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. 2012 Oct 1;153(11):5491–5499. doi: 10.1210/en.2012-1379

Oxidant Stress and Mitochondrial Signaling Regulate Reversible Changes of ERα Expression and Apoptosis in Aging Mouse Glomeruli and Mesangial Cells

Simone Pereira-Simon 1, Xiaomei Xia 1, Paola Catanuto 1, Sharon Elliot 1,✉
PMCID: PMC3473210  PMID: 23027807

Abstract

Estrogen actions are largely dependent on the intracellular estrogen receptor (ER) levels. During aging the decline of estrogens or ER leads to a loss in antiinflammatory protection and an increase in oxidant stress due to changes in mitochondrial function. Estrogens/ER may also coordinate signaling between the nucleus and mitochondria through ERK activation, which paradoxically decreases ER expression. The changes in ER expression and transcriptional activation that occur with aging as well as the mitochondria-to-nuclear signaling pathways have not been studied in the glomerulus. We found that ER expression and transcriptional activation decreased with age. Whereas ER levels decreased by greater than 90%, serum 17β-estradiol levels decreased by less than 30%, suggesting alternative mechanisms for ER decrease. Because we postulated that this was due in part to age-related oxidant stress, we treated mesangial cells (MCs) with ethidium bromide (EtBr) to deplete mitochondria. EtBr treatment resulted in decreased ERK activation and reactive oxygen species, which were associated with increased ERα expression and transcriptional activation in old MCs. EtBr treatment also decreased apoptosis and caspase-9 protein expression in old MCs. These data suggest that loss of several of the functions of 17β-estradiol during aging could be mainly due to decreased ERα expression, that the ER loss is reversible by reducing reactive oxygen species, and that mitochondrial retrograde signaling plays a role in this regulation.

The glomerulus is an estrogen target tissue, and estrogen action is particularly important for maintaining glomerular structure and function and extracellular matrix turnover (1, 2). Glomerular cells express estrogen receptors (ER), predominantly the subtype ERα, which mediates most estrogen/ER actions in mesangial cells (MCs) (1). The effects of estrogens in the kidney are largely dependent on the intracellular ER levels, which are determined and regulated by genetic traits, the hormonal milieu, and environmental factors. For instance, female ROP Os/+ mice, which have an increased susceptibility to glomerulosclerosis, have lower glomerular and mesangial cell ER levels than C57BL/6 mice, a strain that is more resistant to glomerulosclerosis. In addition, estrogen deficiency at a young age accelerates the progression of glomerulosclerosis in female ROP Os/+ (3).

It is well established that aging and estrogen deficiency are associated with increased oxidant stress, which promotes age-related diseases in the renal vasculature (4, 5). Oxidant stress occurs when free radicals, single reactive oxygen species (ROS), and other reactive intermediates, such as advanced glycation endproducts of lipid peroxidation products, overwhelm antioxidant systems (6, 7). Mitochondria, a primary source of ROS in the cell, play an important role in cellular adaptation to oxidant stress (8). The consequences of increased age-related mitochondrial ROS production coupled with a decline in estrogens on the expression of ER, have not been well studied. We found that female C57BL/6 mice develop glomerular lesions as they age (9). We also showed that aged, ovariectomized C57BL/6 mice develop glomerular lesions after exposure to cigarette smoke (a source of oxidant injury and advanced glycation endproducts) (10), which can be ameliorated by 17β-estradiol (E2) replacement therapy (11). Based on these data, we postulated that the level of ER expression and function in the glomerulus decreases with age and may be mediated by age-associated oxidant stress.

In addition, because E2 regulation of mitochondrial function is most likely mediated through ER-dependent mechanisms (12, 13), a decline in ER expression and function in glomerular cells could lead to altered mitochondrial responses such as apoptosis. We found that aging decreases ERα mRNA and protein expression in glomeruli and MCs isolated from female C57BL6 mice. Transcriptional activation was decreased in parallel. Manipulation of oxidant stress in vitro also regulated ER expression. Surprisingly, in aging MCs, decreased ER expression and function were reversed by preventing signaling from the mitochondria through ERK. Finally, we show that increased apoptosis present in aging MCs was also reversed by reducing mitochondrial ROS production.

