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Published ahead of print on June 29, 2005
J Am Soc Nephrol 16: 2615-2625, 2005
© 2005 American Society of Nephrology
doi: 10.1681/ASN.2005020142
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Cell Biology

Dexamethasone Prevents Podocyte Apoptosis Induced by Puromycin Aminonucleoside: Role of p53 and Bcl-2–Related Family Proteins

Takehiko Wada, Jeffrey W. Pippin, Caroline B. Marshall, Sian V. Griffin and Stuart J. Shankland

Division of Nephrology, Department of Medicine, University of Washington, Seattle, Washington

Address correspondence to: Dr. Stuart J. Shankland, Division of Nephrology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195. Phone: 206-543-2346; Fax: 206-685-8661; stuartjs{at}u.washington.edu

Received for publication February 5, 2005. Accepted for publication May 26, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nephrotic-range proteinuria is due to glomerular diseases characterized by podocyte injury. Glucocorticoids are the standard of care for most forms of nephrotic syndrome. However, the precise mechanisms underlying the beneficial effects of glucocorticoids on podocytes, beyond its general immunosuppressive and anti-inflammatory effects, are still unknown. This study tested the hypothesis that the synthetic glucocorticoid dexamethasone directly reduces podocyte apoptosis. Growth-restricted immortalized mouse podocytes in culture were exposed to puromycin aminonucleoside (PA) to induce apoptosis. Our results showed that dexamethasone significantly reduced PA-induced apoptosis by 2.81-fold. Dexamethasone also rescued podocyte viability when exposed to PA. PA-induced apoptosis was associated with increased p53 expression, which was completely blocked by dexamethasone. Furthermore, the inhibition of p53 by the p53 inhibitor pifithrin-{alpha} protected against PA-induced apoptosis. Dexamethasone also lowered the increase in the proapoptotic Bax, which was increased by PA, and increased expression of the antiapoptotic Bcl-xL protein. Moreover, the decrease in p53 by dexamethasone was associated with increased Bcl-xL levels. Podocyte apoptosis induced by PA was caspase-3 independent but was associated with the translocation of apoptosis-inducing factor (AIF) from the cytoplasm to nuclei. AIF translocation was inhibited by dexamethasone. These results show that PA-induced podocyte apoptosis is p53 dependent and associated with changes in Bcl-2–related proteins and AIF translocation. The protective effects of dexamethasone on PA-induced apoptosis were associated with decreasing p53, increasing Bcl-xL, and inhibition of AIF translocation. These novel findings provide new insights into the beneficial effects of corticosteroids on podocytes directly, independent of its immunosuppressive effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The glomerular visceral epithelial cell, also called the podocyte, is a highly specialized and terminally differentiated cell with a limited mitotic capacity. Podocytes therefore have limited ability to be replaced if lost. There is a growing body of literature showing that reduced podocyte number is a critical determinant underlying the development of glomerulosclerosis that leads to progressive renal failure in diabetic and nondiabetic renal disease. Decreased podocyte number is caused by detachment of cells from the glomerular basement membrane and/or by apoptosis (programmed cell death) (1). The mechanisms underlying renal and nonrenal cell apoptosis include the tumor suppressor protein p53 (2–4), Bcl-2–related family proteins (5), and caspases (6). p53 plays a pivotal role in the regulation of the several forms of apoptosis as well as proliferation (3). Increased p53 induces apoptosis or cell-cycle arrest through transcriptional activation of target genes (7,8). Bcl-2 family proteins, which comprise both proapoptotic (e.g., Bad, Bax) and antiapoptotic (e.g., Bcl-2, Bcl-xL) members, act as checkpoints upstream of caspases and mitochondrial dysfunction. Caspases are cysteine proteases that underlie the executionary process of many forms of apoptosis. However, little is known about the molecular mechanisms of podocyte apoptosis.

Glucocorticoids improve renal structure and function in experimental renal diseases characterized by podocyte injury, including murine lupus nephritis (9,10), anti–glomerular basement membrane nephritis (11), the passive Heymann nephritis model of membranous nephropathy (12), and the puromycin aminonucleoside nephrosis (PAN) model of minimal-change disease and focal segmental glomerulosclerosis (FSGS) (13–15). In the clinical setting, glucocorticoids are widely used for the treatment of glomerular diseases, including membranous nephropathy, minimal-change disease, FSGS, and lupus nephritis, which all are characterized by podocyte injury and proteinuria. In addition to its immune-modulating effect, glucocorticoids inhibit the expression of cytokines, adhesion molecules, and enzymes involved in inflammation (16). However, in PAN, an animal model of minimal-change disease and FSGS, which lacks clear evidence for immunologic or inflammatory events, the mechanisms for the therapeutic benefits of glucocorticoids are still unclear. PAN is characterized by podocyte apoptosis (17,18), and puromycin aminonucleoside (PA) causes apoptosis in cultured podocytes (19,20). Several lines of evidence have shown that glucocorticoids have direct effects on the other cell types (21–23); however, it is still unknown whether glucocorticoids have direct effects on podocytes, beyond its immunosuppressive actions.

