Rapamycin-induced autophagy protects proximal tubular renal cells against proteinuric damage through the transcriptional activation of the nerve growth factor receptor NGFR

Rapamycin transcriptionally upregulates NGFR expression in HK-2 cells

As first step of our in vitro study we tested the effects of rapamycin exposure on NGFR levels. HK-2 cells were plated in culture medium and treated with increasing concentrations of rapamycin (from 0.1 to 18 ng/ml). mRNA (Figure 1A) and protein (Figure 1B) expression levels of NGFR were evaluated, after 24 h and 48 h of treatment, by real time PCR and western blotting analysis, respectively. As showed in Figure 1, the treatment with rapamycin significantly induced NGFR mRNA and protein levels at all investigated doses. The transcriptional activation of NGFR was confirmed by transfection assay, using a luciferase reporter plasmid containing the wild-type NGFR promoter region (from −900 to +100 base pairs). After 24 h, transfected cells were treated for 18 h as reported and then luciferase activity was measured. Results showed a significant rapamycin-induced transactivation of the NGFR promoter, starting from the lower doses (Figure 1C). These data provided evidence, for the first time, that in HK-2 cells, the rapamycin exposure, upregulated neurotrophin receptor NGFR expression in a transcriptional dependent-manner.

Rapamycin induces autophagy via NGFR in HK-2 cells

First, we tested the effects of chronic exposure (48 h) to increasing concentrations of rapamycin (from 0.1 to 18 ng/ml) on HK-2 cells viability, by a luminescent cell viability assay. We observed that rapamycin at doses from 0.1 to 1 ng/ml did not elicit growth inhibitory effects. On the contrary, a slight reduction of HK-2 cell viability was observed at the doses of 7 ng/ml and 18 ng/ml respect to untreated cells (Figure 2A left panel). Next, in order to clarify if the reduction of vitality was associated with apoptotic events, in HK-2 treated with 7 ng/ml of rapamycin for 48 h we evaluated the PARP1 cleavage by western blotting and the fragmentation of DNA by a DNA laddering assay. We observed that rapamycin treatment did not induce change in PARP1 cleavage or in the inter-nucleosome fragmentation profile of genomic DNA (Figure 2A central and right panel), which is a diagnostic hallmark of cells undergoing apoptosis, suggesting that the growth inhibition (at doses of 7 ng/ml) did not occur through the apoptotic process, but may involve the autophagic pathway. Analysis of protein content confirmed the activation of autophagy at all doses investigated and an enhanced autophagic flux at the last 2 concentrations as shown by the SQSTM1/p62 decrease (Figure 2B left panel). To better confirm the obtained results, we performed the same experiment also in podocytes treated with rapamycin and/or bafilomycin A₁ (BAF; 25 nM). As expected, the activated autophagic flux, confirmed by upregulation of BECN1/Beclin 1 and LC3-II, (the autophagosome-associated and phosphatidylethanolamine [PE]-conjugated form of LC3-I), as well as by a decrease in SQSTM1, was reversed in the presence of the inhibitor BAF (Figure 2B right panel). Because the most common signaling by which rapamycin exerts its biological effects is mediated by MTOR inhibition, we evaluated the involvement of this pathway using the MTOR activator MHY1485 that potently inhibits autophagy by suppression of fusion between autophagosomes and lysosomes. Therefore, HK-2 cells were cotreated in complete medium with increasing doses of rapamycin with or without 2 µM MHY1485 and then, protein levels of MTOR, NGFR, SQSTM1, BECN1, as well as LC3B-I and LC3B-II were evaluated by western blotting analysis. Results reported in Figure 2C showed that all doses of rapamycin inhibited MTOR, concomitant with an upregulation of both NGFR and BECN1, and induced conversion of LC3-I to LC3-II (Figure 2C left panel). These effects were reversed in cells treated with rapamycin plus MHY1485, suggesting that the proautophagic action of rapamycin occurred through inhibition of MTOR signaling (Figure 2C right panel). In order to confirm the activated autophagic flux in HK-2 cells, the same experiment was performed in the presence of the autophagic inhibitor chloroquine (25 μM). Results showed similar effect like MHY1485 except for MTOR that persisted in the inhibited form and NGFR levels that were mitigated but not completely reversed after chloroquine exposure (Figure 2D). To clarify the involvement of NGFR in autophagy activation, HK-2 cells were transfected with NGFR RNAi for 48 h and then treated for 6 h with increasing doses of rapamycin. Results reported in Figure 2F, showed that in cells silenced for NGFR (Figure 2E), the mRNA (Figure 2F upper panel) and protein (Figure 2F bottom panel) induction of the proautophagic markers BECN1, as well as LC3-II was reversed, highlighting the crucial role of NGFR in mediating rapamycin-induced autophagy.

