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.

Fast method for the simultaneous determination of 2-oxo acids in biological fluids by high-performance liquid chromatography.

A fast and sensitive method for the single-run quantification of various 2-oxo acids including 2-ketoglutarate, glyoxylate and pyruvate is described. It ensures good separation of peaks with minor interference by other substances. The 2-oxo acid derivatives are measured photometrically at 324 nm after derivatization with phenylhydrazine and subsequent isocratic separation with ethanolic phosphate buffer on a C18 reversed-phase HPLC column. Recovery was found to be complete [range: 96.3 +/- 5.6% (pyruvate) to 104.8 +/- 5.2% (2-ketoglutarate)]. Detection limits ranged from less than 0.1 mumol/l (glyoxylate) to 0.25 mumol/l (2-ketoglutarate and pyruvate). Results for all substances examined showed good linearity with correlations (r2) of equal to or better than 0.998.

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.

Improved determination of small amounts of free hydroxyproline in biological fluids.

A fast, reliable, and sensitive (< 20 pmol) method for the quantification of 4-hydroxyproline is described. It ensures good separation of imino acid peaks, eliminates interference by primary amino acids, and guarantees full (96-105%) recovery of hydroxyproline (HYP). Interfering primary amino acids are derivatized by o-phthaldialdehyde and removed from the sample by use of a discardable C18 column. HYP is measured photometrically at 254 nm after a second derivatization with phenylisothiocyanate and isocratic separation on a reversed-phase HPLC column.

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)

Differences in the renal handling of pancreatic and salivary amylase in the rat.

Plasma activity and excretion of pancreatic (P) amylase in the rat was found to be negligible. In contrast, the excretion rate of salivary (S) amylase was substantial and variable, depending on diuresis. P-amylase had a higher isoelectric point, a greater sieving coefficient, and a shorter half-life than S-amylase. A bolus injection of 125I-labelled enzymes was followed by the appearance of 125I-labelled enzyme- as well as protein-free 125I activity in the urine. The enzyme loss was smaller and the fraction of protein-free 125I activity higher following injection of P-amylase. The affinity of P-amylase to paraffin oil exceeded that of S-amylase in partition experiments with water and paraffin oil in vitro. It is concluded that both renal filtration and reabsorption of P-amylase exceed those of S-amylase. This might be due to the higher lipophility of P-amylase in comparison to the salivary type.

Renal handling of 125I-labelled homologous pancreatic lipase and amylase in the rat.

Experiments were carried out in vivo on rats and in vitro on tubular brush border vesicles in order to study the renal mechanisms of the elimination of pancreatic lipase and amylase from the circulation. Highly purified 125I-labelled homologous lipase, amylase or 125I-labelled di-iodo-tyrosine was injected intravenously in a single dose. The sieving coefficients of lipase and amylase were found to be 0.126 and 0.118 respectively. Less than 1% of the lipase activity but more than 10% of the radioactivity were found in the urine in the course of a 120 min experiment. In experiments with amylase, 16% of the enzyme activity and 19% of the radioactivity were present in the urine. Elimination of both enzymes showed first order kinetics and was of the same magnitude (17-24 min). The elimination curves of the radioactivity consisted of at least two components: a fast component immediately after the injection, which was identical with the decrease of the resp. enzyme activity; and a slow component (half-life 106 min), which in both cases proved to be identical with the half-life of di-iodo-tyrosine. In experiments with amylase, the excretion of protein-free 125I-activity started later than with lipase. The radioactivity of 125I-labelled lipase was taken up faster by brush-border-vesicles than that of 125I-amylase. Liberation of protein-free 125I-activity from both enzymes occurred at the same rate. At the end of the experiments the kidneys had no lipase or amylase activity, but they contained 5.4% (lipase), 3.8% (amylase) of the injected radioactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

[Enzymuria following administration of contrast media in hypertensive rats]. / Enzymurie nach Kontrastmittelgabe bei hypertonen Ratten.

