Kidney growth,hypertrophy and the unifying mechanism of diabetic complications

Summary. Michael Brownlee has proposed a ‘Unifying Mechanism’ of hyperglycemia-induced damage in diabetes mellitus. At the crux of this hypothesis is the generation of reactive oxygen species (ROS), and their impact on glycolytic pathways. Diabetes is the leading cause of chronic kidney failure. In the early phase of diabetes, prior to establishment of proteinuria or fibrosis, comes kidney growth and hyperfiltration. This early growth phase consists of an early period of hyperplasia followed by hypertrophy. Hypertrophy also contributes to cellular oxidative stress, and may precede the ROS perturbation of glycolytic pathways described in the Brownlee proposal. This increase in growth promotes hyperfiltration, and along with the hypertrophic phenotype appears required for hyperglycemia-induced cell damage and the progression of downstream diabetic complications. Here we will evaluate this growth phenomenon in the context of diabetes mellitus.     

Polyamines are unevenly distributed within the rat and rabbit kidney

Summary. Aliphatic polyamines have generally been measured on the whole kidney. Since the kidney is composed of a variety of cells, whole organ data are of limited value for the interpretation of the functions of the polyamines. The aim of this study was to establish the distribution pattern of putrescine, spermidine and spermine within the kidneys of male and female rats and rabbits. It is shown that the polyamines are unevenly distributed along the cortico-papillary axis. Each amine exhibited its own distinct distribution pattern. The polyamines are predominantly located in the cortex. Putrescine levels increased gradually from the cortex to the papillary tip in rabbits, whereas, in rats, fluctuations in putrescine level were marked. In the six zones of the rabbit kidney studied, spermidine and spermine concentrations were markedly higher in females than in males. This difference was less marked in rats. Received April 1, 1999, Accepted May 17, 1999     

DNA cleavage function of seryl-histidine dipeptide and its application

Summary. Previously published evidences highlighted the effect of transglutaminase (TG, EC 2.3.2.13) activation on the reduction of the in vitro adhesive and invasive behaviour of murine B16-F10 melanoma cells, as well as in vivo. Here, we investigated the influence of spermidine (SPD) incorporation by TG into basement membrane components i.e. laminin (LN) or Matrigel (MG), on the adhesion and invasion of B16-F10 melanoma cells by these TG/SPD-modified substrates. The adhesion assays showed that cell binding to the TG/SPD-modified LN was reduced by 30%, when compared to untreated LN, whereas the reduction obtained using TG/SPD-modified MG was 35%. Similarly, tumor cell invasion by the Boyden chamber system through TG/SPD modified LN or MG was respectively reduced by 45%, and by 69%. Evaluation of matrix metalloproteinase (gelatinases MMP-2 and MMP-9) activities by gel-zymography showed that MMP-2 activity was unaffected, while MMP-9 activity was reduced by about 32% using TG/SPD-modified substrate. These results strongly suggest that the observed antiinvasive effect of TG activation in the host may be ascribed to the covalent incorporation of polyamines, which led to the post-translational modification of some components of the cell basement membrane. This modification may interfere with the metastatic property of melanoma cells, affecting the proteolytic activity necessary for their migration and invasion activities. Authors’ address: Simone Beninati, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, I-00133 Rome, Italy     

Effects of D-amino acids on lipoperoxidation in rat liver and kidney mitochondria

Summary. The effects of the amino acids D-ser, D-asp, and D-ala on lipoperoxidation under conditions of hypertension, alcoholism, and ammonemia in rat liver and kidney mitochondria were studied. Under normal conditions, D-alanine increased in 54% free radicals production in liver mitochondria (p < 0.05). The D-amino acids had no effect on kidney mitochondria. D-ser and D-ala increased lipoperoxidation in spontaneously hypertensive rats (SHR) as compared with their normotensive genetic control Wistar-Kyoto (WKY) rats (p < 0.05). During hypertension and in oxidative stress in the presence of calcium, only D-ala produced 46% and 29% free radicals in liver and kidney mitochondria (p < 0.05), respectively. During chronic alcoholism, D-ser increased lipoperoxidation in 80% in kidney mitochondria (p < 0.05), as compared to control. During ammonemia, D-ser produced 41% free radicals.     

