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NDT Advance Access originally published online on November 2, 2007
Nephrology Dialysis Transplantation 2008 23(2):510-517; doi:10.1093/ndt/gfm600
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant

Shigeyoshi Oba, Masayo Hino and Toshiro Fujita

Department of Nephrology and Endocrinology, University of Tokyo Graduate School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Correspondence to: Toshiro Fujita, PhD, MD Department of Nephrology and Endocrinology, University of Tokyo Graduate School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Email: fujita-dis{at}h.u-tokyo.ac.jp



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Cell viability
 Discussion
 References
 
Background. We previously reported that puromycin aminonucleoside (PAN) increased adrenomedullin (AM) secretion and AM mRNA expression in podocytes, through overproduction of oxidative stress. To clarify the cytoprotective role of AM as antioxidative and antiapoptotic substance in podocytes, we investigated the effect of exogenous AM and AM antagonist on PAN-induced apoptosis in conditionally immortalized murine podocytes.

Methods. The expression of AM, RAMP 2 and RAMP 3 was investigated using real-time PCR, western blotting analysis and immunofluorescence microscopy. Reactive oxygen species (ROS) production was measured by CM-H2DCFDA fluorescence intensity method. The percentage of apoptotic cells was measured by Hoechst 33342 staining.

Results. PAN (100 µg/ml) significantly (P < 0.01) increased ROS production, associated with an increase in apoptosis; the percentage of apoptotic cells is 5.3% + 0.05% (P < 0.01) with 36 h treatment of PAN compared to 0.24 + 0.16% with no treatment. Several antioxidants could markedly reduce PAN-induced apoptosis in cultured podocytes, suggesting that PAN-induced apoptosis might be attributable to the overproduction of ROS. Accordingly, the administration of exogenous AM (10–6 M) could significantly reduce not only ROS production via a PKA-dependent pathway, but also the resultant apoptosis induced by PAN. AM antagonists, CGRP8-37, augmented PAN-induced apoptosis, associated with increased ROS production, 2.2- and 2.3-Fold, respectively. RAMP 2 and RAMP 3 could be detected in podocytes and glomeruli.

Conclusions. This suggests that ROS-induced up-regulation of AM with PAN could counteract ROS-induced apoptosis, by the suppression of ROS production. Therefore, AM might have the endogenous antioxidant potential to protect against ROS-induced podocyte injury.

Keywords: adrenomedullin; aminonucleoside; antioxidants; apoptosis; oxidative stress; podocyte; proteinuria; puromycin; reactive oxygen species



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Cell viability
 Discussion
 References
 
Glomerular visceral epithelial cells, which are also called podocytes, function as critical size and charge barriers of protein excretion from glomerulus, therefore, podocyte injury induced marked proteinuria [1,2]. There is a growing body of evidence suggesting that podocyte injury plays an important role in not only proteinuria but also the progression to end-stage renal disease in diabetic nephropathy [3] and hypertensive glomerulosclerosis [4]. Since podocytes lack the ability of proliferation, they do not recover from their disappearance induced by podocyte injury, resulting in the decrease of podocytes from the glomerulus, podocytopenia, which is one of the important processes of glomerulosclerosis. Apoptosis is known to be the major course of podocytopenia [5]. Although there are several factors influencing apoptosis in podocytes, oxidative stress is one of the important causative factors. Accordingly, oxidative stress, in vitro, induces podocyte injury, associated with apoptosis of podocytes. Moreover, we recently demonstrated that antioxidants could not only improve podocyte injury but also inhibit proteinuria in salt-loaded Dahl salt-sensitive rats and aldosterone/salt-induced rats [6].

Adrenomedullin (AM) is known to be a potent vasoactive substance produced abundantly in vascular endothelial and smooth muscle cells [7,8]. Moreover, AM is also an antioxidant, since endogenous AM has the potential to protect from oxidative stress-induced vascular damages in angiotensin II/salt-treated rats and mice [9,10]. ROS up-regulates AM gene expression [11,12], and, in turn, AM inhibits ROS production induced by angiotensin II/salt [13]. Thus, vascular AM counteracts angiotensin II/salt as an endogenous antioxidant. Moreover, AM is reported to be expressed in podocytes as well as in the glomerular mesangial cells and tubular epithelial cells [14–16].

