17β-estradiol attenuates diabetic kidney disease via regulating extracellular matrix and transforming growth factor-beta protein expression and signaling

Abstract

We have previously shown that supplementation with 17β-estradiol (E₂) from the onset of diabetes attenuates the development of diabetic renal disease. The aim of this study was to examine if E₂ can also attenuate the disease process once it has already developed. The study was performed in non-diabetic (ND) and streptozotocin (STZ)-induced diabetic (D) Sprague-Dawley rats. E₂ supplementation began after 9 weeks of diabetes for further 8 weeks. Diabetes was associated with an increase in urine albumin excretion (UAE) (ND; 9.4±1.0; D, 56.2±8.9 mg/day; P<0.001), glomerulosclerosis (GSI; ND, 0.13±0.02; D, 1.38±0.09 AU; P<0.01), tubulointerstitial fibrosis (TIFI; ND, 0.17±0.04; D, 1.35±0.10 AU; P<0.01), renal cortical collagen type I (CI; ND, 1.02±0.05; D, 1.83±0.03 ROD; P<0.001), collagen type IV (CIV; ND, 0.97±0.05; D, 1.56±0.03 ROD, P<0.01), laminin (L; ND, 1.03±.0.03; D, 1.80±0.02 ROD; P<0.001), PAI-1 (ND, 0.34±0.08; D, 0.88±0.03 ROD; P<0.01), TIMP-1 (ND, 0.30±0.07; D, 0.93±0.14 ROD; P<0.01), TIMP-2 (ND, 0.40±0.09; D, 0.96±0.11 ROD; P<0.01), TGF-β (ND; 0.70±0.14; D, 1.63±0.08 ROD; P<0.001), TGF-βRI (ND, 0.69±0.23; D, 1.47±0.14 ROD; P<0.05), TGF-βRI (ND, 0.40±0.09; D, 1.07±0.10 ROD; P<0.001), Smad2/3 (ND, 0.68±0.08; D, 1.71±0.03 ROD; P<0.01), pSmad2/3 (ND, 0.28±0.07; D, 0.74±0.12 ROD, P<0.05) and Smad4 (ND, 0.82±.0.24; D, 1.77±0.03 ROD; P<0.05) protein expression and CD68-positive cells (ND, 5.0±0.8; D, 33.8±2.4 cells/mm²; P<0.001). Decreases in MMP-2 (ND, 1.35±0.06; D, 0.98±0.05 ROD; P<0.05) protein expression and activity (MMP-2 act; ND, 0.39±0.09; D, 0.22±0.01 ROD; P<0.05), Smad6 (ND, 0.85±0.10; D, 0.40±0.03 ROD; P<0.001) and Smad7 (ND, 1.41±0.06; D, 1.01±0.05 ROD; P<0.05) protein expression were also associated with D. E₂ supplementation either completely or partially attenuated these changes. We conclude that E₂ attenuates the progression of disease once it has already developed via regulating extracellular matrix, TGF-β and expression of its downstream regulatory proteins.

INTRODUCTION

While the female sex appears to be a protective factor against the development of non-diabetic renal disease (45, 50, 67), this protection is lost in diabetes. The incidence and the rate of progression of renal disease is far greater in diabetic than in non-diabetic women (9, 13, 50), suggesting that the diabetic milieu provides a stage for the development and faster progression of renal disease in women. Our previous studies suggested that the possible reasons for the loss of the female sex as a protective factor in diabetes are reduced levels of plasma estradiol and abnormal regulation of estrogen receptors in the diabetic kidney (40, 74). Furthermore, these studies showed that supplementation with 17β-estradiol (E₂) from the onset of diabetes restored the levels of estradiol to those observed in non-diabetics, and protected against the development of diabetic renal injury (40). One of the mechanisms through which E₂ exerts this renoprotection is through regulating extracellular matrix (ECM) protein expression (39, 40).

Increased extracellular matrix (ECM) protein synthesis and/or decreased ECM degradation contribute to the development of diabetes-associated glomerulosclerosis and tubulointerstitial fibrosis (42, 76, 80). It is now well recognized that the rate of progression of diabetic renal disease correlates with the degree of cortical tubulointerstitial fibrosis (22, 47, 56, 59). Thus, attenuating ECM accumulation and/or enhancing ECM degradation is considered a prime target in the treatment of diabetic renal complications.