Rapamycin transactivates the NGFR promoter via the EGR1 transcription factor

Because we observed a significant role of NGFR in mediating rapamycin-induced autophagy, we aimed to study the consensus sites contained in its promoter (Figure 3A) that could be involved in autophagy activation.

To identify the transcription factor responsible for NGFR promoter transactivation induced by rapamycin exposure, HK-2 cells were transiently transfected for 24 h with a luciferase reporter plasmid containing the wild-type NGFR promoter region (from −900 to +100 base pairs), and with its deletion constructs (NGFR −164 + 100, NGFR −315 +100, NGFR −41 +100), each one lacking multiple consensus sites, and then treated for 18 h with 7 ng/ml of rapamycin.

Luciferase activity measurements showed that the transactivation of the NGFR promoter induced by rapamycin was still maintained using all deletion constructs (Figure 3B), suggesting that the region between −41 +100 could include consensus sites binding a transcription factor involved in the transactivation of the NGFR promoter, leading, in turn, to the activation of the autophagic process.

Promoter analysis for transcription factor binding sites, performed using Alibaba software, identified within the NGFR −41 +100 construct, multiple consensus sites for several transcription factors, as EGR1, NR3C1/GR, SP1, TFAP2A/AP2α, NF1, that act as modulators of genetic transcription, regulating various cellular processes such as cell survival, differentiation, apoptosis, autophagy and so on. Thus, we explored, in HK-2 cells treated with 7 ng/ml of rapamycin for 6 h, the nuclear translocation of above mentioned transcription factors. Results obtained by western blotting analysis reported in Figure 3C, showed that rapamycin exposure induced the nuclear translocation only of EGR1. Concomitantly, we observed that this effect was mediated by NGFR, as EGR1 nuclear translocation was reversed in cells silenced for NGFR (Figure 3D). To better confirm the involvement of MTOR inhibition pathway in activation of rapamycin-induced autophagy, in the same experimental condition, we explored the EGR1 cytosol-nucleus shuttle in HK-2 cells treated with rapamycin plus MHY1485. As reported in Figure 3E, the persistent activation of MTOR triggered by MHY1485 blocked the EGR1 transcription factor in the cytoplasm compartment. Taken together these results underlined that rapamycin induced autophagy through MTOR inhibition and activating EGR1 nuclear translocation that, in turn, transactivated NGFR promoter.

Rapamycin enhanches autophagy in HK-2 cells exposed to albumin overload

In order to clarify the biological role of activated autophagy induced by rapamycin in our experimental model, HK-2 cells were exposed for 6 h to rapamycin with or without 8 mg/ml of human albumin to mime in vitro, the proteinuria. Firstly, we observed, by western blotting analysis, that the upregulation of NGFR was more evident in cells cotreated with albumin plus rapamycin with respect to cells treated with rapamycin alone (Figure 4A). Our results showed that the combined treatment with rapamycin plus albumin significantly upregulated the autophagic marker BECN1 and enhanced the conversion of LC3-I to its PE-conjugated form LC3-II. In our experimental condition, performed for 6 h, albumin alone did not promote autophagy (Figure 4B left panel), according to that reported by Liu et al [13], that detected it at longer time (8 h). Interestingly, these effects were reversed in cells in which the expression of NGFR was silenced (Figure 4B right panel), highlighting that NGFR, activated by rapamycin, triggered the autophagy process, protecting proximal tubular renal cells from proteinuria damage. Next, to further examine autophagic vacuoles we performed transmission electron microscopy (TEM). As reported in Figure 4C, numerous autophagic structures (black arrows) with partially degraded material have been recognized in rapamycin-treated cells (Figure 4C-ii upper panel). Rapamycin-induced autophagy was not accompanied by destructive cytoplasmic changes, while silencing of NGFR drastically blocked rapamycin-induced autophagy (Figure 4C-iv upper panel). In albumin-treated cells, small lipid droplets (LDs) accumulated and became dispersed within the cytoplasm (Fig 4C-i bottom panel). Induction of autophagy has been demonstrated upon treatment of the cells with albumin plus rapamycin (Figure 4C-ii bottom panel). Autophagic vacuoles (black arrows) contained dense, amorphous materials and membranous myelin-like whorls. Interestingly, silencing of NGFR inhibited autophagy although did not attenuate lipid accumulation (Figure 4C-iii e -iv bottom panel).