We previously reported that puromycin aminonucleoside (PAN) induced podocyte injury, associated with increased AM secretion and AM mRNA expression in podocytes, possibly through overproduction of ROS [17]. In order to clarify the cytoprotective role of AM up-regulated by PAN, in the present study, we investigated the effect of exogenous AM and AM antagonist on PAN-induced apoptosis, a marker of podocyte injury, in conditionally immortalized murine podocytes [18].



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Cell viability
 Discussion
 References
 
Reagents
Interferon-{gamma} was purchased from Toyobo (Tokyo, Japan). PAN, rotenone, antimycin A, diphenyleneiodonium chloride (DPI) and N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89) were purchased from Sigma Aldrich (St Louis, MO, USA). The 4'-hydroxy-3'-methoxyacetophenone (apocynin) was purchased from Tokyo Kasei (Tokyo, Japan). Human CGRP8-37 was purchased from Peptide Institute (Osaka, Japan).

Cell culture
We used mouse podocyte cell lines established by Mundel et al. [18] in 1997. Cells were cultured in RPMI1640 with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin in the presence of 10 U/ml recombinant murine interferon-{gamma} at 33°C in 5% CO2/95% air (permissive condition). To differentiate, podocytes were plated on type I collagen at a density of 1 x 104cells/cm2 and cultured with 1% FBS in the absence of {gamma}-interferon at 37°C (non-permissive condition). Two or three days after subculture, the concentration of FBS was reduced to 0.5%. Podocytes maintained under non-permissive condition for 10–14 days were used for experiments [17].

Stimulation of podocytes
Podocytes maintained under non-permissive condition for 10–14 days were exposed to PAN 1, 10 and 100 µg/ml. For ROS experiments, podocytes were treated with H2O2 10 µM for 24 h. For antioxidant experiments, podocytes were pretreated with rotenone 100 µM, antimycin A 1 µM, DPI 5 µM or apocynin 60 µM 40 min before stimulation of PAN. To examine the effects of the addition of AM receptor antagonists, pretreatment of human CGRP8-37 at 10–8, 10–7 and 10–6M was done preceded by the addition of PAN for 40 min. To examine the effects of the addition of a PKA inhibitor, podocytes were treated with H-89 at 10–5M 30 min before stimulation of AM. Detection of apoptosis was analysed by harvesting cells at 36 h, and other experiments were analysed at 24 h.

Real-time quantitive reverse transcription-polymerase chain reaction (real-time quantitive RT-PCR)
Total RNA was extracted using an RNeasy kit (Qiagen K. K., Tokyo) and treated with DNase I (Qiagen) to remove contaminating genomic DNA. The cDNA was synthesized from 1 mg of total RNA with random primers (Promega Corporation, Madison, WI) and Superscript II reverse transcriptase (Invitrogen Corp. Carisbad, CA). Gene expression was quantitively analysed by real-time RT-PCR using an ABI PRISM 7000 (Applied Biosystems, Foster City, CA). TaqMan chemistry and assay by design primers and probe sets were used for mouse AM, RAMP2, RAMP3 and β-actin. PCR was carried out on ABI PRISM 7000 with an initial activation of AmpliTaq Gold DNA polymerase at 95°C for 10 min, then 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. Relative quantification was accomplished with measurement of the threshold cycle and use of the standard curve. Gene expression of the target sequence was normalized to that of β-actin. Transcript level in the control group was arbitrarily expressed as 1 [17].

Measurement of secreted 8-OHdG
Total 7.5 x 104 cells were incubated on a 100 mm collagen I coated dish with 7 ml medium for 24 h with or without the agents. The supernatant was used for the 8-OHdG assay. We used the ELISA kit, New 8-OHdG Check (Japan Institute for the Control of Aging, Nikken SEIL Corp., Shizuoka), according to the protocol [19]. That is, the sample was applied to 8-OHdG-coated microplates then anti-8-OHdG antibody was added and incubated at 37°C for one hour. After washing, the enzyme-labelled antibody was added and incubated at 37°C an hour. After colour fixing under light interception, absorbance at 450 nm was measured. The concentration of 8-OHdG was calculated in comparison to the standard curve of absorbance, using standard samples included in the kit.