Transforming growth factor-beta (TGF-β) is a key regulator of ECM protein synthesis and degradation in the diabetic kidney (7, 8, 11, 27, 28, 72). TGF-β has consistently been shown to be upregulated in renal parenchymal and infiltrating inflammatory cells in the diabetic kidney, both in humans and experimental models (4, 7, 27, 28, 79). A number of factors have been shown to stimulate TGF-β protein expression in the diabetic kidney, including hyperglycemia (31, 63, 73), advanced glycation end-products (52, 75), oxidative stress (68), mechanical stretch (24) and numerous vasoactive peptides such as angiotensin (12, 19, 73) and endothelin (63, 64). TGF-β exerts its effects through firstly binding to the membrane-bound TGF-β type II (TGF-βRII) and subsequent activation of the TGF-β type I receptor (TGF-βRI) (8, 72). This receptor activation is followed by activation of the Smad regulatory pathway; firstly, Smad2 and Smad3 are phosphorylated, form a heteromeric complex with Smad4 and then the complex translocates into the nucleus where it regulates transcription of target genes (21, 31, 46). In contrast, Smad6 and Smad7 are inhibitory Smads that act in a negative feedback loop to inhibit TGF-β activity by preventing phosphorylation of Smad proteins and activation of TGF-βRI (72). Smad7 also inhibits TGF-β signaling by promoting TGF-β receptor degradation involving ubiquitin ligase Smurf2 (20, 70).

Our previous studies have shown that supplementation with E₂ from the onset of diabetes, prevents albuminuria, glomerulosclerosis and tubulointerstitial fibrosis associated with diabetic nephropathy, via regulating ECM synthesis and degradation (39, 40). While these studies show that E₂ is able to attenuate diabetic renal injury when supplemented from the onset of diabetes, little is know about the ability of E₂ to attenuate the progression of the disease once it has already developed. This is of particular importance since the vast majority of patients with diabetes present with either moderate or advanced renal injury, thus attenuating the disease progression is of high clinical relevance. The aim of the current study was to examine if E₂ is also able to attenuate the progression of renal functional and structural changes once they have already been initiated by the diabetic milieu. Specifically, our study examined the effects of E₂ supplementation on ECM protein expression and the expression of TGF-β and its downstream signaling proteins involving Smads and Smurf2 in the STZ-induced model of diabetic nephropathy.

MATERIALS AND METHODS

Animal model

Female Sprague Dawley rats (10 weeks of age) were purchased from Harlan (Madison, WI) and were maintained on a phytoestrogen-free rat chow (Harlan, Madison, WI) and tap water ad libitum. The animals were first randomly divided into 2 treatment groups: Non-diabetic (ND, n=8) and Diabetic (D, n=14) for 9 weeks. The diabetic animals were then further randomly divided into 2 treatment groups: Diabetic (D, n=7) and diabetic with E₂ supplementation (D+E₂, n=7). All animals, including the ND group, were treated for additional 8 weeks. After cumulative 17 weeks of treatment, the animals were weighed, anesthetized with sodium pentobarbitone (40 mg/kg ip) and blood collected (via cardiac puncture) for measurement of plasma E₂ levels. The kidneys were weighed and then either snap frozen in liquid nitrogen for protein analysis or immersion fixed with HistoCHOICE (Amresco, Solon, OH) for morphological and immunohistochemical analysis. The 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.

Induction of diabetes

Following an overnight fast, the animals received either a single ip 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). Diabetic rats were supplemented with insulin (2–4 U sc. every second day, Lantus, Aventis Pharmaceuticals Inc., Kansas City, MO) for the duration of the study to maintain blood glucose levels between 250–450 mg/dl and prevent weight loss. Every 4 weeks, the animals were placed in metabolic cages for 24h for determination of urine output and measurement of UAE.

Estrogen supplementation and plasma Estradiol levels

At the time of induction of diabetes, the animals were subjected to two ip injections (4h apart) of Deslorelin acetate (10 μg/ml, Bachem Chemicals, Torrance, CA). The diabetic animals on E₂ supplementation were injected with 17β-estradiol (5 μg/kg in 200 μl peanut oil, Sigma, St. Louis, MO) every 4 days, 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 (74). Plasma E₂ levels were measured by ELISA (Alpha Diagnostic, San Antonio, TX) according to the manufacturer’s protocol.

Urine albumin excretion (UAE)

Urine albumin concentration was determined using the Nephrat II albumin kit (Exocell, Inc., Philadelphia PA), according to the manufacturer’s protocol. The rate of UAE was calculated from the measurement of urine albumin concentration and output.

Indices of glomerulosclerosis (GSI) and tubulointerstitial fibrosis (TIFI)

Glomerulosclerosis was defined as accumulation of extracellular matrix (ECM) deposits and mesangial expansion (1). The GSI was assessed in PAS-stained sections in 80 randomly selected glomeruli and the degree of sclerosis graded using a semi-quantitative scoring method as previously described (61). Tubulointerstitial fibrosis was defined as tubular atrophy or dilatation, deposition of ECM and presence of inflammatory cells (1). The TIFI was assessed in Masson’s trichrome-stained sections in 60 randomly selected fields of view at the magnification of X400 using a light microscope and the degree of fibrosis graded using a semi-quantitative scoring method as previously described (40).

