Chrysin Ameliorates Cyclosporine-A-Induced Renal Fibrosis by Inhibiting TGF-ß1-Induced Epithelial-Mesenchymal Transition.

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial-mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK1 cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-ß1 signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3ß pathway. Inhibition of both pathways diminished the cytosolic accumulation of ß-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-ß1-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.

Elevated glucose concentration in culture media decreases membrane trafficking of SGLT2 in LLC-PK1 cells via a cAMP/PKA-dependent pathway.

Na+-dependent glucose reabsorption in the renal proximal tubule is dynamically regulated by changes in blood glucose levels. There is, however, a disparity in reports studying the relationship between hyperglycemia and Na+-glucose-linked transporter (SGLT) function and expression. Similarly, manipulation of the glucose content in growth media of cultured renal cells has been shown to influence SGLT activity. In this investigation, SGLT activity was significantly lower in proximal tubule LLC-PK1 cells cultured in medium containing 17.5 than 5 mM glucose. α-Methyl d-glucopyranoside (AMG) transport kinetics showed reduced apparent Vmax and Km in cells grown in 17.5 mM glucose. SGLT2 was identified as the isoform responsible for glucose transport, and protein expression analyses showed decreased apical membrane localization of SGLT2 in cells grown in 17.5 mM glucose, explaining the reduced activity. Multiple signaling pathways have been implicated in regulation of SGLT activity and trafficking. Elevated media glucose decreased intracellular cAMP and PKA activation, leading to decreased SGLT2 trafficking into the plasma membrane, which was reversed after treatment with 1 µM forskolin. The effects of media glucose on SGLT activity were found to be dependent on p38 MAPK activation due to PKA-mediated signaling. Glucose-modulated AMG uptake is reversible and was associated with altered SGLT2 membrane trafficking and cAMP alterations. In summary, elevated glucose concentrations in culture medium decrease SGLT activity in LLC-PK1 cells by reducing membrane trafficking of SGLT2 via decreasing intracellular cAMP, resulting in a lowered PKA-dependent phosphorylation of p38 MAPK.

Acetoacetate enhances oxidative metabolism and response to toxicants of cultured kidney cells.

Cultured kidney cells maintained in conventional growth media with high glucose levels exhibit increased glycolytic activity compared to the cells in vivo. In contrast, renal proximal tubules utilize substrates such as ketone bodies and rely on mitochondrial oxidative phosphorylation. LLC-PK1 cells maintain many features of the proximal tubule but are exposed to glucose concentrations ranging from 17 to 25 mM. This may impact their reliability in predicting mitochondrial toxicity. This study is designed to test the impact of the ketone body acetoacetate on metabolic characteristics of LLC-PK1 cells. Basal respiration, maximal respiration, spare respiratory capacity and ATP-linked respiration were significantly increased in cells grown in growth medium supplemented with 5 mM acetoacetate. In contrast, glycolytic capacity, as well as glycolytic reserve were significantly reduced in the acetoacetate group. There was an increased expression in biomarkers of mitochondrial biogenesis, and an increase in mitochondrial protein expression. Cells grown in medium complemented with acetoacetate displayed a significantly lower LC50 when treated with clotrimazole and diclofenac. There was a marked increase in uncoupled respiration in the presence of diclofenac, while clotrimazole and ciprofibrate significantly decreased respiration in the acetoacetate. The results indicate that acetoacetate complemented media can alter cellular metabolism and increase sensitization to toxicants.

The Toxicology Education Summit: building the future of toxicology through education.

Toxicology and careers in toxicology, as well as many other scientific disciplines, are undergoing rapid and dramatic changes as new discoveries, technologies, and hazards advance at a blinding rate. There are new and ever increasing demands on toxicologists to keep pace with expanding global economies, highly fluid policy debates, and increasingly complex global threats to public health. These demands must be met with new paradigms for multidisciplinary, technologically complex, and collaborative approaches that require advanced and continuing education in toxicology and associated disciplines. This requires paradigm shifts in educational programs that support recruitment, development, and training of the modern toxicologist, as well as continued education and retraining of the midcareer professional to keep pace and sustain careers in industry, government, and academia. The Society of Toxicology convened the Toxicology Educational Summit to discuss the state of toxicology education and to strategically address educational needs and the sustained advancement of toxicology as a profession. The Summit focused on core issues of: building for the future of toxicology through educational programs; defining education and training needs; developing the "Total Toxicologist"; continued training and retraining toxicologists to sustain their careers; and, finally, supporting toxicology education and professional development. This report summarizes the outcomes of the Summit, presents examples of successful programs that advance toxicology education, and concludes with strategies that will insure the future of toxicology through advanced educational initiatives.