Next, we explored which NGFR downstream pathways was involved in the autophagic process rapamycin-induced. Our results, obtained by of western blotting, revealed that the enhanced phosphorylation of MAPK1/ERK2-MAPK3/ERK1 induced by rapamycin, was strengthened in the presence of albumin overload, concomitantly with a significant EGR1 translocation in the nuclear compartment (Figure 4D and Figure 4E left panel). These effects were reversed in cells pretreated for 30 min with PD98059 (10 μM), a specific inhibitors of MAPK pathway (Figure 4D and Figure 4E right panel). Conversely, the pathway of MAPK/JNK was not activated in the same experimental condition (data not shown). Data obtained were in agreement with a luminescent cell viability assay (Figure 4F) that showed a reduction of cell death in HK-2 cells cotreated for 6 h with rapamycin plus albumin respect to cells treated with albumin alone, suggesting that NGFR via MAPK1/3 phosphorylates EGR1 that migrates to the nucleus, exerting its proautophagic effects.

The recruitment of EGR1 at the EGR1 site on the NGFR promoter region regulates rapamycin-induced autophagy in HK-2 cells

Results reported in Figure 3 suggested the crucial role of EGR1 transcription factor on activation of autophagy via NGFR. These data prompted us to investigate the molecular mechanism by which rapamycin induced nuclear EGR1 translocation, transactivating the NGFR promoter. Therefore, HK-2 cells were transfected with WT (NGFR −900 +100) and its shorter deletion (NGFR −41+100) constructs and then treated with rapamycin and albumin alone or in combination for 18 h. Luciferase activity of both constructs showed that the rapamycin-induced transactivation of NGFR was potentiated in cells cotreated as reported above, while no significant effect was observed upon albumin alone (Figure 5A).

The functional role of the EGR1 motif, located in the NGFR promoter, in mediating autophagy was investigated by chromatin immunoprecipitation assay (ChIP). After 6 h of treatment the protein-chromatin complexes were immunoprecipitated using antibodies against EGR1 and the POLR2A subunit of RNA Polymerase II. Results indicated that EGR1 was constitutively bound on the EGR1 site of the NGFR promoter and that its recruitment was increased after rapamycin exposure and to a higher extent, in the presence of rapamycin plus albumin. The same result was obtained using an antibody against POLR2A, highlighting that the cotreatment increased transcription of the NGFR promoter that, in turn, activated the autophagy process (Figure 5B). In accordance with these data, the potentiated transcription of POLR2A was substantiated by re-ChIP assay that showed a reduced recruitment of the chromatin silencer HDAC1 on the EGR1 motif in HK-2 cells treated with rapamycin alone or with albumin (Figure 5B). The crucial role of EGR1 on NGFR promoter transactivation was better demonstrated in HK-2 cells transfected with the construct NGFR −41 +100 and treated with 100 nM mithramycin A, a GC-rich inhibitor that displaces EGR1 binding to DNA. As reported in Figure 5C, exposure to mithramycin A reduced the transactivation of NGFR induced by rapamycin alone or with albumin, concomitantly with a decrease of EGR1 and POLR2A recruitment on the EGR1 site (Figure 5D).