Accordingly, in this study we investigated the direct effect of dexamethasone, a synthetic glucocorticoid, on podocyte apoptosis induced by PA and the mechanisms that underlie this effect. Our results show that dexamethasone reduces PA-induced apoptosis in differentiated podocytes in vitro, which are mediated by p53 and Bcl-2–related family proteins but not by caspase-3. Dexamethasone also inhibited the translocation of apoptosis-inducing factor (AIF) from cytoplasm to nuclei induced by PA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells in Culture
Experiments were performed using early-passage (passages 10 to 18), growth-restricted, conditionally immortalized mouse podocytes as described previously (24). Immortalized podocytes were derived from immortomice produced by crossing transgenic H-2Kb-tsA58 mice (ImmortoMouse; Jackson Laboratory, Bar Harbor, ME). These cells have been characterized by standard criteria (24), including positive staining for WT-1 (Santa Cruz Biotechnology, Santa Cruz, CA), synaptopodin (gift of Peter Mundel, Einstein Collage of Medicine, New York, NY), podocin (gift of Peter Mundel), CD2AP (gift of Andrey Shaw, Washington University, St. Louis, MO), nephrin (gift of Harry Holthofer, University of Helsinki, Helsinki, Finland), ezrin (gift of Heinz Furthmayr, Stanford University, Stanford, CA), and podocalyxin (podocalyxin-like protein 1; Santa Cruz Biotechnology).

Podocytes were grown in RPMI 1640 medium that contained 10% FBS (Summit Biotechnology, Ft. Collins, CO), penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (1 mmol/L; all from Irvine Scientific, Santa Ana, CA), HEPES buffer (10 mmol/L; Sigma Chemical Co., St. Louis, MO), and sodium bicarbonate (0.075%; Sigma Chemical Co.). For passaging cells, podocytes were grown under "growth permissive" conditions, which involved growing cells at 33°C in the presence of IFN-{gamma} (50 U/ml; Roche Diagnostics Co., Indianapolis, IN). For podocytes to acquire a differentiated and quiescent phenotype, cells were grown under "restrictive conditions" at 37°C in 95% air/5% CO2 without IFN-{gamma} for >12 d. In the studies described below, growth-restricted podocytes were used.

Experimental Design and the Assessment of Apoptosis
For examining the effect of glucocorticoid on PA-induced podocyte apoptosis, podocytes grown under growth restrictive conditions for >11 d were plated at a density of 7.5 x 104/cm2 and allowed to attach to tissue culture plates for 24 h. Cells then were incubated with medium that contained 10% FBS in the presence or absence of 1 µM dexamethasone (Sigma Chemical Co.). One hour later, PA was added to the medium at the concentration of 30 µg/ml. The percentage of apoptotic cells was assessed (described below), and cell lysates were harvested at 0, 8, 24, and 48 h for protein extraction (see below). Apoptosis was measured by staining with Hoechst 33342 (Sigma Chemical Co.) as described previously (25). Using transferase-mediated dUTP nick-end labeling staining, we have previously verified that this method is very accurate and reliable. After cells were exposed to the reagents as indicated above, Hoechst 33342 was added to the medium at a final concentration of 10 µM at each time point. Apoptosis was defined as the presence of nuclear condensation on Hoechst staining, and the percentage of the cells with nuclear condensation was calculated on at least 300 consecutive cells. All experiments were performed a minimum of three times.

To determine the activity of caspase-3, we used the BD ApoAlert Caspase Colorimetric Assay Kit (BD Biosciences, Palo Alto, CA), according to the manufacturer’s instructions. This assay uses the spectrophotometric detection of the chromophore p-nitroaniline after its cleavage by caspase-3 from the labeled caspase-specific substrates. Adherent cells together with floating cells in the medium were collected, and a cell lysate was obtained using cell lysis buffer included in the kit. After the cell lysate was incubated with caspase-3 substrate DEVD–p-nitroaniline (final concentration, 50 µM) at 37°C for 1 h, the caspase-3 activities were measured by the absorbance at 405 nm by a Packard Spectracount microplate reader. For determining the role of caspase-3 in PA-induced podocyte apoptosis, the caspase-3 inhibitor Ac-DEVD-CHO (Bachem Bioscience Inc., King of Prussia, PA) was used. Growth-restricted podocytes were incubated with 10% FBS-containing RPMI medium in the presence or absence of Ac-DEVD-CHO at the concentration of 0, 15, 50, and 150 µM for 1 h before the addition of PA at the same concentration described above.

