title: Osmotic expression of aldose reductase in retinal pigment epithelial cells: involvement of NFAT5.
abstract: BACKGROUND
  Diabetic retinopathy is associated with osmotic stress resulting from hyperglycemia and intracellular sorbitol accumulation. Systemic hypertension is a risk factor of diabetic retinopathy. High intake of dietary salt increases extracellular osmolarity resulting in systemic hypertension. We determined the effects of extracellular hyperosmolarity, chemical hypoxia, and oxidative stress on the gene expression of enzymes involved in sorbitol production and conversion in cultured human retinal pigment epithelial (RPE) cells.
  
  
  METHODS
  Alterations in the expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH) genes were examined with real-time RT-PCR. Protein levels were determined with Western blot analysis. Nuclear factor of activated T cell 5 (NFAT5) was knocked down with siRNA.
  
  
  RESULTS
  AR gene expression in RPE cells was increased by high (25 mM) extracellular glucose, CoCl2(150 μM)-induced chemical hypoxia, H2O2(20 μM)-induced oxidative stress, and extracellular hyperosmolarity induced by addition of NaCl or sucrose. Extracellular hyperosmolarity (but not hypoxia) also increased AR protein level. SDH gene expression was increased by hypoxia and oxidative stress, but not extracellular hyperosmolarity. Hyperosmolarity and hypoxia did not alter the SDH protein level. The hyperosmotic AR gene expression was dependent on activation of metalloproteinases, autocrine/paracrine TGF-β signaling, activation of p38 MAPK, ERK1/2, and PI3K signal transduction pathways, and the transcriptional activity of NFAT5. Knockdown of NAFT5 or inhibition of AR decreased the cell viability under hyperosmotic (but not hypoxic) conditions and aggravated the hyperosmotic inhibition of cell proliferation.
  
  
  CONCLUSIONS
  The data suggest that sorbitol accumulation in RPE cells occurs under hyperosmotic, but not hypoxic and oxidative stress conditions. NFAT5- and AR-mediated sorbitol accumulation may protect RPE cells under conditions of osmotic stress.
authors.affiliation: Department of Ophthalmology and Eye Hospital, Faculty of Medicine, University of Leipzig, Liebigstrasse 10-14, D-04103, Leipzig, Germany.
authors.forename: Anica
authors.initials: A
authors.lastname: Winges
authors.affiliation: Department of Ophthalmology and Eye Hospital, Faculty of Medicine, University of Leipzig, Liebigstrasse 10-14, D-04103, Leipzig, Germany.
authors.forename: Tarcyane Barata
authors.initials: TB
authors.lastname: Garcia
authors.affiliation: Department of Ophthalmology and Eye Hospital, Faculty of Medicine, University of Leipzig, Liebigstrasse 10-14, D-04103, Leipzig, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Prager
authors.affiliation: Department of Ophthalmology and Eye Hospital, Faculty of Medicine, University of Leipzig, Liebigstrasse 10-14, D-04103, Leipzig, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Wiedemann
authors.affiliation: Department of Ophthalmology and Eye Hospital, Faculty of Medicine, University of Leipzig, Liebigstrasse 10-14, D-04103, Leipzig, Germany.|Helios Klinikum Aue, Aue, Germany.
authors.forename: Leon
authors.initials: L
authors.lastname: Kohen
authors.affiliation: Department of Ophthalmology and Eye Hospital, Faculty of Medicine, University of Leipzig, Liebigstrasse 10-14, D-04103, Leipzig, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bringmann
authors.affiliation: Department of Ophthalmology and Eye Hospital, Faculty of Medicine, University of Leipzig, Liebigstrasse 10-14, D-04103, Leipzig, Germany. hollbm@medizin.uni-leipzig.de.
authors.forename: Margrit
authors.initials: M
authors.lastname: Hollborn
mesh_terms: D000449:Aldehyde Reductase / Q000096:biosynthesis / Q000235:genetics*; D015153:Blotting, Western; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003930:Diabetic Retinopathy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D007064:L-Iditol 2-Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D050778:NFATC Transcription Factors; D009994:Osmolar Concentration; D018384:Oxidative Stress; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000473:pathology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
chemical_list: C118125:NFAT5 protein, human; D050778:NFATC Transcription Factors; D014157:Transcription Factors; D012313:RNA; D007064:L-Iditol 2-Dehydrogenase; D000449:Aldehyde Reductase
keywords: Diabetic retinopathy; Hypoxia; Osmolarity; Retinal pigment epithelium; Sorbitol