Finally, the crucial role of EGR1 was better assessed by site-directed mutagenesis assay on the EGR1 site. We found that the mutated EGR1 motif abrogated the transactivation of the NGFR −41+100 construct, induced by rapamycin alone or with albumin (Figure 5E upper panel). In vitro data of Chen et al. have provided the first evidence that in chronic obstructive pulmonary disease, the activation of the E2F4 and EGR1 transcription factors by HDAC inhibition induces the expression of LC3B [14]. Therefore, we evaluated the protein levels of LC3B-I and LC3B-II in HK-2 cells treated as indicated above. Results reported in Figure 5F showed that the rapamycin-induced upregulation of LC3-II was enhanced in the presence of albumin overload and that LC3B-II levels were decreased in HK-2 cells treated with mithramycin A.

The chronic exposure to rapamycin protects HK-2 cells from apoptosis via MTOR inhibition

To explore whether the chronic exposure to rapamycin could activate apoptosis, HK-2 cells were treated with rapamycin with or without albumin for 48 h and then PARP1 cleavage was evaluated by western blotting. As evident in Figure 6A, the exposure to rapamycin reduced the apoptosis induced by human albumin alone, concomitant with a persistent autophagy activation. This data fit well with cell viability results (Figure 6B) that showed that the chronic activation of MTOR signaling, mimed by MHY1485 exposure, abrogated the protective effects induced by rapamycin in HK-2 cells exposed to overload of human albumin. These results confirmed the importance of MTOR inhibition in protecting HK-2 cells through autophagy activation.

Cell culture and treatments

Human renal proximal tubular cells, HK-2 (ATCC, CRL-2190), were grown in Keratinocyte-SFM (Invitrogen, 17005–034) containing 5 ng/ml EGF (Invitrogen, 10450-013), bovine pituitary extract (0.5 mg/ml; Invitrogen, 13028-014) and 1 mg/ml penicillin/streptomycin (Invitrogen, 15140-122). Human immortalized podocytes, a gift from professor G. Camussi (Department of Internal Medicine and Center of Experimental Research and Medical Sciences (CERMS), University of Turin, Turin, Italy) [38], were grown in Dulbecco's modified Eagle' medium (Gibco, 41966-029) plus glutamax (Gibco, 25030-024) containing 10% fetal bovine serum (Invitrogen, 10500-064) and 1 mg/ml penicillin-streptomycin. All experiments were performed in complete medium because starvation conditions induce basal autophagy activation. The cells were exposed to the following treatments: rapamycin (from 0.1 to 18 ng/ml; Sigma Aldrich, R8781); albumin from human serum (8 mg/ml; Sigma Aldrich, A1653); chloroquine (25 µM; Sigma Aldrich, C6628), bafilomicyn A₁/BAF (25 nM; Sigma Aldrich, B1793), MHY1485 (2 µM; Sigma Aldrich, SML0810); PD98059 (10 µM; Sigma Aldrich, P215).

Immunoblot analysis

Cells were treated in complete medium as indicated and then subjected to total protein extraction as described previously [8]. Nitrocellulose membrane were probed with: PARP1 (1:1000; Santa Cruz Biotechnology, sc-7150), NGFR (1:300; Santa Cruz Biotechnology, sc-8317), SQSTM1/p62 (1:1000; Santa Cruz Biotechnology, sc-28359), BECN1/Beclin 1 (1:1000; Santa Cruz Biotechnology, sc-48341), MAP1LC3B (1:200; Santa Cruz Biotechnology, sc-28266), pMTOR (Ser2448) (1:500; Cell Signaling Technology, sc-101738), total MTOR (1:500; Santa Cruz Biotechnology, sc-136269), phosphorylated and total MAPK (MAPK1/ERK2/p42 and MAPK3/ERK1/p44) (1:1000; Santa Cruz Biotechnology, sc-7383 and Cell Signaling Technology, 9102) and MAPK/JNK (1:500; pan-phospho-MAPK8/9/10[JNK1/2/3, respectively] Santa Cruz Biotechnology, sc-6254 and pan-nonphospho-MAPK8/9/10 [JNK1/2/3], Santa Cruz Biotechnology, sc-571) antibodies. As internal control, all membranes were subsequently stripped (0.2 M glycine, pH 2.6, for 30 min at room temperature) of the first antibody and reprobed with anti-GAPDH antibody (1:10000; Santa Cruz Biotechnology, sc-25778). The antigen-antibody complex was detected by incubation of the membranes for 1 h at room temperature with peroxidase-coupled goat anti-mouse (1:2000, IgG-HRP; Santa Cruz Biotechnology, sc-2005) or anti-rabbit (1:7000, IgG-HRP; Santa Cruz Biotechnology, sc-2004) and revealed using the enhanced chemiluminescence system (Santa Cruz Biotechnology, sc-2048). Blots were then exposed to film (Santa Cruz Biotechnology).

