Reactive Oxygen Species Alter Gene Expression in Podocytes: Induction of Granulocyte Macrophage-Colony-Stimulating Factor
Stefan Greiber,
Barbara Müller,
Petra Daemisch and
Hermann Pavenstädt
Department of Medicine, Division of Nephrology, University Hospital of Freiburg, Freiburg, Germany.
Correspondence to Hermann Pavenstädt, Medizinische Universitätsklinik IV, Nephrologie, Hugstetter Str. 55, D-79106 Freiburg, Germany. Phone: +49-761-270-3492; Fax: +49-761-270-3245; Email: paven{at}mm41.ukl.uni-freiburg.de
ABSTRACT. It has been suggested that reactive oxygen radicals(ROS) play a crucial role in the pathogenesis of proteinuriaand podocyte injury. It was investigated whether changes ingene expression might account for ROS-induced podocyte dysfunction.Differentiated podocytes were incubated with control media orwith exogenous ROS from the xanthine/xanthine-oxidase reactionfor 4 h. A PCR-based suppressive subtractive hybridization assaywas applied to isolate and clone mRNAs that were differentiallyexpressed by exogenous ROS. One differentially expressed clonewas identified as the proinflammatory cytokine granulocyte macrophage-colony-stimulatingfactor (GM-CSF). Regulation of GM-CSF in podocytes was furtherstudied by Northern analysis and enzyme-linked immunosorbentassay. Exogenous ROS caused a concentration-dependent, >10-foldinduction of GM-CSF mRNA after 4 h. A >50-fold increase inGM-CSF protein release in podocytes that had been stimulatedwith ROS could be detected. Induction of GM-CSF protein wasinhibited by actinomycin D, which indicated that increased mRNAtranscription was involved. The ROS scavengers dimethyl-thio-ureaand pyrrolidone-dithio-carbamate strongly inhibited increasedGM-CSF production induced by ROS. GM-CSF release was also inducedwhen internal ROS production was triggered with NADH, whereasH2O2 had only a small effect. GM-CSF release by podocytes wasalso stimulated by lipopolysaccharide (LPS), interleukin-1 (IL-1),and phorbolester (PMA). Dimethyl-thio-urea significantly inhibitedthe LPS-, IL-1-, and PMA-induced GM-CSF production. Activationof the transcription factor nuclear factor-B (NF-B) but notactivator protein-1 was involved in the upregulation of ROS-inducedGM-CSF production. The data indicate that GM-CSF is differentiallyexpressed by ROS in podocytes. ROS also partially mediate theeffects of PMA and IL-1 on podocyte GM-CSF production. BecauseGM-CSF can enhance glomerular inflammation and induces mesangialproliferation, these data might provide further insight intothe mechanisms of ROS-induced glomerular injury.
The podocyte plays a crucial role in maintaining the permselectivefunction of the glomerular capillary wall (1). Under pathophysiologicconditions, the podocyte contributes to the initiation and progressionof a variety of glomerular diseases. Membranous nephropathy,minimal change disease, and focal segmental sclerosis in particularhave all been related to primary or secondary podocyte injury(2,3). Overproduction of reactive oxygen radicals (ROS) hasbeen found in glomerular diseases in which the podocyte is theprimary target cell of glomerular injury, such as puromycinnephrosis, a model of minimal change disease, Heyman nephritis,a model of membranous nephropathy, and the Mpv 17 (-/-) mouse,a model for steroid-resistant focal segmental sclerosis (3,4).The release of ROS leads to proteinuria by affecting glomerularendothelial, and epithelial cells and disturbing normal glomerularpermselectivity (5,6). In these glomerular diseases, pretreatmentof animals with the antioxidant probucol or ROS scavengers markedlyprevented foot-process effacement and proteinuria. So far, themechanisms by which ROS might contribute to podocyte damageare incompletely understood. Because the majority of cellularprocesses are characterized by changes in gene expression, weused a cell culture model of differentiated podocytes to studychanges in gene expression caused by ROS. Herein, we used aPCR-based suppressive subtractive hybridization (PCR-SSH) toidentify genes in podocytes that are differentially changedby ROS. PCR-SSH is a method based on suppressive PCR that allowscreation of subtracted cDNA libraries for the identificationof genes differentially expressed in response to a stimulus(7,8). PCR-SSH differs from earlier subtractive methods by includinga normalization step that equalizes for the relative abundanceof cDNA within the target population. This modification enhancesthe probability to identify the increased expression of low-abundancetranscripts and represents a potential advantage over othermethods, such as differential display PCR, for identifying differentiallyregulated genes (9). With the PCR-SSH technique, we demonstratethat the granulocyte macrophage-colony-stimulating factor (GM-CSF)is differentially expressed by ROS in podocytes. GM-CSF is acytokine that regulates the survival, growth, and differentiationof hematopoietic progenitor cells (10). In the kidney, GM-CSFexerts its effects primarily on macrophages, where it stimulatestumor necrosis factor and interleukin-1 (IL-1) production ofthese cells. Both cytokines have been suggested to play a majorrole in the pathogenesis of glomerular inflammation and proteinuria(11,12). In addition, GM-CSF might serve as a critical signalfor macrophage migration into the glomerulus (13).