Immunohistochemistry

In 4μm paraffin sections, endogenous avidin and biotin activities were blocked using a kit (Vector, Burlingame, CA). Sections were incubated with 0.1% albumin (for collagen type I and type IV) or with 10% non-immune goat serum (for other proteins) in phosphate buffered saline (PBS, pH 7.4) for blocking of non-specific immunolabeling. Sections were then incubated with antisera against proteins of interest (specific concentrations are given in the table below) at 4°C overnight. After washing with PBS, sections were incubated with biotinylated goat anti-rabbit, anti-mouse or anti-goat IgG (Dakopatts, Glostrup, Denmark) diluted 1:100 in PBS for 1 h at room temperature (RT), followed by incubation with the avidin-biotin complex (Vector, Burlingame, CA) diluted 1:100 with PBS for 1 h at RT. Positive immunoreaction was detected after incubation with 3,3-diaminobenzidine for 2 min at RT and counterstaining with Mayer’s hematoxylin. Sections incubated with 0.1% albumin or 10% goat serum instead of the primary antisera were used as negative controls.

Western blotting

Renal cortical tissue samples were homogenized and the protein concentration determined using a colorimetric assay (BioRad). For collagen type I and type IV, samples were analyzed under non-reducing conditions, while for other proteins, samples were denatured at 95°C for 10 minutes then treated according to the following conditions:

Protein conc.	Ab. dil. IHC	% gel	Ab. dil. Western	Source	Manufacturer
Collagen type I (15 μg)	1:200	4–20	1:1,000	mouse	Sigma, St. Louis. MO
Collagen type IV	1:400	N/A	N/A	goat	Southern Biotech, Birmingham, AL
Collagen type IV(7 μg)	N/A	4–20	1:1,000	mouse	Chemicon, Temecula, CA
Laminin (30 μg)	1:400	7.5	1:1,000	rabbit	Research Diagnostics, Concord, MA
Fibronectin (30 μg)	1:400	7.5	1:1,000	mouse	Research Diagnostics
MMP-2 (50 μg)	1:400	12.5	1:1,000	Mouse	Oncogene, Cambridge, MA
MMP-9 (50 μg)	1:400	12.5	1:1,000	mouse	Oncogene
TIMP-1 (50 μg)	1:100	18	1:100	Mouse	Calbiochem, San Diego, CA
TIMP-2 (50 μg)	1:100	18	1:100	Mouse	Calbiochem
PAI-1 (38 μg)	1:100	15	1:400	goat	Santa Cruz, Santa Cruz, CA
TGF-β (30 μg)	1:400	18	1:500	rabbit	Santa Cruz
TGFβRI (30 μg)	1:200	12.5	1:500	rabbit	Santa Cruz
TGFβRII (30 μg)	1:200	12.5	1:500	rabbit	Santa Cruz
Smad2/3 (30 μg)	1:200	12.5	1:500	goat	Santa Cruz
pSmad2/3 (30 μg)	1:200	12.5	1:500	goat	Santa Cruz
Smad4 (30 μg)	1:200	12.5	1:800	goat	Santa Cruz
Smad6 (30 μg)	1:200	12.5	1:800	goat	Santa Cruz
Smad7 (30 μg)	1:200	12.5	1:800	goat	Santa Cruz
Smurf2 (30 μg)	1:200	12.5	1:1,000	goat	Santa Cruz
CD68	1:400	N/A	N/A	mouse	Serotec, Oxford, UK

Following incubation with primary antisera at 4°C overnight, the membranes were washed, incubated with either goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase and proteins visualized by enhanced chemiluminescence (KPL, Gaithersburg, MD). The densities of specific bands were normalized to the total amount of protein loaded in each well following densitometric analysis of gels stained with Coomassie blue. The densities of specific bands were quantitated by densitometry using the Scion Image beta (version 4.02) software.

Zymography

Renal cortical tissue samples were homogenized and the protein concentration determined using a colorimetric assay (BioRad). The homogenized samples were loaded onto a 10% SDS acrylamide gel containing 1 mg/ml gelatin (BioRad). After electrophoresis, the gel was incubated in renaturing buffer (Invitrogen, Carlsbad, CA), activated in developing buffer (Invitrogen) and gelatinase activity visualized by staining the gel with Coomassie blue. Bands were quantitated by densitometry using the Scion image beta (version 4.02) software.

Quantitative analysis of macrophage number

The number of macrophages (CD68-positive cells) was assessed by counting the number of positive cells in twenty random fields per section in 4 sections per kidney from each treatment group.

Statistical analysis

Data are expressed as means± SEM and were analyzed with a one-way ANOVA followed by the Tukey’s post test, using the Sigma Stat software. Differences were considered statistically significant at P < 0.05.