Materials and Methods

Isolation of glomeruli and glomerular cells

Glomeruli were isolated as previously described (14) for use in mRNA experiments. MCs were isolated from two separate glomerular isolates from each experimental group 7 and 24 months C57BL/6 mice (15). Glomeruli were plated in Nunc dishes (Rochester, NY) in the presence of 10% fetal bovine serum (FBS). MCs were identified by morphology, immunochemical profile (end to end F-actin filaments, smooth muscle actin, and the absence of nephrin and WT-1). Cell lines isolated from at least two individual mice per group were used for all experiments. All cell lines were studied between passages 5 and 15. Serum E2 levels were measured by ELISA as previously described (11).

Transfection studies

MCs from young and old mice and those treated with ethidium bromide (EtBr) were plated in basal medium with 20% charcoal/dextran-treated fetal bovine serum (<5 pg/ml estrogens) in 24-well plates. Twenty-four hours before transfections, MCs were grown in phenol red-free medium containing 0.1% charcoal stripped serum. MCs were transfected using GenePorter (Promega, Madison, WI) with a 4ERE-TATA-Luc reporter gene construct and the β-galactosidase gene pRSV-βgal (0.4 μg/well) to control for transfection efficacy. MCs were subsequently treated with vehicle or E2 (10 nm). After 48 h, MCs were harvested and luciferase and β-galactosidase assays were performed as previously described (1). Briefly, cells were washed two times in PBS and lysed with 100 μl of reporter lysis buffer (Promega) at room temperature for 15 min. Wells were scraped and the lysate transferred to a Microfuge tube, vortexed, and microcentrifuged for 2 min at 4 C. The supernatant was collected and frozen at −70 C until assayed. In some experiments, cells were transfected with a dominant-negative ERK1 or ERK2 plasmid (kind gift of Melanie Cobb, University of Texas Southwestern Medical Center, Dallas, TX).

Polymerase chain reaction

Amplification and measurement of target RNA was performed on the on the Step 1 real time PCR system as previously described (16). The mRNA sequence was obtained from the National Center for Biotechnology Information (Bethesda, MD) to acquire the copy number for each ER subtype. The number of occurrences of each of the four nucleobases was counted and multiplied by its respective molecular weight. These four numbers were then summed together to obtain the mass of 1 mol of each subtype of the ER. The mass of the purified plasmid of each subtype and the unknown samples was calculated by the A260 method on a Molecular Devices SpectraMax PLUS (Ramsey, MI) (17).

Western blot analysis

For protein analysis, cell lysates were extracted and protein assessed using the Pierce BCA protein assay kit (Rockford, IL). Equal amounts of protein were applied to precast sodium dodecyl sulfate polyacrylamide gels (Life Technologies, Grand Island, NY) and analyzed as previously described (2) for ERα (H184), ERK and phospho-ERK (Santa Cruz Biotechnology, Santa Cruz, CA) and caspase-9 (Cell Signaling Technology, Beverly MA). Blots were analyzed as described (17). In some experiments, cells were treated overnight with 40 μm PD98059. Western blots were also exposed to β-actin (Sigma Chemical, St. Louis, MO) to control for protein loading. Human recombinant ERα was used as a control (PanVera, Madison, WI).

Modulation of ROS

In some experiments, MCs were treated overnight with 25 μm N-acetyl-l-cysteine (NAC), an antioxidant, followed by 50 μm hydrogen peroxide (H2O2) for 4 h. Cells were collected as described for Western analysis and ERα protein expression determined.

ROS measurement

ROS was measured using 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate according to the manufacturer's directions (carboxy-H2DCFDA; Molecular Probes, Eugene, OR).