Cell Culture
Conditionally immortalized mouse podocytes were cultured asreported elsewhere (14). In brief, podocytes were maintainedin RPMI 1640 medium (Life Technologies, Eggenstein, Germany)supplemented with 5% fetal calf serum (Boehringer Mannheim,Mannheim, Germany), 100 kU/L penicillin, and 100 mg/L streptomycin(Life Technologies). To propagate podocytes, cells were cultivatedat 33°C on type I collagen (permissive conditions), andthe culture medium was supplemented with 10 U/ml recombinantinterferon- to enhance the expression of the T antigen. To inducedifferentiation, podocytes were maintained on type I collagen(Biochrom, Berlin, Germany) at 37°C without interferon-(nonpermissive conditions). Podocytes between passage 10 and16 were used in all experiments. To examine the effects of ROSon podocyte GM-CSF mRNA expression or GM-CSF release, podocytesfrom one cell pool were plated at a cell density of 104 cells/cm2in media that contained 5% fetal calf serum in six-well platesfor Northern analysis or in 96-well plates for measurementsof GM-CSF protein. Cells were switched to media that contained1% fetal calf serum 24 h before the experiments and then exposedto various treatments.
Generation of a Differentially Expressed cDNA Library
To screen for genes that are differentially expressed by ROSin podocytes, a PCR-SSH approach (7,8) was used (PCR-Select;Clone Tech, Palo Alto, CA). In brief, RNA was isolated fromcontrol cells and cells that had been stimulated with extracellularsuperoxide generated from the xanthine/xanthine-oxidase reaction(X/XO; 50 µM/5 or 50 mU/ml) for 4 h. cDNA synthesis from1 µg of total RNA from each cell population was achievedwith the SMART PCR cDNA synthesis kit (Clone Tech) and subsequentlong-distance PCR. cDNA from ROS-stimulated cells was named"tester-cDNA," and cDNA from control cells was named "driver-cDNA."After column purification, PCR products were subjected to RsaIdigestion to obtain shorter and blunted molecules. The testercDNA was then divided into two portions, and each was ligatedto a different cDNA adapter. Driver cDNA was not ligated toan adapter sequence. Two hybridizations were performed. In thefirst, an excess of denatured driver-cDNA was added to eachdenatured tester population. Hybridization kinetics led to equalizationand enrichment of differentially expressed sequences. In thesecond hybridization, the two primary hybridization sampleswere mixed together without denaturing and denatured drivercDNA was added. The remaining subtracted tester cDNAs couldnow reassociate and form hybrids with different ends that correspondedto the sequences of the two adapters. The differentially expressedcDNA population of this "forward subtraction" was then PCR-amplifiedwith nested primers corresponding to the two different adaptersequences. For further screening steps, a reverse-subtractedprobe with the original tester cDNA as a driver and the drivercDNA as a tester was generated.
Screening of the Differentially Expressed cDNA Library
The subtracted cDNA library was cloned into a 3.9-kb PCRTM 2.1vector (TA-cloning kit; Invitrogen, San Diego, CA). UltracompetentEscherichia coli (INV.F') were transformed and plated onto agarplates that contained 50 µG/ml ampicillin, 50 µG/mlisopropyl-ß-D-thiogalactoside, and 50 µG/ml5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.Ninety-six transformed clones were regrown overnight in 100µl of LB media in 96-well microtiter plates. A PCR withthe nested primer from the adapter sequences was then performedon 1 µl of each bacterial culture, to amplify the clonedcDNA inserts. Five microliters of the PCR reaction were thenspotted onto two duplicate nylon membranes and cross-linkedby ultraviolet irradiation. One nylon filter was then hybridizedwith a 32P-dCTP-labeled part of the forward subtracted library,and the duplicate filter was hybridized with the reverse-subtractedlibrary. Filters were then subjected to autoradiography. Positiveclones that showed differential expression on both blots werefurther analyzed by virtual Northern blotting: cloned insertswere excised from the vector by EcoRI digestion, separated byagarose gel-electrophoresis, and extracted from the gel (QIAEX;Quiagen, Heidelberg, Germany). A total of 25 ng of cDNA werethen labeled with a 32P-dCTP by random priming and used to analyze500 ng of double-stranded c-DNA from each control and X/XO-treatedpodocytes by virtual Northern analysis. Virtual Northern analysisshows that cDNA from clone 48 hybridized to a 1.2-kb transcriptthat was strongly upregulated in cells that had been treatedwith X/XO (Figure 1). The insert was then sequenced with anautomated ALF sequencer and then identified by a computer-basedBlast search of Genbank (15) as part of the mouse GM-CSF cDNAsequence.
