4 ng/min 20 min after the addition of AVP to the perfusate, and then fell towards baseline levels over the next 30 min. 1-Desamino-8-D-AVP (8 pg/ml) caused a comparable increase in kallikrein excretion. Prostaglandin synthesis inhibition with indomethacin did not alter the stimulatory effect of AVP on kallikrein excretion. Parathyroid hormone 1-34 (144 ng/ml) and calcitonin (102 ng/ml) also increased urinary kallikrein. Kallikrein excretion rose from 9.1 +/- 2.0 to 24 +/- 4.5 ng/min in response to calcitonin and from 8.3 +/- 1.6 to 43.7 +/- 3.4 ng/min following the addition of parathyroid hormone to the perfusate. Kallikrein was found to accumulate in the perfusate in a linear fashion. Based on the slope of the relationship between perfusate kallikrein and time, the rate of release of kallikrein into the perfusate was estimated to be 0.79 ng/min in control kidneys. The rate of release of kallikrein into the perfusate in kidneys treated with AVP was the same (0.74 ng/min). Thus while kallikrein is released into the perfusate, this process is not influenced by AVP. In conclusion, AVP stimulates release of kallikrein into the urine (but not the perfusate) independently of systemic events. The effect of AVP is not mediated by prostaglandins. This effect of AVP is mediated via stimulation of the V2 receptor and also occurs in response to two other hormones (calcitonin and parathyroid hormone) that are known to stimulate adenyl cyclase in the rat distal nephron.     

Protective effect of metallothionein on cadmium toxicity in isolated rat hepatocytes.

An isolated rat hepatocyte preparation was used to study the cellular toxicity of cadmium and the protective effects of metallothionein on cadmium-induced toxicity. Exposure of primary suspension cultures of isolated rat hepatocytes to Cd2+ (0-35.7 microM) for 15 min resulted in a dose-dependent reduction in the synthesis of cellular proteins during a subsequent 6 h incubation. Such inhibition could not be correlated with cellular lethality or gross membrane damage. Pre-induction of metallothionein in hepatocytes by zinc treatment in vivo of donor rats protected hepatocytes in vitro from cadmium-induced inhibition of protein synthesis. The protective effects in zinc-pre-induced hepatocytes are not due to alterations in the level of total cellular cadmium, but could be accounted for by the redistribution of intracellular cadmium in the presence of high levels of zinc-metallothionein. The data suggest that metallothionein exerts its protective effect by a kinetic detoxification mechanism, i.e. a decrease in reactive intracellular cadmium.     

Protective effect of lipoic acid on adriamycin induced lipid peroxidation in rat kidney

Adriamycin, which is widely used in the treatment of various neoplastic conditions, exerts toxic effects in many organs. The present study was designed to investigate the effect of lipoic acid upon adriamycin induced peroxidative damages in rat kidney. The increase in peroxidated lipids on adriamycin administration was accompanied by alterations in the antioxidant defense systems. The extent of nephrotoxicity induced by adriamycin was evident from the decreased activities of the enzymes -glutamyl transferase and -glucuronidase in the rat renal tissues. The study was carried out with adult male albino rats of Wistar strain, which comprised of one control and three experimental groups. Group I rats served as controls. GroupII rats received adriamycin (1 mg kg^–1 body wt day^–1) intravenously through the tail vein. Group III rats were given lipoic acid (35 mg kg^–1 body wt day^–1) intraperitoneally. Group IV rats were given lipoic acid 24 h before the administration of adriamycin. Rats subjected to adriamycin administration showed a decline in the thiol capacity of the cell accompanied by high malondialdehyde levels along with lowered activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione metabolizing enzymes (glutathione reductase, glucose-6-phosphate dehydrogenase, glutathione-S-transferase). Lipoic acid pretreatment also restored the activities of -glutamyl transferase and -glucuronidase nearly to control levels thereby suggesting nephroprotection. The study has highlighted the beneficial effects of lipoic acid pretreatment in reversing the damages caused by adriamycin and thereby bringing about an improvement in the oxidative stress parameters.     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

