AT₁R blockade in adverse milieus: role of SMRT and corepressor complexes

Abstract

ANG II type 1 receptor blockade (AT₁R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT₁R-BLK provides podocyte protection through regulation of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and vitamin D receptor (VDR) expression under adverse milieus such as high glucose and human immunodeficiency virus infection. Both AT₁R-BLK and VDR agonists (VDAs) stimulated VDR complex formation that differed not only in their composition but also in their functionality. AT₁R-BLK-induced VDR complexes contained predominantly unliganded VDR, SMRT, and phosphorylated histone deacetylase 3, whereas VDA-VDR complexes were constituted by liganded VDR and CREB-binding protein/p300. AT₁R-BLK-induced complexes attenuated podocyte acetyl-histone 3 levels as well as cytochrome P-450 family 24A1 expression, thus indicating their deacetylating and repressive properties. On the other hand, VDA-VDR complexes not only increased podocyte acetyl-histone 3 levels but also enhanced cytochrome P-450 family 24A1 expression, thus suggesting their acetylating and gene activation properties. AT₁R-BLK- induced podocyte SMRT inhibited expression of the proapoptotic gene BAX through downregulation of Wip1 and phosphorylation of checkpoint kinase 2 in high-glucose milieu. Since SMRT-depleted podocytes lacked AT₁R-BLK-mediated protection against DNA damage, it appears that SMRT is necessary for DNA repairs during AT₁R-BLK. We conclude that AT₁R-BLK provides podocyte protection in adverse milieus predominantly through SMRT expression and partly through unliganded VDR expression in 1,25(OH)₂D-deficient states; on the other hand, AT₁R-BLK contributes to liganded VDR expression in 1,25(OH)₂D-sufficient states.

the histone acetylation/deacetylation status at gene promoters has a significant impact on gene expression due to its effect on chromatin compaction (10). Nucleosomal DNA accessibility and associated gene expression are determined by the presence of corepressor (deaectylator) and coactivator (acetylator) complexes at the promoter region of the gene. In the absence of the ligand, nuclear receptors recruit corepressor complexes that condense chromatin structure (through coiling histone via deacetylation) and make the promoter region inaccessible to transcription factors. Conversely, in the presence of the ligand, receptors dislodge the repressor complexes and recruit coactivators that relax chromatin structures (uncoiling histones through acetylation), thus making the promoter region accessible (permissive) for transcription factors.

The vitamin D receptor (VDR) is a member of the nuclear receptor family of transcription factors (2, 11, 32). Actions of nuclear receptors provide the concept of cyclical gene regulation in which transcription factors oscillate between on and off states (1). However, VDR differs from these classical nuclear receptors by being located in the nucleus even in the absence of its ligand [1,25(OH)₂D; active vitamin D₃] (3). Nonetheless, the presence or absence of the VDR ligand determines the recruitment of activator versus repressor complexes (4, 6, 11, 25) by VDR. Therefore, vitamin D response element (specific DNA sequences for VDR binding at the target genes) sites are biologically relevant in both the presence and absence of the VDR ligand (32). VDR heterodimerizes with the retinoid X receptor (RXR) and forms a VDR-RXR complex, which recruits either repressor [transducin β-like 1 (TBL1)/TBL1 receptor (TBL1R), mSin-3, nuclear corepressor-1/nuclear corepressor-2 (silencing mediator of retinoic acid and thyroid hormone receptor; SMRT), and histone deacetylase (HDAC)1/3] (4, 15, 27) or activator [CREB-binding protein (CBP)/p300, P/CBP-interacting protein (CIP), and steroid receptor coactivator (SRC)-1] complexes. In the absence of the ligand, nuclear VDR recruits corepressor complexes, which repress the transcription; nonetheless, loss of the VDR-RXR corepressor complex not only derepresses target genes but also opens its binding sites for other transcription factors for positive gene regulation (26).

Unliganded VDR (VDR-UL) is continuously degraded via the ubiquitination-proteosome pathway (4, 7, 39), and binding of the ligand [1,25(OH)₂D] halts this degradation. 1,25(OH)₂D accelerates nuclear import of VDR, whereas RXR slows down its export (4, 7, 9, 39). Interestingly, enhanced VDR-UL expression has been reported to be associated with attenuated expression of cytochrome P-450 family 24A1 (CYP24A1) in cancer cells (1). These investigators speculated that this may be an attempt to retard degradation of 1,25(OH)₂D in 1,25(OH)₂D-deficient states. Conversely, liganded VDR enhances the transcription of CYP24A1, which degrades 1,25(OH)₂D and thus serves as negative feedback to prevent 1,25(OH)₂D-induced hypercalcemia.

Phosphorylation of either TBL1R or SMRT destabilizes the corepressor complex and facilitates the translocation of SMRT to the cytosol for proteosomal degradation (13, 24–27), whereas the phosphatase and tensin homolog (PTEN) pathway stabilizes and activates the SMRT complex (34, 37). High-ANG II states, such as high glucose and human immunodeficiency virus (HIV) infection, have been reported to enhance Akt phosphorylation (14, 33), which, in turn, can potentially stimulate SMRT phosphorylation and proteosomal degradation (24, 26). On that account, we hypothesized that both high glucose and HIV would induce derepression of CYP24A1. We further hypothesized that ANG II type 1 receptor (AT₁R) blockade (AT₁R-BLK) has the potential to provide protection to podocytes in adverse milieus (AMs) in multiple ways, including enhanced expression of VDR-UL and SMRT (as a part of repressor complex) and retardation of 1,25(OH)₂D degradation (via repressing the expression of CYP24A1) in 1,25(OH)₂D-deficient states and enhanced expression of liganded VDR in 1,25(OH)₂D-sufficient states.

In the present study, we evaluated the role of de novo activation of corepressor complexes during AT₁R-BLK. We determined whether AT₁R-BLK-induced SMRT expression confers additional podocyte protection in AMs.

MATERIALS AND METHODS

Human podocytes.

Human podocytes (HPs) were conditionally immortalized by introducing temperature-sensitive simian virus 40-T antigen by transfection (29). These cells proliferate at a permissive temperature (33°C) and enter growth arrest after transfer to a nonpermissive temperature (37°C). The growth medium contained RPMI-1640 supplemented with 10% FBS, 1 × penicillin-streptomycin, 1 mM l-glutamine, and 1 × ITS (Invitrogen) to promote the expression of T antigen. The incubation medium contained 5–15 pM 1,25(OH)₂D [normal serum, 50–150 pM 1, 25(OH)₂D]. In all experiments (except those with high glucose), we used RPMI-1640 containing 5 mM glucose.

Production of pseudotyped retroviral supernatant.

Replication-defective viral supernatants were prepared as previously described (14).

Immunofluorescence detection of VDR, SMRT, CYP24A1, DNA strand breaks, and repairs.

Control and experimental podocytes were fixed and permeabilized with buffer containing 0.02% Triton X-100 and 4% formaldehyde in PBS. Fixed cells were washed three times in PBS and blocked in 1% BSA for 30 min at 37°C. Subsequently, cells were labeled with either anti-VDR (Santa Cruz Biotechnology), anti-CYP24A1 (Santa Cruz Biotechnology), and anti-SMRT (Santa Cruz Biotechnology) antibodies. Propidium iodide (Sigma) and 4′,6-diamidino-2-phenylindole were used for nuclear localization. Subsequently, cells were examined under an immunofluorescence microscope. Images of 10 random fields (∼8–10 cells/field) in three sets of experiments were captured. The integrated density of each cell was analyzed by ImageJ. Each dot represents an average of 8–10 cells/field. All data were imported into Microsoft Excel worksheets to give a summated set of results for each experiment. The integrated density of the fluorescent signal forms the y-axis.