For nuclear and cytoplasmic extracts cells were harvested in cold PBS (Sigma-Aldrich, P4417) and then 300 μl of cytosolic buffer (50 mM HEPES, pH 7.5 [Sigma Aldrich, H3375, 150 mM NaCl [Sigma Aldrich, S7653] 1% Triton X-100 [Sigma Aldrich, T8787], 1.5 mM MgCl2 [Sigma Aldrich, M8266], 1 mM EGTA, pH 7.5 [Sigma Aldrich, E3889, 10% glycerol [Sigma Aldrich, G5516], protease inhibitors [Sigma-Aldrich, P8340]) were added. Following centrifugation (14000 g, 4°C, 10 min), the supernatant was referred to as cytoplasmic fraction. The pellet containing nuclei was resuspended in high salt buffer (20 mM HEPES pH 7.9, 25% [v:v] glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA [Sigma Aldrich, E5134], protease inhibitors). Extraction of nuclear proteins was achieved by vortexing this solution thoroughly, incubating overnight with agitation and subsequent centrifugation (14000 g, 4°C, 10 min). The collected supernatant represents the nuclear fraction. The purity of the cytoplasmic and nuclear fractions was confirmed by immunoblotting respectively with an anti-GAPDH antibody (Santa Cruz Biotechnology, sc-25778) and anti-LMNB/Lamin B (1:3000; Santa Cruz Biotechnology, sc-6217). Protein concentration was determined by Bradford Protein Assay (Bio-Rad, 5000006) and then equal amounts (40 μg) of cytosolic and nuclear proteins were prepared, resolved by 8% SDS-PAGE and probed with antibodies directed against: EGR1 (1:300; Santa Cruz Biotechnology, sc-110); NR3C1/GR (1:1000; Santa Cruz Biotechnology, sc-8992); SP1 (1:1000; Santa Cruz Biotechnology, sc-59); TFAP2A/AP2α (1:500; Santa Cruz Biotechnology, sc-12726); NF1 (1:500; Santa Cruz Biotechnology, sc-870). The bands of interest were quantified using the ImageJ densitometry scanning program.

Real-time RT-PCR assays

HK-2 cells were grown in 6-well plates to 70% to 80% confluence and exposed to treatments as indicated. Total RNA was isolated from cells with TRIZOL reagent (Invitrogen, 15596026) according to the manufacturer's protocol. The purity and integrity of the RNA was confirmed both spectroscopically and electrophoretically. Analysis of gene expression was performed using real-time reverse transcription PCR.

cDNA was synthesized from 2 μg of total RNA with random hexamer primers using the High capacity cDNA Archive Kit (Applied Biosystems, 4368813. Real-time PCR was performed in an iCycler iQ Detection System (Bio-Rad, 170–8740, Hercules, CA, USA) using SYBR Green Universal PCR Master Mix (Applied Biosystems, 4309155) with 0.1 mmol/l of each primer in a total volume of a 30-μl reaction mixture following the manufacturer's recommendations. Negative control containing water instead of first-strand cDNA was used. Each sample was normalized on its GAPDH mRNA content.

Primers used for the amplification were:

• NGFR: forward 5’-CGTATTCCGACGAGGCCAACC-3’ and reverse 5’-CCACAAGGCCCACAACCACAGC-3’

• BECN1: forward 5’-GGCCAATAAGATGGGTCTGA-3’ and reverse 5’-GCTTTTGTCCACTGCTCCTC-3’

• MAP1LC3B: forward 5’-GATGTCCGACTTATTCGAGAGC-3’ and reverse 5’-TTGAGCTGTAAGCGCCTTCTA-3’

• EGR1: forward 5’-CTTCAACCCTCAGGCGGACA-3’ and reverse 5’-GGAAAAGCGGCCAGTATAGGT-3’

• (GAPDH): forward 5’-CCCACTCCTCCACCTTTGAC-3’ and reverse 5’-CATACCAGGAAATGAGCTTGACAA-3’

The relative gene expression levels were normalized as previously described [12].