RESULTS

Body and kidney weight, blood glucose and E₂ levels and UAE

No differences in either body or kidney weight were observed between any of the treatment groups (Table 1). Diabetes (D) was associated with increased blood glucose (ND, 109.3±8.9; D, 498.3±30.1 mg/dl; P<0.001), decreased plasma levels of E₂ (ND, 54.9±4.6; D, 40.2±4.1 pg/ml; P<0.05) and increased UAE (ND; 9.4±1.0; D, 56.2±8.9 mg/day; P<0.001), compared with their non-diabetic (ND) counterpart (Table 1). Animals supplemented with E₂ exhibited a similar increase in blood glucose levels as in D (446.6±23.0 mg/dl), while the decrease in plasma levels of E₂ (53.1±6.4 pg/ml) and increase in UAE (39.4±15.7 mg/day) associated with D was completely (plasma E₂) or partially (UAE) attenuated with E₂ supplementation (Table 1).

Table 1.

Effects of E₂ supplementation on metabolic and renal parameters

	ND	D	D+E2
Body weight (g)	258.4±14	250±3.5	223±13
Kidney weight (g)	1.4±0.10	1.5±0.17	1.4±0.10
Blood glucose (mg/dl)	109.3±8.9	498.3±30.1aaa	446.6±23.0aaa
Plasma E2 (pg/ml)	54.9±4.6	40.2±4.1a	53.1±6.4b
UAE9w (mg/day)	6.6±0.4	19.4±3.1aa	N/A
UAE17w (mg/day)	9.4±1.0	56.2±8.9aaa	39.4±15.7aa,b
GSI (AU)	0.13±0.02	1.38±0.09aa	0.20±0.02a,b
TIFI (AU)	0.17±0.04	1.35±0.10aa	0.38±0.07aa

Data are expressed as mean±SEM.

^aP<0.05,

^aaP<0.01,

^aaaP<0.001 vs ND.

^bP<0.05 vs D.

E₂, 17β-estradiol; ND, non-diabetic; D, diabetic, D+E₂, diabetic with 17β-estradiol supplementation; UAE_9w, urine albumin excretion after 9 weeks of diabetes; UAE_17w, urine albumin excretion after 17 weeks of diabetes; GSI, glomerulosclerosis index; TIFI, tubulointerstitial fibrotic index, AU, arbitrary units.

UAE was also measured after the initial 9 weeks of diabetes, i.e. prior to E₂ supplementation. At 9 weeks, increased UAE was already observed with D (ND, 6.6±0.4; D, 19.4±3.1mg/day; P<0.01; Table 1).

Glomerulosclerosis and Tubulointerstitial fibrosis

D was associated with moderate glomerulosclerosis (GSI; ND, 0.13±0.02; D, 1.38±0.09 AU; P<0.01; Fig. 1A, Table 1) and tubulointerstitial fibrosis (TIFI; ND, 0.17±0.04; D, 1.35±0.09 AU; P<0.01; Fig. 1B, Table 1). Supplementation with E₂ attenuated these changes (GSI, 0.20±0.02 AU; TIFI, 0.38±0.078 AU; Fig. 1, Table 1).

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.

Collagen type I, collagen type IV, laminin and fibronectin

In the ND kidney, collagen type I was mainly immunolocalized to tubulointerstitial areas of the renal cortex (Fig. 2), while collagen type IV (Fig. 2), laminin (Fig. 3) and fibronectin (Fig. 3) were predominantly immunolocalized to basement membranes of the glomerulus, proximal and distal tubules. Diabetes was associated with an overall increase in the intensity of immunostaining for collagen type I, collagen type IV (Fig. 3), laminin and fibronectin (Fig. 4), while E₂ supplementation either completely or partially attenuated these changes. Quantiation of changes in collagen type I, collagen type IV, laminin and fibronectin protein expression by western blotting confirmed the immunohistochemical observations. Diabetes was associated with an increase in collagen type I (CI; ND, 1.02±0.05; D, 1.83±0.03 ROD; P<0.001), collagen type IV (CIV; ND, 0.97±0.05; D, 1.56±0.03 ROD, P<0.01) and laminin (L; ND, 1.03±.0.03; D, 1.80±0.02 ROD; P<0.001) protein expression (Fig. 3 and Fig. 4), while no changes in fibronectin protein expression were observed with D (Fig. 4). Supplementation with E₂ partially (CI, 1.26±0.10 ROD; L, 1.45±0.01 ROD) or fully (CIV, 0.86±0.32 ROD) attenuated these changes.

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.

MMPs, TIMPs and PAI-1

Diabetes was associated with no apparent differences in the intensity of immunostaining for either MMP-2 or MMP-9 (Fig. 5) compared with ND, while E₂ supplementation appeared to increase the intensity of immunostaining for MMP-2 above the levels observed in ND. Qunatitative analysis of MMP-2 protein expression and activity by western blotting and zymography, respectively, showed a decrease in MMP-2 protein expression (ND, 1.35±0.06; D, 0.98±0.05 ROD; P<0.05) and activity (ND, 0.39±0.09; D, 0.22±0.01 ROD; P<0.05) associated with D, while no differences in either MMP-9 protein expression or activity were observed (Fig. 5). Supplementation with E₂ increased MMP-2 protein expression and activity to levels above or similar to that observed in ND (Fig. 5).