In parallel sets of experiments, control and experimental cells were labeled for H2AX (double strand breaks, Molecular Probes) and KU80 (DNA repair proteins, Cell Signaling) as previously described (30) and examined under a fluorescence microscope.

Western blot experiments.

Control and experimental cells were harvested, lysed in RIPA buffer containing 50 mM Tris·Cl (pH 7.5), 150 mM NaCl, 1mM EDTA, 1% Nonidet P-40, 0.25% deoxycholate, 0.1% SDS, 1× protease inhibitor cocktail (Cocktail Set I, Calbiochem), 1 mM PMSF, and 0.2 mM sodium orthovanadate. The protein concentration was measured using a Bio-Rad Protein Assay kit (Pierce, Rockford, IL). Total protein lysates (20 μg/lane) were loaded on a 15% polyacrylamide premade gel (Bio-Rad, Hercules, CA) and transferred onto a nitrocellulose membrane using a Bio-Rad miniblot apparatus. Nitrocellulose membranes were processed for immunostaining with primary antibodies against VDR (Santa Cruz Biotechnology), CYP24A1 (Santa Cruz Biotechnology), SMRT (Santa Cruz Biotechnology), Sin3A Rb monoclonal antibody (Abcam, Cambridge, MA), TBL1R (Abcam), phosphorylated (p-)HDAC3 (Ser⁴²⁴, Santa Cruz Biotechnology), p-Akt (Ser⁴⁷³, Cell Signaling), CBP/p300 (K1535, Cell Signaling), and acetyl-histone 3 (Ac-H3; Lys^9/14, Santa Cruz Biotechnology) and subsequently with the appropriate horseradish peroxidase-β-labeled secondary antibodies. Blots were developed using a chemiluminescence detection kit (Pierce) and exposed to X-ray film (Eastman Kodak, Rochester, NY). Equal protein loading and protein transfer were confirmed by immunoblot analysis for the determination of β-actin protein (Santa Cruz Biotechnology) on the same (stripped) Western blots.

Measurement of proteosomal activity.

Control and experimental podocytes were lysed in buffer [20 mM Tris·HCl (pH 7.2), 50 mM NaCl, 2 mM MgCl₂, 0.1% Nonidet P-40, and protease inhibitor cocktail complete (Roche Molecular Biochemicals, Mannheim, Germany)]. After the estimation of protein content, lysates were incubated in triplicate with substrate buffer [20 mM Tris·HCl (pH 7.2), 25 mM KCl, 10 mM NaCl, 1 mM MgCl₂, and 0.1 mM EDTA] containing 100 μM of the synthetic fluorogenic peptide substrate (Suc-LLVY-AMC, Calbiochem) for 30 min at 37°C. Fluorescence was measured with a plate reader (SLT-Lab Instruments, Crailsheim, Germany) using an excitation wavelength of 390 nm and emission spectrum at 460 nm.

RT-PCR analysis.

Control and experimental podocytes were used to quantify mRNA expression of VDR and CYP24A1. RNA was extracted using TRIzol (Invitrogen). For cDNA synthesis, 2 μg of the total RNA were preincubated with 2 nmol of random hexamer (Invitrogen) at 65°C for 5 min. Subsequently, 8 μl of the reverse transcription reaction mixture containing cloned avian myelobalstosis virus reverse transcriptase, 0.5 mmol each of the mixed nucleotides, 0.01 mol DTT, and 1,000 U/ml Rnasin (Invitrogen) were incubated at 42°C for 50 min. For a negative control, a reaction mixture without RNA or reverse transcription was used. Samples were subsequently incubated at 85°C for 5 min to inactivate the reverse transcriptase.

Quantitative PCR was carried out in an ABI Prism 7900HT sequence detection system using the following primer sequences: VDR, forward 5′-GACTTTGACCGGAACGTGCCC-3′ and reverse 5′-CATCATGCCGATGTCCACACA-3′; and CYP24A1, forward 5′-CAAACCGTGGAAGGCCTATC-3′ and reverse 5′-AGTCTTCCCCTTCCAGGATCA-3′.

SYBR green was used as the detector and ROX as a stabilizing dye. Results (means ± SD) represent at least three sets of experiments, as described in the figures. The data were analyzed using the comparative threshold cycle (C_T) method (ΔΔC_T method). Differences in C_T values were used to quantify the relative amount of PCR target contained within each well. Data were expressed as relative mRNA expression in reference to the control, normalized to the quantity of RNA input by performing measurements on an endogenous reference gene (GAPDH).

Immunoprecipitation.

Protein lysates were first immunoprecipitated after the addition of 10 μg monoclonal antibody to VDR/SMRT/TBL1R (Santa Cruz Biotechnology). Immune complexes were then collected using 25 μl protein A + G-Sepharose beads (GE Health Care, Life Science) in RIPA buffer. Immunoprecipitation was carried out at 4°C for 4 h on a rotating platform. After this, protein A + G-precipitated proteins were pelleted down by centrifugation at 4,500 rpm for 10 min at 4°C. Next, the protein pellet was washed three times each with 1 ml cold RIPA lysis buffer followed by centrifugation each time for 10 min at 2,500 rpm in a microfuge. After the washes, the beads were resuspended in 100 μl lysis buffer to which SDS-PAGE sample buffer (50 μl) was added, and samples were boiled at 100°C followed by SDS-PAGE and immunoblotted using the respective antibodies as indicated.

The SMRT/TBLR1 phosphorylation status was determined by immunoblot analysis of Immunoprecipitated lysates and probing the blots using mouse anti-phosphoserine antibody (clone 4A4, 05-100x, Upstate/Millipore, Bellerica, MA).

Statistical analysis.

For comparison of mean values between two groups, an unpaired t-test was used. To compare values between multiple groups, ANOVA was applied and a Bonferroni multiple-range test was used to calculate a P value. Statistical significance was defined as P < 0.05. All values are displayed as means ± SD.

RESULTS

AT₁R-BLK upregulates VDR but downregulates CYP24A1 expression.

To determine the effect of AT₁R-BLK and vitamin D agonist (VDA) on the relationship between VDR and CYP24A1 expression, immunofluorescence experiments were carried out with HPs. HPs were treated with either buffer, VDA (100 pM), or losartan (10⁻⁷ M) for 48 h followed by colabeling for VDR and CYP24A1. Representative microfluorograms are shown in Fig. 1A. Cumulative densitometric data are shown in a scattergram in Fig. 1B. VDA-induced VDR expression (P < 0.05, control vs. VDA) was associated with enhanced expression of CYP24A1 (P < 0.05, control vs. VDA), whereas losartan-induced VDR expression (P < 0.05, control vs. losartan) was associated without any alteration in CYP24A1 expression.

Fig. 1.

