The Role of PKGIα and AMPK Signaling Interplay in the Regulation of Albumin Permeability in Cultured Rat Podocytes.

The permeability of the glomerular filtration barrier (GFB) is mainly regulated by podocytes and their foot processes. Protein kinase G type Iα (PKGIα) and adenosine monophosphate-dependent kinase (AMPK) affect the contractile apparatus of podocytes and influence the permeability of the GFB. Therefore, we studied the interplay between PKGIα and AMPK in cultured rat podocytes. The glomerular permeability to albumin and transmembrane FITC-albumin flux decreased in the presence of AMPK activators and increased in the presence of PKG activators. The knockdown of PKGIα or AMPK with small-interfering RNA (siRNA) revealed a mutual interaction between PKGIα and AMPK and influenced podocyte permeability to albumin. Moreover, PKGIα siRNA activated the AMPK-dependent signaling pathway. AMPKα2 siRNA increased basal levels of phosphorylated myosin phosphate target subunit 1 and decreased the phosphorylation of myosin light chain 2. Podocytes that were treated with AMPK or PKG activators were characterized by the different organization of actin filaments within the cell. Our findings suggest that mutual interactions between PKGIα and AMPKα2 regulate the contractile apparatus and permeability of the podocyte monolayer to albumin. Understanding this newly identified molecular mechanism in podocytes provides further insights into the pathogenesis of glomerular disease and novel therapeutic targets for glomerulopathies.

Hyperglycemia alters mitochondrial respiration efficiency and mitophagy in human podocytes.

Podocytes constitute the outer layer of the renal glomerular filtration barrier. Their energy requirements strongly depend on efficient oxidative respiration, which is tightly connected with mitochondrial dynamics. We hypothesized that hyperglycemia modulates energy metabolism in glomeruli and podocytes and contributes to the development of diabetic kidney disease. We found that oxygen consumption rates were severely reduced in glomeruli from diabetic rats and in human podocytes that were cultured in high glucose concentration (30 mM; HG). In these models, all of the mitochondrial respiratory parameters, including basal and maximal respiration, ATP production, and spare respiratory capacity, were significantly decreased. Podocytes that were treated with HG showed a fragmented mitochondrial network, together with a decrease in expression of the mitochondrial fusion markers MFN1, MFN2, and OPA1, and an increase in the activity of the fission marker DRP1. We showed that markers of mitochondrial biogenesis, such as PGC-1α and TFAM, decreased in HG-treated podocytes. Moreover, PINK1/parkin-dependent mitophagy was inhibited in these cells. These results provide evidence that hyperglycemia impairs mitochondrial dynamics and turnover, which may underlie the remarkable deterioration of mitochondrial respiration parameters in glomeruli and podocytes.

A comprehensive approach to the monitoring of steroidal glycoalkaloids in foods of plant origin.

Steroidal glycoalkaloids (GAs) are toxins produced by solanaceous plants. As there are no fully standardized methods for their extraction and determination in food, the research aimed to: (1) develop and critically compare methods based on gas (GC) and liquid (LC) chromatography, including their coupling with mass spectrometry, and (2) to develop and optimize a universal GA extraction method. Hyphenated techniques proved to be the most useful in GA analysis: LC-MS was the most sensitive one, while GC-MS offered the highest chromatographic resolution. It was proven that quantitative results obtained using different analytical techniques cannot be directly compared. New extraction method that is more efficient than the AOAC method (997.13) was then designed and optimized. It was characterized by higher absolute recovery (99% and 34%, respectively) and allowed to extract much more GAs from the same material (e.g. 21.2 ± 1.4 and 11.82 ± 0.97 mg g-1 of potato tubers, respectively).

Isolation of atropine and scopolamine from plant material using liquid-liquid extraction and EXtrelut® columns.

Tropane alkaloids are toxic secondary metabolites produced by Solanaceae plants. Among them, plants from Datura genus produce significant amounts of scopolamine and hyoscyamine; the latter undergoes racemization to atropine during isolation. Because of their biological importance, toxic properties and commonly reported food and animal feed contamination by different Datura sp. organs, there is a constant need for reliable methods for the analysis of tropane alkaloids in many matrices. In the current study, three extraction and sample-clean up procedures for the determination of scopolamine and atropine in plant material were compared in terms of their effectiveness and repeatability. Standard liquid-liquid extraction (LLE) and EXtrelut® NT 3 columns were used for the sample clean-up. Combined ultrasound-assisted extraction and 24h static extraction using ethyl acetate, followed by multiple LLE steps was found the most effective separation method among tested. However, absolute extraction recovery was relatively low and reached 45-67% for atropine and 52-73% for scopolamine, depending on the compound concentration. The same method was also the most effective one for the isolation of target compounds from Datura stramonium leaves. EXtrelut® columns, on the other hand, displayed relatively low effectiveness in isolating atropine and scopolamine from such a complex matrix and hence could not be recommended. The most effective method was also applied to the extraction of alkaloids from roots and stems of D. stramonium. Quantitative analyses were performed using validated method based on gas chromatography with flame ionization detector (GC-FID). Based on the results, the importance of the proper selection of internal standards in the analysis of tropane alkaloids was stressed out.

Evaluation of four derivatization methods for the analysis of fatty acids from green leafy vegetables by gas chromatography.

Green leafy vegetables are valuable secondary sources of nutrients, including lipids, commonly consumed in developing countries. However, method development for the analysis of fatty acids is usually focused on the animal lipid samples, rarely including natural plant extracts. Hence, the usefulness of four derivatization methods for the gas chromatographic analysis of plant lipids was studied. Methylation using 10% solution of BF3 in methanol and 2.0M solution of (trimethylsilyl)diazomethane (TMSD) in hexane, trimethylsilylation and tert-butyldimethylsilylation were compared using lipid standards and extracts from the leaves of Solanum macrocarpon and S. melongena after saponification. While silylation was found effective and precise using lipid standards, it initially did not perform well in the analysis of plant lipids due to the presence of transesterification products in samples. Optimization of the hydrolysis conditions resulted in an effective analysis of these derivatives, but poor separation of FA(18:0) from unsaturated FA(18:X) compounds and the presence of larger amounts of interferences disqualified the use silylation for the analysis of plant fatty acids in applied analytical conditions. Methylation using TMSD gave more precise quantitative results when compared to BF3/MeOH method. Also, it produced a significantly lower amount of interferences when applied to plant lipid samples. Additionally, the TMSD-based method is simple, safe and less time-consuming when compared to other procedures. Thus, we suggest using TMSD-based methylation as a method of choice in the GC analysis of plant-derived fatty acids.