Western blotting
Western blot analysis was performed to measure the protein levels of adrenomedullin [6]. Adherent cells were treated with lysis buffer [composition: 50 mM Tris–HCl (pH 7.4), 10 mM EDTA, 1% Triton X-100, protease inhibitor], then the proteins (20 µg) were electrophoresised on 12.5% SDS-polyacrylamide gel, and the gels were blotted to the nitrocellulose membrane. Rabbit anti-adrenomedullin antibody (Santa Cruz Biotechnology, Inc., CA) diluted to 1/1000 was incubated at 4°C overnight after blocking with 5% non-fat dry milk powder solved in TTBS buffer (Tris-buffered saline with tween-20) for an hour. After washing with TTBS buffer, the membrane was incubated with an anti-rabbit IgG antibody-conjugated horseradish peroxidase (Amersham Biosciences) diluted to 1/4000 for an hour at room temperature. The resultant bands were detected with chemiluminescence Western blotting reagents (Amersham Biosciences) and exposed to hyperfilm ECL (Amersham Biosciences).

Measurement of CM-H2DCFDA fluorescence intensity
Podocytes differentiated in 6-well dishes were stimulated by the agents 36 h after being cultured with serum-free medium for more than 12 h. After disruption of the cells with trypsin, the number of harvested cells was counted. Then the cells were incubated with 5- (and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Invitrogen), 50 µg/ml, at 37°C for 60 min, and the fluorescence intensity of 100 µl of the sample was measured at 480 nm excitation and 535 nm emission by micorplate reader (WALLAC 1420 ARVO MX/Light, Perkin Elmer) [20,21]. Fluorescence intensities were compensated by their numbers of the cells.

Detection of caspase-3 activity
The samples were exposed to stimuli for 36 h. The caspaTagTM Caspase-3 In Situ Assasy Kit, sulforhodamine (Chemicon), was applied using the fluorochrome inhibitors of caspase (FLICA) method (22). After incubation at 37°C for one hour, cells were added to Hoechst solution at a final concentration of 0.5% for 5 min. After washing three times, the fixation buffer included in the kit was added and observed by microscope for caspase-3 activity at 550 nm excitation and 580 nm emission, and for Hoechst staining, at 351 nm excitation and 460 nm emission with a 420 nm filter. We confirmed that Hoechst-positive apoptotic cells also produced fluorescence of caspase-3 activity by observation of the sample simultaneously with caspase-3 labelling and Hoechst staining.

Hoechst 33342
The sample cells were exposed to stimuli for 36 h after being cultured with serum-free medium >12 h and were incubated with 10 µM Hoechst 33342 solution (Dojindo) at 37°C for 10 min, and fixed by 4% paraformaldehyde, then observed by microscope at 351 nm excitation and 460 nm emission with a 420 nm cutoff filter [23,24]. We judged an apoptotic cell by nucleus fragmentation and aggregation characteristic of apoptosis. Two pathologists examined more than 400 cells in a blinded manner, and calculated the percentages of apoptotic cells. The results by two pathologists were almost same.

Measurement of cell viability
To investigate the cytotoxicity of PAN, AM and CGRP8-37 of the podocytes, MTT assay for cell viability was performed. We used the [3-(4,5-dimethythiazol-2-yl) -2,5-diphenyl tetrazolium bromide] MTT Cell Proliferation Kit I (Roche Diagnostics, IN, USA) to investigate the viability of podocytes. Cells were seeded into 12-well culture plates and incubated with PAN, AM, CGRP8-37 for 24 h. The 100 µl MTT reagent was added to each well and incubated for 4 h in humidified atmosphere (37°C, 5% CO2). The total 500 µl of the solubilization solution was added to each well and 16 h later the colorimetric reaction was measured at 580 nm. Results were expressed as percentage of controls.

