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Am J Physiol Regul Integr Comp Physiol 292: R769-R777, 2007. First published August 24, 2006; doi:10.1152/ajpregu.00375.2006 Free Article
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Sex Differences in Renal and Cardiovascular Function: Physiology and Pathophysiology

17beta-Estradiol supplementation reduces tubulointerstitial fibrosis by increasing MMP activity in the diabetic kidney

Richard W. Mankhey,1 Corinne C. Wells,1 Faizah Bhatti,1 and Christine Maric1,2

1Department of Medicine, Georgetown University Medical Center, Washington, DC; and 2Center for the Study of Sex Differences: in Health, Aging and Disease, Georgetown University Medical Center, Washington, DC

Submitted 31 May 2006 ; accepted in final form 15 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported that supplementation with 17beta-estradiol (E2) attenuates albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis in diabetic nephropathy. The present study examined the mechanisms by which E2 regulates extracellular matrix (ECM) metabolism, a process that contributes to the development of glomerulosclerosis and tubulointerstitial fibrosis. The study was performed in female nondiabetic (ND), streptozotocin-induced diabetic (D), and diabetic with E2 supplementation (D+E2) Sprague-Dawley rats for 12 wk. Diabetes was associated with an increase in the renal expression of collagen {alpha} type IV [ND, 0.22 ± 0.02; D, 0.99 ± 0.09 relative optical density (ROD); P < 0.05] and fibronectin protein (ND, 0.36 ± 0.08; D, 1.47 ± 0.08 ROD; P < 0.05), as measured by Western blot analysis. E2 supplementation partially attenuated this increase in collagen {alpha} type IV (D+E2, 0.47 ± 0.10 ROD) and fibronectin (D+E2, 0.71 ± 0.16 ROD) protein expression associated with D. Diabetes was also associated with a decrease in the expression of matrix metalloproteinase (MMP) isoform MMP-2 (ND, 0.79 ± 0.01; D, 0.62 ± 0.06 ROD; P < 0.05) and MMP-9 protein (ND, 0.49 ± 0.02; D, 0.33 ± 0.03 ROD; P < 0.05). E2 supplementation restored MMP-2 and MMP-9 protein to levels similar or even greater than in the ND kidneys (MMP-2, 0.75 ± 0.06; MMP-9, 0.73 ± 0.01 ROD). The activities of MMP-2 (ND, 7.88 ± 0.44; D, 5.60 ± 0.54 ROD; P < 0.05) and MMP-9 (ND, 29.9 ± 1.8; D, 12.9 ± 2.3 ROD; P < 0.05), as measured by zymography, were also decreased with D. E2 supplementation restored MMP-2 and MMP-9 activity to levels similar to that in ND kidneys (MMP-2, 7.66 ± 0.35; MMP-9, 21.4 ± 2.9 ROD). We conclude that E2 supplementation is renoprotective by attenuating glomerulosclerosis and tubulointerstitial fibrosis by reducing ECM synthesis and increasing ECM degradation.

diabetes; extracellular matrix; tubulointerstitial fibrosis; matrix metalloproteinases

GLOMERULOSCLEROSIS AND TUBULOINTERSTITIAL fibrosis is a prominent feature of progressive renal disease, including diabetic nephropathy (25). Both glomerulosclerosis and tubulointerstitial fibrosis are characterized by accumulation of extracellular matrix (ECM) proteins (25). This accumulation is a result of increased synthesis and/or reduced degradative rates of ECM proteins and is associated with abnormal activity of matrix metalloproteinases (MMPs), enzymes involved in ECM degradation. Thus reducing ECM synthesis and/or increasing ECM degradation is an important target for the attenuation of the progression and development of progressive renal disease, including diabetic nephropathy.