Inhibiting p53
For determining the role of p53 in PA-induced apoptosis and determine whether this was a target for the inhibitory effect of dexamethasone in podocyte apoptosis, growth-restricted podocytes were incubated with p53 inhibitor pifithrin-{alpha} (PFT-{alpha}; EMD Biosciences, San Diego, CA) (26) for 1 h before exposing podocytes to PA. The podocytes were incubated with PFT-{alpha} or vehicle throughout the study period.

Measuring Viable Cell Number
Finally, viable cell number was assessed by methylthiazoletetrazolium (MTT) assay using CellTiter 96 Non-Radioactive Cell Proliferation Assay kit (Promega, Madison, WI) according to the instructions of the manufacturer. This assay identifies living cells and is based on the cellular conversion of a tetrazolium salt into a formazan product, a chromophore, which can be quantified by absorbance at 560 nm.

Western Blot Analysis
Western blot analysis was performed to measure the protein levels of p53 and specific Bcl-2 family proteins in cells that were exposed to PA in the presence and absence of dexamethasone. Cells were washed three times with ice-cold PBS and harvested by trypsin digestion at 37°C for 3 min. Cells were pelleted by centrifugation (1200 rpm for 3 min at 4°C), washed twice with ice-cold PBS, and then suspended in lysis buffer that contained 1% Triton, 10% glycerol, 20 mmol/L HEPES, 100 mM NaCl, and protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). After an overnight freeze–thaw cycle, lysates were cleared by centrifugation at 14,000 rpm for 5 min at 4°C, and protein concentration was determined by BCA Protein Assay Kit (Pierce, Rockford, IL) according to the manufacturer’s protocol. Reducing buffer was added to each protein extract, and samples were boiled for 5 min. Reduced protein sample (5 to 10 µg) then was loaded per lane on a 15% SDS–polyacrylamide gel and subsequently transferred to a polyvinylidene difluoride membrane (PerkinElmer Life Sciences, Boston, MA) by electroblotting at 350 mA for 75 min. After blocking for 30 min in 5% nonfat dried milk, membranes were incubated overnight (4°C) with the following primary antibodies: Anti-p53 mAb (Oncogene Research Products, San Diego, CA), anti-Bax mAb (Santa Cruz Biotechnology), anti–Bcl-xL mAb (Santa Cruz Biotechnology), and anti-tubulin mAb (NeoMarkers, Fremont, CA). After three wash cycles with Tris-buffered saline with 0.1% Tween, membranes were incubated with an anti-mouse alkaline phosphatase-conjugated secondary antibody (Promega) for 1 h at room temperature. The resultant bands were detected with chromagen 5-bromo-4-chloro-3-inodyl phosphate/nitro blue tetrazolium (Sigma Chemical Co.).

Immunocytochemistry
We investigated the possibility that AIF might be involved in a caspase-3–independent pathway in apoptosis induced by PA. To determine the intracellular localization of AIF, we performed immunocytochemical staining on podocytes that were incubated with PA in the presence and absence of dexamethasone. After 11 d of culture under growth-restrictive conditions, temperature-sensitive mouse podocytes were plated at a density of 7.5 x 103/cm2 and allowed to adhere to the culture plates for 24 h. After they were incubated with PA in the presence or absence of dexamethasone as we described above, cells were fixed for 20 min in 10% buffered formalin at room temperature. After a wash in PBS, the cells were incubated overnight at 4°C with a rabbit polyclonal anti-AIF antibody (Chemicon International, Temecula, CA). A secondary biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA) was used, followed by an Alexa Fluor 594–conjugated streptavidin (Molecular Probes, Eugene, OR). Counterstaining for nuclei was performed using DAPI-containing mounting medium (VECTASHIELD mounting medium with DAPI; Vector Laboratories). Negative controls included omitting the primary antibody, or the primary antibody was substituted with an irrelevant antibody. To determine the percentage of the cells in which AIF is localized in nuclei, we counted at least 200 nuclei in triplicate in each experiment.

Statistical Analyses
Statistical analysis was performed using Statview 4.5 software program (Abacus Concepts, Berkeley, CA); statistical significance was evaluated using t test or Mann-Whitney U test. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dexamethasone Reduces Podocyte Apoptosis
We first tested the hypothesis that dexamethasone has an inhibitory effect on PA-induced apoptosis in podocytes. To test this hypothesis, we quantified podocyte apoptosis and measured viable cell number in cultured immortalized podocytes that were grown under restrictive conditions for 12 d and then were injured by exposure to PA. In our pilot studies, we confirmed with FACS analysis that >90% of growth-restricted podocytes were in the G0/G1 phase of the cell cycle at the time of initiation of all experiments and that neither dexamethasone nor PA changed their cell-cycle distribution (data not shown).

We first measured the percentage of apoptotic cells by careful observation of the morphology of nuclei stained with Hoechst 33342. PA induced podocyte apoptosis in a time-dependent manner, and the results are shown in Figure 1. As shown in Figure 1A, there was a 6.5-fold increase in apoptotic cell number after 48 h of exposure to PA (3.79 ± 0.715% at 0 h versus 24.7 ± 3.43% at 48 h; P < 0.001). In contrast, dexamethasone significantly reduced PA-induced apoptosis at 48 h by 64.3% (8.80 ± 1.73%; P < 0.005 versus without dexamethasone).