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.

While TIMP-1 was not detectable by immunohistochemistry, TIMP-2 was mainly immunolocalized to distal tubules (Fig. 5). Quantitative analysis of TIMP-1 and TIMP-2 protein expression by western blotting (Fig. 5) showed that D was associated with an increase in TIMP-1 (ND, 0.30±0.07; D, 0.93±0.14 ROD; P<0.01) and TIMP-2 (ND, 0.40±0.09; D, 0.96±0.11 ROD; P<0.01) protein expression. Supplementation with E₂ completely attenuated these changes.

Similar to TIMPs, the intensity of PAI-1 immunostaining in proximal and distal tubules (Fig. 5) and protein expression (ND, 0.34±0.08; D, 0.88±0.03 ROD; P<0.01), was increased with D. Supplementation with E₂ partially attenuated these changes.

Transforming growth factor beta (TGF-β) and TGF-β receptor (TGF-βRI and TGF-βRII) protein expression

While only weak immunostaining for TGF-β was observed in the ND kidneys, the intensity of immunostaining in glomerular mesangial cells, proximal and distal tubules was increased in the D kidneys (Fig. 6). Supplementation with E₂ attenuated this diabetes-associated increase in the intensity of TGF-β immunostaining. Western analysis confirmed the immunohistochemical findings. Diabetes (D) was associated with an increase in renal cortical expression of TGF-β protein (ND, 0.70±0.14; D, 1.63±0.08 ROD; P<0.001), while E₂ supplementation partially attenuated this increase (D+E₂, 1.27±0.10 ROD), Fig. 7.

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.

In the ND kidney, TGF-βRI was predominantly immunolocalized to glomerular mesangial cells (Fig. 6). D was associated with an apparent increase in the overall intensity of TGF-βRI immunostaining and in addition to being observed in the mesangial cells, TGF-βRI was also evident in distal, and to a lesser extent proximal tubules (Fig. 6). Supplementation with E₂ attenuated this diabetes-associated increase in the intensity of TGF-βRI immunostaining. While TGF-βRII was not detectable in the ND kidneys by immunohistochemistry, it was immunolocalized predominantly to proximal and distal tubules and in the mesangial cells in the D kidneys (Fig. 6). Supplementation with E₂ attenuated this diabetes-associated increase in the intensity of TGF-βRI immunostaining. Western analysis confirmed the immunohistochemical findings. Diabetes (D) was associated with an increase in renal cortical expression of TGF-βRI (ND, 0.69±0.23; D, 1.47±0.14 ROD; P<0.05) and TGF-βRII (ND, 0.40±0.09; D, 1.07±0.10 ROD; P<0.001) protein, while E₂ supplementation partially attenuated this increase (TGF-βRI, 0.97±0.12; TGF-βRII, 0.68±0.04 ROD) Fig. 7.

Smad protein expression

In the ND kidney, no Smad2/3, pSmad2/3 or Smad4 were detected by immunohistochemistry (Fig. 8). In the D kidney, Smad2/3, pSmad2/3 and Smad4 were immunolocalized to glomerular mesangial cells, proximal and distal tubules (Fig. 8). Supplementation with E₂ attenuated this diabetes-associated increase in the intensity of Smad2/3, pSmad2/3 and Smad4 immunostaining. Western analysis confirmed the immunohistochemical findings. Diabetes (D) was associated with an increase in renal cortical expression of Smad2/3 (ND, 0.68±0.08; D, 1.71±0.03 ROD; P<0.01; Fig. 9), pSmad2/3 (ND, 0.28±0.07; D, 0.74±0.12 ROD; P<0.05; Fig. 9) and Smad4 (ND, 0.82±0.24; D, 1.77±0.03 ROD; P<0.05; Fig. 9) protein. Supplementation with E₂ attenuated these changes (Smad2/3, 1.24±0.19; pSmad2/3, 0.24±0.09; Smad4, 1.65±0.03 ROD; Fig. 9).

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.

In the ND kidney, Smad6 was mainly immunolocalized to proximal, and to lesser extent distal tubules (Fig. 10), while Smad7 was immunolocalized predominantly to distal tubules (Fig. 10). In the D kidney, an overall decrease in the intensity of immunolocalization for both Smad6 and Smad7 was observed (Fig. 10). Supplementation with E₂ attenuated this diabetes-associated decrease in the intensity of Smad6 and Smad7 immunostaining. Western analysis confirmed the immunohistochemical findings. Diabetes (D) was associated with a decrease in renal cortical expression of Smad6 (ND, 0.85±0.10; D, 0.40±0.03 ROD; P<0.001) and Smad7 (ND, 1.41±0.06; D, 1.01±0.05 ROD; P<0.05) protein, Fig. 10. Supplementation with E₂ attenuated these changes (Smad6, 0.70±0.07; Smad7, 1.27±0.09 ROD).