There is a growing number of experimental and clinical observational studies suggesting that supplementation with 17beta-estradiol (E2) is renoprotective in a number of renal diseases, including diabetic nephropathy. Supplementation with E2 attenuates age-related renal disease in the Dahl salt-sensitive rat (23), animal models of renal ablation (3), and chronic allograft nephropathy (4). We and others have shown that supplementation with E2 from the onset of diabetes attenuates the development of glomerulosclerosis and tubulointerstitial fibrosis in the kidney of the streptozotocin (STZ)-induced diabetic rat (22) and the db/db mouse (8). Furthermore, hormone therapy using combined estradiol and norgestrel reduces proteinuria and improves creatinine clearance in postmenopausal women with type 2 diabetes, suggesting a renoprotective effect of estrogen (36). The mechanisms by which E2 affords renoprotection have mainly been studied in vitro. In cultured mesangial cells, E2 inhibits apoptosis (5, 10, 31, 26), increases the expression of MMP-2 and MMP-9, and reduces collagen type I and type IV synthesis (12, 30, 11, 33). The aim of the present study was to examine whether E2 exerts a renoprotective effect via similar mechanisms as those observed in vivo. Specifically, we examined the effects of E2 supplementation on the expression of ECM proteins and the expression and activity of MMPs in the kidney of the STZ-induced diabetic rat.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model. Female Sprague-Dawley rats (10 wk of age) were purchased from Harlan (Madison, WI) and were maintained on a phytoestrogen-free rat chow (Harlan, Madison, WI) and allowed free access to tap water. The animals were randomly divided into three treatment groups: Nondiabetic (ND, n = 8), Diabetic (D, n = 6), and Diabetic with 17beta-estradiol supplementation (D+E2, n = 7). After an overnight fast, the animals received either a single intraperitoneal injection of 0.1 M citrate buffer (pH 4.5, ND) or 55 mg/kg streptozotocin (STZ, Sigma, St. Louis, MO) in 0.1 M citrate buffer (D and D+E2). E2 supplementation began at the onset of diabetes. Diabetic rats were supplemented with insulin (2–4 U every second day, Lantus, Aventis Pharmaceuticals, Kansas City, MO) throughout the 12 wk of the study to maintain blood glucose levels between 250 and 400 mg/dl. Every 4 wk, the animals were placed in metabolic cages for 24 h for determination of urine output and measurement of urine albumin excretion (UAE). After 12 wk of treatment, the animals were anesthetized with sodium pentobarbitol (40 mg/kg ip) and blood samples collected, by cardiac puncture, for measurement of plasma levels of E2 and serum creatinine. The right kidney was removed and the renal cortex snap frozen in liquid nitrogen for Western blot analysis and zymography. The left kidney was immersion fixed with HistoCHOICE (Amresco, Solon, OH) for morphological and immunohistochemical analyses. Uterine weights were also measured at the time of death. All experiments were performed according to the guidelines recommended by the National Institutes of Health and approved by the Georgetown University Animal Care and Use Committee.

Estrogen supplementation and plasma estradiol levels. At the time of induction of diabetes, the animals were subjected to two intraperitoneal injections (4 h apart) of deslorelin acetate (10 µg/ml, Bachem Chemicals, Torrance, CA). Deslorelin is a GnRH analog, and its administration has been reported to synchronize the estrous cycle in mammals (24), and our recent study has also shown this to be the case in rats (40). One week before death, this procedure of injection with Deslorelin acetate was repeated, and at the time of death, vaginal smears were taken to verify that all animals were at the same stage of their cycle. The diabetic animals on E2 supplementation were injected with 17beta-estradiol (5 µg/kg in 200 µl peanut oil, Sigma, St. Louis, MO) every 4 days, from the onset of diabetes, to mimic the cyclical nature of estrogen release, while animals not on E2 supplementation were injected with 200 µl peanut oil only. The dose of E2 was chosen based on our previous studies showing that this dose, when injected every 4 days, results in circulating E2 levels that are in the peak physiological range (40). Plasma E2 levels were measured by ELISA (Alpha Diagnostic, San Antonio, TX).

UAE and serum creatinine. Urine albumin concentration was determined using the Nephrat II albumin kit (Exocell, Philadelphia, PA), according to the manufacturer's protocol. UAE was calculated based on urine albumin concentration and 24 h urine output. Serum creatinine concentrations were determined using the Beckman Creatinine Analyzer II (Brea, CA.) with the modified Jaffe rate method as previously described (22).