Immunofluorescence microscopy
An immunofluorescence study was performed according to a previously described method [25]. Cryostat sections were fixed with acetone for one minute and incubated with rabbit anti-RAMP 2, and anti-RAMP 3 (Santa Cruz Biotechnology, Santa Cruz, CA), stained with FITC-conjugated anti-rabbit IgG (Dako, Glostrup, Denmark), and observed with raser microscopy.

Statistical processing
Data were expressed as means ± SD. Statistical analyses were performed by unpaired t-test or analysis of variance and subsequent Tukey's simultaneous multiple comparison. A P value <0.05 was considered to be statistically significant [6]. All experiments were repeated three times.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Cell viability
 Discussion
 References
 
PAN induced AM expression on podocytes (Figure 1)
PAN (100 µg/ml) increased the gene expression of AM mRNA in cultured podocytes (AM/β-actin 15.99 ± 0.77 vs 1.79 ± 0.13 (P < 0.01), which is consistent with the results of the previous study [17]. Western blotting analysis revealed that H2O2 and PAN increased the expression of AM at the protein level (AM/β-actin 1.00 ± 0.09 vs 1.72 ± 0.19 and 2.21 ± 0.41) (P < 0.05) and that AM upregulation by PAN was normalized by the antioxidant, rotenone (2.21 ± 0.41 vs 1.14 ± 0.27 (P < 0.05) (Figure 1).


Figure 1
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  		Fig. 1. Western blotting of AM in podocytes. Podocytes were stimulated with H2O2 1 µM, PAN 100 µg/ml for 24 h and protein was analysed with western blotting. For antioxidant experiments, podocytes were pretreated with rotenone 100 µM 40 min before stimulation of PAN. #H2O2 and PAN predominantly increased the expression of AM at the protein level (AM/β-actin 1.00 ± 0.09 vs 1.72 ± 0.19 and 2.21 ± 0.41 (P < 0.05) and *AM up-regulation by PAN was normalized by rotenone (2.21 ± 0.41 vs 1.14 ± 0.27) (P < 0.05).

 
PAN-induced apoptosis and the effect of antioxidants (Figure 2)
To evaluate the effect of PAN on apoptotic cell death of the podocytes, we measured apoptosis morphologically by nucleus fragmentation or aggregation by staining Hoechst 33342. The percentage of apoptotic cells was significantly increased at the podocyte with 36 h treatment of PAN from 0.24 ± 0.09% without treatment to 4.45% with (Figure 2). To confirm apoptotic cell death, moreover, we examined the activation of caspase-3 in the morphologically apoptotic cells. The signal of activated caspase-3 was positive in cells with nucleus fragmentation and aggregation (insert of Figure 2) and the signal disappeared with fluorochrome inhibitors of caspase (FLICA) (data not shown). To evaluate the effects of antioxidants on apoptosis of podocytes induced by PAN, we used DPI and apocynin as NADPH oxidase inhibitors, and rotenone and antimycin A as mitochondrial antioxidants. All antioxidants could decrease apoptosis of podocytes induced by PAN significantly (Figure 2): rotenone 1.21 ± 0.15% (P < 0.01), antimycin A 0.22 ± 0.01% (P < 0.01), DPI 0.08 ± 0.10% (P < 0.01) and apocynin 1.83 ± 0.13% (P < 0.01).


Figure 2
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  		Fig. 2. PAN-induced apoptosis and the effect of antioxidants. (A) We measured apoptotic cell death of the podocytes morphologically by nucleus fragmentation or aggregation by Hoechst 33342 staining (n = 3±SD) (Cont, control; PAN, the podocytes treated with PAN; r+P, the podocytes treated with rotenone and PAN; antiA+P, the podocytes treated with antimycin A and PAN; DPI+P, the podocytes treated with DPI and PAN; apo+P, the podocytes treated with apocynin and PAN). *The percentage of apoptotic cells was significantly increased at the podocyte with 36 h treatment of PAN from 0.24 ± 0.09% without treatment to 4.45%±0.51% with. #Antioxidants could decrease apoptosis of podocytes induced by PAN significantly: rotenone: 1.21 ± 0.15% (P < 0.01), antimycin A: 0.22 ± 0.01% (P < 0.01), DPI: 0.08 ± 0.10% (P < 0.01) and apocynin: 1.83 ± 0.13% (P < 0.01). (B) The signal of activated caspase-3 was positive in cells with nucleus fragmentation and aggregation (insert of Figure 2).

