Herman Meisner

Ochratoxin A, an in vivo inhibitor of renal phosphoenolpyruvate carboxykinase.

Abstract Ochratoxin A, a nephrotoxin produced as a secondary metabolite by A. ochraceus, is a potent inhibitor of renal PEPCK activity, in vivo. When fed orally to rats for 2 days, renal PEPCK activity is reduced 50% by a total dose of 0.3-0.5 mg toxin. Renal gluconeogenic capacity is reduced only after PEPCK activity is inhibited by 50%. Hepatic PEPCK activity is unaffected up to 1.5-2.0 mg ochratoxin A, which were the highest doses tested. Other enzymes located in proximal convoluted tubules, including phosphatedependent glutaminase, γ-glutamyl transpeptidase, pyruvate carboxylase, and Na,K-ATPase, are not affected. Renal protein synthesis from [3H]phenylalanine or [3H]leucine is inhibited 30–40% by ochratoxin A in vivo. By covalently coupling the toxin to albumin with carbodiimide or mixed anhydride, the inhibitory effect on renal PEPCK activity is retained, but protein synthesis is not affected and cytological evidence of nephrotoxicity is lost. Injection of the ochratoxin A-albumin carbodiimide complex results in a decrease of hepatic PEPCK activity as well. Removal of the phenylalanine group from the toxin prevents the in vivo inhibition of PEPCK activity, as well as protein synthesis. We conclude that the decrease in renal PEPCK activity, in vivo, requires the phenylalanine group of ochratoxin A, and occurs by a mechanism independent of the known nephrotoxicity effects.

Decrease of renal phosphoenolpyruvate carboxykinase RNA and poly(A)+ RNA level by ochratoxin A

Ochratoxin A, a nephrotoxin produced by Aspergillus ochraceus, decreases the activity of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in the cytosol of rat kidneys, as well as inhibits renal gluconeogenesis (Meisner, H., and Meisner, P. (1981) Arch. Biochem. Biophys. 208, 146-153). Ochratoxin A greatly reduces the level of translatable mRNA for PEPCK in kidneys of rats fed the toxin for 2 days, while the efficiency of translation of poly(A)+ RNA is not affected. A species of poly(A)+ RNA coding for a 72,000 Mr protein is increased in relative amount. Although similar in molecular weight to PEPCK, this protein is not precipitated by an antibody to PEPCK, and has a different peptide map. The sequence abundance of PEPCK mRNA, as measured by either Northern blotting or the dot blot technique, is reduced, while the hepatic level of PEPCK mRNA is not changed. The total poly(A)+ RNA level is reduced 50% in kidneys, but not livers, of rats fed a standard dose of ochratoxin A for 3-5 days. In nuclei isolated from toxin-fed rats, the rate of transcription of total RNA or PEPCK mRNA, as measured by incorporation of [32P]UTP, is not reduced by the toxin. Ochratoxin A therefore lowers total renal mRNA concentration, and certain species, notably PEPCK, are reduced to a greater extent than the bulk of the RNA pool.

Energy-dependent uptake of ochratoxin A by mitochondria

Abstract The interaction of ochratoxin A, a mycotoxin produced by Aspergillus ochraceus , with isolated rat liver mitochondria and plasma membranes has been studied. Cell membranes bind [ 14 C]ochratoxin A poorly and do not show saturation in the concentration range examined. The uptake of the toxin by mitochondria is saturable, with an apparent K m at 0 °C of 30 nmol/mg of protein. Sonication or freeze-thawing reduces the extent of incorporation by 88%. Ochratoxin A uptake is energy dependent, resulting in a depletion of intramitochondrial ATP. Uncouplers such as m -chlorocarbonylcyanide phenylhydrazone or the respiratory inhibitors rotenone and antimycin A inhibit uptake 60–85%, while ATP reverses the antimycin and rotenone inhibition. Phosphate transport is sensitive to inhibition by the toxin, as measured by Ca 2+ plus P i stimulated respiration and [ 32 P]P i incorporation. In turn, phosphate inhibits nearly completely [ 14 C]ochratoxin A uptake at 22 °C and causes a concomitant mitochondrial swelling yet is not incorporated into the matrix space. Thus, the saturable uptake of ochratoxin A is accompanied by a decrease in the energy state and inhibition of P i transport, which results in deteriorative changes of the mitochondria, as evidenced by large-amplitude swelling.