Figure 1. Isolation of differentially expressed genes in reactive oxygen radical (ROS)-treated podocytes by PCR suppressive subtractive hybridization (PCR-SSH). PCR-SSH: reverse transcription was performed on RNA from control (driver) and ROS-treated (tester) podocytes. Tester cDNA was divided and ligated to two different adapters. In the subsequent hybridization, excess driver cDNA eliminates cDNA not differentially expressed in the tester cDNA. Differentially expressed tester I + II cDNA eventually hybridizes as D molecules and is exponentially amplified by the subsequent PCR reaction with primers corresponding to two different adapter sequences. Differential screening: after cloning of the PCR reaction, which is enriched for differentially expressed cDNA, clones are spotted on duplicate filters. Several clones, including clone 48 (arrow), produced a stronger autoradiogram signal when hybridized to tester cDNA compared with driver cDNA. This finding suggests that these clones contain cDNA that represent differentially expressed mRNA in ROS-treated podocytes. Virtual Northern analysis: a clone 48 cDNA probe detects a differentially expressed 1.2-kb transcript in xanthine/xanthine-oxidase reaction (X/XO)-treated podocytes. Clone 48 is sequenced and identified as a 420-bp fragment of the granulocyte macrophage-colony stimulating factor (GM-CSF) gene by BLAST search.
Northern Analysis of GM-CSF mRNA in Podocytes
Differentiated podocytes were grown in six-well plates and receivedfresh media 24 h before the experiment. RNA was isolated byacid phenol extraction (16). RNA (10 µg/lane) was size-separatedin agarose/formaldehyde gels and transferred to Hybond nylonmembranes (Pharmacia, Freiburg, Germany). GM-CSF cDNA was labeledwith 32-P-dCTP by use of a random-primer labeling kit (Stratagene,Heidelberg, Germany). Hybridization and washes were performedaccording to the method of Church and Gilbert (17). After analysisof the GM-CSF signal, blots were rehybridized to a 32-P-dCTP-labeledprobe for the housekeeping gene GAPDH, to control for variationin loading and transfer among samples.
Analysis of GM-CSF Protein by Enzyme-Linked Immunosorbent Assay
Quantitative determination of GM-CSF protein release into themedia by podocytes was performed with a mouse GM-CSF immunoassay(Quantikine; R&D Systems, Abingdon, UK), according to themanufacturers protocol. Podocytes were grown in 96-well platesfor scheduled incubation with ROS or other stimulators of GM-CSFrelease, as indicated. Inhibitors and stimulators of GM-CSFrelease were added simultaneously, with the exception of actinomycinD, which was added 1 h before the stimulation with ROS.
Analysis of Nuclear Factor-B and Activator Protein-1 Activation by Electrophoretic Mobility Shift Analysis
Activated nuclear factor-B (NF-B) and activator protein-1 (AP-1)were assayed in nuclear extracts from podocytes, as describedby Ogata et al. (18). Podocytes were treated with stimulantsor vehicle for 20, 60, or 120 min. Thereafter, cells were harvestedfor collection of nuclear extracts. Consensus oligonucleotidesfor NF-B (5'-AGT TGA GGG GAC TTT CCC AGG C-3' and 5'-GCC TGGGAA AGT CCC CTC AAC T-3') were purchased from Santa Cruz (Heidelberg,Germany), and consensus oligonucleotides for AP-1 (5'-CGC TTGATG AGT CAG CCG GAA-3' and 5'-TTC CGG CTG ACT CAT CAA GCG-3')were purchased from Promega (Mannheim, Germany). At least threedifferent analysis were performed for each experimental setup:sample (nuclear extracts and 32P-labeled oligonucleotide probe),negative control (32P-labeled oligonucleotide probe and no nuclearextracts), and specific inhibition (nuclear extracts and 32P-labeledoligonucleotide probes and a 100-fold molar excess of unlabeledoligonucleotide probes).
Statistical Analyses
Data are given as mean ± SEM. Statistical analysis wasperformed by one-way ANOVA for multiple comparisons (Bonferroni’st test). P < 0.05 was considered to be significant.