 
PAN-induced ROS and the effect of exogenous AM and CGRP8-37 (Figure 3–6GoGoGo)
To confirm PAN-induced overproduction of ROS, in this experiment we measured three markers of oxidative stress. First, we measured the concentration of 8-OhdG in the medium of PAN-treated podocytes. The concentration of 8-OhdG was significantly (P < 0.05) increased, from 0.18 ± 0.02 to 0.56 ± 0.12 ng/ml, with the administration of PAN. Secondly, we examined the expression of nitrotyrosine by western blotting which was predominantly enhanced by PAN (data not shown). Thirdly, we measured fluorescence intensity of podocytes incubated with CM-H2DCFDA for 36 h, directly reflecting ROS production. PAN significantly (P < 0.05) increased ROS from 1.00 ± 0.05 to 1.55 ± 0.05 (Figure 3). Figure 4 shows the concentration response curve of the effect of PAN on ROS production. PAN 10 µg/ml and 100 µg/ml could significantly increase dose-dependently ROS from 1.00 ± 0.05 to 1.62 ± 0.25 and 1.53 ± 0.13 (Figure 4).


Figure 3
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  		Fig. 3. PAN-induced ROS and the effect of exogenous AM and CGRP8-37. We measured fluorescence intensity of podocytes incubated with CM-H2DCFDA for 36 h, directly reflecting ROS production (n = 3±SD) (Cont, control; PAN, the podocytes treated with PAN; AM, the podocytes treated with AM; CGRP, the podocytes treated with CGRP8-37; PAN+AM, the podocytes treated with PAN and adrenomedullin; PAN+CGRP, the podocytes treated with PAN and CGRP8-37). *PAN significantly (P < 0.05) increased ROS from 1.00 ± 0.05 to 1.55 ± 0.05. #The administration of exogenous AM (10–6M) could significantly (P < 0.05) reduce PAN-induced ROS production; 1.10 ± 0.03 vs 1.55 ± 0.05. #*AM receptor antagonist, CGRP8-37 increased ROS 2.3-fold; 3.57 ± 0.50 vs 1.55 ± 0.05 (P < 0.01).

 

Figure 4
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  		Fig. 4. Concentration response curve of the effect of PAN on ROS production. Fluorescence intensity of podocytes incubated with CM-H2DCFDA for 36 h (n = 3±SD) (Cont, control; 1, 10 and 100 µg/ml PAN, the podocytes treated with PAN 1, 10 and 100 µg/ml). PAN 10 µg/ml and 100 µg/ml could significantly increase ROS from 1.00 ± 0.05 to 1.62 ± 0.25 and 1.53 ± 0.13 (P < 0.05).

 

Figure 5
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  		Fig. 5. Dose-dependent effect of exogenous AM to PAN-induced ROS. Fluorescence intensity of podocytes incubated with CM-H2DCFDA for 36 h (n = 3±SD) (Cont, control; PAN, the podocytes treated with PAN 100 µg/; PAN+AM10–8, 10–7 and 10–6, the podocytes treated with PAN 100 µg/ml and AM 10–8, 10–7 and 10–6 M). The administration of exogenous AM (10–8, 10–7 and 10–6M) could reduce PAN-induced ROS production dose-dependently. #The administration of exogenous AM (10–7and 10–6M) could significantly (P < 0.05) reduce PAN-induced ROS production; 1.23±0.05 and 1.08 ± 0.13 vs 1.53 ± 0.03.