Index of glomerulosclerosis and tubulointerstitial fibrosis. The glomerulosclerosis (GSI) and tubulointerstitial fibrosis (TIFI) were determined in periodic acid Schiff's (PAS) and Masson's trichrome-stained sections, respectively. Eighty, randomly selected glomeruli, in 10 sections, were assessed and GSI graded using a semi-quantitative scoring method as previously described (22). Sixty, randomly selected fields of view of the renal cortex (at magnification of x200), in 10 sections, were assessed and TIFI graded using a semi-quantitative scoring method as previously described (22).

Immunohistochemistry. Tissue sections were incubated with 10% nonimmune goat serum and then with antibodies against collagen {alpha} type IV (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), fibronectin (1:400, Research Diagnostics, Concord, MA), MMP2 (1:400, Oncogene Science, Cambridge, MA), MMP2 (1:400, Oncogene) at 4°C overnight. The sections were rinsed with phosphate-buffered saline, then incubated with either mouse or rabbit biotinylated IgG, and then with the avidin-biotin complex (Vectastain ABC kit, Vector Laboratories, Burlingame, CA). Positive immunoreaction was identified after incubation with 3'-3'-diaminobenzidine tetrahydrochloride dihydrate and counterstaining with Mayer's hematoxylin. Sections incubated with 10% non-immune goat serum instead of the primary antiserum were used as negative controls.

Western blot analysis. Cortical tissue samples were homogenized in a SDS sample buffer (2% SDS, 10 mM Tris·HCl [pH 6.8], 10% [vol/vol] glycerol) and centrifuged at 10,000 g for 10 min at 4°C, and the protein concentration was determined using a colorimetric assay (Bio-Rad, Hercules, CA). Homogenized samples were loaded on 7.5% SDS-PAGE gels (fibronectin), 4–15% gradient gels (collagen type I and collagen {alpha} type IV), 10% SDS-PAGE gels (MMP-2 and MMP-9), 18% SDS-PAGE gels (TIMP-1 and TIMP-2), and transferred to nitrocellulose membranes (Bio-Rad). The membranes were incubated with 5% nonfat milk in a buffer containing 0.05% Tween 20 and 150 mM NaCl in 10 mM Tris·HCl (pH 7.4) to block nonspecific reactions and then with primary antibodies, diluted in 0.1% bovine serum albumin, against fibronectin (1:1,000), collagen type I (1:1,000, Sigma), collagen {alpha} type IV (1:500), MMP-2 (1:1,000), MMP-9 (1:1,000), TIMP-1 (1:200), and TIMP-2 (1:200) at 4°C overnight. The membranes were washed and incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000 dilution in 5% nonfat milk), and proteins were visualized by enhanced chemiluminescence (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The densities of specific bands were quantitated by densitometry using Scion Image beta (ver. 4.02) software. Band densities were normalized to the total amount of protein loaded in each well, as determined by densitometric analysis of gels stained with Coomassie blue.

Zymography. Cortical tissue samples were homogenized in a buffer containing 50 mM CH3COONa and 200 mM NaCl (pH 8.5), and the protein concentration was determined using a colorimetric assay (Bio-Rad). The homogenized samples were loaded onto a 10% SDS acrylamide gel containing 1 mg/ml gelatin (Bio-Rad). After electrophoresis, the gel was incubated in renaturing buffer (Invitrogen, Carlsbad, CA) for 30 min at room temperature and then activated in developing buffer (Invitrogen) for 24 h at 37°C. Gelatinase activity was visualized by staining the gel with Coomassie blue. Bands were quantitated by densitometry using Scion image beta (ver. 4.02) software.

Statistical analysis. Data are expressed as means ± SE and were analyzed with a one-way ANOVA followed by a Newman-Keuls post-test, using the SigmaStat software. Differences were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Metabolic parameters. Diabetes (D) was associated with a modest decrease in body weight compared with nondiabetic animals (ND) (ND, 311 ± 9; D, 288 ± 11 g, P < 0.05), and this decrease was prevented with E2 supplementation (D+E2, 302 ± 11 g), Table 1. Kidney weight was modestly increased with D (ND, 0.24 ± 0.01; D, 0.27 ± 0.01 g, P < 0.05), while E2 supplementation had no effect on kidney weight (D+E2, 0.26 ± 0.01 g). When expressed as a kidney/body weight ratio, D was associated with an increase in this parameter compared with ND (ND, 0.72 ± 0.02; D, 0.92 ± 0.02 g/g, P < 0.05) and E2 supplementation partially attenuated the increase in kidney/body weight ratio (D+E2, 0.81 ± 0.02 g/g), Table 1.


