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.

Analysis of XBP1 Contribution to Hyperosmolarity-Induced Lipid Synthesis.

The unfolded protein response (UPR) is a complex network of intracellular pathways that transmits signals from ER lumen and/or ER bilayer to the nuclear compartment in order to activate gene transcription. UPR is activated by the loss of ER capacities, known as ER stress, and occurs to restore ER properties. In this regard, glycerolipid (GL) synthesis activation contributes to ER membrane homeostasis and IRE1α-XBP1, one UPR pathway, has a main role in lipogenic genes transcription. Herein, we describe the strategy and methodology used to evaluate whether IRE1α-XBP1 pathway regulates lipid metabolism in renal epithelial cells subjected to hyperosmolar environment. XBP1s activity was hindered by blocking IRE1α RNAse activity and by impeding its expression; under these conditions, we determined GL synthesis and lipogenic enzymes expression.

Sequential and synchronized hypertonicity-induced activation of Rel-family transcription factors is required for osmoprotection in renal cells.

NF-κB and TonEBP belong to the Rel-superfamily of transcription factors. Several specific stimuli, including hypertonicity which is a key factor for renal physiology, are able to activate them. It has been reported that, after hypertonic challenge, NF-κB activity can be modulated by TonEBP, considered as the master regulator of transcriptional activity in the presence of changes in environmental tonicity. In the present work we evaluated whether hypertonicity-induced gene transcription mediated by p65/RelA and TonEBP occurs by an independent action of each transcription factor or by acting together. To do this, we evaluated the expression of their specific target genes and cyclooxygenase-2 (COX-2), a common target of both transcription factors, in the renal epithelial cell line Madin-Darby canine kidney (MDCK) subjected to hypertonic environment. The results herein indicate that hypertonicity activates the Rel-family transcription factors p65/RelA and TonEBP in MDCK cells, and that both are required for hypertonic induction of COX-2 and of their specific target genes. In addition, present data show that p65/RelA modulates TonEBP expression and both colocalize in nuclei of hypertonic cultures of MDCK cells. Thus, a sequential and synchronized action p65/RelA → TonEBP would be necessary for the expression of hypertonicity-induced protective genes.

Coordinate regulation between the nuclear receptor peroxisome proliferator-activated receptor-γ and cyclooxygenase-2 in renal epithelial cells.

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors involved in lipid metabolism and glucose utilization, in cell growth, differentiation and apoptosis, and in the regulation of pro-inflammatory genes expression such as cyclooxygenase-2 (COX-2). PPARγ is the main isoform in the renal inner medulla where it is believed to possess nephroprotective actions. In this kidney zone, COX-2 acts as an osmoprotective gene and its expression is modulated by changes in interstitial osmolarity. In the present work we evaluated whether hyperosmolar-induced COX-2 expression is modulated by PPARγ in renal epithelial cells MDCK subjected to high NaCl medium. The results presented herein show that ligand-activated PPARγ repressed COX-2 expression. But more important, the present findings show that hyperosmolar medium decreased PPARγ protein and increases the PPARγ phosphorylated form, which is inactive. ERK1/2 and p38 activation precedes PPARγ disappearance and induced-COX-2 expression. Therefore, the decrease in PPARγ expression is required for hyperosmotic induction of COX-2. We also found that PGE2, the main product of COX-2 in MDCK cells, induced these changes in PPARγ protein. Our results may alert on the long term use of thiazolidinediones (TZD) since they could affect renal medullary function that depends on COX-2 for cellular protection against osmotic stress.

Environmental hyperosmolality regulates phospholipid biosynthesis in the renal epithelial cell line MDCK.

Hyperosmolality is a key signal for renal physiology. On the one hand, it contributes to the differentiation of renal medullary structures and to the development of the urinary concentrating mechanism. On the other, it is a stress factor. In both cases, hyperosmolality activates processes that require an adequate extension of cellular membranes. In the present work, we examined whether hyperosmolality regulates phospholipid biosynthesis, which is needed for the membrane biogenesis in the renal epithelial cell line Madin-Darby canine kidney (MDCK). Because phospholipids are the structural determinants of all cell membranes, we evaluated their content, synthesis, and regulation in MDCK cultures subjected to different hyperosmotic concentrations of NaCl, urea, or both. Hyperosmolality increased phospholipid content in a concentration-dependent manner. Such an effect was exclusively due to changes in NaCl concentration and occurred at the initial stage of hyperosmolar treatment concomitantly with the expression of the osmoprotective protein COX-2. The hypertonic upregulation of phosphatidylcholine (PC) synthesis, the main constituent of all cell membranes, involved the transcriptional activation of two main regulatory enzymes, choline kinase (CK) and cytidylyltransferase α (CCTα) and required ERK1/2 activation. Considering that physiologically, renal medullary cells are constantly exposed to high and variable NaCl, these findings could contribute to explaining how renal cells could maintain cellular integrity even in a nonfavorable environment.