Identification of the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney

Summary. Glucocorticoid hormones enhance the reabsorptive capacity of filtered amino acids in rat kidney, as it was shown in previous in vivo clearance experiments. In the present study, the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney was investigated using in vivo micropuncture technique. Adult female Wistar rats were treated with dexamethasone (DEX), and fractional excretion of L-glutamine (L-Gln) and L-leucine (L-Leu) were determined and related to inulin after microinfusion into different nephron segments. DEX reduced fractional excretion of both neutral amino acids as a sign of enhanced reabsorptive capacity. The site of main DEX action on L-Leu reabsorption has been localized in the proximal straight tubule. However, in the case of L-Gln, the inhibition of γ-glutamyltranspeptidase (γ-GT) by administration of acivicin indicated the importance of this brush border enzyme in reduced L-Gln excretion. DEX enhanced γ-GT activity by tubular acidification. It can be presumed a DEX-inducible transport system for neutral amino acids mainly localized in proximal straight tubules of rat kidney. Received July 8, 1999     

Polyamine contents in current foods: a basis for polyamine reduced diet and a study of its long term observance and tolerance in prostate carcinoma patients

Summary. Our aim was to develop a liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS²) method to measure free amino acid (FAA) and dipeptide (DP) concentrations in biological fluids. We synthesized chloroformate derivatives of FAA and DP, identified the major precursor ions and used LCMS² to obtain the most intense product ions. Using serial dilutions of unlabeled and labeled standards ([²H₃]-L-Dopa, homoarginine, homophenylalanine, [¹⁵N]-Glutamine and [²H₃]-methionine), we observed linear relationships in MS response that we used to calculate the amounts of FAA and DP in biological samples. This method is sensitive with a limit of detection (LOD) for most of the FAAs and DPs tested in the 0.05–1 pmol range and is linear over 3–5 orders of magnitude when many metabolites were measured simultaneously. Reproducibility and between run or daily variations were <10% for most FAAs and DPs. We applied this method to human samples and quantitatively measured 21 FAAs and 2 DPs in 200 μl CSF, 31 FAAs and 6 DPs in 100 μl plasma, and 23 FAAs and 5 DPs in 200 μl urine. These data demonstrate the potential for using LCMS² to discover changes in FAA and DP metabolic pathways that occur during disease pathogenesis.     

Enhancement of transglutaminase activity and polyamine depletion in B16-F10 melanoma cells by flavonoids naringenin and hesperitin correlate to reduction of the <Emphasis Type="Italic">in vivo</Emphasis> metastatic potential

Summary. The in vitro and in vivo effects of two flavonons, naringenin (NG) and hesperitin (HP) on the proliferation rate of highly metastatic murine B16-F10 melanoma cell were investigated. NG or HP treatment of melanoma cells produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, spermidine and spermine and the enhancement of transglutaminase (TGase, EC 2.3.2.13) activity. Orally administered NG or HP in C57BL6/N mice inoculated with B16-F10 cells affected the pulmonary invasion of melanoma cells in an in vivo metastatic assay. The number of lung metastases detected by a computerized image analyzer was reduced, compared to untreated animals, by about 69% in NG-treated mice and by about 36% in HP-treated mice. Survival studies showed that 50% of the NG-treated animals died 38 ± 3.1 days after tumor cell injection (control group: 18 ± 1.5 days) and HP-treated mice died 27 ± 2.3 days after cell inoculation. Taken together, these findings provide further evidences for the potential anticancer properties of dietary flavonoids as chemopreventive agents against malignant melanoma.     