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  		Figure 1. Dexamethasone prevents puromycin aminonucleoside (PA)-induced apoptosis. (A) The percentage of apoptotic cells, measured by Hoechst 33342 staining, increased at 48 h in the cells that were exposed to PA without dexamethasone (DEX; {square}), whereas cells that were treated with DEX were resistant to PA-induced apoptosis ({blacksquare}). *P < 0.005 versus DEX(–)PA(+). (B) The methylthiazoletetrazolium (MTT) assay showed that the incubation with PA (30 µg/ml) caused the loss of viable podocytes ({square}) and that DEX ({blacksquare}) prevented this. *P < 0.0001 versus DEX(–)PA(+). (C) The MTT assay showed that DEX alone did not affect viable podocyte number.

 
We next measured viable podocyte number after PA exposure in the presence or absence of dexamethasone, by MTT assay. The results shown in Figure 1B demonstrate that after exposure to PA alone, podocyte viability is reduced. However, significantly more podocytes survived in the presence of dexamethasone at 48 h compared with control cells that were given PA without dexamethasone (0.291 ± 0.0130 at 560 nm with dexamethasone versus 0.184 ± 0.0226 without dexamethasone; P < 0.0001). We also performed the MTT assay to measure viable cell number of podocytes that were incubated with dexamethasone alone, to ensure that dexamethasone alone did not alter podocyte number. As shown in Figure 1C, there was no difference in viable podocyte number between dexamethasone-treated podocytes and control podocytes. These results suggest that dexamethasone is a survival and antiapoptotic factor for podocytes in response to PA and thereby maintains podocyte viability.

Dexamethasone Suppresses Upregulation of p53 Induced by PA
To elucidate the mechanisms of PA-induced apoptosis in podocytes and the potential effects of dexamethasone on specific apoptotic pathways, we performed Western blot analysis to measure the protein expression of p53. p53 plays a central role in ultraviolet-induced apoptosis, but the role of p53 is not well understood in podocyte apoptosis. Western blot analysis showed that incubation of the podocytes with PA (30 µg/ml) markedly increased p53 protein levels at 24 and 48 h (Figure 2A). In contrast, dexamethasone completely prevented the increase in p53 induced by PA (Figure 2B). These results demonstrate that incubation of cultured podocytes with PA increased the protein levels of p53 and that addition of dexamethasone prevented podocytes from increasing p53 levels in response to PA. This suggests that dexamethasone may inhibit podocyte apoptosis induced by PA by preventing the upregulation of p53.



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  		Figure 2. DEX reduces PA-induced p53. (A) Western blot analysis showed that PA (30 µg/ml) increased the protein levels for p53 in immortalized mouse podocytes at 24 and 48 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for protein loading. (B) DEX (1 µM) prevented the increase in p53 induced by PA.

 
Inhibition of p53 Prevents Podocytes from Undergoing PA-Induced Apoptosis
To prove that the induction of p53 by PA is central to the induction of apoptosis in podocytes, we evaluated apoptosis in podocytes that were exposed to the p53 inhibitor PFT-{alpha}. First, we confirmed that PFT-{alpha} inhibited p53 expression in podocytes that were exposed to PA. As shown in Figure 3A, PFT-{alpha} blocked the upregulation of p53 after exposure to PA for 24 and 48 h. We next asked whether inhibiting p53 alters podocyte number. Because p53 is central to certain forms of apoptosis in nonrenal cells, we measured apoptosis in the presence and absence of the p53 inhibitor PFT-{alpha}. Our results showed that PFT-{alpha} significantly decreased the number of apoptotic podocytes induced by PA by 2.40-fold (P < 0.005 versus control; Figure 3B). As shown in Figure 3C, the MTT assay results confirmed that PFT-{alpha} suppressed the loss of viable cells in a dose-dependent manner (73.13% decrease of the absorbance at 560 nm at 0 µg/ml versus 36.2% decrease at 15 µg/ml; P < 0.0001). These results show that PA-induced podocyte apoptosis is p53 dependent and supports the notion that dexamethasone exerts an antiapoptotic effect on cells that are exposed to PA through the downregulation of p53.



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  		Figure 3. p53 is required for podocyte apoptosis. For determining the role of p53 in PA-induced apoptosis, podocytes were grown in the absence (–) or presence (+) of pifithrin-{alpha} (PFT-{alpha}), a specific p53 inhibitor. (A) Western blot analysis shows that the increase in p53 induced by PA was prevented by PFT-{alpha}. GAPDH was used as a control for protein loading. (B) Quantification of Hoechst staining used to measure apoptosis showed that PFT-{alpha} reduced PA-induced apoptosis in a dose-dependent manner. *P < 0.005 versus 0 µM; #P < 0.05 versus 0 µM. (C) MTT assay showed that PFT-{alpha} increased the number of viable podocytes after exposure to PA. The effect was dose dependent from 0 to 15 µM, with a maximal effect at 15 µM. *P < 0.0001 versus 0 µM.