Hormonal Regulation of Phosphoenolpyruvate Carboxykinase Gene Expression

Glucose homeostasis in all vertebrates involves the integration of a number of metabolic processes that are under hormonal control. A key element in this process is hepatic and renal gluconeogenesis, which provides the major source of blood glucose during starvation.(1) Tissues such as the red blood cells, brain, and kidney medulla are metabolically dependent on a continued source of glucose, a fact that underlines the importance of proper regulation of gluconeogenesis for the survival of the organism. It is not therefore surprising that a number of hormones, including insulin, glucagon, glucocorticoids, and thyroid hormone, all play a role in coordinating glucose homeostasis by controlling gluconeogenesis.

Cyclic AMP and the synthesis of phosphoenolpyruvate carboxykinase (GTP) mRNA

The regulation of cytosolic phosphoenolpyruvate carboxykinase RNA by cyclic-AMP-dependent hormones is discussed, with special emphasis on transcriptional control in isolated nuclei. A model is proposed to account for stimulation of phosphoenolpyruvate carboxykinase RNA synthesis by cyclic AMP.

Effect of gentamicin on the subcellular distribution of renal β-N-acetylglucosaminidase activity

Abstract The effect of gentamicin on lysosomal enzyme activity in renal cortex of Sprague-Dawley rats was examined. In rats given 60 mg gentamicin/kg subcutaneously for 8 days, renal β - N -acetylglucosaminidase activity increased by 57 per cent while cathepsin B 1 and dipeptidylpeptidase I activities did not change. The specific activity of glucosaminidase in partially purified lysosomes, however, was only 10 per cent higher. The subcellular distribution of glucosaminidase activity in gentamicin-treated rats shifted towards the less dense mitochondrial and 10,000 g supernatant fractions, which showed a 55 and 80 per cent increase, respectively, of total activity. It is concluded that gentamicin caused an increased activity, and reduced latency, of lysosomal glucosaminidase.

Displacement of palmitate from albumin by chlorophenoxyisobutyrate

The effect of chlorophenoxyisobutyrate, a hypolipidemic drug that decreases plasma free fatty acids, triglycerides, and cholesterol, on the partitioning of [14C]-palmitate between hexane and bovine serum albumin was studied at 37°. In this system, hexane served as a hydrophobic trap for free fatty acids displaced from BSA by chlorophenoxyisobutyrate, allowing less than 0.3% to remain in the aqueous phase. As the concentration of chlorophenoxy isobutyrate was raised from 0.4 to 3.2 mM, there was a progressive displacement of palmitate from the [14C]-palmitate-BSA complex into hexane, the magnitude being dependent on the initial V value (moles palmitate bound/mole BSA). Beginning with [14C]-palmitate in hexane, chlorophenoxyisobutyrate (2 mM) decreased the moles palmitate bound/mole of BSA by 16% at V = 0.2, and 34% at V = 3.0.

Changes of renal mRNA species abundance by ochratoxin A

Ochratoxin A is a nephrotoxin produced by certain species of Aspergillus and Penicillium. We have found previously that renal but not hepatic P-enolpyruvate carboxykinase, and the mRNA for this enzyme, are rapidly decreased in rats and swine fed 0.1 to 1 mg/kg body weight for a few days. In the present study, we isolated kidney mRNA from rats fed ochratoxin A for 2-5 days. By screening a rat kidney cDNA library with [32P]RNA, we have identified several renal mRNAs whose concentration is changed within 2 days by the toxin. The transcription rate of each mRNA was measured in nuclei isolated from kidneys of rats fed ochratoxin A. The incorporation of [32P]UMP into P-enolpyruvate carboxykinase mRNA and the synthesis of other RNAs were not affected. Therefore, the toxin changes mRNA abundance at the post-transcriptional level.