Protective effect of gamma-aminobutyric acid (GABA) against cytotoxicity of ethanol in isolated rat hepatocytes involves modulations in cellular polyamine levels

Summary. Gamma-aminobutyric acid (GABA) is considered to be a multifunctional molecule with various physiological effects throughout the body. It is also evident that the liver contains GABA and its transporter. However, the functions of GABA in liver have not been well documented. In this study, the cytoprotective effect of GABA against ethanol-induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. Addition of ethanol induced decrease of cell viability in a dose-dependent manner. However, treatment with GABA resulted in a dose-dependent recovery from ethanol (150 mM)-induced cytotoxicity. GABA reversed the ethanol-induced decrease in intracellular polyamine levels. Furthermore, the addition of polyamines also reversed the ethanol-induced decrease of cell viability. These results suggest that GABA is protective against the cytotoxicity of ethanol in isolated rat hepatocytes and this effect may be modulated by the maintenance of intracellular polyamine levels.     

Mathematical modeling of the onset of capillary formation initiating angiogenesis

It is well accepted that neo-vascular formation can be divided into three main stages (which may be overlapping): (1) changes within the existing vessel, (2) formation of a new channel, (3) maturation of the new vessel. In this paper we present a new approach to angiogenesis, based on the theory of reinforced random walks, coupled with a Michaelis-Menten type mechanism which views the endothelial cell receptors as the catalyst for transforming angiogenic factor into proteolytic enzyme in order to model the first stage. In this model, a single layer of endothelial cells is separated by a vascular wall from an extracellular tissue matrix. A coupled system of ordinary and partial differential equations is derived which, in the presence of an angiogenic agent, predicts the aggregation of the endothelial cells and the collapse of the vascular lamina, opening a passage into the extracellular matrix. We refer to this as the onset of vascular sprouting. Some biological evidence for the correctness of our model is indicated by the formation of teats in utero. Further evidence for the correctness of the model is given by its prediction that endothelial cells will line the nascent capillary at the onset of capillary angiogenesis. Received: 27 May 1999 / Revised version: 28 December 1999 / Published online: 16 February 2001     

Effects of amino acid-derived luminal metabolites on the colonic epithelium and physiopathological consequences

Summary. Depending on the amount of alimentary proteins, between 6 and 18 g nitrogenous material per day enter the large intestine lumen through the ileocaecal junction. This material is used as substrates by the flora resulting eventually in the presence of a complex mixture of metabolites including ammonia, hydrogen sulfide, short and branched-chain fatty acids, amines; phenolic, indolic and N-nitroso compounds. The beneficial versus deleterious effects of these compounds on the colonic epithelium depend on parameters such as their luminal concentrations, the duration of the colonic stasis, the detoxication capacity of epithelial cells in response to increase of metabolite concentrations, the cellular metabolic utilization of these metabolites as well as their effects on colonocyte intermediary and oxidative metabolism. Furthermore, the effects of metabolites on electrolyte movements through the colonic epithelium must as well be taken into consideration for such an evaluation. The situation is further complicated by the fact that other non-nitrogenous compounds are believed to interfere with these various phenomenons. Finally, the pathological consequences of the presence of excessive concentrations of these compounds are related to the short- and, most important, long-term effects of these compounds on the rapid colonic epithelium renewing and homeostasis.     

Polyamines and abiotic stress: recent advances

Summary. In this review we will concentrate in the results published the last years regarding the involvement of polyamines in the plant responses to abiotic stresses, most remarkably on salt and drought stress. We will also turn to other types of abiotic stresses, less studied in relation to polyamine metabolism, such as mineral deficiencies, chilling, wounding, heavy metals, UV, ozone and paraquat, where polyamine metabolism is also modified. There is a great amount of data demonstrating that under many types of abiotic stresses, an accumulation of the three main polyamines putrescine, spermidine and spermine does occur. However, there are still many doubts concerning the role that polyamines play in stress tolerance. Several environmental challenges (osmotic stress, salinity, ozone, UV) are shown to induce ADC activity more than ODC. The rise in Put is mainly attributed to the increase in ADC activity as a consequence of the activation of ADC genes and their mRNA levels. On the other hand, free radicals are now accepted as important mediators of tissue injury and cell death. The polycationic nature of polyamines, positively charged at physiological pH, has attracted the attention of researchers and has led to the hypothesis that polyamines could affect physiological systems by binding to anionic sites, such as those associated with nucleic acids and membrane phospholipids. These amines, involved with the control of numerous cellular functions, including free radical scavenger and antioxidant activity, have been found to confer protection from abiotic stresses but their mode of action is not fully understood yet. In this review, we will also summarize information about the involvement of polyamines as antioxidants against the potential abiotic stress-derived oxidative damage. Authors’ address: Dr. María Patricia Benavides, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Buenos Aires 1113, Argentina     

