Cytoprotective effect of isotonic mannitol at low oxygen tension.

The beneficial effect of mannitol infusion in postischemic kidneys remains unresolved. Contradictory reports may have originated from at least 3 different mechanisms: a) arterial vasodilation, b) osmotic support of hypoxic cellular volume regulation, and c) scavenging of hydroxyl radicals in the reoxygenation period. To exclude vascular effects we tested at 37 degrees C in hypoxic (PO2 less than 1 mmHg) and reoxygenated isolated tubular cells of rat kidney cortex cellular function, i.e. intracellular K+ accumulation (K+), posthypoxic lactate gluconeogenesis (GNG), loss of membrane-bound tau-Glutamyltransferase (tau GT), and formation of a lipid peroxidation product, malondialdehyde (MDA). Mannitol (M) was added to a Ringer incubation medium in variable concentration either without (hypertonic M) or with omission of equiosmolar amounts of NaCl (isotonic M). K+ and GNG were significantly supported, and tau GT-loss markedly suppressed in a range of 10-50 mmol/l hypertonic and isotonic M. At higher concentrations no improvement (isotonic) or even deleterious effects (hypertonic) of M occurred. Beneficial effects of lower concentrations of M (10 mmol/l) were not correlated to anaerobic glycolysis, and 1 as well as 10 mmol/l M induced a comparable and significant reduction in posthypoxic MDA-formation. This effect was most pronounced when M was only added in hypoxia, indicating leakiness of cellular membranes in hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Support of hypoxic renal cell volume regulation by glycine.

PO2 declines to less than 10 mm Hg in local regions of the renal cortex. Amino acids seem to modify the hypoxic tolerance of renal cells. It was suggested that glycine may support renal function in hypoxia. Aim of the present study was to test the effect of glycine on renal cellular hypoxic tolerance. We isolated tubules of the rat kidney cortex (ITS) by collagenase treatment and measured cellular function at different levels of extracellular oxygen tensions (1, 2.5, 5, 10, 40, and 100 mm Hg) both with and without glycine in the incubation medium. No significant effects were observed in the "physiological" range at an extracellular PO2 = 100-10 mm Hg. With no glycine in the incubation medium, the outer tubular diameter of ITS rose at lower oxygen tensions, at PO2 of 1 mm Hg by about 170%, and the loss of 4 marker enzymes increased about 2-4 fold. Hypoxic lactate formation increased at extracellular oxygen tensions less than 10 mm Hg. Intracellular K+ fell in parallel to about one third of the aerobic control values. Addition of glycine to the incubation medium did not significantly change intracellular K+ or anaerobic lactate formation. In contrast, the loss of marker enzymes was significantly suppressed by glycine, lysosomal APase and mitochondrial GlDH by about 30%, cytoplasmatic LDH and brush border tau GT by about 50%. Accordingly, at PO2 = 1 mm Hg the hypoxic swelling of renal cells was suppressed in the presence of glycine by about 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobin induces cytotoxic damage of glycine-preserved renal tubules.

In isolated tubular segments (ITS) of rat kidney cortex, we studied the effect of hemoglobin (Hb) on reoxygenation damage. All tubules were suspended in Ringer's solution containing 5-mm glycine and oxygenated for 30 min with 95% O(2):5% CO(2), followed by a 30-min period with 95% N(2):5% CO(2), and final reoxygenation for 60 min. Untreated tubules served as controls. Different concentrations of free Hb and equivalent amounts of intact erythrocytes were added to the incubation medium. Secondly, we added deferoxamine (DFO) to Hb and erythrocytes. Membrane leakage and lipid peroxidation were measured by lactate dehydrogenase and glutamate dehydrogenase and the development of thiobarbituric acid reactive substances. Cell function was quantified by gluconeogenesis and intracellular potassium accumulation. Hb exerted concentration-dependent cytotoxic effects indicated by significantly increased enzyme leakage rates, lipid peroxidation and a significantly decreased cell function (P < 0.05), in ITS during hypoxia, and subsequent reoxygenation. Moreover, we found that toxicity of both Fe(2+) and Fe(3+) ions increased with rising concentration. However, Fe(2+) showed a higher tissue toxicity than Fe(3+). DFO reduced significantly the reoxygenation damage of free Hb and iron ions. Our data clearly demonstrate a pronounced cytotoxic effect of free Hb in ITS, which critically depended on the reduction state of the iron ions.