 

Figure 6
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  		Fig. 6. Dose-dependent effect of exogenous CGRP8-37 to PAN-induced ROS. Fluorescence intensity of podocytes incubated with CM-H2DCFDA for 36 h (n = 3±SD) (Cont, control; PAN, the podocytes treated with PAN 100 µg/ml; PAN+10–8, 10–7 and CGRP10–6, the podocytes treated with PAN 100 µg/ml and CGRP8-37 10–8, 10–7 and 10–6 M). The administration of CGRP8-37 (10–8, 10–7 and 10–6M) could increase PAN-induced ROS production dose dependently. #The administration of CGRP8-37 10–6M could significantly increase PAN-induced ROS production; 3.42 ± 0.46 vs 1.62 ± 0.23 (P < 0.01).

 
The administration of exogenous AM (10–8, 10–7 and 10–6M) could reduce PAN-induced ROS production dose-dependently and the reduction from AM 10–7 and 10–6M was significant: 1.23±.0.05 and 1.08 ± 0.13 vs 1.53 ± 0.03 (Figure 5). To investigate the role of endogenous AM up-regulated by PAN, we added AM receptor antagonist, CGRP8-37 (10–8, 10–7 and 10–6M), on PAN-induced podocytes. CGRP8-37 could increase PAN-induced ROS production dose-dependently and CGRP8-37 10–6M could significantly increase ROS by 2.3 times; 3.57 ± 0.50 vs 1.55 ± 0.05 (P < 0.01), suggesting that endogenous AM up-regulated by PAN might inhibit overproduction of ROS with PAN (Figure 3 and 6). To understand the role of AM at the physiological state, we investigated the effects of AM and CGRP8-37 on ROS production of the cells without PAN treatment. Administration of CGRP8-37 and AM could not influence the ROS production in the cells without PAN treatment.

AM inhibits PAN-induced ROS generation via the PKA-dependent pathway (Figure 7)
To determine whether the antioxidant effect of AM to PAN-induced ROS is mediated via the PKA-dependent pathway, we tested the effects of the PKA inhibitor, H-89. The inhibitory effect of AM (10–6M) on ROS generation by PAN (10 µg/ml) was predominantly abolished by H-89 (10–5 M); 1.10 ± 0.14 vs 1.47 ± 0.31 (P < 0.05).


Figure 7
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  		Fig. 7. AM inhibits PAN-induced ROS generation via the PKA-dependent pathway. Fluorescence intensity of podocytes incubated with CM-H2DCFDA for 36 h (n = 3±SD) (Cont, control; H-89, the podocytes treated with H-89 10–5M; PAN, the podocytes treated with PAN 100 µg/ml; PAN+AM, the podocytes treated with PAN 100 µg/ml and AM 10–6M; PAN+AM+H-89, the podocytes treated with PAN 100 µg/ml, AM 10–6M and H-89–5M. #The administration of H-89 could significanty abolish the inhibitory effect of AM on ROS generation by PAN; 1.10 ± 0.14 vs 1.47 ± 0.31 (P < 0.05).

 
The effect of exogenous AM and CGRP8-37 on PAN-induced apoptosis (Figure 8)
To evaluate cytoprotective role of endogenous AM, we studied the effect of CGRP8-37 on apoptosis of the podocytes by Hoechst 33342 stain. According to the changes in ROS production, CGRP8-37 increased PAN-induced apoptosis by 2.2 times; 4.50 ± 0.09% vs 2.02 ± 0.29% (P < 0.01). Moreover, the addition of exogenous AM could reduce PAN-induced apoptosis significantly; 1.01 ± 0.08% vs 2.02 ± 0.29% (P < 0.01).


Figure 8
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  		Fig. 8. The effect of exogenous AM and CGRP8-37 on PAN-induced apoptosis. (A) We studied the effect of CGRP8-37 on apoptosis of the podocytes by Hoechst 33342 stain (n = 3±SD) (Cont, control; PAN, the podocytes treated with PAN; AM, the podocytes treated with AM; P+AM, the podocytes treated with PAN and AM; CGRP, the podocytes treated with CGRP8-37; P+CGRP, the podocytes treated with PAN and CGRP8-37). #The addition of exogenous AM could reduce PAN-induced apoptosis significantly; 1.01 ± 0.08% vs 2.02 ± 0.29% (P < 0.01). *CGRP8-37 increased PAN-induced apoptosis by 2.2 times; 4.50 ± 0.09% vs 2.02 ± 0.29% (P < 0.01). (B) Hoechst 33342 stain of podocytes stimulated with PAN (a) and PAN+CGRP8-37 (b). Arrow: apoptotic cells with nucleus fragmentation and aggregation.