ANG II type 1 receptor (AT₁R) blockade (AT₁R-BLK) enhances podocyte vitamin D receptor (VDR) expression and downregulates cytochrome P-450 family 24A1 (CYP24A1) expression. A: human podocytes (HPs) were incubated in media containing either buffer, EB1089 [vitamin D agonist (VDA), 25 pM], or losartan (LOS; 10⁻⁷ M) for 24 h followed by colabeling for VDR and CYP24A1 (n = 3). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). Representative microfluorograms are shown. B: cumulative data (n = 3) of cells prepared for the protocol described in A (n = 3) are shown as a scattergram. Results are shown as means ± SD. *P < 0.05 compared with control; **P < 0.05 compared with LOS; ***P < 0.05 compared with control; #P < 0.05 compared with control; ##P < 0.05 compared with LOS. C: HPs were incubated in media containing either buffer, LOS (10⁻⁷ M), or EB1089 (VDA, 100 pM) for 48 h (n = 3). Subsequently, cells were harvested, and cytosolic and nuclear fractions were separated. Protein blots were probed for VDR and reprobed for actin. Gels are shown, and their densitometric data are shown as bar graphs. D: HPs were incubated in media containing variable concentrations of EB1089 (VDA, 0–10 nM) for 48 h. Protein blots were probed for VDR and reprobed for actin. Gels are shown, and densitometric data are shown in bar graphs. E: HPs were incubated in media containing variable concentrations of EB1089 (0–100 nM) for 48 h. Protein blots were probed for CYP24A1 and reprobed for actin. Gels along with densitometric data are shown. F: HPs were incubated in media containing variable concentrations of LOS (0 and 10⁻⁸–10⁻⁵ M) for 48 h. Protein blots were probed for VDR and reprobed for actin. Gels along with densitometric data are shown. G: HPs were incubated in media containing either buffer (0, control) or variable concentrations of LOS (10⁻⁸–10⁻⁶ M) for 24 h (n = 4). Subsequently, RNA was extracted, cDNA was amplified with VDR-specific primers, and mRNA expression was assayed by real-time PCR. *P < 0.05 compared with control and LOS (10⁻⁸ M); **P < 0.05 compared with other variables. H: HPs were incubated in media containing either buffer (0, control) or variable concentrations of LOS (10⁻⁸–10⁻⁶ M) for 24 h (n = 4). Subsequently, RNA was extracted, cDNA was amplified with CYP24A1-specific primers, and mRNA expression was quantified by real-time PCR. *P < 0.05 compared with control.

To confirm the cellular localization of VDR expression, HPs were treated with either buffer, VDA (100 pM), or losartan (10⁻⁷ M) for 48 h (n = 3) followed by separation and isolation of cytosolic and nuclear fractions. Lysates of cytosolic and nuclear fractions were probed for VDR and reprobed for actin (Fig. 1C). Densitometric data are shown as bar diagrams in Fig. 1C. More than 70% VDR was located in nuclei under control (relatively low vitamin D state), losartan-stimulated, and VDA-stimulated states. Thus, we confirmed that VDR is a nuclear receptor in podocytes in both vitamin D-deficient and vitamin D-sufficient states.

To determine the dose-response effect of VDA on VDR and CYP24A1 expression, HPs were incubated in media containing variable concentrations of VDA (0–100 nM). Lysates were analyzed for VDR or CYP24A1 protein levels by immunoblot analysis with specific antibodies. In both cases, equal protein loading was confirmed by reprobing the blots with antibodies to actin. VDA enhanced both podocyte VDR expression (Fig. 1D) as well as CYP24A1 expression (Fig. 1E) in a dose-dependent manner. Densitometric data are shown in a bar diagram.

To determine the dose-response effect of losartan on VDR protein expression, HPs were treated with either buffer or variable concentrations of losartan (10⁻⁵–10⁻⁸ M) for 48 h. Subsequently, protein blots were probed for VDR and reprobed for actin. Gels and densitometric data are shown in Fig. 1F. Losartan enhanced podocyte VDR protein levels in a dose-dependent manner.

To determine the dose-response effect of losartan on the transcription of VDR, HPs were treated with either buffer or variable concentrations of losartan (10⁻⁸–10⁻⁶M) for 24 h. Subsequently, RNA was extracted, and cDNA amplified with VDR-specific primers, and mRNA expression was assayed by real-time PCR. Losartan also enhanced podocyte VDR transcription in a dose-dependent manner (Fig. 1G).

To evaluate the effect of losartan on transcription of CYP24A1, HPs were treated with either buffer or variable concentrations of losartan (10⁻⁸–10⁻⁶ M) for 24 h. Subsequently, RNA was extracted, cDNA was amplified with CYP24A1-specific primers, and mRNA expression was quantified by real-time PCR. Losartan attenuated the transcription of CYP24A1 (Fig. 1H). Taken together, these data indicate that while both AT₁R-BLK and VDA caused upregulation of VDR expression, they had opposite effects on CYP24A1 expression. Losartan treatment downregulated CYP24A1 transcription, whereas VDA enhanced CYP24A1 expression.

AT₁R-BLK inhibits HIV-induced podocyte VDR downregulation.

We next wanted to determine the effect of AT₁R-BLK on VDR expression in HIV and high-glucose milieu. Control (empty vector) and HIV-transduced HPs were treated with buffer or losartan (10⁻⁷ M) and then labeled for VDR and propidium iodide (nuclear localization) in immunofluorescence experiments. Representative microfluorograms are shown in Fig. 2A. Cumulative data are shown in a scattergram in Fig. 2B. HIV alone downregulated podocyte VDR expression (P < 0.05, control vs. HIV); however, losartan enhanced podocyte VDR expression under both control (P < 0.05, control vs. losartan) and HIV-stimulated states (P < 0.05, HIV vs. HIV/losartan). In parallel sets of experiments, HPs were treated with normal glucose (control) or high glucose and then labeled for VDR and propidium iodide. Representative microfluorograms are shown in Fig. 2C. Cumulative data are shown in a scattergram in Fig. 2D. High glucose downregulated VDR expression (P < 0.05, control vs. high glucose).

Fig. 2.

AT₁R-BLK inhibits human immunodeficiency virus (HIV)-induced podocyte VDR downregulation. A: empty vector (control) or HIV-transduced HPs were incubated in media containing either buffer or LOS (10⁻⁷ M) for 48 h followed by labeling for VDR. Nuclei were stained with propidium iodide (PI; n = 3). Representative microfluorograms are shown. HIV downregulated podocyte VDR expression, whereas LOS enhanced podocyte VDR expression both under control and HIV-stimulated states. B: cumulative data of the protocol described in A (n = 3) are displayed in a scattergram. Results are shown as means ± SD. *P < 0.05 compared with control; **P < 0.05 compared with LOS; ***P < 0.05 compared with HIV/LOS; #P < 0.05 compared with control; ## P < 0.05 compared with HIV and LOS alone. C: HPs were incubated in media containing either buffer or high glucose (GLU) for 48 h followed by labeling for VDR (n = 3). Nuclei were stained with PI. Representative microfluorograms are shown. D: cumulative data of the protocol described in C (n = 3) are displayed in a scattergram. Results are shown as means ± SD. *P < 0.05 compared with control. E: HPs were incubated in media containing variable concentrations of ANG II (0 and 10⁻⁸–10⁻⁵ M) for 48 h. Protein blots were probed for VDR. The same blots were reprobed for actin. Gels are shown, and densitometric data are shown in bar graphs.

Since both HIV and high glucose have been reported to induce downstream effects through ANG II generation in podocytes (5, 8, 17, 28, 35), we evaluated the dose-response effect of ANG II on podocyte VDR expression. HPs treated with varying concentrations of ANG II (10⁻⁸–10⁻⁵ M) were analyzed for changes in VDR expression by immunoblot analysis. ANG II attenuated podocyte VDR expression in a dose-dependent manner (Fig. 2E). Densitometric data are represented as bar graphs.

Podocyte CYP24A1 expression is inversely related to SMRT and VDR-UL.