GM-CSF is Induced by the X/XO Reaction in Podocytes
To identify genes that are induced by ROS, we used a PCR-basedcDNA subtractive hybridization strategy to generate a cDNA libraryof genes that are differentially expressed by ROS in podocytes(Figure 1). One hundred clones from the differentially expressedcDNA library were further analyzed by differential screening,and 27 clones generated an increased hybridization signal whenhybridized to RNA from X/XO-treated podocytes compared withRNA from control cells (Figure 1). Out of these 27 clones, clone48 detected a 1.2-kb transcript that was highly upregulatedin podocytes that had been stimulated with ROS (Figure 1). ThiscDNA clone was sequenced and identified by BLAST analysis asa 420-bp fragment of the mouse GM-CSF cDNA.
GM-CSF mRNA is Upregulated by X/XO after 4 and 12 h
To further characterize the induction of GM-CSF mRNA by ROS,a time course of GM-CSF mRNA expression in podocytes was investigated.Induction of GM-CSF mRNA in podocytes was strongest detectedat 4 h and was markedly declined but still detectable after12 h after incubation of podocytes with X/XO (Figure 2A). Noinduction of GM-CSF mRNA was seen when podocytes were incubatedwith xanthine or xanthine-oxidase alone (data not shown). Wealso stimulated podocytes with media that contained X/XO foronly 30 min and then exchanged the media with fresh media thatcontained no X/XO. The time course of GM-CSF mRNA inductionwas not different from the cells that had been stimulated withX/XO for the whole incubation period, but the total increasein GM-CSF mRNA was reduced (Figure 2B). We also found that increaseof intracellular superoxide production by diamide (19), causeda four- to fivefold induction of GM-CSF mRNA (data not shown).
Figure 2. GM-CSF mRNA is time dependently expressed in ROS-treated podocytes. (A) The time course of GM-CSF mRNA induction by ROS was studied in control podocytes or in podocytes that had been incubated with X/XO (50 µM/50 mU/ml) for indicated times. (B) The time course of GM-CSF mRNA induction by ROS was studied in control podocytes or in podocytes that had been incubated with X (50 µM/ml)/XO (50 mU/ml) for 30 min.
Podocytes Release GM-CSF Protein in Response to ROS
To investigate whether the increased GM-CSF mRNA levels in responseto ROS results in a subsequent increase of protein release,GM-CSF protein concentration was measured in the media of ROS-treatedand control podocytes by enzyme-linked immunosorbent assay.After 24 h of incubation with X/XO (50 µM/50 mU), GM-CSFlevels increased from 0.5 ± 0.3 to 156 ± 20 pg/ml(n = 30). Figure 3 shows the time and concentration dependencyof 50 µM X and different concentrations of XO (1 to 100mU/ml) on GM-CSF release. A threshold concentration of 5 mU/mlXO was required to induce GM-CSF release in podocytes. After4 h of incubation with X/XO, a first significant GM-CSF releasewas observed. After 8 h of incubation, XO concentrations (5mU) induced a maximal GM-CSF release that stayed elevated overcontrols even after 24 h of incubation (Figure 3).
Figure 3. GM-CSF protein is time dependently expressed by ROS. Podocytes were incubated with control media or media that contained X (50 µM) and XO (1 to 100 mU) for indicated times. Data are mean ± SEM from six experiments. *, P < 0.05 versus control.
To test the hypothesis that intracellularly generated ROS caninduce GM-CSF release, additional experiments were performedwith the use of NADH (5 mM), a substrate for the podocyte NAD(P)Hoxidases (20). NADH (5 mM) strongly increased GM-CSF release,whereas the effect of hydrogen peroxide (H2O2, 100 µM)was less pronounced (Figure 4). Effects of NADH and H2O2 weredose-dependent (data not shown), with NADH 5 mM and H2O2 100µM, respectively, resulting in the strongest increasein GM-CSF protein release. At NADH concentrations >10 mM,GM-CSF release began to decline.
Figure 4. GM-CSF production is also stimulated by H2O2 and activation of endogenous NAD(P)H-oxidases. Podocytes were incubated with control media or media that contained X (50 µM)/XO (50 mU), H2O2 (100 µM), or NADH (5 mM), respectively, for 24 h. Data are mean ± SEM from 12 experiments. *, P < 0.05 versus control.
ROS Scavengers Dimethyl-Thio-Urea and Pyrrolidone-Dithio-Carbamate Inhibit the GM-CSF Release Induced by ROS
To further investigate whether X/XO-induced effects were attributableto the generation of ROS, we used oxygen radical scavengersto inhibit the effects of ROS. Incubation of podocytes withthe oxygen radical scavenger dimethyl-thio-urea (DMTU, 10 mM)inhibited GM-CSF release from podocytes by 81 ± 5% (n= 12), whereas equimolar urea that was used as a nonscavengercontrol had no effect on X/XO-induced GM-CSF release (Figure 5).Another thiol compound and radical scavenger, pyrrolidone-dithio-carbamate(PDTC), has been shown to inhibit activation of the transcriptionfactor NF-B, which is involved in the regulation of CSF-1 inother cell lines (21). Incubation of podocytes with PDTC (50µM) completely inhibited the X/XO-induced GM-CSF releasefrom podocytes (n = 12) (Figure 5).