 
The expression of AM receptors, RAMP 2 and RAMP 3, in podocytes and glomeruli (Figure 9)
We examined the expression of the AM receptors RAMP 2 and RAMP 3 in podocytes and glomeruli of control and PAN treated podocytes and glomeruli. The mRNA of RAMP 2 and RAMP 3 could be detected in podocytes, however PAN could not influence the expression of mRNA (data not shown). The investigation with immunofluorescence microscopy revealed that the signal of RAMP 3 was detected in glomeruli more intensively than RAMP 2 (Figure 9), however, PAN could not influence the intensity of signals.


Figure 9
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  		Fig. 9. The expression of AM receptors RAMP 2 and RAMP 3 in glomeruli. The frozen sections were stained with rabbit anti-RAMP 2 and anti-RAMP 3. The signals of both RAMP 2 and RAMP 3 could be detected in glomeruli and the signals of RAMP 3 were more intensive than RAMP 2. Original magnifications: x400.

 


   Cell viability
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Cell viability
Discussion
 References
 
Incubation for 24 h of AM 10–6M and CGRP8-37 10–6M did not change cell viability (AM 0.98 ± 0.05, CGRP 0.99 ± 0.03), however, the concentration of 100 µg/ml PAN was a subtoxic dosage (0.65 ± 0.02, P < 0.01).



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Cell viability
 Discussion
References
 
In the present study, we demonstrated that the administration of antioxidants such as rotenone, antimycin A, DPI and apocynin could markedly reduce PAN-induced apoptosis measured by Hoechst 33342 staining in conditionally immortalized murine podocytes, which is consistent with the results of previous studies indicating that PAN-induced apoptosis of podocytes might be attributable mainly to overproduction of oxidative stress. Correspondingly, the addition of exogenous AM significantly reduced PAN-induced apoptosis, associated with the decreased PAN-induced overproduction of ROS measured by CM-H2DCFDA fluorescence. Moreover, the administration of the AM receptor antagonist CGRP8-37 could augment not only ROS production but also apoptosis induced by PAN. This suggests that AM might protect against ROS-induced apoptosis with PAN, through the inhibition of ROS production.

There is a growing body of evidence that oxidative stress plays a critical role in the progression to end-stage renal failure as well as the development of cardiovascular disease. Hypertension, dyslipidaemia, diabetes, obesity and smoking cause the incidence of chronic kidney disease and renal failure, through overproduction of oxidative stress. There are several factors producing oxidative stress. Vasoactive substances such as angiotensin II and aldosterone also produce oxidative stress, resulting in atherosclerosis and renal damage. Indeed, angiotensin II and aldosterone caused podocyte injury [26], associated with proteinuria, and the administration of tempol, a membrane-permeable SOD mimetic, could correct not only podocyte injury but also proteinuria [27]. In the present study, we used puromycin aminonucleoside (PAN), since the podocyte is the main target of the injury by PAN. Many studies focused on the mechanism of PAN-induced nephropathy. Among them, some studies showed that podocyte injury induced by PAN was solely mediated by oxidative stress [28,29]. In the present study, therefore, in order to clarify the mechanism for ROS-induced podocyte injury with PAN, we firstly studied the effects of antioxidants on PAN-induced apoptosis and podocyte injury. In this report, we showed that exogenous AM inhibited PAN-induced apoptosis by about 50%, although AM completely inhibited ROS production increased by PAN. One of the reasons may be that induction of apoptotic cell death needs a certain amount of ROS, and so AM could inhibit apoptosis by decreasing ROS to less than the level which could induce apoptosis. According to the results of previous studies, the present experiment showed that PAN induced apoptosis, a marker of podocyte injury. It should be noted that podocytes lack the ability of proliferation because of the dominance of CDK-inhibitors as compared to cyclin-dependent kinases [30]. As a result, the podocytes that disapper can never be replaced, leading to podocytopenia, the loss of podocytes from glomerulus, which is one of the important processes of glomerulosclerosis. Apoptosis is well known to be the major course of podocytopenia leading to podocyte injury. Several studies revealed the involvement of oxidative stress in apoptosis of podocytes. Suzuki showed that PAN induced overproduction of ROS and apoptosis of podocytes, in vitro, using podocyte sieving from glomeruli [31]. Moreover, Sanwal et al. [32] demonstrated that the administration of ROS scavengers such as SOD and DMTU could suppress apoptosis induced by PAN. Consistently, in the present study the administration of antioxidants could ameliorate PAN-induced apoptosis.