To determine whether AT₁R-BLK has an effect on the expression of the corepressor complex, HPs were treated with variable concentrations of losartan (10⁻⁸–10⁻⁶ M). Protein blots were probed for SMRT and reprobed for CYP24A1 and actin. Gels and densitometric data are shown in Fig. 3A. Losartan downregulated CYP24A1 expression in podocytes, consistent with previous results, but, in addition, SMRT expression was induced.

Fig. 3.

Podocyte CYP24A1 expression is inversely related to silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and unliganded VDR (VDR-UL). A: HPs were incubated in media containing variable concentrations of LOS (0 and 10⁻⁸–10⁻⁶M) for 48 h. Protein blots were probed for SMRT. The same blots were reprobed for CYP24A1 and actin. Gels along with densitometric data are shown. LOS enhanced podocyte expression of SMRT but decreased podocyte expression of CYP24A1. Densitometric analysis data are shown in bar graphs. B: HPs were transfected with VDR plasmid (VDR/HPs). Protein blots of control and VDR/HPs were probed for VDR, and the same blots were reprobed for CYP24A1 and actin (n = 4). Gels shown here displayed three observations only. Densitometric data are shown in bar graphs (n = 4). *P < 0.01 compared with the respective control; **P < 0.05 compared with the respective control. C: Control HPs and VDR/HPs were incubated in media containing either buffer or LOS (10⁻⁷ M) for 48 h (n = 4). Protein blots were probed for VDR. The same blots were probed for CYP24A1 and actin. Representative gels are shown, and cumulative densitometric data are shown in bar graphs. *P < 0.01 compared with control; **P < 0.05 compared with VDR/LOS. D: protein blots of empty vector (EV)-transduced HPs (EV/HPs; control) and HIV-transduced HPs (HIV/HPs) were probed for SMRT (n = 3). The same blots were reprobed for VDR, CYP24A1, and actin. Representative gels are shown, densitometric data are shown as bar graphs. *P < 0.05 compared with control (SMRT); **P < 0.05 compared with control (VDR); ***P < 0.05 compared with control (CYP24A1). E: HPs grown on coverslips were incubated in media containing either buffer (control), EB1089 (VDA, 100 pM), or LOS (10⁻⁷ M) for 48 h (n = 3). Subsequently, cells were colabeled for VDR and SMRT. Representative microfluorograms are shown. F: cumulative data (VDR/SMRT, integrated density) of the protocol described in E (n = 3) are shown in a scattergram. Results are presented as means ± SD. *P < 0.05 compared with control; **P < 0.05 compared with LOS; ***P < 0.05 compared with control; #P < 0.05 compared with control; ##P < 0.05 compared with VDA.

Since levels of VDR-UL are expected to be much higher in losartan treatment versus VDA treatment, we wanted to examine whether VDR-UL may account for CYP24A1 downregulation. To determine the relationship between VDR-UL and CYP24A1 expression, HPs were transfected with a plasmid overexpressing VDR. Protein blots of control and VDR-transfected HPs were probed for VDR, and the same blots were reprobed for CYP24A1 and actin sequentially (n = 4). Gels and densitometric data are shown in Fig. 3B. VDR-transfected HPs displayed enhanced (P < 0.01) expression of VDR but attenuated (P < 0.05) expression of CYP24A1 (Fig. 3B).

We then asked whether losartan could further enhance VDR expression in VDR-transfected HPs and modulate CYP24A1 expression. HPs and VDR-transfected HPs were incubated in media containing buffer with or without losaratan (10⁻⁷ M) for 48 h. Protein blots were probed for VDR and reprobed for CYP24A1 (n = 4). Gels and densitometric data are shown in Fig. 3C. Both VDR-transfected HPs and losartan-treated HPs displayed enhanced (P < 0.01) expression of VDR and downregulation of CYP24A1; however, losartan did not enhance VDR expression further in VDR-transfected HP cells.

To determine the effect of HIV on the interrelationship among podocyte expression of SMRT, VDR, and CYP24A1, protein blots of HPs and HIV-transduced HPs were probed for SMRT and reprobed for VDR, CYP24A1, and actin (n = 3). Gels and cumulative densitometric data are shown in Fig. 3D. HIV-transduced HPs displayed downregulation (P < 0.05) of both SMRT and VDR but upregulation (P < 0.05) of CYP24A1. These findings suggest that AMs such as HIV downregulate corepressor complex formation, leading to a rebound in podocyte CYP24A1 expression.

To confirm the relationship between VDR and SMRT, HPs were treated with buffer, VDA (100 pM), or losartan (10⁻⁷ M) and then colabeled for VDR and SMRT. Representative microfluorograms are shown in Fig. 3E, and cumulative data are shown in Fig. 3F. VDA enhanced expression of VDR (P < 0.05, VDA vs. control) but did not increase expression of SMRT (Fig. 3, E and F). On the other hand, losartan enhanced nuclear expression of both VDR (P < 0.05, control vs. losartan) and SMRT (P < 0.05, control vs. losartan). These findings suggest that the presence of nuclear SMRT is a marker of the activation of corepressor complex containing VDR-UL.

Both VDA and losartan enhance VDR expression but display disparate effects on podocyte CYP24A1 expression in AMs.

HPs were treated with buffer, ANG II (10⁻⁷ M), and high glucose either with or without VDA (100 pM) for 48 h. Additionally, HIV-transduced HPs were treated with either buffer or VDA (100 pM) for 48 h (n = 5). Protein blots were probed for VDR and reprobed for actin. Representative gels and cumulative densitometric data are shown in Fig. 4A. AMs downregulated podocyte VDR expression; however, VDA enhanced VDR expression in both control and AMs (Fig. 4A).

Fig. 4.

Both VDA and LOS enhance VDA expression but display a disparate effect on podocyte CYP24A1 expression in adverse milieus (AMs). A: HPs were incubated in media containing either buffer (containing 5 mM glucose), ANG II (10⁻⁸ M), or GLU (30 mM) with or without EB1089 (VDA, 100 pM) for 48 h (n = 5). Additionally, HIV/HPs were incubated with or without EB1089 (100 pM) for 48 h (n = 5). Protein blots were probed for VDR and reprobed for actin. Representative gels along with cumulative densitometric data are shown. *P < 0.005 compared with control; **P < 0.01 compared with ANG II; ***P < 0.04 compared with HIV; #P < 0.01 compared with GLU. B: HPs were incubated in media containing either buffer (with 5 mM glucose), ANG II (10⁻⁸ M), or GLU (30 mM) with or without LOS (10⁻⁷ M) for 48 h (n = 4–5). Additionally, HIV/HPs were incubated with or without LOS (10⁻⁷ M) for 48 h (n = 4–5). Protein blots were probed for VDR and reprobed for actin. Representative gels along with cumulative densitometric data are shown. *P < 0.05 vs. control; **P < 0.01 vs. ANG II; ***P < 0.01 vs. HIV; #P < 0.02 vs. GLU. C: HPs were incubated in media containing either buffer (with 5 mM glucose), ANG II (10⁻⁸ M) or GLU (30 mM) with or without LOS (10⁻⁷ M) (n = 3). Protein blots were probed for SMRT (C1) and reprobed for CYP24A1 (C2) and actin. Representative gels and cumulative densitometric analysis (bar graphs in C1 and C2) are shown. In C1, *P < 0.05 compared with control and **P < 0.05 compared with respective HIV alone, GLU alone, and ANG II alone; in C2, *P < 0.05 compared with control, **P < 0.05 compared with control, and ***P < 0.05 compared with respective HIV, GLU, and ANG II alone.