Figure 5. ROS scavengers dimethyl-thio-urea (DMTU) and pyrrolidone-dithio-carbamate (PDTC) inhibit the GM-CSF release induced by X/XO. Podocytes were incubated with control media or media that contained X (50 µM)/XO (50 mU) with or without inhibitors (DMTU, 10 mM; urea, 10 mM; and PDTC, 50 µM). In an additional set of experiments, podocytes were incubated with an inhibitor of RNA transcription, actinomycin D (0.1 µg/ml), as indicated in the Materials and Methods section. Data are mean ± SEM from 12 experiments. *, P < 0.05 versus X/XO.
Increased GM-CSF Production by ROS Requires New RNA Transcription
To further delineate the regulation of GM-CSF production byROS in podocytes, we tested whether mRNA transcription was requiredfor increased GM-CSF release. When podocytes were preincubatedwith actinomycin D (0.1 µg/ml) before stimulation withX/XO, increased GM-CSF release was reduced from 172 (15 pg/ml)to control levels (2 ± 1 pg/ml) (n = 12). Figure 5 showsthe effect of actinomycin D on GM-CSF release from podocytes.
IL-1, Phorbolester, and Lipopolysaccharide Increase GM-CSF Release in Podocytes
Next we examined whether GM-CSF release is regulated by agentsthat have been implicated to participate in glomerular inflammationsuch as IL-1 (11), phorbolester (PMA) (22), and lipopolysacharide(LPS) (12,23). Stimulation of GM-CSF was induced by as littleas 1 pg/ml IL-1. The effects of IL-1 plateaued at 100 pg/ml.PMA caused an significant increase in GM-CSF release at doses10 to 9 M: the maximal effect was seen at 10 to 7 M. The maximaleffect of PMA on GM-CSF release was about twice the effect ofIL-1. LPS, when used in concentrations from 0.1 to 100 µg/ml,also had a dose-dependent effect on GM-CSF release: the maximaleffect was seen at a dose of 1 µg/ml (data not shown).
Figure 6 shows that IL-1 (50 pg/ml), PMA (0.1 µM), andLPS (1 µg/ml) induced a time-dependent increase in GM-CSFrelease in podocytes (n = 6). A significant increase of GM-CSFrelease was seen after 2 h of incubation with PMA and IL-1 andafter 4 h of incubation with LPS. With all three agents, GM-CSFrelease was still elevated compared with controls, even after24 h.
Figure 6. Phorbolester (PMA), interleukin-1 (IL-1), and lipopolysaccharide (LPS) cause a time-dependent increase in GM-CSF release. Podocytes were incubated in control media or media with PMA (0.1 µM), IL-1 (50 pg/ml), and LPS (1 µg/ml) for indicated times before GM-CSF release into the media was measured. Data are mean ± SEM of six experiments. *, P < 0.05 versus control.
Stimulation of GM-CSF Release in Podocytes by PMA, IL-1, and LPS is Partially Mediated by ROS
In another set of experiments, we tried to determine whetherthe induction of GM-CSF release by PMA, IL-1, and LPS was ROS-dependent.Figure 7 shows the effect of ROS scavengers DMTU (10 mM), PDCT(50 µM), and N-acetylcystein (NAC, 10 mM) on PMA-, IL-1-,and LPS-induced GM-CSF release by 64 ± 1, 59 ±2, and 58 ± 2%, respectively. Addition of PDCT to podocytesreduced PMA-, IL-1-, and LPS-induced GM-CSF by 74 ± 3,78 ± 4, and 85 ± 1%, respectively. Glutathioneperoxidase represents an important intracellular defense mechanismagainst ROS. Glutathione peroxidase does not directly scavengesuperoxide but is necessary in the subsequent detoxificationof hydrogen peroxide, which is generated by the superoxide dismutasefrom the superoxide anion. Addition of NAC increases intracellularglutathione, which is the substrate for the glutathione peroxidaseduring the detoxification of hydrogen peroxide (24). NAC suppressedthe increase of GM-CSF release in response to IL-1 and PMA butonly marginally attenuated the effects of LPS (Figure 7).
Figure 7. Induction of GM-CSF release by PMA, IL-1, and LPS is mediated by reactive oxygen species. Podocytes were incubated in control media or media with PMA (0.1 µM), IL-1 (50 pg/ml), and LPS (1 µg/ml) with or without ROS scavengers, as indicated (DMTU, 10 mM; PDTC, 50 µM; and NAC 10 mM) for 24 h before GM-CSF release into the media was measured. Data are mean ± SEM of 12 experiments. *, P < 0.05 versus no inhibitor.