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  		Table 1. Effects of E2 supplementation on metabolic and renal parameters

 
Diabetes was associated with an increase in blood glucose levels compared with ND (ND, 104.0 ± 8; D, 387 ± 21.3 mg/dl, P < 0.01) (Table 1). Blood glucose was similarly increased in the D+E2 group (348 ± 19.8 mg/dl).

Plasma estradiol levels were decreased in the D group compared with the ND (37.2 ± 4.8; D, 25.0 ± 6.4 mg, P < 0.05), while E2 supplementation increased plasma estradiol to similar levels as in the ND group (D+E2, 32.8 ± 5.2 mg) (Table 1). Similarly, uterine weights were decreased in the D group compared with the ND (ND, 421.2 ± 24.5; D, 284.3 ± 36.9 mg, P < 0.05), while E2 supplementation increased uterine weights to similar levels as in the ND group (D+E2, 412.7 ± 54.3 mg) (Table 1).

UAE and serum creatinine. Diabetes was associated with an increase in UAE compared with ND (ND, 0.28 ± 0.05; D, 4.7 ± 0.7 mg/day, P < 0.001), and this increase was prevented with E2 supplementation (D+E2, 0.38 ± 0.06 mg/day) (Table 1). No differences in serum creatinine levels were observed in any of the treatment groups (ND, 0.59 ± 0.03; D, 0.64 ± 0.07; D+E2, 0.62 ± 0.05 mg/dl) (Table 1).

Glomerulosclerosis and tubulointerstitial fibrosis. The diabetic kidneys were characterized by mild glomerulosclerosis and tubulointerstitial fibrosis, as evidenced by mesangial expansion (Fig. 1A) and deposition of extracellular matrix and the presence of inflammatory infiltrates in the tubulointerstitial spaces (Fig. 1B), respectively. Quantitative analyses of these changes showed that diabetes was associated with an increase in the GSI (ND, 0.42 ± 0.04; D, 1.26 ± 0.08 AU, P < 0.05) and TIFI (ND, 0.43 ± 0.03; D, 1.68 ± 0.20 AU, P < 0.05). E2 supplementation attenuated these changes (GSI, 0.62 ± 0.06; TIFI, 0.47 ± 0.08 AU).


Figure 1
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  		Fig. 1. Glomerulosclerosis and tubulointerstitial fibrosis in the nondiabetic (ND) and diabetic (D) rat kidney. A: periodic acid Schiff-stained sections of the renal cortex. Diabetic kidneys were characterized by moderate glomerulosclerosis (g), as evidenced by mesangial expansion (arrowhead). These changes were attenuated with E2 supplementation (D+E2). B: Masson's trichrome-stained sections of the renal cortex. Diabetic kidneys were characterized by moderate tubulointerstitial fibrosis, as evidenced by the presence of extracellular matrix deposits (blue staining, arrows) surrounding the tubules and also inflammatory infiltrates within the extracellular spaces (arrow). These changes were attenuated with E2 supplementation. Original magnification x400.

 
Collagen type I, collagen {alpha} type IV and fibronectin. In the ND kidney, collagen {alpha} type IV was immunolocalized to basement membranes of proximal and distal tubules and collecting ducts, as well as in the mesangial areas in the glomerulus (Fig, 2A). Diabetes was associated with an apparent increase in the intensity of immunostaining for collagen {alpha} type IV in these areas, and most intensely in the expanded mesangial areas. E2 supplementation resulted in a similar pattern of immunolocalization for collagen {alpha} type IV as observed in the ND kidneys (Fig. 2A). To quantitate the changes in collagen {alpha} type IV expression, we used Western blot analysis. Collagen type {alpha} type IV was detected as a monomer (41 kDa) and dimer (82 kDa). The expression of the 82 kDa band was increased in the D compared with the ND kidneys (ND, 0.22 ± 0.02; D, 0.99 ± 0.09 ROD, P < 0.05), while E2 supplementation partially attenuated this increase in collagen {alpha} type IV protein expression associated with D (D+E2, 0.47 ± 0.10 ROD). Collagen type 1 protein expression was increased in the D compared with the ND kidneys (ND, 1.75 ± 0.01; D, 2.98 ± 0.05 ROD, P < 0.01), while E2 supplementation partially attenuated this increase (D+E2, 1.85 ± 0.02 ROD).