To evaluate the effect of losartan on VDR expression, HPs were treated under control or AMs in the presence or absence of losartan (10⁻⁷ M) for 48 h (n = 4–5). Additionally, HIV-transduced HPs were treated with either buffer or losartan (10⁻⁷ M) for 48 h (n = 3). Protein blots were probed for VDR and reprobed for actin. Gels and cumulative densitometric data are shown in Fig. 4B. Losartan enhanced VDR expression in both control and HIV/ANG II milieus (Fig. 4B).

To determine the effect of losartan on podocyte SMRT expression in AMs, HPs were treated under the above-mentioned conditions (n = 3). Protein blots were probed for SMRT; the same blots were reprobed for CYP24A1 and actin. Gels and cumulative densitometric data are shown in Fig. 4C. AMs downregulated (P < 0.05) podocyte SMRT expression, whereas losartan enhanced (P < 0.05) SMRT expression in both control and AMs (Fig. 4C). On the other hand, AMs upregulated (P < 0.05) podocyte expression of CYP24A1. However, losartan downregulated (P < 0.05) podocyte expression of CYP24A1 under basal and AM conditions (Fig. 4C). These findings confirm that losartan enhances the expression of VDR-UL.

Losartan enhances VDR/SMRT expression and provides protection against DNA damage by inhibiting proteosomal degradation.

VDR is a short-lived nuclear receptor that is continuously degraded through ubiquitination and subsequent proteosomal degradation (11). We asked whether AT₁R-BLK enhanced podocyte VDR expression through inhibition of proteosomal activity. HPs were treated with either buffer, ANG II (10⁻⁷ M), or high glucose with or without losartan (10⁻⁷ M) for 48 h (n = 4). Additionally, HIV-transduced HPs were treated with either buffer or losartan (10⁻⁷ M) for 48 h (n = 4). Cellular lysates were assayed for proteosomal activity. AMs enhanced (P < 0.05) podocyte proteosomal activity (Fig. 5A). However, losartan attenuated (P < 0.05) AM-induced podocyte proteosomal activity (Fig. 5A).

Fig. 5.

LOS enhances VDR expression and provides protection against DNA damage by inhibiting proteosomal degradation A: HPs were incubated in media containing either buffer (with 5 mM glucose), ANG II (10⁻⁸ M), or GLU (30 mM) with or without LOS (10⁻⁷ M) for 48 h (n = 4). Additionally, HIV/HPs were incubated with or without LOS (10⁻⁷ M) for 48 h (n = 4). Cellular lysates were analyzed for proteosomal activity. Cumulative data from 4 sets of experiments are shown. *P < 0.05 compared with control; **P < 0.05 compared with control and LOS; ***P < 0.05 compared with respective HIV, GLU, and ANG II alone. B: HPs were incubated in media containing either buffer or GLU (30 mM) in the presence or absence of phosphatidylinositol 3-kinase inhibitor [PI3Ki (LYZ94002); 10 μM] for 48 h (n = 3). Subsequently, cellular lysates were analyzed for proteosomal activity. Cumulative data from 3 sets of experiments are shown. *P < 0.05 compared with other variables. C: HPs were incubated in media containing either buffer or ANG II (10⁻⁸ M) in the presence or absence of MG132 (10⁻⁸ M, a proteosomal degradation inhibitor) for 48 h (n = 3–7). Protein blots were probed for VDR and reprobed for actin. Representative gels along with densitometric analysis in bar graphs are shown. *P < 0.05 vs. control; **P < 0.01 vs. control and ANG II alone; ***P < 0.05 vs. control; #P < 0.01 vs. ANG II alone. D: HPs grown on coverslips were treated with either buffer or ANG II (10⁻⁸ M) with or without MG132 (10⁻⁸ M) for 48 h (n = 3). Cells were colabeled for H2AX and KU80 and examined under a fluorescence microscope. Representative microfluorograms are shown. HPs treated with ANG II displayed attenuated expression of KU80 (DNA repair) and enhanced expression of H2AX (double strand breaks). E: cumulative data (number of H2AX foci) from the protocol described in D (n = 3) were summarized and are shown in a scattergram. Results are presented as means ± SD. *P < 0.05 compared with control; **P < 0.05 compared with control; ***P < 0.05 compared with ANG II; #P < 0.05 compared with control and MG132; ##P < 0.05 compared with ANG II/MG132. F: cumulative data (integrated density, KU80) from the protocol described in D were summarized and are shown in a scattergram. Results are shown as means ± SD. *P < 0.05 compared with control; **P < 0.05 compared with ANG II; #P < 0.05 compared with control. G: HPs grown on coverslips were transfected with SMRT small interfering (si)RNA. Control HPs and SMRT siRNA-transfected HPs were incubated in media containing either buffer or LOS (10⁻⁷ M) for 48 h (n = 3). Cells were colabeled for H2AX and KU80. Representative microfluorograms are shown. H: cumulative data (number of H2AX foci) from the protocol described in G (n = 3) are shown in a scattergram. Results are shown as means ± SD. *P < 0.05 compared with control; **P < 0.05 compared with LOS; #P < 0.05 compared with control; ##P < 0.05 compared with LOS. I: cumulative data (integrated density, KU80) from the protocol described in G (n = 3) were summarized and are shown in a scattergram (means ± SD). *P < 0.05 compared with control; #P < 0.05 compared with LOS.

We then asked whether the phosphatidylinositidyl 3-kinase (PI3K)-Akt pathway contributed to high glucose-induced proteosomal degradation. HPs were incubated in media containing either buffer or high glucose in the presence or absence of PI3K inhibitor [LYZ94002 (10 μM), Sigma] for 48 h followed by an assay for proteosomal activity (n = 3). Cumulative data are shown in Fig. 5B. High glucose enhanced (P < 0.05) proteosomal activity, but this effect of high glucose was inhibited by PI3K inhibitor.

We next asked if ANG II attenuated podocyte VDR expression through an increase in proteosomal activity. In this scenario, inhibition of proteosomal activity should enhance podocyte VDR expression under ANG II-stimulated states. HPs were treated with buffer or ANG II (10⁻⁷ M) in the presence or absence of a proteosomal activity inhibitor [MG132 (10⁻⁸ M)] for 48 h (n = 3–7). Subsequently, protein blots were probed for VDR and actin. Representative gels and densitometric data are shown as bar graphs in Fig. 5C. ANG II attenuated (P < 0.05) expression of podocyte VDR; however, MG132 enhanced VDR expression in both the presence and absence of ANG II. These findings suggest that ANG II-induced downregulation of VDR is mediated through proteosomal degradation of VDR. In parallel sets of experiments, protein blots of podocytes treated under the above-mentioned conditions were probed for SMRT and actin. ANG II downregulated podocyte SMRT expression, but MG132 inhibited this effect (data not shown). These findings indicate that ANG II-induced downregulation of SMRT is also mediated through proteosomal activity.

We hypothesized that SMRT expression will also provide protection against AM-induced podocyte DNA damage. HPs were treated under the conditions described above and colabeled for H2AX and KU80. H2AX foci are markers for DNA damage-induced double strand breaks, and the tyrosine phosphorylation state of H2AX helps to determine whether DNA repair or proapoptotic factors are recruited to chromatin; KU80 is a marker for DNA double strand break repair. Representative microfluorograms are shown in Fig. 5D. Quantitative analysis of both H2AX phosphorylation and KU80 foci are shown in scattergrams in Fig. 5, E and F. ANG II (10⁻⁷M) treatment attenuated (P < 0.05) DNA repair and enhanced (P < 0.05) DNA damage, whereas MG132 attenuated (P < 0.05) this effect by enhancing (P < 0.05) DNA repair (Fig. 5, D–F).

