Role of L-lactate as an energy substrate in primary rat podocytes under physiological and glucose deprivation conditions.

Lactate has long been acknowledged to be a metabolic waste product, but it has more recently been found as a fuel energy source in mammalian cells. Podocytes are an important component of the glomerular filter, and their role in maintaining the structural integrity of this structure was established. These cells rely on a constant energy supply and reservoir. The utilization of alternative energy substrates to preserve energetic homeostasis is a subject of extensive research, and lactate appears to be one such candidate. Therefore, we investigated the role of lactate as an energy substrate and characterize the lactate transport system in cultured rat podocytes during sufficient and insufficient glucose supplies. The present study, for the first time, demonstrated the presence of lactate transporters in podocytes. Moreover, we observed modified the amount of these transporters in response to limited glucose availability and after l-lactate supplementation. Simultaneously, exposure to l-lactate preserved cell survival during insufficient glucose supply. Interestingly, during glucose deprivation, lactate exposure allowed the steady flow of glycolysis and prevented glycogen reserves depletion. Summarizing, podocytes utilize lactate as an energy substrate and possess a developed system that controls lactate homeostasis, suggesting that it plays an essential role in podocyte metabolism, especially during fluctuations of energy availability.

Insights into the regulation of podocyte and glomerular function by lactate and its metabolic sensor G-protein-coupled receptor 81.

Podocytes and their foot processes are an important cellular layer of the renal filtration barrier that is involved in regulating glomerular permeability. Disturbances of podocyte function play a central role in the development of proteinuria in diabetic nephropathy. The retraction and effacement of podocyte foot processes that form slit diaphragms are a common feature of proteinuria. Correlations between the retraction of foot processes and the development of proteinuria are not well understood. Unraveling peculiarities of podocyte energy metabolism notably under diabetic conditions will provide insights into the pathogenesis of diabetic nephropathy. Intracellular metabolism in the cortical area of podocytes is regulated by glycolysis, whereas energy balance in the central area is controlled by oxidative phosphorylation and glycolysis. High glucose concentrations were recently reported to force podocytes to switch from mitochondrial oxidative phosphorylation to glycolysis, resulting in lactic acidosis. In this review, we hypothesize that the lactate receptor G-protein-coupled receptor 81 (also known as hydroxycarboxylic acid receptor 81) may contribute to the control of podocyte function in both health and disease.

The cAMP Inducers Modify N-Acetylaspartate Metabolism in Wistar Rat Brain.

Neuronal N-acetylaspartate production appears in the presence of aspartate N-acetyltransferase (NAT8L) and binds acetyl groups from acetyl-CoA with aspartic acid. Further N-acetylaspartate pathways are still being elucidated, although they seem to involve neuron-glia crosstalk. Together with N-acetylaspartate, NAT8L takes part in oligoglia and astroglia cell maturation, myelin production, and dopamine-dependent brain signaling. Therefore, understanding N-acetylaspartate metabolism is an emergent task in neurobiology. This project used in in vitro and in vivo approaches in order to establish the impact of maturation factors and glial cells on N-acetylaspartate metabolism. Embryonic rat neural stem cells and primary neurons were maturated with either nerve growth factor, trans-retinoic acid or activators of cAMP-dependent protein kinase A (dibutyryl-cAMP, forskolin, theophylline). For in vivo, adult male Wistar rats were injected with theophylline (20 mg/kg b.w.) daily for two or eight weeks. Our studies showed that the N-acetylaspartate metabolism differs between primary neurons and neural stem cell cultures. The presence of glia cells protected N-acetylaspartate metabolism from dramatic changes within the maturation processes, which was impossible in the case of pure primary neuron cultures. In the case of differentiation processes, our data points to dibutyryl-cAMP as the most prominent regulator of N-acetylaspartate metabolism.

Neither Excessive Nitric Oxide Accumulation nor Acute Hyperglycemia Affects the N-Acetylaspartate Network in Wistar Rat Brain Cells.

The N-acetylaspartate network begins in neurons with N-acetylaspartate production catalyzed by aspartate N-acetyltransferase from acetyl-CoA and aspartate. Clinical studies reported a significant depletion in N-acetylaspartate brain level in type 1 diabetic patients. The main goal of this study was to establish the impact of either hyperglycemia or oxidative stress on the N-acetylaspartate network. For the in vitro part of the study, embryonic rat primary neurons were treated by using a nitric oxide generator for 24 h followed by 6 days of post-treatment culture, while the neural stem cells were cultured in media with 25-75 mM glucose. For the in vivo part, male adult Wistar rats were injected with streptozotocin (65 mg/kg body weight, ip) to induce hyperglycemia (diabetes model) and euthanized 2 or 8 weeks later. Finally, the biochemical profile, NAT8L protein/Nat8l mRNA levels and enzymatic activity were analyzed. Ongoing oxidative stress processes significantly affected energy metabolism and cholinergic neurotransmission. However, the applied factors did not affect the N-acetylaspartate network. This study shows that reduced N-acetylaspartate level in type 1 diabetes is not related to oxidative stress and that does not trigger N-acetylaspartate network fragility. To reveal why N-acetylaspartate is reduced in this pathology, other processes should be considered.