EtBr treatment

MCs were treated with 50 ng/ml EtBr to block mitochondrial DNA replication (18). Two cell lines of both young and old MCs were passaged and maintained in medium supplemented with 0.1% uridine and 1% sodium pyruvate and EtBr for 3 wk. These conditions allowed the cells to continue exponential growth while reducing mitochondrial DNA by 50% at each doubling. Sister cell lines were grown in parallel in exactly the same medium without EtBr as controls. Depletion of mitochondria was assessed by measurement of ROS and respiration. Cell viability was assessed by trypan blue.

Cell respiration and OxPhos activity

Oxygen consumption was measured polarographically in 0.3 m mannitol, 10 mm KCl, 5 mm MgCl2, 1 mg/ml BSA, 10 mm KH2PO4 (pH 7.4), with a Clark oxygen electrode (Hansatech Instruments, Norfolk, UK). After measuring intact cell endogenous respiration, antimycin A was added to block respiration at complex III. Ascorbate-N, N, N′, N′-tetramethyl-p-phenylenediamine was then be added to drive complex IV respiration and subsequently inhibited with 0.7 mm KCN.

Mito Tracker staining

Control and EtBr-treated MCs were plated on two-well chamber slides for 24 h (Lab-Tek; Nunc). The medium was replaced to remove FBS, and 25 nm of Mito Tracker Green FM and Mito Tracker Red CMXRos (Invitrogen, Carlsbad, CA) was added for 15 min followed by 4′,6′-diamino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA) for 5 min. The slides were washed, mounted, and visualized on a LSM700 confocal using a ×63 Plan Apochromat 63 × 1.4 oil NA objective.

DNA fragmentation by in situ cell staining and flow cytometry

Staining was performed using Apoptag fluorescence (ApopTag terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling fluorescein in situ apoptosis detection kit; Mllipore, Billerica, MA) according to manufacturer's directions. Then 1 × 106 control and EtBr-treated cells were seeded onto a fibronectin-coated flask. Forty-eight hours later, cells were washed with 0.5 ml PBS, resuspended in equilibration buffer, and centrifuged to remove the supernatant. Cells were incubated with terminal deoxynucleotidyl transferase for 30 min at 37 C. After a stop reaction was added, cells were washed and stained with propidium iodide for 15 min at room temperature. For flow cytometry analysis, the violet annexin V/dead cell apoptosis kit (Invitrogen) was used according to the manufacturer's directions. Briefly, MCs were exposed to the pacific blue and SYTOX stains for 30 min. Analysis was performed by two-color flow cytometry assays using flow cytometer (FACs Calibur) and CellQuest software (BD Biosciences, San Jose, CA).

Statistics

In vitro assays were performed in triplicate. One-way ANOVA and the Dunnett multiple-comparison post hoc test or Student's t test were performed for the statistical analysis. (GraphPad Prism; GraphPad Software Inc., San Diego, CA). Statistical significance was set at P < 0.05.

Results

ERα expression

Glomeruli were isolated from female C57BL/6 mice at different stages of reproductive life: young (7 months old) at which time estrous cycles are regular and approximately 4 d long (reproductive period); during the period of persistent vaginal cornification, 14 months of age (perimenopause); and between 20 and 30 months of age anestrous or rodent menopause (19). The glomerular ERα copy number was decreased at 27 months of age compared with 7 months of age (Fig. 1A, *, P < 0.05). This correlated with 30% decline in serum E2 levels at 27 months of age (Fig. 1B). In addition, ERα mRNA (young, 79 copies; old, 43 copies) and protein expression in MCs isolated from old mice decreased approximately 50% compared with young mice (Fig. 2A, **, P < 0.005).

Fig. 1.

Fig. 1.

