J Am Soc Nephrol 13: 86–95, 2002 Reactive Oxygen Species Alter Gene Expression in Podocytes: Induction of Granulocyte Macrophage–Colony- Stimulating Factor STEFAN GREIBER, BARBARA MU¨ LLER, PETRA DAEMISCH, and HERMANN PAVENSTA¨ DT Department of Medicine, Division of Nephrology, University Hospital of Freiburg, Freiburg, Germany. Abstract. It has been suggested that reactive oxygen radicals volved. The ROS scavengers dimethyl-thio-urea and pyrroli- (ROS) play a crucial role in the pathogenesis of proteinuria and done-dithio-carbamate strongly inhibited increased GM-CSF podocyte injury. It was investigated whether changes in gene production induced by ROS. GM-CSF release was also in- expression might account for ROS-induced podocyte dysfunc- duced when internal ROS production was triggered with tion. Differentiated podocytes were incubated with control NADH, whereas H2O2 had only a small effect. GM-CSF media or with exogenous ROS from the xanthine/xanthine- release by podocytes was also stimulated by lipopolysaccha- oxidase reaction for 4 h. A PCR-based suppressive subtractive ride (LPS), interleukin-1 (IL-1), and phorbolester (PMA). Di- hybridization assay was applied to isolate and clone mRNAs methyl-thio-urea significantly inhibited the LPS-, IL-1–, and that were differentially expressed by exogenous ROS. One PMA-induced GM-CSF production. Activation of the tran- differentially expressed clone was identified as the proinflam- scription factor nuclear factor–␬B (NF-␬B) but not activator matory cytokine granulocyte macrophage–colony-stimulating protein–1 was involved in the upregulation of ROS-induced factor (GM-CSF). Regulation of GM-CSF in podocytes was GM-CSF production. The data indicate that GM-CSF is dif- further studied by Northern analysis and enzyme-linked im- ferentially expressed by ROS in podocytes. ROS also partially munosorbent assay. Exogenous ROS caused a concentration- mediate the effects of PMA and IL-1 on podocyte GM-CSF dependent, Ͼ10-fold induction of GM-CSF mRNA after 4 h. A production. Because GM-CSF can enhance glomerular inflam- Ͼ50-fold increase in GM-CSF protein release in podocytes mation and induces mesangial proliferation, these data might that had been stimulated with ROS could be detected. Induc- provide further insight into the mechanisms of ROS-induced tion of GM-CSF protein was inhibited by actinomycin D, glomerular injury. which indicated that increased mRNA transcription was in- The podocyte plays a crucial role in maintaining the perms- permselectivity (5,6). In these glomerular diseases, pretreat- elective function of the glomerular capillary wall (1). Under ment of animals with the antioxidant probucol or ROS scav- pathophysiologic conditions, the podocyte contributes to the engers markedly prevented foot-process effacement and pro- initiation and progression of a variety of glomerular diseases. teinuria. So far, the mechanisms by which ROS might Membranous nephropathy, minimal change disease, and focal contribute to podocyte damage are incompletely understood. segmental sclerosis in particular have all been related to pri- Because the majority of cellular processes are characterized by mary or secondary podocyte injury (2,3). Overproduction of changes in gene expression, we used a cell culture model of reactive oxygen radicals (ROS) has been found in glomerular differentiated podocytes to study changes in gene expression diseases in which the podocyte is the primary target cell of caused by ROS. Herein, we used a PCR-based suppressive glomerular injury, such as puromycin nephrosis, a model of subtractive hybridization (PCR-SSH) to identify genes in minimal change disease, Heyman nephritis, a model of mem- podocytes that are differentially changed by ROS. PCR-SSH is Ϫ Ϫ branous nephropathy, and the Mpv 17 ( / ) mouse, a model a method based on suppressive PCR that allows creation of for steroid-resistant focal segmental sclerosis (3,4). The release subtracted cDNA libraries for the identification of genes dif- of ROS leads to proteinuria by affecting glomerular endothe- ferentially expressed in response to a stimulus (7,8). PCR-SSH lial, and epithelial cells and disturbing normal glomerular differs from earlier subtractive methods by including a normal- ization step that equalizes for the relative abundance of cDNA within the target population. This modification enhances the Received February 10, 2000. Accepted July 12, 2001. probability to identify the increased expression of low-abun- Correspondence to Hermann Pavensta¨dt, Medizinische Universita¨tsklinik IV, dance transcripts and represents a potential advantage over Nephrologie, Hugstetter Str. 55, D-79106 Freiburg, Germany. Phone: ϩ49-761- 270-3492; Fax: ϩ49-761-270-3245; Email: [email protected] other methods, such as differential display PCR, for identifying differentially regulated genes (9). With the PCR-SSH tech- 1046-6673/1301-0086 Journal of the American Society of Nephrology nique, we demonstrate that the granulocyte macrophage–col- Copyright © 2001 by the American Society of Nephrology ony-stimulating factor (GM-CSF) is differentially expressed by J Am Soc Nephrol 13: 86–95, 2002 Reactive Oxygen Species Alter Gene Expression in Podocytes 87 ROS in podocytes. GM-CSF is a cytokine that regulates the Screening of the Differentially Expressed survival, growth, and differentiation of hematopoietic progen- cDNA Library itor cells (10). In the kidney, GM-CSF exerts its effects pri- The subtracted cDNA library was cloned into a 3.9-kb PCRTM 2.1 marily on macrophages, where it stimulates tumor necrosis vector (TA-cloning kit; Invitrogen, San Diego, CA). Ultracompetent factor and interleukin-1 (IL-1) production of these cells. Both Escherichia coli (INV.