Polyamines and thiols in the cytoprotective effect of L-cysteine and L-methionine on carbon tetrachloride-induced hepatotoxicity

Summary. The relationship between cellular glutathione (GSH), protein-SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamines on the cytoprotective ability of L-cysteine and L-methionine, the most important components in the sulfur amino acid metabolic pathway, in carbon tetrachloride (CCl₄)-induced toxicity in isolated rat hepatocytes was studied. CCl₄ induced a LDH release and decreased cellular thiols and polyamines levels but treatment with L-cysteine and L-methionine reversed these decreases. Treating with methylglyoxal bis-(guanylhydrazone), MGBG, an irreversible inhibitor of S-adenosylmethionine decarboxylase, which is a key enzyme in spermidine and spermine biosynthesis, and therefore used to deplete cellular polyamines, prevented the protective effect of L-cysteine and L-methionine, but the addition of exogenous polyamines inhibited the influence of MGBG. These results suggest that the cytoprotective effect of L-cysteine and L-methionine in CCl₄-induced toxicity were via maintenance of cellular polyamines, GSH and protein-SH concentrations and prevention of LDH leakage. Received September 1, 1999, Accepted January 11, 2000     

Transport of L-proline,L-proline-containing peptides and related drugs at mammalian epithelial cell membranes

Summary. Membrane transport of L-proline has received considerable attention in basic and pharmaceutical research recently. Of the most recently cloned members of the solute carrier family, two are “proline transporters”. The amino acid transporter PAT1, expressed in intestine, kidney, brain and other organs, mediates the uptake of proline and derivatives in a pH gradient-dependent manner. The Na⁺-dependent proline transporter SIT1, cloned in 2005, exhibits the properties of the long-sought classical IMINO system. Proline-containing peptides are of interest for several reasons. Many biologically important peptide sequences contain highly conserved proline residues. Xaa-Pro peptides are very often resistant to enzymatic hydrolysis and display, in contrast to Pro-Xaa peptides, a high affinity to the H⁺/peptide cotransporter PEPT1 which is expressed in intestinal, renal, lung and biliary duct epithelial cells. Furthermore, several orally available drugs are recognized by PEPT1 as Xaa-Pro analogues due to their sterical resemblance to small peptides.     

Amino acid transport in the renal proximal tubule

Summary. In the kidney the proximal tubule is responsible for the uptake of amino acids. This occurs via a variety of functionally and structurally different amino acid transporters located in the luminal and basolateral membrane. Some of these transporters show an ion-dependence (e.g. Na⁺, Cl⁻ and K⁺) or use an H⁺-gradient to drive transport. Only a few amino acid transporters have been cloned or functionally characterized in detail so far and their structure is known, while little is known about a majority of amino acid transporters. Only few attempts have been untertaken looking at the regulation of amino acid transport. We summarized more recent information on amino acid transport in the renal proximal tubule emphasizing functional and regulatory aspects. Received August 8, 1999; Accepted April 20, 2000     

Ectopic expression of maize knotted1 results in the cytokinin-autotrophic growth of cultured tobacco tissues