 
Dexamethasone Prevents Downregulation of Bcl-xL Caused by PA
To explore other potential mechanisms that contribute to the antiapoptotic effect of dexamethasone beyond p53, we performed Western blot analysis to measure the protein levels of Bcl-2–related family proteins, which were quantified by densitometric analysis. Bcl-xL is an antiapoptotic molecule, whereas Bax is a proapoptotic molecule.

We first examined the expression of Bcl-2 family proteins in podocytes that were exposed to PA (Figure 4, A through C). As shown in Figure 4B, Bax levels were dramatically upregulated at 8 h (8.72-fold versus control) and 24 h (27.2-fold versus control). In addition, Bcl-xL levels were reduced in response to PA (18.4% at 24 h, 41.8% at 48 h; Figure 4C). We next determined whether dexamethasone had any affects on the levels of specific Bcl-2 family proteins (Figure 4, D through F). Incubation with PA increased levels of the proapoptotic Bax by 1.62-fold at 8 h, 2.55-fold at 24 h, and 2.56-fold at 48 h. However, Figure 4E shows that the increase in the levels of Bax induced by PA was substantially decreased in podocytes by dexamethasone at 48 h (1.98-fold). Figure 4F shows that Bcl-xL was reduced in podocytes that were exposed to PA alone. However, our results show that after injury induced by PA, the protein levels of Bcl-xL was 3.88-fold higher at 48 h in the presence of dexamethasone compared with podocytes that were exposed to PA alone (Figure 4F). Taken together, these results suggest that PA-induced apoptosis is due to increased Bax and reduced Bcl-xL and that these are direct targets of dexamethasone in mediating the reduction of apoptosis.



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  		Figure 4. DEX alters Bax and Bcl-xL expression. (A) Western blot analysis for Bax and Bcl-xL in podocytes in the absence (–) and presence (+) of PA, without DEX. Tubulin was used as a control for protein loading. (B) Densitometric analysis showed that proapoptotic Bax increased markedly in podocytes that were exposed to PA at 8 h (8.72-fold versus control) and 24 h (27.2-fold versus control). (C) Densitometric analysis showed that antiapoptotic Bcl-xL levels were reduced 41.8% in podocytes that were exposed to PA compared with control cells that were given PA alone. (D) Western blot analysis of for Bax, Bcl-xL, and the housekeeping protein tubulin in podocytes that were exposed to PA in the presence (+) and absence (–) of DEX. (E) Densitometric analysis showed the increase in Bax by PA was reduced by DEX at early time points (2.15-fold at 0 h; 1.76-fold at 8 h). (F) Densitometric analysis showed that Bcl-xL levels were markedly higher in the podocytes that were treated with DEX (3.88-fold versus untreated cells) after exposure to PA.

 
Inhibition of p53 Increased Bcl-xL Levels
As described above, we observed that dexamethasone suppressed the increase in p53 induced by PA, and dexamethasone also prevented the decrease in the antiapoptotic Bcl-xL levels induced by PA. On the basis of these results, we hypothesized that the suppression of p53 expression caused the upregulation of the antiapoptotic Bcl-xL and that this pathway was implicated in the antiapoptotic effect of dexamethasone in podocytes. To test this hypothesis, we examined the expression levels of Bcl-xL in the podocytes in which p53 levels were suppressed with the treatment with the p53 inhibitor PFT-{alpha} by Western blot analysis. As shown in Figure 5, after 48 h of exposure to PA, podocytes that were exposed to PFT-{alpha} demonstrated a 70% increase in levels of Bcl-xL compared with basal levels, whereas the control cells showed a 23% decrease of Bcl-xL in response to PA. These results suggest that p53 may facilitate podocyte apoptosis through the downregulation of Bcl-xL, and suppression of p53 by dexamethasone decreases podocyte apoptosis induced by PA through upregulation of Bcl-xL.



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  		Figure 5. Effect of p53 inhibition on the expressions of Bax and Bcl-xL. (A) Western blot analysis for Bax and Bcl-xL in podocytes that were exposed to PA in the presence (+) and absence (–) of the p53 inhibitor PFT-{alpha}. Tubulin was used as a control for protein loading. (B) Bcl-xL levels in the podocytes that were exposed to PA were markedly higher in the presence of PFT-{alpha} (2.21-fold versus in the absence of PFT-{alpha}).