Figure 2
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  		Fig. 2. Collagen type I and {alpha} type IV in the nondiabetic and diabetic rat kidney. A: in the nondiabetic kidney, collagen {alpha} type IV (brown staining) was observed in the basement membranes of proximal tubules (pt), distal tubules (dt), collecting ducts (cd), and mesangial areas (arrows) in the glomeruli (g). In the D kidney, the intensity of immunostaining was increased in the basement membranes of all tubules and the glomeruli with increased deposition in the expanded mesangium (arrows). This deposition was attenuated by D+E2. Original magnification x400. B: in the renal cortex of the ND kidneys, collagen {alpha} type IV protein, as measured by Western blot analysis, was detected as a monomer (41 kDa) and dimmer (82 kDa). In the D group, collagen {alpha} type IV protein expression was increased by 4.5-fold compared with the ND. This increase was attenuated with D+E2. C: in the D group, collagen type I protein expression was increased by 1.7-fold compared with the ND. This increase was attenuated with D+E2. B and C, top: representative gels of collagen type I and collagen {alpha} type IV protein expression, respectively. B and C, bottom: densitometry summaries in relative optical density (ROD) of collagen type I and collagen {alpha} type IV protein (82-kDa band) from top. Data are expressed as means ± SE.

 
Similar to collagen {alpha} type IV, fibronectin was immunolocalized to basement membranes of proximal and distal tubules and collecting ducts, and the mesangial areas in the glomerulus (Fig, 3A). In the D kidneys, an overall increase in the intensity of immunostaining for fibronectin was observed, especially in the tubulointerstitial areas, while E2 supplementation resulted in a similar pattern and intensity of immunostaining for fibronectin as observed in the ND kidneys (Fig. 3A). Western blot analysis supported the immunohistochemical observations and showed that D was associated with an increase in fibronectin protein expression (220 kDa) compared with ND (ND, 0.36 ± 0.08; D, 1.47 ± 0.08 ROD, P < 0.05), while E2 supplementation partially attenuated this increase in fibronectin protein expression associated with D (D+E2, 0.71 ± 0.16 ROD).


Figure 3
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  		Fig. 3. Fibronectin immunolocalization and protein expression in the ND and D rat kidney. A: in the ND kidney, fibronectin (brown staining) was observed in the basement membranes of pt, dt, cd, and in the Bowman's capsule (arrowheads) of the g. In the D kidney, the intensity of immunostaining was increased in the basement membranes of all tubules and the glomeruli with increased deposition in the tubulointerstitium (arrows). This deposition was attenuated by D+E2. Original magnification x400. B: in the renal cortex of the D kidneys, fibronectin protein expression was increased by 4.1-fold compared with the ND. This increase was attenuated with D+E2. Top: representative gels of fibronectin protein expression. Bottom: densitometry summary in relative optical density (ROD) of fibronectin protein (220 kDa band) from top. Data are expressed as means ± SE.

 
MMPs and tissue inhibitors of metalloproteinases. In the ND kidney, MMP-2 was immunolocalized to proximal and distal tubules, collecting ducts and the mesangial areas in the glomerulus (Fig. 4A). A similar pattern of immunolocalization for MMP-9 was also observed in the ND kidney (Fig. 4B). Diabetes was associated with an apparent overall reduction in the intensity of immunolocalization for MMP-2 and MMP-9, though in the mesangial areas, the intensity of immunostaining for MMP-9 was apparently increased in D. In the D rats receiving E2 supplementation, the pattern of immunostaining for both MMP-2 and MMP-9 was similar to that observed in the ND kidneys.