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.

Smurf2 expression

Weak immunostaining in distal tubules was observed in ND kidney, while in D, there was an overall increase in the intensity of staining for Smurf2, and in addition to distal tubules, staining was also observed in isolated cells in proximal tubules (Fig. 11). This increase in the intensity of immunostaining for Smurf2 was attenuated with E₂ supplementation. While there was a trend towards an increase in Smurf2 protein expression in the D compared with ND kidneys and a decrease with E₂ supplementation, this did not reach statistical significance (Fig. 11).

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.

Renal inflammation

The D kidney was characterized by the presence of CD68-positive cells in the glomerulus and cortical tubulointerstitium (Fig. 12), suggesting the presence of macrophages. Quantitative analysis showed that D was associated with an increase in the abundance of CD68-positive cells compared with ND (ND, 5.0±0.8; D, 33.8±2.4 cells/mm², P<0.001) and that E₂ supplementation completely reversed this increase in abundance of CD68-positive cells associated with D (D+E₂, 6.6±0.7 cells/mm²), Fig. 12.

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.

DISCUSSION

We have previously shown that E₂, when supplemented from the onset of diabetes, is renoprotective by preventing albuminuria, glomerulosclerosis and tubulointerstitial fibrosis (39, 40). The present study demonstrates that when supplemented after 9 weeks of diabetes, E₂ is also able to attenuate albuminuria and structural changes associated with diabetes through regulating ECM protein and TGF-β protein expression and signaling.

Increased UAE is a hallmark of diabetic renal disease (10). In our study, increased UAE was observed as early as 9 weeks of diabetes, and increased further for the duration of the study (17 weeks). E₂ supplementation initiated after 9 weeks of diabetes, at a time when albuminuria has already developed, reduced this diabetes-associated increase in UAE. These observations suggest that in addition to being able to attenuate, E₂ also reverses the decline in albuminuria associated with diabetes. Variable effects of E₂ supplementation on UAE have been reported in other studies. While in the OLETF diabetic rat E₂ had no effect (71), in type II diabetic patients E₂ supplementation from the onset of diabetes reduced proteinuria (69). However, no studies to date have examined the ability of E₂ to reverse diabetes-related albuminuria.

Our previous studies showed that E₂ supplementation from the onset of diabetes prevents the development of glomerulosclerosis and tubulointerstitial fibrosis (39, 40). Our previous study also shows that the predominate receptor type through which E₂ exerts its effects in the diabetic kidney is ERα (74). The present study shows that when supplemented after 9 weeks of diabetes, E₂ also attenuates the progression of development of glomerulosclerosis and tubulointerstitial fibrosis. Specifically, we show that 17 weeks of diabetes is associated with an upregulation of collagen type I, collagen type IV and laminin and fibronectin protein expression, confirming observations of previous studies from this and other laboratories (32, 38, 39). Supplementation with E₂ either partially (collagen type IV and laminin) or fully (collagen type I) attenuated these increases, but did not affect fibronectin protein expression. Most studies to date mainly examined the ability of E₂ to regulate renal ECM protein expression in vitro. These studies have demonstrated that E₂ inhibits: 1) catecholamine-induced collagen synthesis in human mesangial cells (16, 78), 2), collagen type IV accumulation in mesangial cells derived from glomerulosclerosis-resistant mice (58), and 3) collagen type I and type IV synthesis in mesangial cells derived from SJL/J (H-2S) mice (65, 66). The few in vivo studies, albeit, mostly performed in non-diabetic models, support the observation of the present study that E₂ downregulates ECM protein expression. In the ROP Os/+ (18), Alb/TGF-beta transgenic mice (6) and the Dahl salt sensitive rat (41), E₂ decreases synthesis of interstitial and basement membrane collagens and laminin. In ovariectomized db/db (14) and TGF-β1 transgenic mice (5), treatment with E₂ for 8 and 6 weeks, respectively, attenuates diabetes-induced fibronectin and collagen protein expression. The finding of the inability of E₂ to attenuate the diabetes-associated increase in fibronectin protein expression was surprising given that our previous study showed that when supplemented from the onset of diabetes, E₂ attenuated fibronectin protein expression (39). One of the possible explanations for this observation may be the inability of E₂ to adequately upregulate enzymes involved in fibronectin metabolism. While E₂ upregulates expression of MMP-2, fibronectin degradation, with advanced diabetes, may be more dependent on other MMPs, including MMP-9, which are not responsive to treatment with E_2.