To confirm the role of SMRT in DNA repair, HPs were transfected with SMRT small interfering (si)RNA. Control HPs and SMRT siRNA-transfected HPs were treated with either buffer or losartan (10⁻⁷M) and then colabeled for H2AX and KU80. Representative microfluorograms are shown in Fig. 5G. Cumulative data are shown in scattergrams in Fig. 5, H and I. Losartan-treated podocytes displayed diminished (P < 0.05) DNA damage and increased (P < 0.05) DNA repairs compared with control cells. SMRT-depleted podocytes, however, displayed increased (P < 0.05) levels of DNA damage, while DNA repair markers were barely visible. Losartan did not modulate DNA repair in SMRT-silenced podocytes (Fig. 5, G–I). These findings indicate that the presence of SMRT is required for DNA repair during AT₁R-BLK.

Losartan induces histone deacetylation through corepressor formation, whereas VDA promotes histone acetylation via coactivator complex formation.

We next examined the mechanism of AT₁R-BLK-induced transcriptional repression. Deacetylation of histones has been reported to induce gene repression through chromatin compaction (10). SMRT associates with HDAC3, whose activity is enhanced by phosphorylation (37). We proposed that losartan may enhance phosphorylation of HDAC3 in the corepressor complex, thereby downregulating CYP24A1. To determine the effect of losartan on corepressor complex formation, HPs were treated with either buffer, losartan (10⁻⁷ M), or VDA (100 pM) for 48 h, and levels of VDR, SMRT, mSin3A, p-HDAC3, CBP/p300, Ac-H3, and actin were analyzed by Western blot analysis. Gels and densitometric data are shown in bar diagrams in Fig. 6A. As expected, losartan enhanced expression of VDR and SMRT, and, in addition, p-HDAC3 levels were also elevated. VDA-treated podocytes, on the other hand, showed increased Ac-H3 levels along with VDR.

Fig. 6.

Effect of LOS and VDA on the deacetylation and acetylation of histones via corepressor and coactivator complex formation. A: HPs were incubated in media containing either buffer, LOS (10⁻⁷ M), or EB1089 (VDA, 100 pM) for 48 h (n = 3). Subsequently, proteins were extracted from cell lysates, and protein blots were probed for VDR (A1). The same blots were reprobed for SMRT (A2), Sin3A (A3), phosphorylated (p)-histone deacetylase 3 (HDAC3; A4), CREB-binding protein (CBP)/p300 (A5), acetyl-histone 3 (Ac-H3; A6), and actin. Densitometric analyses (protein/actin) are shown in bar diagrams (A1–A6). In A1, *P < 0.01 vs. control and **P < 0.001 vs. control; in A2, *P < 0.01 vs. control and **P < 0.05 vs. control; in A3, *P < 0.05 vs. control or LOS; in A4, *P < 0.01 vs. control and **P < 0.01 vs. LOS; in A5, not significant; in A6, *P < 0.05 vs control. B: immunoprecipitation (IP) of cell lysates from the protocol described in A with VDR antibody was carried out (n = 3). IP fractions were probed for VDR (B1), SMRT (B2), p-HDAC3 (B3), CBP/p300 (B4), and Ac-H3 (B5). IgG labeling is shown to display the loading of proteins. Densitometric analyses (protein/IgG) are shown in bar diagrams (B1–B5). In B1, *P < 0.01 vs. control; in B2, *P < 0.05 vs. control or VDA; in B3, P < 0.05 vs. control and **P < 0.05 vs. LOS; in B4, *P < 0.05 vs. other variables; in B5, *P < 0.05 vs. other variables. C: proposed mechanisms of binding of VDR-UL to repressor or liganded VDR to activator complexes. In the absence of the ligand, VDR-UL is bound with the repressor complex [transducin β-like 1 receptor (TBL1R), Sin3A, and SMRT], which recruits HDACs. The latter deacetlylates histones (H3 and H4). Deacetylation of histone tails initiates chromatin compaction and repression of gene activation, a consequence of which is inaccessibility of DNA. In the presence of 1,25(OH)₂D or VDA, the AF2 region of VDR triggers the release of corepressors from the VDR. 1,25(OH)₂D- or VDA-bound VDR form a coactivator complex [CBP/p300, SRC-1, p-p300/CBP-associated factor (p-CAF), and p-CBP-interacting protein (p-CIP)] containing histone acetyl transferase activity, which initiates acetylation of histones (H3 and H4) and thus induces chromatin decompaction and gene activation as a consequence of availability of accessible DNA. D: HPs were incubated in media containing either buffer or GLU (30 mM) in the presence or absence of LOS (10⁻⁷ M) for 48 h (n = 3). Subsequently, cell lysates were immunoprecipitated with anti-SMRT antibody. Immunoprecipitates were probed for p-SMRT and reprobed for SMRT. Gels are shown, and densitometric analyses are shown in bar diagrams. *P < 0.05 vs control; **P < 0.001 vs. control, LOS alone, or GLU/LOS; ***P < 0.01 vs. LOS. E: HPs were incubated in media containing either buffer or GLU (30 mM) in the presence or absence of LOS (10⁻⁷ M) for 48 h (n = 3). Protein blots were probed p-Akt (Ser⁴⁷³). The same blots were reprobed for actin. Gels are shown, and densitometric analyses are shown in bar graphs. *P < 0.01 vs. control; *P < 0.05 vs. LOS. F: HPs were incubated in media containing either buffer or GLU (30 mM) in the presence or absence of LOS (10⁻⁷ M) for 48 h. Cell lysates were immunoprecipitated with anti-TBL1R antibody (n = 3). Immunoprecipitates were probed for TBL1R and reprobed for phosphorylation (serine) status. IgG labeling is shown to display the loading of proteins. Gels and densitometric analyses in bar graphs are shown. *P < 0.05 vs. control; **P < 0.01 vs. control or LOS. G: proposed mechanism of disintegration of the corepressor complex. Phosphorylation of TBL1R induces the disintegration of the corepressor complex. Phosphorylation of Akt or MAPK3 leads to the phosphorylation of SMRT; p-SMRT translocates to the cytosol and undergoes proteosomal degradation.

To further investigate the formation of corepressor or coactivator complexes upon losartan/VDA treatment, cellular lysates of HPs treated with either buffer, losartan (10⁻⁷ M), or VDA (100 pM) were immunoprecipitated with anti-VDR antibody. These complexes were subjected to Western blot analysis with antibodies to VDR, SMRT, p-HDAC3, CBP/p300, and Ac-H3. Gels and densitometric data are shown in Fig. 6B. VDR immunoprecipitates from losartan- and VDA-treated samples displayed increased levels of VDR (P < 0.01) compared with the control (Fig. 6B). However, only losartan-treated VDR complexes displayed increased levels of SMRT and p-HDAC3 compared with the control. On the other hand, VDR immunoprecipitates from VDA-treated samples displayed enhanced levels of CBP/p300 and Ac-H3, but levels of SMRT and p-HDAC3 were significantly diminished (Fig. 6B). Taken together, these observations indicate that losartan induces VDR corepressor complexes that contain SMRT and p-HDAC3. Conversely, VDA treatment inhibits the formation of corepressor complexes and instead recruits the coactivator CBP/p300 to VDR complexes, also enhancing the association with acetylated histones.

