Diabetes mellitus increases endothelin-1 gene transcription in rat kidney

Background Mesangial cell hypertrophy and increased extracellular matrix (E CM) contribute to mesangial expansion in early progressive diabetic nephrop athy. Previous studies suggest that the growth factor endothelin-1 (ET-1) i s not only upregulated in diabetes, but may mediate the effects of hypergly cemia on mesangial cell hypertrophy and ECM synthesis. In models of diabete s mellitus, the mechanisms underlying increased ET-1 peptide and mRNA remai n unknown. Therefore. our purpose is to determine whether ET-1 gene activit y 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 p rotein, and urinary ET-1 excretion were recorded. All rats were sacrificed at 12 weeks postinjection. Prepro-ET-1 mRNA from whole kidneys was determin ed using both RNase protection and reverse transcription-polymerase chain r eaction (RT-PCR). The abundance of ET-1 peptide in primary cultured mesangi al cells was detected by indirect immunofluorescence following treatment wi th 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 exposu re to increasing concentrations of glucose for 24 hours. Rat mesangial cell s 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. 513.75 +/- 15.08 g), had increased urinary volume (158.6 +/- 24.32 vs. 5.38 +/- 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 pe ptide increased 26.4-fold in diabetic versus control rats (17.5083 +/- 5.40 5 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 immunofluoresce nce labeling of ET-1 peptide and ET-1 mRNA in response to increasing concen trations 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 re lative to those treated with 5.6 mmol/L glucose. Conclusion. 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 enhanc es ET-1 promoter activity. The greater relative increase in peptide compare d with transcription suggests the potential participation of other mechanis ms such as increased mRNA stability, protein stability, and/or enhanced tra nslational efficiency.