␣F') were transformed and plated onto agar cytokines have been suggested to play a major role in the plates that contained 50 ␮G/ml ampicillin, 50 ␮G/ml isopropyl-␤-D- ␮ ␤ pathogenesis of glomerular inflammation and proteinuria thiogalactoside, and 50 G/ml 5-bromo-4-chloro-3-indolyl- -D-ga- (11,12). In addition, GM-CSF might serve as a critical signal lactopyranoside. Ninety-six transformed clones were regrown over- night in 100 ␮l of LB media in 96-well microtiter plates. A PCR with for macrophage migration into the glomerulus (13). the nested primer from the adapter sequences was then performed on 1 ␮l of each bacterial culture, to amplify the cloned cDNA inserts. Five microliters of the PCR reaction were then spotted onto two Material and Methods duplicate nylon membranes and cross-linked by ultraviolet irradiation. Cell Culture One nylon filter was then hybridized with a ␣32P-dCTP-labeled part Conditionally immortalized mouse podocytes were cultured as of the forward subtracted library, and the duplicate filter was hybrid- reported elsewhere (14). In brief, podocytes were maintained in RPMI ized with the reverse-subtracted library. Filters were then subjected to 1640 medium (Life Technologies, Eggenstein, Germany) supple- autoradiography. Positive clones that showed differential expression mented with 5% fetal calf serum (Boehringer Mannheim, Mannheim, on both blots were further analyzed by virtual Northern blotting: Germany), 100 kU/L penicillin, and 100 mg/L streptomycin (Life cloned inserts were excised from the vector by EcoRI digestion, Technologies). To propagate podocytes, cells were cultivated at 33°C separated by agarose gel-electrophoresis, and extracted from the gel on type I collagen (permissive conditions), and the culture medium (QIAEX; Quiagen, Heidelberg, Germany). A total of 25 ng of cDNA was supplemented with 10 U/ml recombinant interferon-␥ to enhance were then labeled with a ␣32P-dCTP by random priming and used to the expression of the T antigen. To induce differentiation, podocytes analyze 500 ng of double-stranded c-DNA from each control and were maintained on type I collagen (Biochrom, Berlin, Germany) at X/XO-treated podocytes by virtual Northern analysis. Virtual North- 37°C without interferon-␥ (nonpermissive conditions). Podocytes be- ern analysis shows that cDNA from clone 48 hybridized to a 1.2-kb tween passage 10 and 16 were used in all experiments. To examine the transcript that was strongly upregulated in cells that had been treated effects of ROS on podocyte GM-CSF mRNA expression or GM-CSF with X/XO (Figure 1). The insert was then sequenced with an auto- release, podocytes from one cell pool were plated at a cell density of mated ALF sequencer and then identified by a computer-based Blast 2 104 cells/cm in media that contained 5% fetal calf serum in six-well search of Genbank (15) as part of the mouse GM-CSF cDNA plates for Northern analysis or in 96-well plates for measurements of sequence. GM-CSF protein. Cells were switched to media that contained 1% fetal calf serum 24 h before the experiments and then exposed to various treatments. Northern Analysis of GM-CSF mRNA in Podocytes Differentiated podocytes were grown in six-well plates and re- ceived fresh media 24 h before the experiment. RNA was isolated by Generation of a Differentially Expressed cDNA Library acid phenol extraction (16). RNA (10 ␮g/lane) was size-separated in To screen for genes that are differentially expressed by ROS in agarose/formaldehyde gels and transferred to Hybond nylon mem- podocytes, a PCR-SSH approach (7,8) was used (PCR-Select; Clone branes (Pharmacia, Freiburg, Germany). GM-CSF cDNA was labeled ␣32 Tech, Palo Alto, CA). In brief, RNA was isolated from control cells and with -P-dCTP by use of a random-primer labeling kit (Stratagene, cells that had been stimulated with extracellular superoxide generated Heidelberg, Germany). Hybridization and washes were performed from the xanthine/xanthine-oxidase reaction (X/XO; 50 ␮M/5 or 50 according to the method of Church and Gilbert (17). After analysis of ␣32 mU/ml) for 4 h. cDNA synthesis from 1 ␮g of total RNA from each cell the GM-CSF signal, blots were rehybridized to a -P-dCTP–labeled population was achieved with the SMART PCR cDNA synthesis kit probe for the housekeeping gene GAPDH, to control for variation in (Clone Tech) and subsequent long-distance PCR.