To test whether induction of GM-CSF release by PMA, IL-1, andLPS also requires new mRNA synthesis, stimulation of podocyteswith these agents were performed in the presence of actinomycinD. Figure 8 demonstrates that actinomycin D completely suppressedthe stimulation of GM-CSF release caused by PMA, LPS, and IL-1.Dexamethasone (1 µM), which not only represses AP-1- andNF-B-mediated transcriptional activation (25,26) but also mayserve as an antioxidant, also significantly suppressed the effectsof PMA, IL-1, and LPS on GM-CSF release (Figure 8).
Figure 8. Inhibition of RNA synthesis blunts induction of GM-CSF by PMA, IL-1, and LPS. Podocytes were incubated in control media or media with PMA (0.1 µM), IL-1 (50 pg/ml), or LPS (1 µg/ml) with or without actinomycin D (AmD, 0.1 µg/ml) or dexamethasone (Dex, 1 µM), as indicated, for 24 h before GM-CSF release into the media was measured. Data are mean ± SEM of 12 experiments. *, P < 0.05 versus no inhibitor.
ROS, PMA, IL-1, and LPS Induce NF-B Activation in Podocytes
Because indirect evidence from inhibitor experiments suggestedthat activation of NF-B as well as AP-1 might be involved inthe activation of GM-CSF, we directly analyzed activation ofthese transcription factors in response to different stimuli.Electrophoretic mobility shift analysis showed that podocytesendogenously contained a small amount of active NF-B in thenucleus without any stimulus. Figure 9 shows that treatmentwith ROS, PMA, IL-1, and LPS for 20, 60, or 120 min increasedNF-B/DNA binding activity (n = 4). The addition of 100-foldexcess of unlabeled NF-B-oligonucleotide probe as a competitoreliminated the NF-B/DNA binding from treated and untreated cells.ROS as well as the other agents tested elicited an early, two-to threefold increase of NF-B/DNA binding activity that wasdetectable after 20 min of stimulation. Increase NF-B/DNA bindingactivity was visible for 120 min. After 240 min, NF-B/DNA-bindingactivity had returned to nearly control levels in ROS- and PMA-treatedcells, whereas upregulation seemed to be more prolonged in LPS-and IL-1-treated cells (data not shown).
Figure 9. ROS, IL-1, LPS, and PMA induce nuclear factor-B (NF-B) binding activity in podocytes. (A) Effect of H2O2 (250 µM), LPS (1 µg/ml), X/XO (50 µM/50 mU/ml), PMA (100 nM), and IL-1 (50 pg/ml) on NF-B activation in podocytes. C indicates unlabeled probe. (B) Summary of experiments. Data are mean ± SD from four independent experiments. *, P < 0.05 versus control.
ROS Do Not Induce AP-1 Activation in Podocytes
A basal AP-1/DNA binding activity was detectable in untreatedpodocytes (Figure 10). An increased AP-1/DNA binding activitycompared with control cells was detectable in PMA-, IL-1-, andLPS-treated podocytes after 60 min (n = 4). In contrast, X/XOand H2O2 did not induce an increase in AP-1/DNA binding activity(n = 5, Figure 10).
Figure 10. ROS, IL-1, LPS, and PMA induce AP-1 binding activity in podocytes. Effect of H2O2 (250 µM), LPS (1µg/ml), X/XO (50 µM/50 mU/ml), PMA (100 nM), and IL-1 (50 pg/ml) on AP-1 activation in podocytes. C indicates unlabeled oligonucleotide probe. (B) Summary of experiments. Data are mean ± SD from four to five independent experiments. *, P < 0.05 versus control.
ROS are not only produced by infiltrating neutrophils and monocytesbut are also generated by mesangial cells and podocytes duringglomerular damage. For example, in the Heymann nephritis modelof glomerulonephritis, cytochrome b558, a major component ofthe NADPH oxidoreductase complex, is localized in podocytes,and cytochrome b558 subunits have been demonstrated in culturedhuman podocytes (27). In addition, an increase in glomerularXO activity, due to a conversion of xanthine dehydrogenase tothe oxidase form, seems to be responsible for ROS productionin this model of glomerular damage (28). Recent studies haveprovided further evidence that oxidative damage is involvedin the pathophysiology of human renal disease; Gröne etal. (29) demonstrated oxidatively modified proteins in podocytes,mesangial cells, and basal membranes in kidney biopsies of patientswith membranous nephropathy. In patients with focal segmentalglomerulosclerosis, increased expression of the NAD(P)H-oxidaseprotein cytochrome b558 was found (30). However, despite thecompelling evidence for the contribution of ROS to the progressionof experimental glomerular disease and especially podocyte injury,the clinical benefit derived from these insights so far hasyet to come. One reason for the weak efficiency of ROS scavengersin reversing established glomerular injury may be that ROS itselfchange several signaling cascades of podocytes, which will thenmaintain podocyte injury by mechanisms distinct from ROS. Therefore,because ROS might directly change podocyte function for thelonger term, it is of interest to identify ROS-mediated effectsof podocyte function.
