17beta-Estradiol attenuates diabetic kidney disease by regulating extracellular matrix and transforming growth factor-beta protein expression and signaling

Abstract

We previously showed that supplementation with 17beta-estradiol (E2) from the onset of diabetes attenuates the development of diabetic renal disease. The aim of the present study was to examine whether E2 can also attenuate the disease process once it has developed. The present study was performed in nondiabetic and streptozotocin-induced diabetic Sprague-Dawley rats. E2 supplementation began after 9 wk of diabetes and continued for 8 wk. Diabetes was associated with an increase in urine albumin excretion, glomerulosclerosis, tubulointerstitial fibrosis, renal cortical collagen type I and IV, laminin, plasminogen activator inhibitor-1, tissue inhibitors of metalloproteinase-1 and -2, transforming growth factor (TGF)-beta, TGF-beta receptor type I and II, Smad2/3, phosphorylated Smad2/3, and Smad4 protein expression, and CD68-positive cell abundance. Decreases in matrix metalloproteinase (MMP)-2 protein expression and activity and decreases in Smad6 and Smad7 protein expression were also associated with diabetes. E2 supplementation completely or partially attenuated all these changes, except Smad4 and fibronectin, on which E2 supplementation had no effect. These data suggest that E2 attenuates the progression of diabetic renal disease once it has developed by regulating extracellular matrix, TGF-beta, and expression of its downstream regulatory proteins. These findings support the notion that sex hormones in general, and E2 in particular, are important regulators of renal function and may be novel targets for the treatment and prevention of diabetic renal disease.

Fig. 1

Glomerulosclerosis (A) and tubulointerstitial fibrosis (B). A. PAS-stained sections of the renal cortex. Abbreviations: glomerulus (g), mesangial expansion (arrow head). B. Masson’s trichrome-stained sections of the renal cortex. Original magnification X400.

Fig. 2

Collagen type I and type IV renal cortical immunolocalization and protein expression. Immunohistochemistry: abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g), mesangial cells (arrow head). Original magnification X400. Western blotting: top panel, representative gels of collagen type I and type IV protein expression. Bottom panel, densitometric scans of collagen type I and type IV protein levels in relative optical density (ROD) expressed as a ratio of collagen type I/Coomassie blue and collagen type IV/Coomassie blue. Data are expressed as mean±SEM.

Fig. 3

Laminin and fibronectin renal cortical immunolocalization and protein expression. Immunohistochemistry: abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g), mesangial cells (arrow head), tubulointerstitial areas (arrow). Original magnification X400. Western blotting: top panel, representative gels of laminin and fibronectin protein expression. Bottom panel, densitometric scans of laminin and fibronectin protein levels in relative optical density (ROD) expressed as a ratio of laminin/Coomassie blue and fibronectin/Coomassie blue. Data are expressed as mean±SEM.

Fig. 4

Matrix metalloproteinase-2 (MMP-2) and MMP-9 renal cortical immunolocalization, protein expression and activity. Immunohistochemistry: abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g). Original magnification X400. Western blotting and zymography: top panel, representative gels of MMP-2 and MMP-9 protein expression. Bottom panel, densitometric scans of MMP-2 and MMP-9 protein levels in relative optical density (ROD) expressed as a ratio of MMP-2/Coomassie blue and MMP-9/Coomassie blue. Data are expressed as mean±SEM.

Fig. 5

Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), TIMP-2 and plasminogen activator inhibitor-1 (PAI-1) renal cortical immunolocalization and protein expression. Immunohistochemistry: abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g). Original magnification X400. Western blotting: top panel, representative gels of TIMP-1, TIMP-2 and PAI-1 protein expression. Bottom panel, densitometric scans of TIMP-1, TIMP-2 and PAI-1 protein levels in relative optical density (ROD) expressed as a ratio of TIMP-1/Coomassie blue, TIMP-2/Coomassie blue and PAI-1/Coomassie blue. Data are expressed as mean±SEM.

Fig. 6

TGF-β, TGF-βRI and TGF-βRII immunolocalization. Abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g), mesangial cells (arrow head). Original magnification X400.

Fig. 7

TGF-β, TGF-βRI and TGF-βRII protein expression. Top panel, representative gels of TGF-β, TGF-βRI and TGF-βRII protein expression. Bottom panel, densitometric scans of TGF-β, TGF-βRI and TGF-βRII protein levels in relative optical density (ROD) expressed as a ratio of TGF-β/Coomassie blue, TGF-βRI/Coomassie blue and TGF-βRII/Coomassie blue. Data are expressed as mean±SEM.

Fig. 8

Smad2/3, pSmad2/3 and Smad4 immunolocalization. Abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g), mesangial cells (arrow head). Original magnification X400.

Fig. 9

Smad2/3, pSmad2/3 and Smad4 protein expression. Top panel, representative gels of Smad2/3, pSmad2/3 and Smad4 protein expression. Bottom panel, densitometric scans of Smad2/3, pSmad2/3 and Smad4 protein levels in relative optical density (ROD) expressed as a ratio of Smad2/3/Coomassie blue, pSmad2/3/Coomassie blue and Smad4/Coomassie blue. Data are expressed as mean±SEM.

Fig. 10

Smad6 and Smad7 immunolocalization and protein expression. Immunohistochemistry: abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g). Original magnification X400. Western blotting: top panel, representative gels of Smad6 and Smad7 protein expression. Bottom panel, densitometric scans of Smad6 and Smad7 protein levels in relative optical density (ROD) expressed as a ratio of Smad6/Coomassie blue and Smad7/Coomassie blue. Data are expressed as mean±SEM.

Fig. 11

Smurf2 protein expression. A. Immunohistochemistry: abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g), selected cells in the proximal tubule positive for Smurf2 (arrow head). Original magnification X400. B. Western blotting: top panel, representative gels of Smurf2 protein expression. Bottom panel, densitometric scans of Smurf2 protein levels in relative optical density (ROD) expressed as a ratio of Smurf2/Coomassie blue. Data are expressed as mean±SEM.

Fig. 12

CD68 immunostaining and abundance. A. Immunohistochemistry: abbreviations: proximal tubule (pt), distal tubule (dt), glomerulus (g). Original magnification X400. B. Quantitative analysis of CD68-positive cells expressed as the number of CD68-positive cells per mm². Data are expressed as mean±SEM.