Figure 4
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  		Fig. 4. Immunolocalization of MMP-2 and MMP-9 in the ND and D rat kidney. Top: in the ND kidney, MMP-2 (brown staining) was immunolocalized to pt, dt, cd, and mesangial areas (arrows) in the g. In the D kidney, the intensity of immunostaining was dramatically decreased in the tubules but not in the glomeruli. D+E2 restored the intensity of immunostaining for MMP-2 in the D similar to that in the ND group. Original magnification x400. Bottom: in the ND kidney, MMP-9 (brown staining) was immunolocalized to pt, dt, and cd and not detectable in the and g. In the D kidney, the intensity of immunostaining was modestly decreased in the tubules but increased in the glomeruli. D+E2 restored the intensity of immunostaining for MMP-9 in the D similar to that in the ND group. Original magnification x400.

 
Diabetes was associated with a decrease in the expression of the active form of MMP-2 protein (62 kDa) compared with ND (ND, 0.79 ± 0.01; D, 0.62 ± 0.06, ROD, P < 0.05), Fig. 5A. Similarly, MMP-9 protein (95 kDa) was decreased in the D compared with the ND kidneys (ND, 0.49 ± 0.02; D, 0.33 ± 0.03, ROD, P < 0.05), Fig. 5B. While supplementation with E2 restored MMP-2 protein to levels similar to that observed in the ND kidneys (MMP-2, 0.75 ± 0.06), MMP-9 protein levels in the D+E2 kidneys were even greater than that in the ND kidneys (MMP-9, 0.73 ± 0.01 ROD). In addition to examining the protein expression of MMPs, we also examined the levels of their activity by zymography. Diabetes (D) was associated with a decrease in the activity of MMP-2 compared with ND (ND, 7.88 ± 0.44; D, 5.60 ± 0.54 ROD, P < 0.05), Fig. 6A, while E2 supplementation attenuated this decrease (D+E2, 7.66 ± 0.35 ROD). Similarly, MMP-9 activity was decreased with D compared with ND (ND, 29.9 ± 1.8; D, 12.9 ± 2.3 ROD, P < 0.05), Fig. 6B, and E2 supplementation partially attenuated this decrease (D+E2, 21.4 ± 2.9 ROD).


Figure 5
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  		Fig. 5. Western blot analysis of MMP and TIMP expression in the ND and D rat renal cortex. In the D group, MMP-2 (A) and MMP-9 (B) protein expression, as measured by Western blot analysis, was decreased by 1.3-fold and 1.5-fold, respectively, compared with the ND. This decrease in both MMP-2 and MMP-9 protein expression was partially attenuated with D+E2. In the D group, TIMP-1 (C) and TIMP-2 (D), protein expression was increased by 2.8-fold and 2.1-fold, respectively, compared with the ND. This increase in both TIMP-1 and TIMP-2 protein expression was partially attenuated with D+E2. A–D, top: representative gels of MMP-2, MMP-9, TIMP-1, and TIMP-2 protein expression, respectively. A–D, bottom: densitometry summaries in ROD of MMP-2 (62 kDa band) and MMP-9 (95 kDa band), TIMP-1 and TIMP-2 protein from top. Data are expressed as means ± SE.

 

Figure 6
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  		Fig. 6. MMP-2 and MMP-9 activity in the ND and D rat kidney. In the renal cortex of the D group, MMP-2 (A) and MMP-9 (B) activity, as measured by zymography, was decreased by 1.4-fold and 2.3-fold, respectively, compared with the ND. This decrease in both MMP-2 and MMP-9 activity was completely (MMP-2) and partially (MMP-9) attenuated with D+E2. A and B, top: representative gels of MMP-2 and MMP-9 zymograms. A and B, bottom: densitometry summaries in ROD of MMP-2 and MMP-9 from top. Data are expressed as means ± SE.