Protective effect of hypothermia during ischemia in neural cell cultures

Hypothermia offers protection from the effects of ischemia in small animals. We have recently shown that similar to small animals, hypothermia may also be protective in an astrocytic model of simulated ischemia in cell culture. This study was designed to look at the protective effects of hypothermia in cultures of cerebellar granular (glutamatergic) and cortical (GABAergic) neurons. We used LDH release into the medium as an indicator for neuron damage. Experiments were all done in sister cultures, in groups of six cultures at two temperatures (37 and 32 degrees Celsius). The duration of ischemia was three hours in cerebellar granular neuronal cell cultures and six hours in cortical neurons. LDH release was measured immediately after the insult. Hypothermia protected both granular and cortical neurons. In granular cells, LDH release was 62+/–18 at 32 degrees and 212+/–15 at 37 degrees (p=0.02). Cortical neurons showed LDH release of 15+/–2 at 32 degrees and 32+/–2 at 37 degrees (p=0.005). Our study suggests that similar to astrocytes, the protective effects of hypothermia are evident in neuronal cell cultures from the cerebellum and the cerebral cortex. Cell culture systems should prove useful techniques in understanding mechanisms of hypothermic protection during simulated ischemia in neurons from different sites.     

Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.     

The effect of hypothermia on the rat radioresistance

The cooling of Wistar rats up to 15-19 degrees C under a condition hypoxia-hypercapnia increased the radioresistance with a dose reduction factor (DRF) of 1.4. To elucidate the mechanisms of hypothermia radioprotective effect was evaluated the functional state of rat neocortex using a electroencephalogram (EEG) as well as was studied the lipid composition of neocortex under the conditions of both normothermia and hypothermia. At 19-20 degrees C the activity within a wide range of frequencies in EEG was suppressed; the nonregular slow waves were recorded against a background of "silence". The reduction of EEG spectrum with increasing temperature began with the low frequencies. At 26-28 egresC the contribution of theta-rhythm (an indicator of brain activity level) in EEG reaches the normothermia value, from this point the rat brain starts to functionate as a whole system. At normothermia the similarity of neocortex lipid composition in nonhibernators (rats) and hibernators (ground squirrels) mammalians was noted. The difference is only in a higher content of phosphatidylinositol in rats. Rats falling into hypothermia state as well as ground squirrels into torpor is followed by a decrease of cholesterol content and the absence of significant changes of the phospholipid composition in neocortex tissues.     

Metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney.

The metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney was investigated. For this purpose LTC4, LTD4 or LTE4 were studied in separate experiments. The isolated perfused rat kidney metabolized all cysteinyl leukotrienes to the final metabolite N-acetyl-LTE4. In the presence of 5% albumin 50% of LTC4 was metabolized to LTD4 (22%), LTE4 (15%) and N-acetyl-LTE4 (13%) within 60 min. Excretion of radioactivity into urine was less than 1%. In contrast, in the absence of albumin, LTC4 was completely metabolized within 45 min to N-acetyl-LTE4, the sole and final metabolite of LTC4 found in the perfusion medium as well as in urine. After 60 min 19% and 42% of total radioactivity were found in the perfusion medium and in urine, respectively. Isolated glomeruli metabolized LTC4 to LTD4 and to LTE4 but not to N-acetyl-LTE4 at a rate comparable to the rate observed by the isolated perfused kidney in the absence of albumin. In contrast to isolated glomeruli isolated tubuli metabolized LTE4 to N-acetyl-LTE4 at a rate comparable to that observed by the isolated perfused kidney in the absence of albumin. The present study shows that the isolated perfused rat kidney metabolizes cysteinyl leukotrienes to the sole and final metabolite N-acetyl-LTE4. In the presence of albumin metabolism is slowed down and excretion of N-acetyl-LTE4 into urine is prevented.     

Inhibition of renin secretion in the isolated rat kidney by angiotensin I.

The effect of angiotensin I on renal perfusion pressure, and on basal and isoprenaline stimulated renin secretion, was examined in the isolated perfused rat kidney. The increase in prefusion pressure associated with intrarenal infusion of angiotensin I suggested conversion of the peptide to angiotensin II within the kidney. Basal renin secretion and the stimulatory response to isoprenaline were significantly suppressed by angiotensin I. The converting enzyme inhibitor SQ 20,881, infused at 1,600 X dose of angiotensin I, partially reversed the vasoconstrictor effect of angiotensin I without altering the degree of suppression of renin secretion.     