DNA laddering

HK-2 cells were plated in 10-cm dishes to 70% confluence and exposed to 7 ng/ml rapamycin for 48 h. Cells were pelleted at 125 × g for 5 min, washed in PBS and resuspended in 0.5 ml of extraction buffer (50 mmol/L Tris-HCl, pH 8, 10 mmol/L EDTA, 0.5% SDS) for 20 min in rotation at 4°C. DNA was extracted with a phenol-chloroform protocol. The aqueous phase was used to precipitate nucleic acids with 0.1 volumes of 3 M sodium acetate and 2.5 volumes cold ethanol overnight at −20°C. The DNA pellet was resuspended in 15 µl of H₂O treated with RNase A (Invitrogen, 12091039) for 30 min at 37°C. The absorbance of the DNA solution at 260 and 280 nm was determined by spectrophotometry (Eppendorf BioPhotometer® D30, 6133000001, Hamburg Germany). The extracted DNA was subjected to electrophoresis on 1.8% agarose gel.

Transfection assay

HK-2 cells were transfected using Lipofectamine 2000 (Invitrogen, 11668019), according to the procedure outlined in the product manual, in complete medium without penicillin/streptomycin with 100 nM of validated stealth NGFR targeted for the human NGFR mRNA sequence RNAi (Invitrogen, ID: 1299001), or with a negative control RNAi (Invitrogen, 452001) that does not match with any currently known human mRNA (scrambled siRNA). Efficiency of knockdown for each gene and the negative control was determined by western blot analyses (data not shown). After 48 h with the respective siRNA complexes the cells were treated as indicated and experiments were performed.

The WT NGFR promoter (NGFR −900 +100) and its deletion constructs (NGFR −164+100, NGFR −315+100, NGFR −41 +100) were a gift from E. Valli (University of Bologna, Department of Biology, Bologna, Italy) [39].

Functional studies were performed in HK-2 cells transfected for 24 h with WT and deletion constructs (0.5 µg) containing the NGFR promoter region, concomitant with 5 ng of pRL-CMV (Promega, E2261), which expresses Renilla luciferase enzymatically distinguishable from firefly luciferase by the strong cytomegalovirus enhancer/promoter, using the Fugen 6 reagent (Promega, E2692). Then HK-2 cells were treated as reported for 18 h. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo Reagent to the same tube. The Stop & Glo Reagent also produces a stabilized signal from the Renilla luciferase, which decays slowly over the course of the measurement. The integrated format of the DLR™ Assay provides rapid quantification of both reporters either in transfected cells or in cell-free transcription and translation reactions. The firefly luciferase values of each sample were normalized by Renilla luciferase activity used as internal control.

Site-directed mutagenesis

The NGFR promoter plasmid containing the EGR1-responsive element-mutated site was created by site-directed mutagenesis using a mutagenesis kit (Thermofisher, F541), according to the manufacturer's method using as template the shorter construct (−41 +100). Primers utilized are the following: (mutations are shown as lowercase letters): Fw: 5’-CGCTGCCGCGGGAGttagtCGATGGGGCAGGTGC-3’ and Rv: 5’-GCACCTGCCCCATCGactaaCTCCCGCGGCAGCG-3’. The mutations were confirmed by DNA sequencing.

Next, the mutated construct (mutated NGFR^EGR1 promoter) was transfected in HK-2 cells as reported above.

Cell viability assay

The CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7571) is a homogeneous method to determine the number of viable cells in culture based on quantification of ATP, which signals the presence of metabolically active cells. The amount of ATP is directly proportional to the number of cells present in culture. Briefly, HK-2 cells (20,000 cells per well) were grown in 96-well plates and exposed to treatments as indicated in complete culture medium. A volume of CellTiter-Glo® Reagent equal to the volume of cell culture medium was added to each well, cells were placed in a shaker for 2 min to induce lysis and incubated at room temperature for 10 min to stabilize the luminescent signal. The luminescence was read by a luminometer (Lumat LB 9507 Tube Luminometer, 2334, Berthold Detection Systems GmbH Pforzheim Germany) and results were reported as fold respect to untreated cells.

