ABSTRACT
Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.
Subject(s)
Calcium/metabolism , Cell Membrane/drug effects , Epithelial Cells/drug effects , Lysophospholipids/pharmacology , Plasma Membrane Calcium-Transporting ATPases/genetics , Receptors, Lysophosphatidic Acid/genetics , Animals , Cell Fractionation , Cell Membrane/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Estrenes/pharmacology , Gene Expression Regulation , Ion Transport/drug effects , Isoxazoles/pharmacology , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Naphthalenes/pharmacology , Phosphorylation/drug effects , Plasma Membrane Calcium-Transporting ATPases/antagonists & inhibitors , Plasma Membrane Calcium-Transporting ATPases/metabolism , Primary Cell Culture , Propionates/pharmacology , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/genetics , Protein Kinase C/metabolism , Pyrrolidinones/pharmacology , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysophosphatidic Acid/metabolism , Signal Transduction , Swine , Tetradecanoylphorbol Acetate/pharmacology , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/genetics , Type C Phospholipases/metabolismABSTRACT
The mechanisms involved in renal repair by mesenchymal stromal cells (MSCs) are not entirely elucidated. The paracrine secretion of bioactive molecules has been implicated in the protective effects. Besides soluble mediators, MSCs have been shown to release extracellular vesicles (EVs), involved in renal repair process for different injury models. EVs have been shown to mediate communication between cells through the transference of several molecules, like protein, bioactive lipids, mRNA, and microRNAs (miRNAs). The miRNAs are noncoding RNAs that posttranscriptionally modulate gene expression and are involved in the regulation of several cellular processes, including those related to repair. The aim of the present study was to investigate the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTECs) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion. In this model we evaluated whether changes in miRNA expression were dependent on direct miRNA transfer or on transcription induction by MSC-EVs. The obtained results showed an enhanced incorporation of MSC-EVs in injured PTECs with protection from cell death. This biological effect was associated with EV-mediated miRNA transfer and with transcriptional modulation of miRNAs expressed by injured PTECs. Prediction of miRNA targets showed that miRNAs modulated in PTECs are involved in process of renal recovery with downregulation of coding-mRNAs associated with apoptosis, cytoskeleton reorganization, and hypoxia, such as CASP3 and 7, SHC1 and SMAD4. In conclusion, these results indicate that MSC-EVs may transfer and modulate the expression of several miRNAs involved in the repair and recovery process in PTECs.