 
PA-Induced Podocytes Apoptosis Is Caspase-3 Independent
The caspase cascade is a critical effector in mediating many forms of apoptosis, and recent studies have shown that caspase-3 is one of the final downstream proteins required for apoptosis. However, not all apoptosis requires caspase-3 activation (27). To determine whether caspase-3 underlies PA-induced podocyte apoptosis and whether the antiapoptotic effect of dexamethasone involves suppression of caspase-3 activation, we performed a caspase-3 activity assay on podocytes that were exposed to PA. Our results showed that PA did not increase caspase-3 activity (Figure 6A), and, as expected, dexamethasone therefore had no effect on caspase-3. The absence of increased caspase-3 was not a false negative, because the positive control, comprising ultraviolet-irradiating podocytes, showed a significant increase in caspase-3 activity by more than three-fold (Figure 6B). Moreover, we measured apoptosis and viable cell number of the podocytes that were exposed to the caspase-3 inhibitor Ac-DEVD-CHO after exposure to PA for 48 h. As shown in Figure 6, C and D, inhibition of caspase-3 activity with Ac-DEVD-CHO did not have any effect on PA-induced podocyte apoptosis or viability of podocytes. These results clearly demonstrated that podocyte apoptosis induced by PA was caspase-3 independent.



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  		Figure 6. PA-induced apoptosis in podocytes is caspase-3 independent. (A) Caspase-3 activity was not altered in podocytes that were exposed to PA (+; {blacksquare}) compared with control cells that were not exposed to PA (–; {square}). (B) Ultraviolet irradiation ({blacksquare}) of podocytes increased caspase-3 activity substantially. {square}, negative control without substrate for caspase-3. (C) Apoptosis was measured at 48 h in podocytes in the presence (+) and absence (–) of PA at a range of concentrations of the caspase-3 inhibitor Ac-DEVD-CHO. Inhibiting caspase-3 had no effect on PA-induced podocyte apoptosis. (D) The number of viable podocytes was measured by MTT assay at 48 h in podocytes in the presence (+) and absence (–) of PA at a range of concentrations of the caspase-3 inhibitor Ac-DEVD-CHO. Inhibiting caspase-3 had no effect on podocyte viability.

 
Dexamethasone Prevents Translocation of AIF to Nucleus
Because PA-induced apoptosis is caspase-3 independent, we measured AIF, which induces apoptosis independent of caspase activation. AIF is a 57-kD flavoprotein located in the mitochondrial intermembrane space. Translocation of AIF from the mitochondria to the nucleus in response to apoptotic stimulation causes chromatin condensation and DNA fragmentation. Accordingly, we performed immunocytochemical staining on the podocytes that were incubated with PA and dexamethasone to determine whether AIF is implicated in the caspase-3–independent apoptosis.

As shown in Figure 7, AIF staining localized mainly to the cytoplasm in the control podocytes that were not incubated with PA or dexamethasone. In contrast, the incubation with PA increased the number of podocytes in which AIF staining localized to the nuclei (41.3 ± 5.82%; P < 0.0001 versus control). It is interesting that dexamethasone significantly reduced the number of nuclei that stained positive for AIF after exposure to PA (26.5 ± 3.64%; P < 0.001 versus dexamethasone and PA). These results suggest that AIF is implicated in PA-induced podocyte apoptosis and is likely the mechanism underlying the caspase-3–independent pathway. Reducing the translocation of AIF to the nucleus therefore may provide an additional mechanism whereby dexamethasone decreases PA-induced podocyte apoptosis.



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  		Figure 7. DEX prevents PA-induced translocation of apoptosis-inducing factor (AIF). (A) Immunostaining with anti-AIF antibody and DAPI showed that AIF staining localized to the cytoplasm in control podocytes that were not exposed to DEX or PA (DEX-PA–). Podocytes that were exposed to PA (30 µg/ml for 48 h) exhibited translocation of AIF to nuclei (DEX-PA+), which was reduced by the co-incubation with DEX (DEX+PA+; 1 µM of DEX). (B) The percentage of the control podocytes in which AIF is located in nuclei was 13.2 ± 0.994%; however, it was significantly higher after exposure to PA (41.3 ± 5.82%; P < 0.0001 versus DEX-PA–). DEX reduced AIF nuclear staining (26.5 ± 3.64%; P < 0.001 versus DEX-PA+).

 
Dexamethasone Does Not Reduce Podocyte Apoptosis Induced by Reactive Oxygen Species
Previous studies have shown that one of the mechanisms underlying PA injury to podocytes is through the release of reactive oxygen species (ROS). To determine whether dexamethasone reduces apoptosis in podocytes by ROS, we exposed cultured podocytes to H2O2 (0, 600, 800, and 1000 µM) in the presence and absence of dexamethasone. As expected, H2O2 decreased viable podocyte number (86.6% reduction at 1000 µM of H2O2 after 48 h); however, dexamethasone had no effect (86.6% reduction without dexamethasone versus 81.5% reduction with dexamethasone; no significant difference; data not shown). Taken together, it is unlikely that dexamethasone alters PA-induced apoptosis by inhibiting ROS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A reduction in podocyte number is closely associated with the development of glomerulosclerosis in experimental and human glomerular disease. Kriz et al. (28) proposed that podocyte depletion as a result of apoptosis and/or detachment and the inability of podocytes to replicate cause glomerulosclerosis and the eventual destruction of the glomerular tuft. This pathway has been verified in diabetic (29,30) and nondiabetic human glomerular diseases (31). Seminal studies by Wiggins and Bottinger (18,32) showed that podocyte apoptosis is a major factor causing a decrease in podocyte number. Recent studies have also shown that angiotensin II (33), ROS (19,20), mechanical stretch (34), cyclosporin A (35), TGF-{beta}, and specific signaling pathways (32) cause podocyte apoptosis.