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Glomerular ERα copy number declines during aging (A) and correlates with a decline in serum E2 (B). ERα copy number was determined by real-time RT-PCR of glomeruli isolated from 7-, 14-, and 27-month-old female C57BL6 mice (n = 5/group). *, P < 0.05, 27 months compared with other ages.

Fig. 2.

Fig. 2.

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ERα protein expression and transcriptional activation are decreased in MCs isolated from C57BL/6 mice during anestrus (24 months). A, Western analysis was performed as described in Materials and Methods. Shown are representative Western blots of ERα protein expression in young (7 months) and old (24 months) MCs and β-actin loading control. The arrow denotes the specific 66-kDa ERα band just less than the 75-kDa molecular weight marker. Data are graphed as the mean ± sem of n = 3 experiments on cell lines isolated from two individual mice. **, P < 0.005. B, Transcriptional activation decreased in MCs isolated from C57BL/6 mice during anestrus (24 months). MCs were grown in phenol red-free DMEM-F12 supplemented with 20% charcoal-stripped FBS for 24 h. After transfection, MCs were treated with either vehicle (V) or 10 nm E2 for 48 h in phenol red-free medium containing 10% charcoal-stripped FBS. Data are expressed as the percentage of young (7 months) MCs. Shown are means ± se of three independent experiments performed in triplicate wells on two cell lines isolated from two individual mice. *, P < 0.05 compared with young vehicle control. rhER, Recombinant human ERα protein.

ER transcriptional activation

To determine whether ER function declined in parallel with ER protein expression, MCs from old and young were transfected in phenol red-free medium containing 10% charcoal-stripped serum with 0.3 μg/well of a luciferase-based reporter construct containing four estrogen response elements. Stimulation with E2 increased transcriptional activation 2-fold in young cells (Fig. 2B, *, P < 0.05), whereas the there was no significant change in MCs from old mice.

ROS modulation of ERα expression

Because we hypothesized that changes in ERα expression and function were possible due to age-associated ROS, we induced ROS by treating cells with H2O2. We found a 45% decrease in ERα expression in young and 65% in old MCs after treatment. Overnight treatment with NAC, an antioxidant, prevented the H2O2 decrease in ERα expression of both young and old MCs (Fig. 3A).

Fig. 3.

Fig. 3.

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ERα expression is regulated by ROS through the ERK pathway. MCs from young (7 months) and old (24 months) C57BL/6 female mice were treated with either vehicle (V), hydrogen peroxide (H2O2), NAC, or a combination (NAC + H2O2) (A) or in a separate experiment with PD98059 (B). Cell lysates were subjected to Western analysis for ERα protein as described in Materials and Methods. Each cell line was normalized to its own vehicle control. Data are graphed as the mean ± sem. *, P < 0.05 compared with vehicle control; **, P < 0.005 compared with old vehicle control (n = 3 experiments). Inset is a representative set of Western blots showing ERα expression after vehicle or PD treatment and β-actin loading control. The arrow denotes the specific ERα band at 66 kDa just less than the 75-kDa molecular weight marker. rhER, Recombinant human ERα protein; PD, PD98059.

ERK phosphorylation, ERα expression, and ER transcriptional activation

Increased MAPK activation has been reported to regulate ERα expression (20, 21). To determine whether this was one of the signaling pathways responsible for age-associated changes in ER expression, we treated MCs from young and old mice with PD98059. Preventing ERK phosphorylation either by PD98059 (Fig. 3B, *, P < 0.05) or DN ERK (Supplement Fig. 1, **, P < 0.005, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org) increased ERα protein expression in old MCs.

Altering retrograde signaling between mitochondria and the nucleus could disrupt pathways important for regulation of ER expression; therefore, MCs were depleted of mitochondrial DNA by treatment with EtBr for 3 wk. Cells were viable as assessed by trypan blue. As expected, mitochondrial depletion resulted in a decrease in respiration (Fig. 4A) and a decrease in production of ROS (Fig. 4B). These data were also confirmed using Mito Tracker Green and Red (Invitrogen) to visualize both mitochondrial number and activity respectively (Fig. 4C) of young and old MCs. At baseline, young cells had more mitochondria and less activity (Fig. 4, B and C) compared with old cells (Fig. 4, J and K). EtBr treatment reduced mitochondrial activity in both young and old cells (Fig. 4, H and P).