A schematic diagram displaying the constituents of corepressor and coactivator complexes is shown in Fig. 6C.

Since the SMRT complex is inactive in CYP24A1 repression in AMs, we investigated the mechanism underlying this inactivation. We asked whether losartan may influence the upstream events leading to SMRT complex inactivation. Phosphorylation of SMRT induces its translocation to the cytosol, which results in its degradation (24, 27). To determine the SMRT phosphorylation status in AMs such as high glucose, lysates of control HPs and high glucose-treated HPs treated with or without losartan (10⁻⁷ M) were immunoprecipitated with SMRT antibody. Immunoprecipitated fractions were probed for p-serine and SMRT. Gels and densitometric data are shown as bar diagrams in Fig. 6D. High glucose enhanced serine and threonine phosphorylation of SMRT. Interestingly, the increase in SMRT phosphorylation was inhibited by losartan treatment.

Since Akt phosphorylation has been reported to induce phosphorylation of SMRT (24, 27), we asked whether high glucose may induce upstream Akt phosphorylation, which, in turn, may be responsible for phosphorylation of SMRT and corepressor disintegration. HPs were treated with either buffer or high glucose in the presence or absence of losartan (10⁻⁷ M) for 24 and 48 h. Protein blots were probed for p-Akt and total Akt. Gels and densitometric data are shown as bar graphs in Fig. 6E. High glucose-treated podocytes displayed enhanced phosphorylation of Akt (Fig. 6E), an effect that was attenuated by losartan. These findings indicate that high glucose has the potential to phosphorylate SMRT via Akt phosphorylation.

Phosphorylation of TBL1R, a component of the SMRT corepressor complex, has also been demonstrated to lead to the disintegration of the corepressor complex (24); therefore, we evaluated the effect of high glucose on podocyte TBL1R phosphorylation. HPs were treated with high glucose in the presence or absence of losartan. Cellular lysates were immunoprecipitated with anti-TBL1R antibody, and immunoprecipitated samples were sequentially probed for TBL1R and serine phosphorylation (p-TBL1R). Gels and densitometric data are shown in Fig. 6F. High glucose enhanced phosphorylation of TBL1R at the serine site (Fig. 6F); however, this effect of glucose was attenuated by losartan. Taken together, these data demonstrate that multiple phosphorylation events, including Akt, TBL1R, and SMRT, could cause SMRT disintegration. Losartan treatment diminishes phosphorylation of these factors, thereby playing an important role in preventing SMRT complex disintegration.

A schematic diagram of the disintegration of the co-repressor complex is shown in Fig. 6G.

AT₁R-BLK downregulates proapoptotic gene expression through SMRT expression.

We examined the apoptotic pathways leading to podocyte injury in AMs and the role of losartan in influencing these pathways. AMs are known to induce podocyte injury through DNA damage and activation of the p53 pathway (12, 30). p53 induces expression of proapoptotic genes such as BAX through downstream signaling via Wip1 and dephosphorylation of checkpoint kinase 2 (Chk2) (12, 20, 21). Since Chk2 has been shown to recruit SMRT to downregulate proapoptotic gene expression (12, 20), we asked whether AT₁R-BLK has the potential to phosphorylate Chk2 through downregulation of Wip1 in high glucose milieu. To establish a causal relationship between downregulation of proapoptotic genes such as BAX and SMRT during AT₁R-BLK, protein blots of control HPs and HPs treated with high glucose with or without losartan were probed for p53 and sequentially reprobed for Wip1, p-Chk2, SMRT, BAX, and actin. Gels and densitometric data are shown in Fig. 7A. Glucose enhanced expression of BAX but downregulated expression of SMRT and p-Chk2 (Fig. 7A). However, this effect of high glucose was attenuated by losartan. Losartan not only enhanced phosphorylation of Chk2 but also downregulated expression of BAX. Since losartan partially attenuated expression of p53, some of the effects of losartan in BAX downregulation may be through this effect. Nonetheless, to confirm the role of SMRT in the downregulation of BAX during AT₁R-BLK, control HPs or HPs silenced for SMRT were treated with buffer or losartan. Protein blots were probed for SMRT and reprobed for Wip1, p-Chk2, BAX, and actin, sequentially. Gels and densitometric data are shown in Fig. 7B. HPs lacking SMRT displayed enhanced expression of Wip1 and BAX. In control HPs, losartan enhanced SMRT expression and downregulated Wip1 and BAX expression. However, in SMRT-depleted HPs, the effect of losartan on Wip1 and Bax expression was abrogated, indicating that losartan-mediated upregulation of SMRT does contribute to the downregulation of BAX expression.

Fig. 7.

AT₁R-BLK downregulates proapoptotic gene expression through SMRT expression. A: HPs were incubated in media containing either buffer (with 5 mM glucose) or GLU (30 mM) with or without LOS (10⁻⁷ M) for 48 h (n = 3). Protein blots were probed for p53 (A1). The same blots were reprobed sequentially for p-checkpoint protein 2 (p-Chk2; A2), Wip1 (A3), SMRT (A4), BAX (A5), and actin. Gels and densitometric analyses in bar graphs (A1–A5) are shown. In A1, *P < 0.01 vs. control, **P < 0.001 vs. control, ***P < 0.001 vs. LOS, #P < 0.001 vs. LOS, and ##P < 0.01 vs. GLU; in A2, *P < 0.05 vs. control or LOS, **P < 0.01 vs. control, ***P < 0.05 vs. LOS, and #P < 0.001 vs. GLU; in A3, *P < 0.01 vs. control, **P < 0.01 vs. LOS, ***P < 0.05 vs. LOS, and #P < 0.05 vs. GLU; in A4, *P < 0.01 vs. control, **P < 0.05 vs. LOS, and ***P < 0.05 vs. GLU; in A5, *P < 0.001 vs. control, **P < 0.001 vs. LOS, and ***P < 0.001 vs. GLU. B: control HPs or HPs transfected with SMRT siRNA were incubated in media containing buffer (control) with or without LOS (10⁻⁷M) for 48 h (n = 3). Protein blots were probed for SMRT (B1), Wip1 (B2), p-Chk2 (B3), BAX (B4), and actin, sequentially. Gels and densitometric analyses in bar graphs (B1–B4) are shown. In B1, *P < 0.05 vs. LOS; in B2, *P < 0.05 vs. LOS; in B3, *P < 0.01 vs. control, **P < 0.001 vs. control, and ***P < 0.01 vs. LOS; in B4, *P < 0.01 vs. control, **P < 0.01 vs. SMRT siRNA; and ***P < 0.01 vs. LOS/SMRT siRNA.

The proposed mechanism of AT₁R-BLK-induced repression of target genes is shown schematically in Fig. 8.

Fig. 8.

Proposed mechanism of VDR (UL)/SMRT in the suppression of proapoptotic gene expression and enhanced DNA repair. AMs work through podocyte generation of ANG II. Angiotensin receptor blockers (ARBs) block the effects of ANG II and stimulate the formation of corepressor complexes by enhancing expression of VDR and SMRT. Corepressor complexes downregulate CYP24A1 transcription; since CYP24A1 inhibits the degradation of 1,25(OH)₂D and the later enhances the expression of CYP24A1, net CYP24A1 expression would depend on the presence of 1,25(OH)₂D in the milieu. AMs through ANG II will also induce DNA damage. The latter activates the p53 pathway and expression of proapoptotic genes, such as BAX. SMRT would downregulate expression of proapoptotic gene expression through inhibition of Wip1 and phosphorylation of Chk2. Additionally, SMRT would directly enhance DNA repair and thus provide protection against DNA damage occurring in AMs. VDR-L, liganded VDR.