The ectopic expression of knotted homologues has cytokinin-like effects on plant morphology. The functional relationship between knotted and cytokinins was investigated in cultures of leaf tissue established from tobacco (Nicotiana tabacum L. cv. Havana 425) plants transformed with the maize knotted1 (kn1) gene regulated by cauliflower mosaic virus 35S RNA expression signals. In contrast to leaf tissues of untransformed plants, leaf tissues of kn1 transformants were capable of sustained, cytokinin-autotrophic growth on auxin-containing medium and resembled the tobacco cytokinin-autotrophic mutants Hl-1 and Hl-2. The concentration of 18 cytokinins was measured in cultures initiated from leaves of three independent kn1 transformants and the Hl-1 and Hl-2 mutants. Although cytokinin contents were variable, the content of several cytokinins in Kn1, Hl-1 and Hl-2 tissue lines was at least 10-fold higher than that of wild-type tobacco tissues and in the range reported for other cytokinin-autotrophic tobacco tissues. These results suggest that the cytokinin-autotrophic growth of Kn1 lines could result from elevated steady-state levels of cytokinins. Received: 7 July 1999 / Accepted: 10 November 1999     

When quinones meet amino acids: chemical, physical and biological consequences

Summary. Quinones and amino acids are usually compartmentally separated in living systems, however there are several junctions in which they meet, react and influence. It occurs mainly in wounded, cut or crushed plant material during harvest, ensiling or disintegrating cells. Diffusing polyphenols are oxidized by polyphenol oxidases (PPOs) to quinonic compounds, which associate reversibly or irreversibly with amino acids and proteins. The reaction takes place with the free nucleophilic functional groups such as sulfhydryl, amine, amide, indole and imidazole substituents. It results in imine formation, in 1,4-Michael addition via nitrogen or sulphur and in Strecker degradation forming aldehydes. The formation and activity of quinone–amino acids conjugates influences the colour, taste, and aroma of foods. Physical and physiological phenomena such as browning of foods, discoloration of plants during processing, alteration of solubility and digestibility, formation of humic substances, germicidal activity, cytotoxicity and more occur when quinones from disintegrating cells meet amino acids. The mechanisms of toxicity and the pathways by which PCBs may be activated and act as a cancer initiator include oxidation to the corresponding quinones and reaction with amino acids or peptides. Sclerotization of insect cuticle is a biochemical process involving also the reaction between quinones and amino acid derivatives.     

Antizyme and antizyme inhibitor activities influence cellular responses to polyamine analogs

Summary. Close structural analogs of spermidine and spermine, polyamine mimetics, are potential chemotheraputic agents as they depress cellular polyamines required for tumor growth. Specific mimetic analogs stimulate synthesis of the regulatory protein antizyme (AZ), which not only inactivates the initial enzyme in polyamine biosynthesis but also inhibits cellular uptake of polyamines. The role of AZ induction in influencing cellular uptake of representative analogs was investigated using three analogs produced by Cellgate Inc., CGC-11047, CGC-11102, and CGC-11144, which exhibit markedly distinct AZ-inducing potential. An inverse correlation was noted between the AZ-inducing activity of a compound and the steady-state levels accumulated in cells. As some tumor cells over express AZI as a means of enhancing the polyamines required for aggressive growth, analog sensitivity was examined in transgenic CHO cells expressing exogenous antizyme inhibitor protein (AZI). Although AZI over expression increased cell sensitivity to analogs, the degree of this affect varied with the analog used.     

Development of an expression macroarray for amine metabolism-related genes

Summary. Cationic amino acids are the precursors of biogenic amines, histamine from histidine, and putrescine, spermidine and spermine from arginine/ornithine (and methionine), as well as nitric oxide. These amines play important biological roles in inter- and intracellular signaling mechanisms related to inflammation, cell proliferation and neurotransmission. Biochemical and epidemiological relationships between arginine-derived products and histamine have been reported to play important roles in physiopathological problems. In this communication, we describe the construction of an expression macroarray containing more than 30 human probes for most of the key proteins involved in biogenic amines metabolisms, as well as other inflammation- and proliferation-related probes. The array has been validated on human mast HMC-1 cells. On this model, we have got further support for an inverse correlation between polyamine and histamine synthesis previously observed on murine basophilic models. These tools should also be helpful to understand the amine roles in many other inflammatory and neoplastic pathologies. The first two authors contributed equally to this work.