Fig. 4.

Fig. 4.

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Preventing mitochondrial replication decreases both respiration and ROS production. MCs from young (7 months) and old (24 months) C57BL/6 female mice were treated with medium containing uridine in the presence or absence (control) of EtBr to prevent mitochondrial replication. Respiration (A) measured polarographically and ROS (B) measured using 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate were decreased after treatment. #, P < 0.005, @, P < 0.05 compared with young control; ***, P < 0.005 compared with old control, Data are the mean ± sem of n = 3 independent experiments on two different cell lines isolated from two individual mice. C, MCs were incubated with DAPI (A, E, I, and M) or Mito Tracker Green FM (B, F, J, and N) to detect mitochondrial number or Mito Tracker Red CXRos (C, G, and K) to detect mitochondrial activity. Localization and activity were merged using confocal microscopy as described in Materials and Methods (D, H, L, and P). EtBr treatment reduced mitochondrial activity in both young (G) and old MCs (O), although to a greater extent in old cells. Magnification, ×63 oil.

Blocking mitochondrial transcription/replication by EtBr treatment had no effect on ERK phosphorylation in MCs from young mice, whereas it decreased phosphorylation in MCs isolated from old mice (Fig. 5, *, P < 0.05). Importantly, it increased MC ERα protein expression 2.7-fold (Fig. 6A, *, P < 0.05) and mRNA expression almost 6-fold compared with MCs from young mice (young, 11 copies; old, 63 copies). In addition, transcriptional activation was approximately 50% higher (1 and 10 nm) in old MCs compared with that found in young MCs (Fig. 6B).

Fig. 5.

Fig. 5.

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ERK activation is reduced by EtBr treatment in MCs isolated from old (24 months) female C57BL/6 mice. MCs were treated with EtBr as described in Materials and Methods. Representative Western blots of total ERK, phospho-ERK, and β-actin loading control are shown. Data are the mean ± sem of the ratio of p42 to total ERK graphed as a percentage of control treated MCs (n = 3 independent experiments on two different cell lines isolated from two individual mice). *, P < 0.05 compared with old control MCs. C, Control.

Fig. 6.

Fig. 6.

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Inhibition of mitochondrial replication increases ERα expression (A) and transcriptional activation (B) in MCs from old (24 months) but not young (7 months) female C57BL/6 mice. MC protein was treated with EtBr as described in Materials and Methods. Western analysis was performed for ERα. A, Representative Western blots of ERα and β-actin as loading control. Data are the mean of n = 4 experiments expressed as a percentage of its own control-treated cells. rhER, Recombinant human ER protein; C, control. Arrow denotes specific ERα band at 66 kDa just less than 75-kDa molecular weight marker. B, MCs were treated with medium containing 1% uridine in the presence of EtBr and transfected with an estrogen-responsive reporter plasmid as described in Materials and Methods. After transfection, MCs were further treated with either vehicle (V) or 0–10 nm E2 for 48 h in phenol red-free medium containing 10% charcoal-stripped FBS. Data shown are expressed as luciferase reporter activity (percentage of young EtBr treated cells). Shown are mean ± se of three independent experiments performed in triplicate wells on two cell lines isolated from two individual mice. *, P < 0.05 compared to control. **, P < 0.005 compared with vehicle.

