Tl(I) and Tl(III) induce reticulum stress in MDCK cells.

The effects of the exposure of proliferating MDCK cells to thallium [Tl(I) or Tl(III)] on cell viability and proliferation were investigated. Although Tl stopped cell proliferation, the viability was > 95%. After 3 h, two autophagy markers (SQSTM-1 expression and LC3ß localization) were altered, and at 48 h increased expression of SQSTM-1 (60%) and beclin-1 (50-100%) were found. At 24 h, the expression of endoplasmic reticulum (ER) stress markers ATF-6 and IRE-1 were increased in 100% and 150%, respectively, accompanied by XBP-1 splicing and nuclear translocation. At 48 h, major ultrastructure abnormalities were found, including ER enlargement and cytoplasmic vacuolation which was not prevented by protein synthesis inhibition. Increased PHB (85% and 40% for Tl(I) and Tl(III), respectively) and decreased ß-tubulin (45%) expression were found which may be related to the promotion of paraptosis. In summary, Tl(I) and Tl(III) promoted ER stress and probably paraptosis in MDCK cells, impairing their proliferation.

Dynamics of differentiated-renal epithelial cell monolayer after calcium oxalate injury: The role of cyclooxygenase-2.

AIMS: Calcium oxalate (Oxa), constituent of most common kidney stones, damages renal tubular epithelial cells leading to kidney disease. Most in vitro studies designed to evaluate how Oxa exerts its harmful effects were performed in proliferative or confluent non-differentiated renal epithelial cultures; none of them considered physiological hyperosmolarity of renal medullary interstitium. Cyclooxygenase 2 (COX2) has been associated to Oxa deleterious actions; however, up to now, it is not clear how COX2 acts. In this work, we proposed an in vitro experimental system resembling renal differentiated-epithelial cells that compose medullary tubular structures which were grown and maintained in a physiological hyperosmolar environment and evaluated whether COX2 â†’ PGE2 axis (COX2 considered a cytoprotective protein for renal cells) induces Oxa damage or epithelial restitution. MAIN METHODS: MDCK cells were differentiated with NaCl hyperosmolar medium for 72 h where cells acquired the typical apical and basolateral membrane domains and a primary cilium. Then, cultures were treated with 1.5 mM Oxa for 24, 48, and 72 h to evaluate epithelial monolayer restitution dynamics and COX2-PGE2 effect. KEY FINDINGS: Oxa completely turned the differentiated phenotype into mesenchymal one (epithelial-mesenchymal transition). Such effect was partially and totally reverted after 48 and 72 h, respectively. Oxa damage was even deeper when COX2 was blocked by NS398. PGE2 addition restituted the differentiated-epithelial phenotype in a time and concentration dependence. SIGNIFICANCE: This work presents an experimental system that approaches in vitro to in vivo renal epithelial studies and, more important, warns about NSAIDS use in patients suffering from kidney stones.

TAG synthesis and storage under osmotic stress. A requirement for preserving membrane homeostasis in renal cells.

Hyperosmolarity is a controversial signal for renal cells. It can induce cell stress or differentiation and both require an active lipid metabolism. We showed that hyperosmolarity upregulates phospholipid (PL) de novo synthesis in renal cells. PL synthesis requires fatty acids (FA), usually stored as triglycerides (TAG). PL and TAG de novo synthesis utilize the same initial biosynthetic route: sn-glycerol 3P (G3P) → phosphatidic acid (PA) → diacylglycerol (DAG). In the present work, we evaluate how such pathway contributes to PL and TAG synthesis in renal cells subjected to hyperosmolarity. Our results show an increase in PA and DAG formation under hyperosmotic conditions; augmented DAG production, due to lipin enzyme activity, lead to the increase of both TAG and PL. However, at early stages (24 and 48 h), most of the de novo synthesized DAG was directed to PL synthesis; longer treatments downregulated PL synthesis and the DAG formed was mainly driven to TAG synthesis. Hyperosmolarity induced ACC and FASN transcription which mediated FA de novo synthesis. New FA molecules were stored in TAG. Silencing experiments revealed that hyperosmotic-induction of lipin-1 and -2 was mediated by SREBP1. Interestingly, SREBP1 knockdown also dropped SREBP2, indicating a modulatory action between both isoforms. Impairing SREBP activity leads to a decline in TAG levels but not PL. Membrane homeostasis is maintained through the adequate PL synthesis and renewal and constitute a protective mechanism against hyperosmolarity. The present data reveal the relevance of TAG synthesis and storage for PL synthesis in renal cells.