Substrate support for renal functions during hypoxia in the perfused rat kidney.

Do the substrates that can be utilized for anaerobic ATP production by cytosolic (G, glycolytic) and/or mitochondrial (M) metabolic pathways support renal function during marked hypoxia? The isolated rat kidney was perfused at 38 degrees C, pH 7.4, at a mean pressure of 120 mmHg for 110 min with a Krebs-Ringer bicarbonate solution containing 6 g/100 ml substrate-free albumin (SFA0). After substrate-free aerobic (PO2 = approximately 646 mmHg) internal control observations were made, the perfusate was gassed with 95% N2/5% CO2 (n = 15) and substrates (each 5 mM) were added (G, glucose, K, alpha-ketoglutarate, A, aspartate), or SFA0 perfusion was continued. Perfusion flow rate (PFR) increased 20-43% during hypoxia; thus there was no limitation in substrate delivery to the kidney. Although GFR decreased during all hypoxic perfusions, due to the variations in GFR, the reductions in GFR were not significant. Fractional Na+ reabsorption (%TNa+) was reduced in the hypoxic kidney but the decreases in %TNa+ in the presence of G or M substrates were significantly smaller (-26 to -36%) than the decreases observed during hypoxic SFA0 perfusion (-44%). Free water clearance decreased markedly during substrate-free hypoxic perfusion; by contrast, addition of G or M substrates either increased or maintained CH2O. G increased hypoxic CH2O (+194% to +440% of internal control) more than did M substrates. It is postulated that the increases in %TNa+ in the presence of substrates during hypoxia results in the increases in GFR. By making substrates available that can be oxidized anaerobically in cytosol or in mitochondria, the kidney can better maintain a portion of its tubular functions during severe hypoxia.

Glucagon induced functional changes of isolated perfused rat kidney.

The influence of glucagon on renal function and hemodynamics was studied on isolated perfused rat kidneys. During perfusion with solutions containing no vasoactive substances glucagon increased total renal perfusion flow, while the GFR remained unchanged. The autoregulation of blood flow under these conditions was completely abolished. Na-excretion was slightly reduced under the action of glucagon, due to an increased fractional Na-reabsorption. However, increased fractional Na-reabsorption was not due to a direct influence of glucagon on tubular transport mechanism as comparison of TNa at identical Na-loads demonstrated. In presence of angiotensin II renal perfusion flow was markedly reduced and doubled almost after starting glucagon infusion. GFR under these conditions rose by about 100%. It is concluded from the results that changes of kidney function following glucagon infusion are mainly due to a reduced vascular resistance.

Hippurate metabolism as a hydroxyl radical trapping mechanism in the rat kidney.

Hippurate (Hip) is considered to be the end product of benzoate (BA) metabolism. However, the kidney is able to metabolize Hip. Although only Hip but no BA is present in the blood, rat urine contains under normal conditions less Hip (about 0.4 mM) than BA (about 4.5 mM) and of hydroxylated derivatives of BA (hydroxy-BAs = HB and dihydroxy-BAs = DHB). Generation of HBs and DHBs is the result of radical substitution by free OH radicals (*OH). Thus, rate of synthesis of HBs and DHBs may reflect the production rate of *OH in the kidney *OH generation is elevated following ischemic stress. Therefore, production of HBs and DHBs can be expected to be elevated in postischemic injury. The validity of this assumption was tested in vitro on isolated tubular segments and in vivo in the rat. Metabolism of Hip at 0.1 mmol x l(-1 ) (0.1 mM) as well as of BA resulted in enlarged production of both HBs (especially 3-HB and 4-HB) and of DHBs (especially 2,6-DHB). Production of 2,3- and especially of 2,5-DHB was elevated in the presence of high concentration (1.0 mM) of salicylate (2-HB) only. In vivo both in acute (120 min) and in chronic (5 days) experiments ligation of one renal artery for 30 respectively 60 min resulted in enlarged excretion of HBs and DHBs, especially of 2,6- and 3,5-DHB. This finding is noteworthy since (a) formation of 2,6-DHB necessitates as precursor salicylate which could not be detected in our experiments and (b) the spontaneous attack of *OH upon the benzol ring would prefer the positions 2,3- 2,5- and 3,4-. Therefore, the existence of regulating factor(s) guiding OH groups to definite positions is a distinct possibility. These results indicate that metabolism of Hip leading to hydroxylated BAs may be a renoprotective mechanism against attack of *OH in reoxygenated renal tissue.