DNA fragmentation and apoptosis

Because mitochondria play a significant role in apoptosis, the effect of removal of mitochondria by EtBr treatment was examined. The increase in DNA fragmentation was partially abolished in MCs from old mice (Fig. 7A, panel G). There was no change in young MCs. We also performed flow cytometry (Fig. 7B) and confirmed that there were fewer apoptotic cells at baseline in young vs. old MCs (less than one vs. three cells), which was reduced by at least 80 ± 10% in old MCs (less than one cell, n = 3 individual experiments). In parallel, we found that EtBr treatment decreased caspase-9 protein expression approximately 30% in old (Fig. 7C) but not young MCs.

Fig. 7.

Fig. 7.

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EtBr treatment represses age-related apoptosis and caspase-9 expression in mesangial cells from old (24 months) female C57BL/6 mice. A, Young (7 months, panels A–D) and old (24 months, panels E–H) MCs were exposed to apoptag to detect apoptotic cells (panels A, C, E, and G). MCs were also stained with DAPI (panels B, D, F, and H). Old MCs exhibited increased apoptosis compared with young MCs. EtBr treatment abolished apoptosis coinciding with a reduction in mitochondrial signaling. Representative panels from two individual experiments are shown. B, Representative graphs of young (upper panels) and old (lower panels) control and EtBr-treated MCs exposed to the violet annexin V/dead cell apoptosis kit (Invitrogen). Flow cytometry was performed according to manufacturer's directions and showed decreased apoptosis after EtBr treatment in old MCs (n = 3 individual experiments). Live cells are shown in quadrant 3 (Q3) and apoptotic cells in quadrant 4 (Q4), C, Caspase-9, one of the caspases involved in the apoptosis cascade, is decreased after EtBr treatment of old (24 months) but not young (7 months) MCs. Inset shows a representative Western blot of caspase-9 (49 kDa) and β-actin as loading control. Data are graphed as a percentage of control-treated cells mean ± sem (n = 3 independent experiments on two cell lines isolated from two individual mice). *, P < 0.05.

Discussion

The effect of aging on glomeruli and glomerular cell ERα expression has not been previously described, although there have been studies in the kidney, brain. and inner ear of mice (22–24). In this report, we show for the first time that glomerular ERα mRNA expression decreases with increasing age in glomeruli isolated from female C57BL/6 mice. In addition, ERα protein expression of MCs isolated from young and old mice follow the same pattern. This is in contrast to a study on total kidney isolated from female AK mice, which reported an increase in ERα mRNA and protein expression with age (23). These conflicting results are very likely due to differences in mouse strains and/or the fact that the glomerulus represents a small fraction of total kidney and therefore could have a different expression pattern. We have previously shown that ERα expression levels correlate with the degree of glomerulosclerosis (GS), and we postulated that decreased ERα expression may promote age-related GS in mice as previously described (9). In fact, we show in the current study that transcriptional activation of ER is also decreased in an age-related fashion correlating with the decrease of ERα expression.

Several mechanisms have been reported for regulation of the ER including posttranscriptional destabilization of ERα mRNA (25) and destruction of ERα protein through the ubiquitin-proteasome pathway (26). Because the ERα mRNA levels in our study decreased in parallel with the protein expression, we believe that other mechanisms are more likely. For example, an age-related increase in the hypermethylation of CpG islands in the ER promoter region, which blocks the expression of the ERα gene, has been shown in human vascular smooth muscle cells and aortic endothelial cells (27–30). In addition, oxidant stress activates several transcription factors, including nuclear factor-κB, and MAPK (31, 32), which can regulate ER expression levels. We have previously shown that MCs isolated from mice that develop GS such as db/db and ROP Os/+ mice (33) have decreased ERα expression induced by hyperactivation of MAPK, which is also true for breast cancer cells (21, 34). Our current study suggests that ERK signaling regulates ER expression in old MCs. In addition, it is probable that ERK signaling to and from the mitochondria in old MCs led to the down-regulation of ERα, although this is likely not the sole source of glomerular/mesangial cell ER down-regulation associated with aging.