Transmission electron microscopy

For transmission electron microscopy (TEM), samples were fixed in 3% glutaraldehyde prepared in 0.1 M phosphate buffer for 2 h at 4°C, postfixed in 3% osmium tetroxide, dehydrated in graded acetone, and embedded in Araldite (Fluka, 10951). Ultrathin sections were prepared using a diamond knife, collected on copper grids (EMS, G 300 Cu) and then examined with a Jeol JEM 1400 Plus electron microscope (Peabody, Massachusetts, USA) at 80 kV.

Chromatin immunoprecipitation (ChIP) assay

HK-2 cells were treated for 6 h as indicated and cross-linked in PBS containing 1X formaldehyde for 7 min at 37°C. Then cells were pelletted and resuspendend in a lysis buffer (1% SDS, 10 mmol/L EDTA, 50 mmol/L Tris-HCl, pH 8.1), and left on ice for 10 min. The HK-2 cells were sonicated 4 times for 10 sec at 30% of maximal power (Vibra Cell 500 W; Sonics and Materials, Inc., NEWTOWN, Connecticut, USA) and collected by centrifugation at 4°C for 10 min at 11,000 × g. Sample (30 µl) was used as input (non immunoprecipitated). The samples were diluted in immunoprecipitation buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mmol/L EDTA, 16.7 mmol/L Tris-HCl, pH 8.1, 16.7 mmol/L NaCl), precleared with 60 µl of protein A/G Plus (Santa Cruz Biotechnology, SC-2003) for 1 h at 4°C.

Precleared chromatin was immunoprecipitated with anti-EGR1 or anti-POLR2A antibodies (Santa Cruz Biotechnology, sc-899) overnight at 4°C and reimmunoprecipitated with antibody against HDAC1 (Santa Cruz Biotechnology, sc-8410). The next day 60 µl of protein A/G Plus was added, and precipitation was further continued for 2 h at 4°C. Immunocomplexes DNA-proteins were washed sequentially for 5 min with the following buffers: Wash A (0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl, pH 8.1, 150 mmol/L NaCl); Wash B (0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl, pH 8.1, 500 mmol/L NaCl]; and Wash C [0.25 M/L LiCl, 1% NP-40 [Sigma Aldrich, I3021], 1% sodium deoxycholate [Sigma Aldrich, 30970], 1 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 8.1), and then twice with 10 mmol/L Tris, pH 8.0, 1 mmol/L EDTA. Samples were eluted with elution buffer (1% SDS, 0.1 M/L NaHCO₃). 5 M NaCl was added to the ChIP samples overnight at 65°C followed by digestion with proteinase K (0.5 mg/ml; Bioline, BIO-37037) at 45°C for 1 h. DNA was extracted using phenolchloroform-isoamyl alcohol. Two microliters of 10 mg/ml yeast tRNA (Sigma Aldrich, R4018) were added to each sample, DNA was precipitated with 95% ethanol for 24 h at −20°C, washed with 70% ethanol and resuspended in 20 µl of 10 mmol/L Tris, pH 8.0, 1 mmol/L EDTA buffer. Purified DNA (0.5 µl) was used as a template for real-time PCR detection using primers flanking the EGR1 site sequence present in the human NGFR promoter: Fw: 5’-GGACCGAGCTGGAAGTCG-3’ and Rv: 5’-GCTAACACTCACCCCCAGAA- 3’.

Results were normalized in comparison to total input DNA.

Statistical analysis

All experiments were performed in, at least, triplicate per treatment and repeated in 3 independent experiments. Real Time RT-PCR and Luciferase activity results are presented as fold induction over basal condition. Optical densities were measured using the ImageJ 1.47T software and their results are presented as fold induction respect to control. All results are presented as mean ± SD of data from 3 combined experiments. Data were analyzed by the Student t test using the GraphPad Prism 4 software program. P < 0.05 was considered as statistically significant.