This study demonstrates that GM-CSF mRNA protein can be inducedin podocytes and characterizes the regulation of GM-CSF by ROSand other proinflammatory agents. Induction of GM-CSF mRNA wasnot immediate, because it required >1 h to be detectable,but was relatively long-lasting, given that elevated GM-CSFmRNA was still detectable 12 h after podocytes had been exposedto exogenous ROS. There was a time-dependent activation of GM-CSFrelease in podocytes in response to ROS, which was already evidentwith 5 mU/ml XO. Activation of GM-CSF release in podocytes wastriggered from both exogenous X/XO and endogenous ROS. ActivatedNAD(P)H-oxidases or diamide, as well as H2O2, all increasedendogenous ROS, although the latter compound had a much smallereffect. We could also demonstrate that LPS and IL-1, as wellas activation of the protein kinase C-pathway by PMA, inducedGM-CSF and that these effects were also partially mediated byROS: the ROS scavenger DMTU diminished GM-CSF production byonly 50% to 60%. Surprisingly, NAC suppressed the increase ofGM-CSF in response to IL-1 and PMA but had only marginal effectson LPS-related effects. One explantation for this observationis that LPS might induce downstream effectors other than ROSsuch as tumor necrosis factor-, given that it has been describedin human umbilical vein endothelial cells that were cocultivatedwith LPS-treated peripheral blood mononuclear cells (31).
Because the increase in protein release was completely inhibitedby actinomycin D, our data indicate that regulation of GM-CSFexpression by ROS involves translation of protein from newlytranscribed mRNA. Transcriptional regulation of genes by ROShas been attributed to enhanced expression and/or DNA bindingof transcription factors in response to ROS. Numerous transcriptionfactors have been identified to be ROS-sensitive, includingfos, jun, myc, erg-1 NF-B, heat shock protein, and T cell factor/stemcell factor (32). GM-CSF production was nearly completely inhibitedwith PDTC in response to PMA, IL-1, and LPS. Because PDTC hasbeen shown to suppress activation of NF-B (33,34), these datapoint to the fact that activation of this transcription factormight be involved in the regulation of GM-CSF in podocytes.Indeed, promoter analysis of the murine CSF-1 gene did demonstratethe presence of a NF-B binding site (35). Direct analysis ofNF-B/DNA binding by electrophoretic mobility shift analysisin podocytes revealed an increase after stimulation with ROS,LPS, IL-1, and PMA, which supports the hypothesis that NF-Bis involved in the regulation of GM-CSF production in podocytes.Transactivation of the phorbol-responsive transcription factorAP-1 by oxygen radicals might have also been involved in theregulation of GM-CSF in podocytes. This hypothesis would havebeen supported by the fact that dexamethasone, which is knownto blunt AP-1 as well as NF-B-mediated transcriptional activation,significantly suppressed the effects of PMA as well as IL-1and LPS on GM-CSF release. In electrophoretic mobility shiftassays, a moderate increase in AP-1 activity in podocytes wasobserved after stimulation with LPS, IL-1, and PMA. In contrast,AP-1 activity was not elevated over controls in podocytes stimulatedwith X/XO or H2O2, which indicates that AP-1 is not involvedin the regulation of ROS-induced GM-CSF production in podocytes.In several cultured cells, ROS have been shown to activate AP-1(for review, see Ref. [(36]). On the other hand, several studieshave reported that ROS-mediated activation of NF-B is not necessarilyassociated with activation of AP-1: in smooth-muscle cells,ROS generation induced by cisplatin leads to an activation ofNF-B but not AP-1 (37), and ROS-induced expression of epithelialNa+ channels in lung epithelial cells is associated with NF-Bbut not AP-1 activation (38). In addition, in alveolar macrophages,the activity of NF-B, in contrast to that of AP-1, was activatedafter stimulation with LPS (39). It has been discussed thatthere is no single redox paradigm into which AP-1 can be fitted.O2-related molecules do not just interact with AP-1 itself—theyregulate other molecules in the signal transduction pathway.Therefore, there is no consensus whether AP-1 mediates or countersoxidative stresses (36).
Of interest is also the prolonged time course of GM-CSF mRNAincrease in response to even short-term stimulation with ROS,which indicates that a short-lived exposure of podocytes toROS induces relatively long-lived sequel. This observation mighthelp to explain why there has so far been no convincing clinicalevidence for the usage of oxygen radical scavengers in humanglomerular diseases: because the short-lived ROS cause relativelylong-lived alterations of transcriptional patterns, a treatmentwith ROS scavengers might come too late because (1) the transcriptionalcascade has been already initiated and (2) even a short periodof suboptimal scavenging during dosage intervals might be sufficientto reinitiate the altered transcription pattern.