DISCUSSION

Loss of podocytes as a result of apoptotic or nonapoptotic pathways has been reported to cause proteinuria in diabetic and HIV-infected patients (16, 31, 36). Both glucose and HIV have been reported to induce podocyte injury via generation of ANG II (5, 8, 28, 30, 33). On that account, AT₁R-BLK has been used to provide protection to podocytes in high glucose and HIV milieus (35, 38). In a recent study (28), glucose-induced podocyte apoptosis was rescued by the upregulation of VDR both in vitro and in vivo, suggesting that VDA-induced downregulation of the renin-angiotensin system contributed to this protective effect. Similarly, HIV-induced downregulation of VDR was associated with an induction of apoptosis, but the use of VDA not only upregulated VDR expression but also mitigated podocyte apoptosis (5). In the latter study, HIV-induced downregulation of VDR was incriminated in the activation of the renin-angiotensin system in podocytes. In the present study, we demonstrated that AT₁R-BLK provides podocyte protection through the induction of corepressor complexes that downregulate the expression of CYP24A1. Additionally, AT₁R-BLK enhances expression of SMRT, which augments DNA repair mechanisms and downregulates the expression of proapoptotic genes.

Phosphorylation of TBL1R or SMRT induces the disintegration of the corepressor complex (24–27). For example, phosphorylation of TBL1R destabilizes the corepressor complex and induces the release of SMRT and its translocation to the cytosol; similarly, phosphorylation of either Akt or MAPK3 may lead to phosphorylation of SMRT and translocation to the cytoplasm, where it undergoes proteosomal degradation. On the other hand, a negative regulator of Akt, PTEN, has been proposed to enhance activity of corepressors (34). PTEN induces the formation of d-myo-inositol-1,3,4,5-tetrakisphosphate, which stabilizes the SMRT/HDAC3 complex; the latter is essential for the deacetylase activity of the corepressor complex. In the present study, AMs seemed to destabilize corepressor complexes, as manifested by attenuated expression of SMRT and VDR. This was further confirmed by increased CYP24A1 expression in the presence of AMs as a manifestation of derepression of corepressor-modulated gene transcription. In the present study, high glucose could have destabilized corepressor complexes through phosphorylation of TBL1R, Akt, or both. Additionally, AMs enhanced proteosomal degradation. We have previously demonstrated that HIV enhances Akt phosphorylation in podocytes (14). In the present study, losartan inhibited phosphorylation of both TBL1R and Akt in high-glucose milieu; thus, it appears that losartan-induced nuclear expression of SMRT might be the outcome of the stabilization of corepressor complexes.

Normally, 1,25(OH)₂D and VDA enhance transcription of their targeted genes, such as CYP24A1, via coactivator complexes (23). CYP24A1 induces the degradation of 1,25(OH)₂D and thus serves as a negative feedback to keep a check on hypercalcemic effects of 1,25(OH)₂D (11). On the other hand, VDR-UL expression decreases the expression of CYP24A1 (through activation of the corepressor complex), which may decrease the degradation of 1,25(OH)₂D and, therefore, may also serve as negative feedback response to VDR-UL-induced formation of corepressor complexes.

In the present study, losartan-induced VDR upregulation was associated with downregulation of CYP24A1 transcription, thus indicating that corepressor complexes are stabilized. We further confirmed that AT₁R-BLK enhanced expression of VDR-UL. These findings indicate that AT₁R-BLK has the potential to provide partial protection to podocytes through attenuating the degradation of 1,25(OH)₂D in 1,25(OH)₂D-deficient states.

In diabetic nephropathy models, supplementation of VDA with AT₁R-BLK has been reported to slow down the progression of renal lesions more effectively (38). Zhang et al. (38) demonstrated that AT₁R-BLK enhanced renal tissue renin expression in a diabetic mouse model. These investigators proposed that addition of a vitamin D analog provided additional beneficial effects through downregulation of renin during AT₁R-BLK. Since in the presence of 1,25(OH)₂D-sufficient states AT₁R-BLK-induced VDR will be liganded, it would be contributing to the activation of activator complexes optimally. On that account, in 1,25(OH)₂D-sufficient states, AT₁R-BLK alone should be as effective as combination therapy. It would be important to evaluate this aspect and will be the basis of our future studies.

Marshall et al. (22) used molecular modeling software to study the affinity of ANG II receptor blockers to VDR, predicting that ANG II receptor blockers could be used as immunomodulatory agents. In the prsent study, losartan enhanced podocyte expression of VDR-UL and downregulated expression of CYP24A1. Since CYP24A1 degrades 1,25(OH)₂D, its downregulation is likely to enhance the accumulation of 1,25(OH)₂D. Thus, the findings in the present study are consistent with predictions of molecular modeling.

Chk2 serves as a regulator of the DNA damage response and modulates the cellular response (18, 20). Wip1 regulates Chk2 stability through its dephosphorylation, thus affecting the timing of Chk2 activation and carrying out DNA repairs (19). In response to DNA damage, Chk2 recruits SMRT to the promoter site of proapoptotic genes (12). In the present study, SMRT interacted with Wip1 and Chk2 in high-glucose milieu and AT₁R-BLK. Since the presence of SMRT was critical for DNA repairs in podocytes, it appears that AT₁R-BLK enhances DNA repairs in AMs through enhanced expression of SMRT and stabilization of corepressor complexes.

SMRT acts as a repressor of Wip1, a phosphatase, which dephosphorylates Chk2 (12, 19). DNA damage, p53, and lack of SMRT enhance expression of Wip1. In the present study, high glucose enhanced podocyte expression of Wip1, whereas losartan inhibited high glucose-induced podocyte expression of Wip1. Similarly, high glucose dephosphorylated podocyte Chk2, whereas AT₁R-BLK inhibited high glucose-induced dephosphorylation of Chk2. Derepression of Wip1 by SMRT knockdown has been demonstrated to be associated with increased caspase activation (12, 19). In the present study, AT₁R-BLK was associated with increased expression of SMRT and downregulation of BAX expression. Since knockdown of SMRT enhanced expression of BAX during AT₁R-BLK, it appears that SMRT contributes to the downregulation of proapoptotic gene expression during AT₁R-BLK.

In conclusion, AT₁R-BLK prevents podocyte damage in AMs in multiple ways in both 1,25(OH)₂D-deficient and -sufficient states. In 1,25(OH)₂D-deficient states, it enhances expression of VDR-UL, which would attenuate degradation of 1,25(OH)₂D and leads to stabilization of VDR to some extent. AT₁R-BLK also enhances expression of SMRT, which would provide podocyte protection directly by enhancing DNA repairs and indirectly through downregulation of proapoptotic genes. On the other hand, in 1,25(OH)₂D-sufficient states, AT₁R-BLK acts by contributing to VDA-induced VDR expression.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-084910, RO1-DK-083931, and 1-R01-DK-098074 (to P. C. Singhal).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: T.S., K.A., P.R., N.C., S.H., and R.L. performed experiments; T.S., K.A., P.R., N.C., S.H., R.L., M.H., V.V., A.C., H.V., M.A.S., G.D., P.N.C., A.M., L.G.M., and P.C.S. approved final version of manuscript; M.H., V.V., A.C., H.V., G.D., and A.M. analyzed data; A.C. and H.V. prepared figures; M.A.S., P.N.C., and L.G.M. interpreted results of experiments; P.C.S. edited and revised manuscript.