 
Diabetes (D) was also associated with an increase in the expression of TIMP-1 (ND, 0.38 ± 0.06; D, 1.05 ± 0.09 ROD, P < 0.05), and TIMP-2 (ND, 0.88 ± 0.15; D, 1.89 ± 0.03 ROD, P < 0.05), Figs. 5, C and D, respectively. Supplementation with E2 restored TIMP-1 and TIMP-2 protein to levels similar to that observed in the ND kidneys (TIMP-1, 0.53 ± 0.09; TIMP-2, 1.52 ± 0.24 ROD).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our previous study showed that supplementation with E2 from the onset of diabetes in ovariectomized nondiabetic and STZ-induced diabetic rats attenuates albuminuria, glomerulosclerosis and tubulointerstitial fibrosis (22). Furthermore, our recent study showed that the STZ-induced diabetic rat exhibits low levels of circulating estradiol and an imbalance in the expression of renal estrogen receptors compared with its nondiabetic counterpart (40). The reduced levels of circulating estradiol in diabetes were also observed in the present study and may explain why women with diabetes "lose" the protective effect of the female gender and exhibit prevalence and progression of renal disease greater than nondiabetic women (27, 14). The major conclusions from these studies are that sex hormones play an important role in the pathophysiology of diabetic renal disease and that supplementation with E2, to restore the levels of estradiol to those observed under normal physiological conditions, is renoprotective in diabetic nephropathy. What remained unanswered in these studies were the mechanisms by which E2 supplementation affords renoprotection to the diabetic kidney. Thus the aim of the current study was to examine the effect of E2 on ECM metabolism (synthesis and degradation), as one of the major cellular processes that are adversely regulated and contribute to the development and progression of glomerulosclerosis and tubulointerstitial fibrosis associated with diabetic nephropathy.

As observed in our previous study (18), E2 supplementation attenuated albuminuria, glomerulosclerosis and tubulointerstitial fibrosis associated with diabetes. Our original study that examined the effect of E2 supplementation on the diabetic kidney was performed in the ovariectomized STZ-induced diabetic rat (22). However, since we observed that diabetes itself is associated with reduced levels of estradiol, we performed the present study in intact animals, to more resemble the hormonal status of diabetic, premenopausal women. These women, although exhibit reduced levels of plasma estradiol, are still cycling (9); thus examining the effects of E2 supplementation in a diabetic animal model with intact ovaries, we felt, would produce more valuable information regarding the potential use of E2 supplementation in diabetic women. It has been reported that women with type 1 diabetes reach menopause quicker than nondiabetic women (9). Thus E2 supplementation in these women may not only attenuate the progression of diabetes-associated end-organ complications, but also prevent premature menopause, which in itself is a risk factor for the development of diabetic cardiac and renal complications (18, 6).

Increase in ECM synthesis and/or decrease in ECM degradation are major contributors to the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in progressive renal disease, including diabetic nephropathy (20, 25). Thus one of the targets in attenuating and treating diabetic nephropathy is attenuating ECM synthesis and/or increasing ECM degradation. The current study confirms our previous report that diabetes is associated with mild glomerulosclerosis and tubulointerstitial fibrosis and that these changes are attenuated with E2 supplementation. Our study also shows that supplementation with E2 from the onset of diabetes both decreases ECM synthesis and increases ECM degradation, thus having a dual renoprotective role relating to ECM metabolism. One of the major ECM proteins that are upregulated in the diabetic kidney are collagen types I and IV and fibronectin (21, 15, 16). Our study shows that E2 supplementation decreases both collagens type I and type IV and fibronectin protein expression. Previous studies in cultured mesangial cells have shown that E2 reduces collagen type IV (31) and type I protein expression (29). Tamoxifen, a selective estrogen receptor modulator (SERM), also reduces collagen type IV protein expression in cultured mesangial cells (28). 2-Hydroxyestradiol, a metabolite of estradiol reduces immunostaining for collagen type IV in puromycin aminonucleoside nephropathy (37), while 2-methoxyestradiol, another estradiol metabolite attenuates collagen type IV synthesis in renal injury induced by chronic nitric oxide synthase inhibition (38). However, no study to date has reported the ability of E2 to reduce collagens type I and IV protein expression in diabetic nephropathy. In the db/db mouse, a model of type 2 diabetes, E2 and raloxifene, another SERM, reduces fibronectin expression in the diabetic kidney (8). Other ECM proteins, including laminin, also contributes to glomerulosclerosis and tubulointerstitial fibrosis associated with diabetic nephropathy (32). Our previous study has shown that E2 supplementation in the kidney of the aging Dahl salt sensitive rat reduces laminin protein expression (23). Further studies are needed to determine if E2 supplementation regulates the expression of laminin in the diabetic kidney. Collectively, studies from this and other laboratories support the role for E2 in regulating ECM synthesis in vivo and in vitro.

