title: Diabetes mellitus increases endothelin-1 gene transcription in rat kidney.
abstract: BACKGROUND
  Mesangial cell hypertrophy and increased extracellular matrix (ECM) contribute to mesangial expansion in early progressive diabetic nephropathy. Previous studies suggest that the growth factor endothelin-1 (ET-1) is not only up-regulated in diabetes, but may mediate the effects of hyperglycemia on mesangial cell hypertrophy and ECM synthesis. In models of diabetes mellitus, the mechanisms underlying increased ET-1 peptide and mRNA remain unknown. Therefore, our purpose is to determine whether ET-1 gene activity increases in kidneys of streptozotocin (SZT)-treated rats.
  
  
  METHODS
  Male Sprague-Dawley rats were injected with either SZT or vehicle. Parameters including glucose, body weight, 24-hour urine volume, urinary protein, and urinary ET-1 excretion were recorded. All rats were sacrificed at 12 weeks postinjection. Prepro-ET-1 mRNA from whole kidneys was determined using both RNase protection and reverse transcription-polymerase chain reaction (RT-PCR). The abundance of ET-1 peptide in primary cultured mesangial cells was detected by indirect immunofluorescence following treatment with 5.6, 11.2, or 22.5 mmol/L D-glucose for 24 hours. Cellular ET-1 mRNA was measured using RT-PCR in control cells at time 0 and also following exposure to increasing concentrations of glucose for 24 hours. Rat mesangial cells were transfected with a luciferase reporter construct containing the rat ET-1 promoter (pET1. Luc), and relative ET-1 promoter activity was measured after a 24-hour exposure to 5.6 and 22.5 mmol/L of D- or L-glucose.
  
  
  RESULTS
  After 12 weeks of hyperglycemia, diabetic rats gained less weight (344 +/- 23.9 vs. 548.75 +/- 15.08 g), had increased urinary volume (158.6 +/- 24.32 vs. 8.38 +/- 1.56 mL/day), and had marked proteinuria (101.7 +/- 12.2 vs. 14.1 +/- 2.8 mg/day) compared with controls. Total urinary ET-1 peptide increased 26.4-fold in diabetic versus control rats (17.5083 +/- 5.405 vs. 0.6635 +/- 0.343 ng/day). ET-1 mRNA extracted from whole rat kidneys was increased 2.1-fold in diabetic versus control animals. Primary cultured rat mesangial cells demonstrated a significant increase in immunofluorescence labeling of ET-1 peptide and ET-1 mRNA in response to increasing concentrations of glucose. Furthermore, transfected mesangial cells exposed to 22.5 mmol/L D-glucose showed a 1.6-fold increase in ET-1 promoter activity relative to those treated with 5.6 mmol/L glucose.
  
  
  CONCLUSIONS
  Glucose increases ET-1 gene expression in the kidney of the SZT-treated rat model of diabetes mellitus. Furthermore, high glucose induces ET-1 expression in primary cultured rat mesangial cells and directly enhances ET-1 promoter activity. The greater relative increase in peptide compared with transcription suggests the potential participation of other mechanisms such as increased mRNA stability, protein stability, and/or enhanced translational efficiency.
authors.affiliation: Departments of Medicine and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: G M
authors.initials: GM
authors.lastname: Hargrove
authors.forename: J
authors.initials: J
authors.lastname: Dufresne
authors.forename: C
authors.initials: C
authors.lastname: Whiteside
authors.forename: D A
authors.initials: DA
authors.lastname: Muruve
authors.forename: N C
authors.initials: NC
authors.lastname: Wong
mesh_terms: D000818:Animals; D001786:Blood Glucose; D001835:Body Weight; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000503:physiopathology* / Q000652:urine; D003928:Diabetic Nephropathies / Q000503:physiopathology* / Q000652:urine; D019332:Endothelin-1 / Q000235:genetics* / Q000652:urine; D016232:Endothelins / Q000235:genetics; D005109:Extracellular Matrix / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D006943:Hyperglycemia / Q000503:physiopathology / Q000652:urine; D008297:Male; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D011498:Protein Precursors / Q000235:genetics; D011507:Proteinuria / Q000503:physiopathology / Q000652:urine; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection; D014556:Urine; D014661:Vasoconstriction / Q000502:physiology
chemical_list: D001786:Blood Glucose; D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D012333:RNA, Messenger; D005947:Glucose