Renal complications may occur in risk patients with pre-existing kidney damage if x-ray contrast media are administered. Nothing is known of the exact mechanisms of these reactions. We examined in rats with long-term renovascular hypertension and resulting nephrosclerosis the low osmolar contrast media ioxaglate, iohexol and iopamidol in respect of their renal tolerance compared with an osmotically equivalent mannite solution. Under the conditions of intravenous pyelography, we observed directly after the administration of all substances (3.5 mg/kg body weight) an enhanced enzymuria of gamma-glutamyl transpeptidase and N-acetyl-beta-D-glucosaminidase. The loss of enzyme is mainly due to the osmotic effect of the preparations. The nonionic contrast media iohexol and iopamidol produce a slighter enzymuria than the ionic ioxaglate.

The role of the kidney in the elimination of pancreatic lipase and amylase from blood.

Two clinical observations indicate that the kidney plays the main role in the elimination of lipase and amylase from the circulation: 1. in patients with uncomplicated acute pancreatitis the decrease of the activity of both enzymes in the serum ran almost in parallel. The half life for lipase was found to be 6.9-13.7 h, and somewhat higher figures (9.3-17.7 h) were calculated for amylase; 2. in patients with reduced glomerular filtration rate the serum activity of either or of both enzymes was distinctly elevated. The contribution of the kidney to the elimination of lipase and amylase from blood was studied in the rat. After an intravenous bolus injection of homologous lipase and amylase, the serum activity of both enzymes decreased rapidly. The half-life of lipase was 18.1 min, that of amylase 20.5 min. Up to 30% of the injected amylase but only traces of lipase activity were recovered in the urine. In animals with ligated kidneys the serum half-life of both enzymes was 3 times longer. Our results indicate that lipase as well as amylase are removed from the serum mainly by glomerular filtration at nearly the same rate. Reabsorption of lipase is almost complete, in contrast to that of amylase. It is suggested that the differences in the renal handling of both enzymes are due to their differing affinities for hydrophilic and hydrophobic surfaces.

Effect of castration on the experimental renal hypertension of the rat. Blood pressure, nephrosclerosis, long-chain fatty acids, and N-acetylation of PAH in the kidney.

Rats were rendered hypertensive by clamping one renal artery. Both kidneys remained in situ ('two-kidney one-clip Goldblatt hypertension'). Half of the animals were simultaneously castrated. 18-24 weeks after the operation both castrated females and males had a lower level of hypertension than the uncastrated controls. The kidneys of castrates contained less connective tissue (measured as the content of hydroxyproline) and long-chain (C-18) fatty acids and had a higher specific activity of the enzyme N-acetylating p-aminohippurate (N-acetyltransferase) than those of uncastrated rats. Thus, castration seems to alleviate some renal effects of the Goldblatt hypertension. In all animals the clamped kidney contained more hydroxyproline and C-18 fatty acids and had a lower N-acetyltransferase activity than the contralateral untouched organ. These results are in accordance with the theory that renal fatty acid concentration interferes directly with the N-acetyltransferase activity of the kidney. The enhanced hydroxyproline content of the kidneys (nephrosclerosis) inhibits N-acetylation most probably indirectly by raising the tissue concentration of C-18 fatty acids.

Effect of castration and testosterone substitution on the urinary output of gamma-glutamyl transpeptidase and of N-acetyl-beta-D-glucosaminidase of male rats with renovascular hypertension.

The effect of castration of male rats with experimental renal hypertension ('two-kidney Goldblatt hypertension') was studied on the height of the hypertension and on the urinary output of gamma-glutamyl transpeptidase (gamma GT) and of N-acetyl-beta-D-glucosaminidase (NAG). Castration was carried out immediately after clamping one renal artery. Some of the castrates received testosterone substitution from the 3rd postoperative week onwards. Uncastrated hypertensive males served as controls. The experiments were carried out 8-18 weeks after eliciting high blood pressure. Hypertension as well as enzymuria were less expressed in castrates than in uncastrated males or in testosterone-substituted rats. In all animals studied the gamma GT excretion rate showed a positive correlation with the blood pressure. The output of gamma GT and of NAG as well as the specific gamma GT activity of the renal membrane fraction was lower in castrates than in uncastrated males or in substituted castrates. In uncastrated males and in testosterone-substituted castrates the daily NAG output showed a direct correlation with the renal hydroxyproline content. No such correlation was found in castrated males. The kidneys of castrates and of testosterone-substituted castrates contained less hydroxyproline than those of uncastrated males.

