Differential effects of continuous and intermittent 17beta-estradiol replacement and tamoxifen therapy on the prevention of glomerulosclerosis: modulation of the mesangial cell phenotype in vivo

Abstract

Female ROP Os/+ mice are partially protected by endogenous estrogens against progressive glomerulosclerosis (GS) during their reproductive period; however, ovariectomy accelerates GS progression. We examined the effects of continuous and intermittent 17beta-estradiol (E(2)) replacement and tamoxifen therapy on the development of GS in ovariectomized (Ovx) ROP Os/+ mice. Continuous E(2) replacement (CE(2)) throughout 9 months prevented microalbuminuria and excess extracellular matrix accumulation in Ovx ROP Os/+, not only compared to placebo-treated Ovx mice but also in comparison to intact female ROP Os/+. Tamoxifen had a similar effect, but of lesser magnitude. Intermittent 3-month on-off-on E(2) did not reduce the kidney changes. Mesangial cells (MCs) from CE(2) mice maintained their estrogen responsiveness. E(2) in vitro prevented transforming growth factor-beta1 stimulation of a Smad-responsive reporter construct and increased MMP-2 expression and activity in MCs isolated from CE(2) mice. MCs from mice on either placebo or intermittent E(2) treatment did not respond to added E(2), consistent with a stable alteration of their estrogen responsiveness. Tamoxifen protection against GS was less pronounced in ROP Os/+ mice. Thus, prolonged estrogen deficiency promotes GS and renders MCs insensitive to subsequent estrogen treatment. This underscores the importance of continuous estrogen exposure for maintaining glomerular function and structure in females susceptible to progressive GS.

Figure 1-6943

Treatment scheme for mice receiving E₂. Arrow indicates time of ovariectomy (Ovx). Top of figure denotes 9-month continuous treatment with E₂ and bottom denotes 9-month intermittent treatment with E₂.

Figure 2-6943

Effect of continuous and intermittent E₂ replacement and tamoxifen therapy on UAE. ROP Os/+ mice were Ovx at 3 months of age and subsequently received either continuous or intermittent E₂ (0.05 mg/pellet), tamoxifen (Tam, 0.5 mg/pellet), or placebo (Pla) via subcutaneously implanted 90-day release pellets as described in the Materials and Methods section. A: Time course of UAE of Ovx mice on placebo (Pla, square), continuous E₂ (CE₂, triangle), intermittent E₂ (IE₂, inverted triangle), and tamoxifen (Tam, circle) replacement. Data are the mean ± SEM of each group of animals from 3 months of age until sacrifice at 12 months of age. B: UAE at the time of sacrifice of Ovx ROP Os/+ mice on placebo (Pla, square), continuous E₂ (CE₂, triangle), intermittent E₂ (IE₂, inverted triangle), and tamoxifen (Tam, circle) replacement. Compared to Pla-treated mice: ***P < 0.001, **P < 0.01. Each point on the graph represents an individual mouse.

Figure 3-6943

Histology: methacrylate-embedded sections were stained with periodic acid-Schiff silver methanamine. Renal cortex sections of 12-month-old female ROP Os/+ mouse are shown after mice underwent sham operation (A) or ovariectomy at 3 months of age and subsequently received placebo (B), CE₂ (C), or tamoxifen (D). Original magnifications, ×200.

Figure 4-6943

Morphometric analysis. Glomerular area (A) and volume (B) were smaller in mice on CE₂ (triangles) than in animals receiving placebo (Pla, squares) (P < 0.005) or sham-operated mice. Intermittent E₂ replacement (IE₂, inverted triangles) or tamoxifen (Tam, circles) did not reduce either of these parameters. Percentage of vascular space (C) increased with CE₂ and Tam therapy. Each point on the graph represents an individual mouse. *P < 0.05, **P < 0.005.

Figure 5-6943

Immunofluorescence staining of laminin. Representative kidney sections of Ovx ROP Os/+ mouse on placebo (A), CE₂ (B), or tamoxifen (C) at the time of sacrifice at 12 months of age. Original magnifications, ×400.

Figure 6-6943

TGF-β1 responsiveness. MCs were isolated and propagated from Ovx ROP Os/+ mice that had received either placebo (A) or CE₂ therapy (B) in vivo. Cells were transfected with the TGF-β1-responsive luciferase-based reporter construct p3TP-Lux, which contains three Smad-binding elements in the promoter region. After 24 hours, MCs were treated with vehicle or TGF-β1 (50, 125, and 500 pg/ml) in the absence (white bars) or presence of 10 nmol/L E₂ (black bars). Data are expressed as percentage of vehicle control. Shown are means ± SEM of cell lysates collected from two individual cell lines obtained from each treatment group. Triplicate wells were collected for each concentration of TGF-β1 (**P < 0.05 compared to control of same treatment, ^##P < 0.05 compared to untreated at the same dose, n = 3 individual collections).

Figure 7-6943

MMP-2 promoter activation. MCs were isolated from mice that had received either placebo (A) or CE₂ (B) and were transfected with an MMP-2 promoter linked to a luciferase reporter gene as described in Materials and Methods. After 24 hours, MCs were treated with vehicle or E₂ (0.1, 1, or 10 nmol/L). E₂ elicited a dose-dependent increase in luciferase activity but only in MCs isolated from mice that were on CE₂in vivo (B, black bars). TGF-β1 (500 pg/ml) abolished the estrogen-mediated increase in luciferase activity in MCs derived from mice on CE₂in vivo (B, white bars). Data are expressed as percentage of vehicle control. Shown are means ± SEM of cell lysates collected from two individual cell lines for each mouse treatment group. Triplicate wells were collected for each concentration of E₂ (*P < 0.05, n = 4 individual collections).

Figure 8-6943

MMP-2 activity. Supernatants of MCs isolated from mice on placebo or CE₂ were collected for zymography as described in Materials and Methods. A: Representative zymogram of supernatants of MCs that were treated with vehicle (V), E₂ (0.1 and 1 nmol/L), ICI (1 μmol/L), or ICI + E₂ (1 nmol/L). B: Graph depicting the mean ± SEM of duplicate experiments of MMP-2 activity in the supernatant of MCs isolated from mice on placebo after in vitro E₂ stimulation. C: Graph depicting the mean ± SEM of duplicate experiments of MMP-2 activity after in vitro E₂ stimulation of MCs isolated from mice on CE₂. Data are expressed as percentage of vehicle control. *P < 0.05.