Major regulators of ECM degradation in the kidney are MMP-2 and MMP-9 (20). Our study shows that E2 supplementation increases the expression and activity of both MMP-2 and MMP-9 in the diabetic kidney. Other studies have shown that E2 increase MMP-2 (12) and MMP-9 (30) protein expression and activity in cultured mesangial cells and MMP-2 and MMP-9 protein expression in the aging Dahl salt sensitive rats (23). However, no studies to date have shown the ability of E2 to increase MMP expression and activity in the diabetic kidney. The activity of MMPs is regulated by tissue inhibitors of metalloproteinases (TIMPs) (41). An increase in TIMP expression, thought to contribute to the decrease in ECM degradation by inhibiting the expression and activity of MMPS, has been demonstrated in diabetic nephropathy (13, 35). Our study confirms the findings that both TIMP-1 and TIMP-2 protein expression are upregulated in the diabetic kidney. Our findings, that E2 supplementation reduces TIMP protein expression, provide further evidence that E2 is not only renoprotective by reducing ECM synthesis but also by increasing ECM degradation.

Although studies from this and other laboratories have reported beneficial effects of E2 on attenuating glomerulosclerosis and tubulointerstitial fibrosis, there are studies that report the opposite. A study in the obese Zucker rat, a model of glomerulosclerosis and renal failure in the setting of hyperlipidemia, found that E2 supplementation worsens albuminuria and increases glomerular expression of desmin and type IV collagen (34). In the OLETF rats, E2 ameliorates mesangial expansion but fails to attenuate proteinuria and glomerulosclerosis (39). These conflicting findings may be explained by the fact that these studies were performed in different models of diabetes, and there may be other genetic factors in these animal models that contribute to the inability of E2 to exert its renoprotective effect observed in our study. Most importantly, E2 supplementation has recently been reported to attenuate urine albumin excretion in diabetic and nondiabetic postmenopausal women (1, 2), strongly supporting the concept of a renoprotective effect of E2 in diabetes. The fact that both clinical and experimental studies are still producing conflicting findings with respect to beneficial effects of E2 only suggests that our understanding of its actions are not completely understood and that further studies are needed to evaluate its potential as a therapeutic agent in the prevention and treatment of disease processes, including diabetic nephropathy.

Estradiol has recently been reported to protect pancreatic beta-cells from apoptosis and prevent insulin-deficient diabetes mellitus in mice (19). This study suggests that estradiol sustains insulin production and prevents diabetes. In women with coronary heart disease, combined estrogen and progestin therapy reduces the incidence of diabetes by 35% (17). Other studies have also shown that low doses of E2 improve insulin sensitivity in postmenopausal women by increasing hepatic insulin clearance (7). Although our present study did not examine the effects of E2 on improving insulin secretion, it is unlikely that we would have observed this effect, since both the diabetic untreated and E2-treated groups were supplemented with insulin to maintain body weight and regulate glucose to the same level. Further studies are warranted to examine the protective effects of E2 in diabetic nephropathy through regulating insulin secretion.

Although our studies have only examined the contribution of estradiol in the pathophysiology of diabetic nephropathy, it is likely that other sex hormones, including testosterone and progesterone, also contribute to the disease process. Our unpublished observations show that testosterone levels are also reduced in diabetes, suggesting that there is a general imbalance in sex hormone levels associated with diabetes. Further studies are warranted to determine the contribution of testosterone in the pathophysiology of diabetes and its associated end-organ complications.

In summary, the present study demonstrates that supplementation with E2 from the onset of diabetes attenuates glomerulosclerosis and tubulointerstitial fibrosis associated with diabetic nephropathy, via reducing ECM deposition and increasing ECM degradation. These findings suggest that E2 supplementation is beneficial in preventing the development of diabetic renal complications and stresses the importance of examining the contribution of other sex hormones in the development and treatment of diabetic renal disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Research Award from the American Diabetes Association and the Carl W. Gottschalk Award from the American Society of Nephrology to C. Maric.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Maric, Center for the Study of Sex Differences: in Health, Aging and Disease, Georgetown Univ. Medical Center, 394 Bldg D, 4000 Reservoir Rd., NW, Washington, DC, 20057 (e-mail: cm255{at}georgetown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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