In addition to increased synthesis, diabetes-related glomerulosclerosis and tubulointerstitial fibrosis is associated with a decrease in ECM degradation by MMPs (23, 30, 36, 42, 55). Our study shows that 17 weeks of diabetes is associated with a decrease in MMP-2 but not MMP-9 protein expression and activity and that E₂ supplementation attenuates diabetes-induced MMP-2 protein expression and activity. Variable levels of MMP-2 and MMP-9 protein expression and activity have been observed in the diabetic kidney, depending on the duration and model of diabetes. In early diabetes, a decrease in MMP-2 and MMP-9 protein expression and activity has been reported (26, 39, 44) while in longer-term diabetes, in C57BL/6J mice made diabetic by a high fat diet (60) or the STZ-induced diabetic rat (15), no changes in MMP-9 activity were observed. It is conceivable that compensatory mechanisms are activated in response to prolonged diabetes that reverse MMP-9 expression and activity observed in early stages of the disease. Moreover, studies in the MMP-9 knockout mice showed that MMP-9 does not appear to have a discernible role in the progression of glomerulonephritis in a mouse model of Alport syndrome (2), suggesting compensation orredundancy of MMP-9 in this model. MMP-9 may similarly be redundant or compensated in the diabetic kidney especially after prolonged hypreglycemia. Similar to ECM protein expression, very few in vivo studies have examined the ability of E₂ to regulate renal ECM metabolism, especially in the setting of diabetes. In cultured mesangial cells, E₂ increases MMP-2 (25, 34) and hyperglycemia-induced downregulation of MMP-9 (57) protein expression and activity. These observations support the findings of the present study that E₂ is renoportective by upregulating MMP expression and activity.

One of the mechanisms by which MMP activity is regulated is by altering the expression of its inhibitors, namely TIMPs and PAI-1. Diabetic nephropathy, in both humans and experimental models, is associated with increased TIMP-1 (44), TIMP-2 (26) and PAI-1 (36, 51, 54) protein expression, consistent with the findings of the present study. While E₂ supplementation partially (TIMP-1) or fully (TIMP-2) attenuated the diabetes-associated increase in TIMPs, no effect of E₂ on PAI-1 expression was observed. In previous studies, E₂ was shown to attenuate TIMP-2 and PAI-1 expression in the Alb/TGF-beta transgenic mice (6), and attenuate TIMP-1 and TIMP-2 in the diabetic kidney (39). Furthermore, combined estrogen and medroxyprogesterone treatment in post-menopausal diabetic women reduces serum PAI-1 levels (53). These studies clearly demonstrate the ability of E₂ to regulate PAI-1 expression; however, it appears that this regulation is ineffective once diabetes has progressed, at least in the kidney. But despite the inability of E₂ to regulate PAI-1 expression directly, its effects on other ECM regulatory pathways appear to be sufficient to effectively attenuate the progression of glomerulosclerosis and tubulointerstitial fibrosis in the diabetic kidney.

Numerous studies have demonstrated the importance of TGF-β in the pathophysiology of renal disease, including diabetic nephropathy. The diabetic kidney is characterized by temporal overexpression of TGF-β mRNA and protein in both experimental models (4, 27, 28) and in humans (7, 79). Our previous (40) and current study confirm the finding that TGF-β is overexpressed in glomerular mesangial cells, proximal and distal tubules in the STZ-induced diabetic rat after 9 weeks of diabetes. Our present study also shows that supplementation with E₂ for 8 weeks following the 9-week period of diabetes attenuates TGF-β protein expression, suggesting that one of the mechanisms by which E₂ exerts its renoprotective effects in the diabetic kidney is through regulating the expression of locally active cytokines, such as TGF-β. A similar renoprotective effect of E₂ has been observed in the 5/6 remnant kidney model, in which E₂ supplementation inhibits tubulointerstitial fibrosis via reducing TGF-β expression (3, 33). In Alb/TGF-β1 transgenic mice overexpressing TGF-β, supplementation with E₂ reverses renal injury (6). In cultured human embryonic kidney carcinoma cells (43) and in cultured mesangial cells (49), E₂ inhibits TGF-β protein expression under high glucose conditions, further providing support that E₂ is renoprotective in the face of increased TGF-β expression.

The present study supports the previous observations from this and other laboratories that TGF-β protein is upregulated in the diabetic kidney and that this upregulation is associated with glomerulosclerosis and tubulointerstitial fibrosis (40, 42, 76, 80). Numerous studies have demonstrated that TGF-β promotes renal cell growth, stimulates synthesis of key ECM proteins including, type I and type IV collagen, laminin and fibronectin and inhibits ECM degradation via decreasing activity of matrix metalloproteinases (MMPs) (7, 8, 11, 27, 28, 72). Our study also shows that increased TGF-β protein expression in the diabetic kidney coincides with glomerulosclerosis, tubulointerstitial fibrosis, increase in collagen type I, collagen type IV and laminin protein expression and decrease in MMP-2 activity (see accompanying manuscript). The present study also shows that E₂ supplementation attenuated all these changes. In the ROP Os/+ mice, E₂ prevents tubulointerstitial fibrosis by decreasing synthesis of interstitial and basement membrane collagens (18), while in cultured mesangial cells, E₂ increases the activity of MMPs (57) via inhibiting TGF-β expression (48). Collectively, these findings support the role for E₂ in attenuating the progression, and possibly reversing diabetes-associated glomerulosclerosis and tubulointerstitial fibrosis via regulating TGF-β protein expression.