The Impact of Acetyl-CoA and Aspartate Shortages on the N-Acetylaspartate Level in Different Models of Cholinergic Neurons.

N-acetylaspartate is produced by neuronal aspartate N-acetyltransferase (NAT8L) from acetyl-CoA and aspartate. In cholinergic neurons, acetyl-CoA is also utilized in the mitochondrial tricarboxylic acid cycle and in acetylcholine production pathways. While aspartate has to be shared with the malate-aspartate shuttle, another mitochondrial machinery together with the tricarboxylic acid cycle supports the electron transport chain turnover. The main goal of this study was to establish the impact of toxic conditions on N-acetylaspartate production. SN56 cholinergic cells were exposed to either Zn2+ overload or Ca2+ homeostasis dysregulation and male adult Wistar rats' brains were studied after 2 weeks of challenge with streptozotocin-induced hyperglycemia or daily theophylline treatment. Our results allow us to hypothesize that the cholinergic neurons from brain septum prioritized the acetylcholine over N-acetylaspartate production. This report provides the first direct evidence for Zn2+-dependent suppression of N-acetylaspartate synthesis leading to mitochondrial acetyl-CoA and aspartate shortages. Furthermore, Zn2+ is a direct concentration-dependent inhibitor of NAT8L activity, while Zn2+-triggered oxidative stress is unlikely to be significant in such suppression.

Insufficient harmonization of antibiotics assays - Polish experience with an external quality assessment program in the years 2011-2018.

INTRODUCTION: Treatment with vancomycin and gentamycin requires strict monitoring of its serum concentration for proper dosage optimization. This study aimed to assess the quality and the harmonization of antibiotics assays in Polish laboratories. MATERIALS AND METHODS: 413 results of vancomycin and 148 results of gentamycin assays obtained from Polish laboratories in 30 international external quality assessment (EQA) surveys carried out from March 2011 to May 2018 were analyzed. RESULTS: Interlaboratory robust coefficients of variation (rCVs) in particular surveys comprised between 1.3 and 47.2% for vancomycin, and between 1.8 and 34.2% for gentamycin. The percentage of the results with the difference above acceptable limit ±10% from the target value established for own method group was 25.7% for vancomycin and 25.6% for gentamycin. When the difference was established according to target value for all methods, the percentage of results outside the acceptable limit was 2-fold higher on average (54.8% for vancomycin and 43.2% for gentamycin). The comparison of target values for methods revealed statistically significant differences between analytical systems used (p < .0001). The highest difference was 40% for vancomycin and 12% for gentamycin. CONCLUSIONS: The present analysis revealed high dispersion of the antibiotics assays results in Polish laboratories. Moreover, vancomycin and gentamycin results differed significantly in a way dependent on the analytical system used. There appear to be an urgent need for harmonization of methods used for vancomycin and gentamycin measurement.

Forced Degradation Studies of Ivabradine and In Silico Toxicology Predictions for Its New Designated Impurities.

All activities should aim to eliminate genotoxic impurities and/or protect the API against degradation. There is a necessity to monitor impurities from all classification groups, hence ivabradine forced degradation studies were performed. Ivabradine was proved to be quite durable active substance, but still new and with insufficient stability data. Increased temperature, acid, base, oxidation reagents and light were found to cause its degradation. Degradation products were determined with the usage of HPLC equipped with Q-TOF-MS detector. Calculations of pharmacological and toxicological properties were performed for six identified degradation products. Target prediction algorithm was applied on the basis of Hyperpolarization-activated cyclic nucleotide-gated cation channels, as well as more general parameters like logP and aqueous solubility. Ames test and five cytochromes activities were calculated for toxicity assessment for selected degradation products. Pharmacological activity of photodegradation product (UV4), which is known as active metabolite, was qualified and identified. Two other degradation compounds (Ox1 and N1), which were formed during degradation process, were found to be pharmacologically active.

A comparative quantitative structure-retention relationships study for lipophilicity determination of compounds with a phenanthrene skeleton on cyano-, reversed phase-, and normal phase-thin layer chromatography stationary phases.

The phenanthrene skeleton is an important moiety in medical chemistry as it is present in steroidal drugs used as anti-inflammatory and anti-asthmatic agents as well as synthetic hormones or potassium sparing diuretics. Chromatographic properties of 14 derivatives containing the phenanthrene skeleton in their structure with known lipophilicity have been studied. NP, RP, and cyano-bonded silica stationary phases with three binary mobile phases (acetonitrile-water, acetone-water, and acetone-petroleum ether) were tested. Obtained chromatographic data were correlated with the lipophilicity expressed as values of log partition coefficient (P). The presented study was undertaken to find the best TLC system and chromatographic data processing method in order to predict log P values. Correlations between chromatographic data and measurements of lipophilicity of compounds were presented as results of established quantitative structure-retention relationships. Principal component analysis and cluster analysis were used to investigate the similarities among chromatographic systems.