What is the possible contribution of GM-CSF to glomerular injury?Local glomerular macrophage proliferation has been consideredas an important mechanism of macrophage accumulation duringthe development of severe nonimmune renal injury, such as inthe rat remnant kidney. In this model, a tight association oflocal macrophage proliferation within areas of renal injury,such as glomerular segmental lesions and a correlation betweenlocal macrophage proliferation and progressive renal injury,has been demonstrated. Podocyte injury has been suggested toinitiate and maintain the progression of glomerulosclerosisin the rat remnant kidney; thus, cytokine-mediated interactionbetween podocytes and macrophages may contribute to the pathogenesisof podocyte injury in this model (3). Podocyte release of GM-CSFmay therefore play a crucial role in the inflammatory eventsby functionally activating mature leukocytes at an inflammatoryside, inhibiting their migration away from the focus, and enhancingthe proliferation and differentiation of progenitor cells. Inaddition, GM-CSF release by podocytes may not only alter functionsof macrophage but also modulate cellular properties of glomerularendothelial cells in a paracrine fashion. GM-CSF possesses angiogenicactivity in vivo, and it stimulates proliferation and repairof mechanically injured cultured endothelial cells. Hattoriet al. (40) demonstrated that increased GM-CSF release mightalso contribute to glomerular injury in other nonimmunologicmodels of renal disease. In a model of lipid-induced glomerularinjury, they could demonstrate by immunohistochemistry and insitu hybridization that, before macrophage infiltration in theglomerulus, a significant upregulation of GM-CSF was observedin glomerular podocytes and mesangial cells.
Data from renal biopsy studies have also demonstrated a possiblerole of increased GM-CSF expression for the pathogenesis ofglomerular disease such as mesangial proliferative glomerulonephritis:in situ hybridization and immunohistochemistry data have clearlyshown a positive correlation between GM-CSF protein expressionand glomerular proliferation, macrophage infiltration, and thedegree of proteinuria (41).
In a recent investigation by Huang et al. (13), the requirementof GM-CSF for leukocyte-mediated glomerulonephritis was investigatedin GM-CSF -/- knockout mice by use of models of heterologousand homologous anti-GBM glomerulonephritis. GM-CSF -/- miceshowed a significant reduction in proteinuria and neutrophilsinflux in the heterologous model, as well as reduction of crescentformation, glomerular accumulation of CD4+ T cells, and serumcreatine in the homologous model compared with control animals.This is of particular interest, because crescents are best understoodas a mixture of glomerular epithelial cells, most likely ofparietal origin, and macrophages (42). The results of our studynow point to the podocyte as a likely source of GM-CSF in thistype of glomerular diseases.
In conclusion, we have demonstrated that podocytes can be thesource of increased GM-CSF production in response to severalstress factors and that increased GM-CSF production is at largemediated by ROS. This finding, in combination with the investigationsof other researchers who elucidated the role of GM-CSF in glomerulardiseases, further underlines the critical importance of ROSand the podocyte in the initiation and maintenance of glomerularinjury.
Acknowledgments
Podocyte cells were a generous gift from Peter Mundel, AlbertEinstein College of Medicine, New York, NY. This study was supportedby Deutsche Forschungs gemeinschaft PA 483/5-1.
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Received for publication February 10, 2000.
Accepted for publication July 12, 2001.
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Top
Abstract
Introduction
B (NF-
to enhance the expression of the T antigen. To induce differentiation, podocytes were maintained on type I collagen (Biochrom, Berlin, Germany) at 37°C without interferon-
F') were transformed and plated onto agar plates that contained 50 µG/ml ampicillin, 50 µG/ml isopropyl-ß-D-thiogalactoside, and 50 µG/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside. Ninety-six transformed clones were regrown overnight in 100 µl of LB media in 96-well microtiter plates. A PCR with the nested primer from the adapter sequences was then performed on 1 µl of each bacterial culture, to amplify the cloned cDNA inserts. Five microliters of the PCR reaction were then spotted onto two duplicate nylon membranes and cross-linked by ultraviolet irradiation. One nylon filter was then hybridized with a 
10 to 9 M: the maximal effect was seen at 10 to 7 M. The maximal effect of PMA on GM-CSF release was about twice the effect of IL-1. LPS, when used in concentrations from 0.1 to 100 µg/ml, also had a dose-dependent effect on GM-CSF release: the maximal effect was seen at a dose of 1 µg/ml (data not shown). 
120 min. After 240 min, NF-