Observations from the present study indicate that both TGF-βRII and TGF-βRI protein expression is upregulated in the diabetic kidney, supporting the observations from previously reported studies (27, 28, 35). Our study also shows that E₂ supplementation reduces TGF-βRII and TGF-βRI protein expression, suggesting that E₂ exerts its renoprotective effects through regulating the expression of both the ligand (TGF-β) and its receptors. While both in vivo (3, 6, 33) and in vitro (49) studies have shown that E₂ downregulates TGF-β in the setting of diabetes/high glucose, no studies to date have reported the effects of E₂ on TGF- βRII and TGF-βRI protein expression. Our observations thus demonstrate a novel aspect of E₂ regulation of the TGF-β pathway in the diabetic kidney.

Evidence suggests that TGF-β exerts its profibrotic effect through activation of Smad2/3 which then forms a complex with Smad4 that is then translocated into the nucleus to upregulate transcription of profibrotic genes (46, 72). Our study found that diabetes was associated with an increase in the renal cortical expression of Smad2/3 protein as well as its phosphorylated form, pSmad2/3, indicating increased activation of Smad2/3 in the setting of diabetes. Other studies have also reported increases in renal cortical Smad2/3 and Smad4 in the diabetic kidney (21, 29, 31). Our study shows that E₂ supplementation attenuates diabetes-associated increases in Smad2/3 and pSmad2/3 protein expression, while no effects on Smad4 were observed. In cultured human embryonic kidney carcinoma cells, E₂ also decreases Smad3 protein expression (43), further providing support that E₂, in addition to being able to directly regulate TGF-β protein expression, it also suppresses TGF-β activity via downregulating its profibrotic regulatory proteins Smad2/3. Although E₂ did not affect Smad4 protein expression, it is conceivable that E₂ suppresses the profibrotic TGF-β effect through downregulating Smad2/3 protein expression and its phosphorylation only without an effect on Smad4.

TGF-β activity can be downregulated in part by: 1) a feedback mechanism involving Smad7, which prevents phosphorylation of Smad2/3 by binding to the TGF-βRI (72, 77) and by 2) activation of transcriptional repressor function of Smad6 (62). Our study shows that diabetes is associated with a decrease in renal cortical Smad6 and Smad7 protein expression, indicating that this may be one of the mechanisms by which the profibrotic effect of TGF-β is exerted in the diabetic kidney. Interestingly, out study demonstrates that E₂ supplementation attenuates diabetes-associated decrease in both Smad6 and Smad7 protein expression, suggesting that this may be one of the pathways by which E₂ attenuate the profibrotic effects of TGF-β in the diabetic kidney. These observations provide evidence for a dual role for E₂ in regulating TGF-β activity in the diabetic kidney: via downregulating the expression of profibrotic signaling molecules (Smad2/3) and via upregulating antifibrotic signaling molecules (Smad6 and Smad7).

Studies thus far have reported contradictory evidence for the role for Smurf2 in regulating TGF-β signaling and activity. Smurf2 has been reported to induce ubiquitin-dependent degradation of Smad2, induce nuclear export of Smad7 with subsequent increase in TGF-βRI degradation thus negatively regulate TGF-β signaling (17, 37). In contrast, in the unilateral ureteral obstruction model of progressive tubulointerstitial fibrosis, Smurf2 expression is increased in very early stages of the disease (20), at a time when TGF-β protein expression is also upregulated, suggesting that Smurf2 may in fact potentiate TGF-β activity. While our study only observed a trend towards an increase in Smurf2 protein expression at a time when TGF-β protein is upregulated, this change did not reach statistical significance and may only suggest that Smurf2 may not play a role in TGF-β signaling in the STZ-induced diabetic kidney disease, at least not in our hands.

In summary, our study demonstrates that in the STZ-induced diabetic rat, E₂ is renoprotective by attenuating albuminuria and ECM protein expression associated with diabetic glomerulosclerosis and tubulointerstitial fibrosis. One of the mechanisms by which E₂ exerts its renoprotective effects in diabetes is through regulating the expression of TGF-β and its downstream signaling pathway involving members of the Smad family of proteins. These findings suggest that E₂ supplementation may be beneficial in the preventing the development and progression of diabetic renal disease.