Phenylalanine hydroxylation in isolated rat kidney tubules

1. Phenylalanine hydroxylation has been demonstrated to occur in isolated rat kidney tubules under physiological conditions. 2. The hydroxylation flux response is hyperbolic with apparent Km and Vmax values of ca 85 microM phenylalanine and 49 nmol tyrosine formed/mg dry wt per hr respectively. 3. Hydroxylation in kidney tubules is substantially less sensitive to effectors of cyclic AMP turnover and Ca2+ mobilization than phenylalanine hydroxylation in isolated liver cells.     

Metabolic activities of the isolated perfused rat kidney

1. A technique for perfusing the isolated rat kidney is described. It is primarily designed for the study of renal metabolism but is also suitable for studying some aspects of the secretory function; this was normal with respect to minimal glucosuria. The glomerular filtration rate as measured by creatinine clearance was lower than in vivo and slowly decreased with time. 2. Gluconeogenesis from a variety of precursors was rapid and similar to that in kidney-cortex slices, in contrast with liver where the perfused organ is more effective than slices. Whereas the maximal rates of gluconeogenesis from glycerol and pyruvate were similar in liver and kidney, the rates from succinate, malate and fumarate were 14–20 times, and those from glutamate and aspartate about three times, as high in the kidney. 3. The oxygen consumption of the perfused organ was about twice that of cortex slices, presumably because of the secretory work done in the perfused organ but not in slices. 4. The rate of acetoacetate oxidation was about the same in the perfused organ and in slices but, because of the higher rate of oxygen consumption, the percentage contribution of acetoacetate to the fuel of respiration was lower in the perfused organ. The results suggest that acetoacetate can supply energy for the basal requirements and for gluconeogenesis but not for the secretory work. 5. Glutamine was formed at a high rate from glutamate and at a lower rate from aspartate. The high rates indicate that, in the rat, the kidney is a major source of body glutamine.     

Effects of L-glucose on sodium reabsorption in the isolated perfused rat kidney.

In the isolated perfused rat kidney, sodium reabsorption is enhanced in the presence of 5.5 mM D-glucose. However, it is unclear whether this effect is metabolic or whether it is due to a requirement for sodium transport in the process of glucose reabsorption. A third possibility is solvent drag. In an attempt to differentiate between these possibilities, kidneys were perfused with the D-glucose isomer, L-glucose (L-G), a nonmetabolizable hexose. At a perfusate concentration of 5.5 mM L-G, per cent L-G reabsorption was approximately 30. Inhibition of L-G reabsorption by D-glucose suggests carrier-mediated transport. In the presence of 5.5 mM L-G, sodium reabsorption approximated 92% during the course of perfusion. When L-G was omitted from perfusate, sodium reabsorption ultimately declined to 85%. Since significant metabolism of L-G was not observed, the results are compatible with the hypothesis that enhanced sodium reabsorption may be brought about by some still to be defined aspect of glucose transport.     

Kinetic characterization of phosphofructokinase isolated from rat kidney cortex.

1. Phosphofructokinase from rat kidney cortex has been purified by affinity chromatography to a final specific activity of 15 units per mg of protein, measured at 25 degrees C and pH 8. 2. This lower spec. act., compared with that of the enzyme from other sources, shows the enzyme in proximal tubules to be less active, which would account for the main gluconeogenic role of these nephron sections. 3. The binding of fructose-6-phosphate to the enzyme is co-operative. ATP increases the Hill coefficient and produces a marked allosteric inhibition on the activity. 4. Fructose-2,6-bis-phosphate is a potent activator of the enzyme from this source. It reduces the Hill coefficient of the enzyme and the inhibition constant of ATP. A marked difference between this and the liver enzyme is that the activation is not co-operative.     

19-nor-DOC biosynthesis in the isolated perfused rat kidney

19-nor-deoxycorticosterone (19-nor-DOC) is a potent salt retaining and hypertensinogenic mineralocorticoid that is excreted in the urine. While the precursor of 19-nor-DOC, 19-oxo-DOC, is produced by the adrenal cortex, conversion to 19-nor-DOC does not occur in the adrenal gland. We have examined the hypothesis that 19-nor-DOC is synthesized from precursors in the kidney. 19-oxo-DOC was added to the perfusate of isolated rat kidney preparations (n = 5) at a concentration of 10 μM. During 1 h of perfusion following addition of 19-oxo-DOC, 71 ± 6% of the precursor was converted to 19-oic-DOC, an immediate precursor of 19-nor-DOC, and 8.3 ± 1.8% was converted to 19-nor-DOC. This represents the first definitive evidence that 19-nor-DOC is produced in the kidney from adrenal precursors.