Different renal oxidation rates of intramolecular carbon atoms in lactate and pyruvate.

In order to get more detailed information on the different renal modes of utilization of individual intramolecular carbon atoms, formation rates of 14CO2 and metabolic products from single-carbon labelled exogenous substrates have been studied in isolated tubular fragments from rat kidney cortex. The results showed that carboxyl groups from acetate, lactate, and pyruvate were converted to CO2 at a much higher rate than substrate-2-C or 3-C. This effect was not due to a physico-chemical splitting. The corresponding C-2 or C-3-atoms were incorporated into newly formed glucose to a higher extent than carboxyl groups. Apparently, the maximum chance to be converted to CO2 for an intramolecular carbon of an exogenous substrate lies at the stereospecific site of action of decarboxylating enzymes. With rising distance from this point the probability to be built into glucose (i.e. to escape the oxidative pathway) increases. Due to the different share of the intramolecular substrate carbon (in the total CO2 formation) without the use of single-carbon labelled substrates one cannot calculate the oxygen requirements for an observed 14CO2 formation.

Contribution of long chain fatty acids to the energy supply of the rat kidney cortex.

Tubular fragments from rat kidney cortex were isolated by collagenase and suspended in an incubation medium containing a combination of several renal substrates. Substrate concentrations were in the physiological range. O2 uptake, total CO2 production, and the 14CO2 production from U-14C-labeled palmitate and oleate were measured. During the first minutes of incubation the CO2 production from palmitate and oleate was 10.5% or 6.3%, respectively, of the total CO3 produced. The RQ was 0.897. A subsequent decrease of the total CO2 production at a constant uptake of oxygen indicated a rising contribution of fatty acids to the fuel of respiration. The renal preference for substrates other than longchain fatty acids is discussed.

Ammonia production from hippurate by the rat kidney in vitro.

Hippurate is known to be synthesized from benzoate and glycine in the liver and kidney. It takes part in renal ammoniagenesis by modulating the activity of gamma-glutamyl transpeptidase (gamma GT). Due to its chemical structure, however, hippurate might also serve as a substrate of renal ammoniagenesis. Hippurate may yield ammonia either having been cleaved by hippuricase or by Erlenmeyer's reaction after condensation with an aldehyde. In order to elucidate the possibility of hippurate being a substrate of renal ammoniagenesis, experiments were carried out on cortical kidney slices and on isolated tubular segments of the rat. The incubation medium (pH 7.1) was enriched with 10 mmol/l hippurate spiked with 15N-hippurate, some of the known competitive inhibitors of hippuricase, acivicin and different aldehydes. Factors known to affect hippuricase or gamma GT did not interfere with renal ammonia production. Glyceraldehyde (up to 1.0 mmol/l) but not glycerate had a stimulating effect, especially on the ammoniagenesis from hippurate. In normal rats fed a vegetarian diet, 1% of the added 15N moiety was found to be 15NH3. Renal 15NH3 production was significantly greater if, prior to the experiments, the animals were either acidotic or had a reduced renal mass or were fed animal proteins. These results indicate that hippurate may, to a certain extent, serve as substrate for ammoniagenesis.