Interdependence of O2 consumption, renal NEFA pattern and N-acetylation of PAH in the isolated perfused rat kidney. Effect of long-chain NEFA and of PGF2 alpha on the renal N-acetyltransferase activity.

Tissue concentration of nonesterified fatty acids (NEFA) exerting an inhibitory effect on renal N-acetyltransferase activity was determined in the isolated perfused rat kidney. Concurrently, O2 consumption and the N-acetylation rate of p-aminohippurate (PAH) were measured. In a second series of experiments, the mode of inhibition of NEFA on acetylating enzyme(s) was studied. Amount and pattern of NEFA as well as N-acetylation rate of PAH in the kidney were related to the O2 consumption: lower O2 supply corresponded to a higher tissue NEFA concentration as well as lower N-acetylation rate, increased O2 supply resulted in a low tissue NEFA concentration and an increased N-acetylation rate of PAH. Decreasing O2 supply elevated the tissue concentration of linoleate especially. NEFA with a carbon chain length of C16-C20 inhibited renal N-acetyltransferase activity in vitro competitively according to the sequence C16, C18, C18:1, C18:2, C18:3 = C20. It is inferred that hypoxia interferes with the N-acetylation of PAH in the rat kidney by increasing the content and changing the pattern of fatty acids, thereby inhibiting the N-acetylating enzyme(s).

[On the etiology of water excretion inhibition in Addison's disease]. / Zur Atiologie der Wasserausscheidungsstörung bei Morbus Addison.

Micropuncture experiments, performed on adrenalectomized rats disclosed an impairment of the hypertonic sodium chloride transport of the thick ascending limb of Henle's loop (the diluting segment). This transport inhibition was documented by a delayed time course of NaCl-concentration decrease within the diluting segment. Assuming a pump leak model of the hypertonic NaCl-transport, we calculated a 50% inhibition of the NaCl-transport velocity compared with controls. Dexamethasone treatment for 3 days led to a normalization of the impaired transport velocity. Both, the reduced GFR as well as the impaired free water clearance are functionally linked to the diminished transport capacity of the diluting segment by the tubuloglomerular feedback mechanism and the ADH mechanism respectively. A new hypothesis on the pathogenesis of the water excretion inhibition in adrenal insufficiency is discussed.

Enzymuria (the output of gamma-glutamyl-transpeptidase and of N-acetyl-beta-D-glucosaminidase) in the course of experimental renovascular hypertension.

The urinary output of gamma-glutamyl-transpeptidase (gamma GT) and of N-acetyl-beta-D-glucosaminidase (NAG) was studied with "two-kidney Goldblatt hypertension' 3-6, 16-19 and 30-33 weeks after eliciting high blood pressure. gamma GT excretion rate of normotensive males was higher than that of females, while the activity of the renal tissue was on the same level. gamma GT output of hypertensive males was elevated in the early and in the middle stages of the disease, it was subnormal in the late stage. In females gamma GT output increased in animals with excessively high blood pressure (less than 200 mm Hg) only. gamma GT output correlated with the tissue activity in males only. In all animals there was an inverse, linear correlation between tissue gamma GT activity and the hydroxyproline content. The pattern of the NAG output was similar to that of gamma GT, however, excretion of NAG showed no sex differences and remained high in the late stage of the disease, too. Nephrosclerosis was less pronounced in female Goldblatt rats than in males.

Quantitative structure-pharmacokinetic relationships derived on antibacterial sulfonamides in rats and its comparison to quantitative structure-activity relationships.

Quantitative structure-pharmacokinetic relationships have been derived for a series of substituted 2-sulfapyridines. Pharmacokinetic parameters, such as elimination rate constant (ke), clearance (Cl), and protein-binding constant (Kassoc), have been determined in rats. The observed variation is statistically significant, explained by changes in the lipophilic (deltaRm), electronic (pKa), and steric effects (I, ES) of the substituents. The obtained correlations are discussed with respect to the previously derived correlations for the antibacterial activity of these compounds. A scale up of the results opens up the possibility of a rational synthesis of highly active sulfonamides with special pharmacokinetic properties because lipophilicity influences strongly the pharmacokinetic properties, whereas no influence on the degree of antibacterial effect is observed. Steric substituent influence is opposite on specific binding to bacterial enzymes and unspecific binding to serum proteins.