Glucocorticoids are widely used for the treatment of many forms of glomerular diseases characterized by podocyte injury and proteinuria. It has been speculated that glucocorticoids exert therapeutic effects through their immunosuppressive and anti-inflammatory mechanisms. However, several lines of evidence have suggested that glucocorticoids have additional direct pleiotropic effects that may alter cell biology. Previous studies have shown that glucocorticoids have beneficial effects on animals with experimental PAN, a model of FSGS, which lacks evidence for immunologic or inflammatory events (14,15,36). Moreover, glucocorticoids have been shown to have direct effects on podocytes (37,38). Mathieson’s group (39) described that dexamethasone augmented human podocyte proliferation, differentiation, and survival in long-term culture. They also showed that dexamethasone did not cause apoptosis in human podocytes (39) and that dexamethasone caused the redistribution and upregulation of the glucocorticoid receptor in vitro (40). On the basis of these findings, we reasoned that glucocorticoids might have a direct effect on the podocyte beyond effects on immune modulation and contribute to podocyte survival and therefore ultimately to the preservation of podocyte number.

In our study, the first major finding was that dexamethasone significantly protected podocytes from apoptosis induced by PA. This is interesting, because glucocorticoids induce apoptosis in many cells of the blood system (41–43). In contrast, glucocorticoids inhibit apoptosis in other cell types, such as the mouse mammary gland (44), human granulose cells (21), and human ovarian follicular cells (22). Although the beneficial effects of glucocorticoids on proteinuria and renal morphology in PAN rats have been described in earlier studies (14,15), our study is the first to demonstrate the preventive effect of dexamethasone on podocyte apoptosis, which has potentially important clinical consequences. We established the concentration of dexamethasone on the basis of the clinical doses used for the treatment of nephrotic syndrome. A study by Frey et al. (45) showed that the mean concentrations of total prednisolone for 24 h after intravenous injection of prednisolone at the bolus dose of 0.8 mg/kg in control subjects and in patients with nephrotic syndrome were 216 ± 25 and 170 ± 23 ng/ml, which are approximately 0.080 and 0.063 µM at the equivalent dexamethasone concentrations, respectively. A study on liver transplant recipients who had received 1 g of methylprednisolone-hemisuccinate by a 1-h intravenous infusion for the treatment of acute rejection demonstrated that maximum serum concentrations of methylprednisolone were from 16 to 46 µmol/L, which correspond to the equivalent dexamethasone concentrations from 3.2 to 9.2 µM (46). A pharmacokinetic analysis using RIA showed that the peak plasma concentration of dexamethasone was 0.0882 ± 0.0153 µM after intramuscular injection of 3.0 mg of dexamethasone (47). On the basis of these data, we tested the effect of dexamethasone at the concentrations from 0.1 to 10 µM. In our pilot studies, we observed similar preventive effects of all of the doses that we tested on PA-induced apoptosis. Taken together, we consider the dose of dexamethasone that we used in this study (1 µM) to be clinically relevant and reasonable.

To explore the mechanisms of PA-induced podocyte apoptosis and the mechanisms underlying the antiapoptotic effect of dexamethasone, we focused on the tumor suppressor p53. The second major finding in this study was that PA increased the levels of p53 in cultured podocytes and that inhibition of p53 by the p53 inhibitor PFT-{alpha} protected podocytes from PA-induced apoptosis. Furthermore, dexamethasone completely suppressed the upregulation of p53 induced by PA. Taken together, these results suggest that PA-induced podocyte apoptosis is p53 dependent and that dexamethasone may protect podocytes from PA-induced podocytes by inhibiting p53. Previous studies showed that p53 is increased in PAN rats in vivo (48,49). We recently described an increase in p53 in podocytes in experimental membranous nephropathy (50). Our study is the first to demonstrate that p53 may be a critical target whereby dexamethasone suppresses apoptosis in podocytes. Further studies are needed to define the mechanisms underlying this effect.

