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.
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 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.