It is well known that the function of ERs can be modulated by the redox state of the cell (35). Variations in gene expression by both direct and indirect pathways occur due to changes in cellular redox state especially during aging (36). Aging increases oxidant stress as measured by increased levels of endogenous lipid peroxides [8-isoprostane levels in blood and urine (37, 38)]. Young men have higher levels of markers of oxidative stress compared with premenopausal age-matched women (39, 40), but parameters of oxidative stress increase in women after menopause (4). We have evidence that carboxy-methyl-lysine, a marker of oxidant injury (41), is increased with aging in the serum of female C57BL6 mice (Elliot, S. E., and H. Vlassara unpublished data); therefore, we investigated whether ER expression could be manipulated by changing the oxidant stress status of the cell. In fact, treatment of the cells with H2O2 decreased ER, whereas treatment with NAC prevented the decrease. Alternatively, to reduce ROS, we treated young and old MCs with EtBr to block mitochondrial transcription/replication. Confirmation of the effectiveness of the EtBr treatment was confirmed by a decrease in cellular respiration. Reducing mitochondrial ROS decreased ERK phosphorylation in old cells with a corresponding increase of ERα expression and transcriptional activation in old cells. We also found a parallel increase in ERα mRNA expression, suggesting a direct effect of ROS/mitochondrial signaling on the promoter of the ERα gene.

Mitochondria play an important role in cellular adaptation via a mitochondria-to-nucleus signaling pathway called retrograde regulation (36, 42). The retrograde response reacts in a continuous manner to the changing metabolic needs of the cell (43). Low ROS concentrations can act to oxidize redox-sensitive proteins phosphatases or kinases and modify the phosphorylation state of receptors and transcription factors, which in turn could influence the mitochondrial response. In addition, mitochondrial calcium release leads to multiple signaling cascades including activation of MAPK, c-Jun terminal kinase, and p38 MAPK (44). To our knowledge, the mitochondria-to-nuclear signaling pathways involved in the glomerulus have not been determined before our studies. Interestingly, in an effect opposite to that of the old MCs, ERK activation remained unchanged in the young MCs, suggesting different regulation of signaling patterns between old and young MCs.

Because mitochondria are central to the initiation of apoptosis, we investigated whether regulation of mitochondrial signaling might play a role in protection against apoptosis. We used apoptosis as a marker of aging because it is well established that apoptosis increases with age in the kidney and can be prevented by reduction in oxidant stress (45, 46). In fact, EtBr treatment effectively reduced both caspase-9 expression and apoptotic cell number in old mesangial cells.

In summary, ERα expression and transcriptional activation in glomeruli and MCs were decreased in aging. This reduction is reversible by manipulation of ROS and/or ERK activation through changes in mitochondrial signaling. These data suggest a role not only for oxidant stress per se in the regulation of ERα but also for retrograde signaling. In addition, it appears that ERα expression levels have the potential to be modulated, and it is possible that changes in mitochondrial signaling alter repressors of transcription and/or translation. Future studies will extend these findings to determine whether ERK and other relevant signaling pathways increase antioxidant enzymes or other protective mechanisms and whether ERα action is important for regulation of these pathways in the glomerulus.

Supplementary Material

Supplemental Data
supp_153_11_5491__index.html (973B, html)

Acknowledgments

We acknowledge the skilled assistance of the Flow Cytometry Core Facility of the Sylvester Comprehensive Cancer Center (University of Miami, Miller School of Medicine) for the provision of sophisticated fluorescence analysis and cell sorting services. The authors also thank Gabriel Gaidosh (Department of Ophthalmology, University of Miami, Miller School of Medicine) for help with the confocal microscopy.

This work was supported by National Institutes of Health Grant RO1 AG17170-11 (to S.E.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
DAPI
4′,6′-Diamino-2-phenylindole
E2
17β-estradiol
ER
estrogen receptor
EtBr
ethidium bromide
FBS
fetal bovine serum
GS
glomerulosclerosis
MC
mesangial cell
NAC
N-acetyl-l-cysteine
ROS
reactive oxygen species.

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