Estrogen-related abnormalities in glomerulosclerosis-prone mice: reduced mesangial cell estrogen receptor expression and prosclerotic response to estrogens

Abstract

The development and progression of glomerulosclerosis (GS) is determined by the genetic background. The incidence of end-stage renal disease is increased in postmenopausal women, suggesting that estrogen deficiency may play a role in the accumulation of extracellular matrix by mesangial cells (MCs), which are primarily responsible for the synthesis and degradation of this matrix. Using mouse models that are prone or resistant to the development of GS, we compared the expression of estrogen receptor (ER)-alpha and ER-beta subtypes in GS-prone and GS-resistant glomeruli and isolated MCs, and examined the effects of estrogens on ER, collagen, and matrix metalloproteinase (MMP) expression in MCs. Glomeruli and MCs from GS-prone mice had decreased expression of ER-alpha and ER-beta subtypes and ER transcriptional activity was also decreased in their MCs. Importantly, although 17 beta-estradiol treatment resulted in decreased collagen accumulation and increased MMP-9 expression and activity in MCs from GS-resistant mice, there was, paradoxically, no effect on collagen accumulation and decreased MMP-9 expression and activity in MCs from GS-prone mice. Thus, GS susceptibility is associated with diminished ER expression in MCs. The renal protective effects of estrogens, including decreased collagen accumulation and increased MMP-9 expression, seem to be blunted in GS-prone MCs.

Figure 1.

Glomeruli isolated from GS-prone mice express lower levels of ER-α and ER-β subtype mRNA than glomeruli isolated from GS-resistant mice by real-time PCR. Total RNA was obtained from 100 glomeruli. ER-α, ER-β, and 18S transcripts were analyzed by real-time PCR. Graphs show ER-α and ER-β mRNA expression, normalized to 18S. Data are expressed as percentage of glomeruli isolated from the B6 strain. Shown are means ± SEM of samples run in duplicate from four animals for each strain. *, P < 0.05; **, P < 0.01.

Figure 2.

MCs isolated from GS-prone mice express lower levels of ER-α and ER-β subtypes than MCs isolated from GS-resistant mice. A: Total RNA was collected from MCs grown in DMEM/F12 medium supplemented with 20% FBS. ER-α, ER-β, and GAPDH transcripts were analyzed by RT-PCR. Representative amplicons of ER-α, ER-β, GAPDH, and molecular weight standards (M) were run in parallel. Graphs show ER-α (left) and ER-β (right) mRNA expression normalized to GAPDH. Data are expressed as percentage of MCs isolated from B6SJL mouse. Shown are means ± SEM of three independent experiments. ***, P < 0.001. B: Mouse MC homogenates (ER-α) or immunoprecipitates (ER-β) were analyzed by Western blotting using the ER-α MC-20 antiserum and the ER-β Y-19 antiserum, respectively. Twenty μg of MC homogenates or 25 μl of ER-β immunoprecipitates were loaded, respectively. The 66-kd human recombinant ER-α peptide (10 ng and 5 ng) and the 53-kd human recombinant ER-β peptide (1 μg and 0.5 μg) served as positive controls (lanes 1 and 2). Signals corresponding to the molecular weight of the wild-type ∼67-kd mouse ER-α, the ∼55-kd ER-β for B6SJL-derived MCs (lanes 3 and 4), and from ROP-derived MCs (lanes 5 and 6) were detected in the immunoblots. Graphs show ER-α (left) and ER-β (right) protein expression. Data are expressed as percentage of MCs isolated from B6SJL mouse. Shown are means ± SEM of three independent experiments. Statistical significance is indicated by * and ** (P < 0.05 and P < 0.01, respectively) when compared to MCs isolated from B6SJL mouse.

Figure 3.

Higher E₂ concentration was required to initiate transcriptional activity of ER in MCs from GS-prone mice. MCs were grown in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS for 4 days. After transfection, MCs were treated with 0 or 0.1 nmol/L of E₂, 10 nmol/L of E₂, or 1 μmol/L of ICI for 24 hours in phenol red-free medium containing 10% charcoal stripped FBS. Data are expressed as percentage of vehicle-treated MCs from three independent experiments performed in triplicate.

Figure 4.

E₂ down-regulated collagen type IV accumulation in MCs from GS-resistant mice, but not in MCs from GS-prone mice. MCs were grown for 3 days in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. The medium was replaced with phenol red-free DMEM/F12 medium supplemented with 0.1% charcoal-stripped FBS and E₂ (1 nmol/L) for 24 hours. Collagen type IV accumulation in the presence of 1 nmol/L of E₂ in MCs isolated from B6SJL mice and from ROP mice. Data are expressed as percentage of vehicle-treated MCs (0.001% ethanol, open bars) for each cell type and shown are means ± SEM of three independent experiments. Statistical significance is indicated by * (P < 0.05) for comparison to vehicle-treated MCs.

Figure 5.

E₂ up-regulated MMP-9 mRNA expression and activity in MCs from GS-resistant mice, but down-regulated them in MCs from GS-prone mice. MCs were grown for 3 days in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. The medium was replaced with phenol red-free DMEM/F12 medium supplemented with 0.1% charcoal-stripped FBS and E₂ (0, 0.1, or 1 nmol/L) for 24 hours. A: MMP-9 mRNA expression in the presence of E₂ in MCs isolated B6SJL and from ROP mice. B: MMP-9 activity in the presence of E₂ in MCs from B6SJL and ROP MCs. Data are expressed as percentage of vehicle-treated MCs (0.001% ethanol, open bars) for each cell type and shown are means ± SEM of three independent experiments. Statistical significance is indicated by *, **, and *** (P < 0.05, P < 0.01, and P < 0.001, respectively) for comparison to vehicle-treated MCs.

Figure 6.

E₂ effects on MMP-9 activity are modulated by ER-α. Treatment of MCs with THC, an ER-α agonist, down-regulated MMP-9 activity in MCs isolated from GS-prone mice but up-regulated MMP-9 activity in GS-resistant MCs. MCs were grown for 3 days in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. The medium was replaced with phenol red-free DMEM/F12 medium supplemented with 0.1% charcoal-stripped FBS and THC (1 μmol/L) for 24 hours. Data are expressed as percentage of vehicle-treated MCs (0.001% EtOH, open bars) for each cell type and shown are means ± SEM of three independent experiments. Statistical significance is indicated by *, **, and *** (P < 0.05, P < 0.01, and P < 0.001, respectively) for comparison to vehicle-treated MCs.