AM is reported to be produced in not only vascular endothelial and smooth muscle cells but also in podocytes, mesangial cells and tubular cells. We demonstrated that AM is synthesized and secreted in the glomerular podocyte cell line established by Mundel et al. AM may play a regulatory role in the podocytes in an autocrine manner. Indeed, cAMP, one of the main second messengers of the AM signalling cascade, was indicated to regulate podocyte function [33]. Alternatively, AM secreted from the podocytes may regulate glomerular function by acting on glomerular endothelial or mesangial cells in a paracrine manner [34].

Several investigators demonstrated, in vitro, that oxidative stress induced AM secretion and AM gene expression in mesangial cells and vascular smooth muscle cells [35]. Moreover, we previously found that AM mRNA expression was enhanced by ROS generators such as H2O2, TNF-{alpha}, human serum albumin and PAN [17]. In addition, AM mRNA up-regulation by PAN was normalized by antioxidants. The result of western blotting analysis in this report was consistent with the previous study. Then, it can be assumed that PAN generates ROS, which activates injurious signalling, resulting in apoptosis, on one hand, and stimulates AM secretion to counteract the podocyte injury at the same time.

AM acts by binding the calcitonin receptor-like receptor (CRLR), whose interaction with the subtypes 2 and 3 of a family of receptor activity-modifying proteins (RAMP) gives rise to two distinct AM receptors, CRLR/RAMP 2 and CRLR/RAMP 3 receptors [36]. AM may prevent PAN-induced apoptosis by up-regulation of its functional receptor. We examined the expression of RAMP 2 and RAMP 3 in podocytes and glomeruli of vehicle and PAN-treated podocytes and glomeruli. We could detect the expression of RAMP 2 and RAMP 3 in podocytes and glomeruli, however, PAN cannot increase the expression of RAMP 2 and RAMP 3. In this report, we showed that the PKA inhibitor, H-89, could abolish the inhibitory effect of AM (10–6M) on ROS generation by PAN, which is consistent with the results of previous studies that AM inhibited angiotensin II-induced oxidative stress in rat endothelial cells via a PKA-dependent pathway. [37].

It is well known that AM is not only a vasoactive substance but also an antioxidant [38–40]. AM has been reported to counteract the deleterious effects of angiotensin II [10,33]. Moreover, we previously reported that endogenous AM exerts protective effects against angiotensin II- or cuff-induced vascular injury through the inhibition of oxidative stress using AM knockout mice [10,41,42]. Accordingly, our recent study has demonstrated that renal damage by unilateral urethral occlusion, UUO, was enhanced in AM knockout mice through overproduction of ROS (manuscript in preparation), suggesting that AM may protect against ROS-induced renal damage with UUO. Moreover, several investigators demonstrated that AM has an anti-apoptotic effect in endothelial cells [43]. Consistently, in the present study, the administration of exogenous AM ameliorated not only PAN-induced ROS overproduction but also the resultant apoptosis of podocytes. Moreover, AM receptor antagonist CGRP8-37 augmented PAN-induced apoptosis in cultured podocytes, associated with the enhanced ROS production. Therefore, it may be plausible to hypothesize that upregulated AM in the podocytes with PAN protects against ROS-induced apoptosis, through the inhibition of ROS.



   Acknowledgments
 
The authors thank Dr Peter Mundel (Mount Sinai School of Medicine, One Gustavo L. Levy Place, New York, USA) for the podocyte cell line.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Cell viability
 Discussion
 References
 

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