We next examined the levels of Bcl-2 family proteins Bax (proapoptotic) and Bcl-xL (antiapoptotic) to explore further the mechanisms of the survival and antiapoptotic effect of dexamethasone in podocytes. It has been shown that Bax mediates podocyte apoptosis induced by TGF-{beta} (32). Bcl-xL is an antiapoptotic member of the Bcl-2 family and has been shown to mediate the antiapoptotic effect of hepatocyte growth factor (HGF) on podocyte apoptosis induced by cyclosporin A (35). Cybulsky’s group (51) demonstrated increased expression of Bcl-xL and decreased expression of Bax in podocytes that were cultured on collagen substratum compared with those that were cultured on plastic, and this was involved in prosurvival effect of collagen. A recent study described that IGF-1 protected podocytes from etoposide-induced apoptosis and that Bad phosphorylation as well as the activation of the phosphatidylinositol 3'-kinase pathway were associated with the antiapoptotic effect (52). In this study, we showed that exposure of podocytes to PA increased levels of Bax, which is consistent with findings from previous in vivo studies in the PAN rat model of FSGS (48). Our data also showed that PA reduces the antiapoptotic protein Bcl-xL. However, the third major finding in this study was that dexamethasone alters the expression of specific Bcl-2–related proteins. Our results showed that dexamethasone reduced the increase in Bax induced by PA and that dexamethasone also prevented the decrease in Bcl-xL after exposure to PA. Taken together, these data support the notion that dexamethasone protects podocytes from apoptosis by regulating the balance of both pro- (Bax) and anti- (Bcl-xL) apoptotic proteins.

Several lines of evidence have demonstrated that Bax and Bcl-xL are regulated by p53. The transcriptional expression of Bax is induced by p53 and mediates the proapoptotic effect of p53 in nonrenal cells (53). Studies have also demonstrated that Bcl-xL is regulated during p53-mediated apoptosis (54,55). However, those results have varied depending on cell types and types of stimulation. Accordingly, we asked whether the increase in p53 induced by PA in podocytes underlies any increases in Bcl-2–related proteins. Our results showed that directly inhibiting p53 with PFT-{alpha} was followed by an increase in Bcl-xL levels. These data support the notion that in response to PA, p53 may directly reduce levels of Bcl-xL, and the decrease in this survival protein causes podocytes to apoptose.

Finally, the role of caspase-3 in the podocyte apoptosis induced by PA was also investigated. Caspase-3 is an effector caspase and involved in many forms of apoptosis (6). However, caspase-3 is still dispensable for certain forms of apoptosis (27). Our results showed that PAN did not increase the activity of caspase-3 and that the caspase-3 inhibitor Ac-DEVD-CHO had no effect on PA-induced podocyte apoptosis, which suggests that PA-induced apoptosis in podocytes is caspase-3 independent. Our data differ from that of Suzuki et al. (20), who showed that PA caused activation of caspase-3 in podocytes; however, those results were obtained from much higher doses of PA, and their results also showed that the caspase-3 activity was not increased above basal levels at the concentration that we used for this study. Because our findings suggest the possibility that caspase-3–independent pathways are involved in PA-induced podocyte apoptosis, we investigated the intracellular localization of AIF, which causes apoptosis in a caspase-independent manner by translocating from the cytoplasm to the nucleus (56). Our results showed that indeed AIF translocated to the nucleus in response to PA, which was reduced by dexamethasone. Given that the translocation of AIF can be caused by the direct induction of p53 by adenovirus vectors in Apaf1-deficient neurons in vitro (57), the upregulation of p53 may regulate AIF in podocytes. This study therefore is the first to implicate AIF translocation in podocyte apoptosis.

Because previous studies that used free radical scavengers have shown that oxidative stress is implicated in podocyte injury induced by PA, we investigated the antioxidant effect of dexamethasone in podocyte injury induced by H2O2. Our results showed that dexamethasone did not rescue podocytes that were exposed to H2O2. This suggests that glucocorticoids do not have an antioxidant effect under the conditions tested and that glucocorticoids exert a cytoprotective effect on the other pathobiological mechanisms rather than oxidative stress.

In summary, our data show that dexamethasone prevents podocyte apoptosis induced by PA. We now can add dexamethasone to the list of known factors that enhance podocyte survival, which include nephrin, CD2AP, attachment to the glomerular basement membrane, and vascular endothelial growth factor. Dexamethasone exerts its antiapoptotic effect at the level of p53, Bcl-2–related family proteins, and AIF. Further studies are required to delineate other molecular mechanisms of podocyte apoptosis and how dexamethasone has a direct protective effect on podocytes. These findings contribute to the exploration of the pathobiological mechanisms of glomerular diseases characterized by podocyte injury and provide a novel rationale for the use corticosteroids in clinical practice characterized by podocyte injury beyond immunosuppression.


   Acknowledgments
 
This work was supported by National Institutes of Health Grants DK60525, DK56799, and DK51096 (to S.J.S.). S.J.S. is also an Established Investigator of the American Heart Association. T.W. was supported by the Uehara Memorial Foundation and is supported by the National Kidney Foundation. S.V.G. is supported by the American Heart Association.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication February 5, 2005. Accepted for publication May 26, 2005.




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