Gene expression patterns in glucose-stimulated podocytes q Seung Hyeok Han a , Sanghwa Yang c , Dong Sub Jung b , Jin Ji Li d , Jin Ju Kim b , Seung Jae Kwak b , Dong Ki Kim a , Sung Jin Moon a , Jung Eun Lee a , Dae-Suk Han a , Shin-Wook Kang a, * a Department of Internal Medicine, Yonsei University College of Medicine, 134 Shinchon-Dong Seodaemoon-Gu, Seoul 120-752, Republic of Korea b Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Republic of Korea c Cancer Metastasis Research Center, Yonsei University College of Medicine, Seoul, Republic of Korea d Department of Internal Medicine, Nephrology and Dialysis Unit, The Affiliated Hospital, YanBian University Medical College, JiLin, China article info Article history: Received 22 March 2008 Available online 3 April 2008 Keywords: Podocyte Gene expression Diabetic nephropathy abstract To explore the mechanisms of podocyte injury under diabetic conditions, we performed an expression profile in glucose-stimulated podocytes. Differential gene expression profiles between conditionally immortalized mouse podocytes cultured in medium containing 5.6 and 30 mM glucose were measured with oligonucleotide microarrays. Of the genes identified, heme oxygenase-1, vascular endothelial growth factor-A, and thrombospondin-1 showed a consistently increased pattern, whereas angioten- sin-converting enzyme-2 and peroxisomal proliferator activator receptor-c were down-regulated. These results were validated using real-time PCR and western blotting in podocytes, and with immunohisto- chemistry on renal tissues from streptozotocin-induced diabetic rats. Not only is this the first report of gene expression profiling of podocyte injury under diabetic conditions, but the identified genes are prom- ising targets for future diabetes research. Ó 2008 Elsevier Inc. All rights reserved. Numerous recent studies have indicated that podocyte injury is an early feature of kidney disease. To date, reactive oxygen species, anigotensin II, and TGF-ß have been implicated in podocyte injury [1]. Molecular mechanisms underlying podocyte injury, however, are still largely unknown. Microarray-based global gene expression profiling is a powerful tool for obtaining gene expression information, and it has been ap- plied to the analysis of diabetic disease. Most of the previous re- ports, however, have examined gene expression in the whole kidney [2,3]. These should be interpreted with caution when eluci- dating specific genes associated with diabetic glomerulopathy, be- cause glomeruli account for less than 10% of the whole kidney. Although gene expression profiling of high glucose-stimulated mesangial cells has been reported [4], similar studies for podocytes are lacking, despite the fact that podocyte injury is the main early event in diabetic nephropathy. In this study, gene expression pro- filing was applied to an in vitro model—of podocytes stimulated by high glucose concentration in order to reveal the underlying molecular changes associated with diabetic nephropathy. Materials and methods Podocyte culture. Conditionally immortalized mouse podocytes, kindly provided by Dr. Peter Mundel (Albert Einstein College of Medicine, Bronx, NY), were cultured as previously described [5]. Briefly, podocytes were grown under permissive conditions at 33 °C with RPMI 1640 containing 10% FBS, 50 U/ml c-interferon, and 100 U/ml of penicillin/streptomycin. The c-interferon concentration was gradually reduced to 10 U/ml in successive passages. Cells were then trypsinized and subcul- tured without c-interferon and were allowed to differentiate at 37 °C for 14 days with media changes on alternate days. After differentiation of podocytes and serum restriction for 24 h, medium was changed to serum-free RPMI containing normal glucose (5.6 mM, LG), high glucose (30 mM, HG) or LG + mannitol (24.4 mM, LG + M). At 2, 6, 24, and 48 h after the media change, cells were harvested and either the RNA or protein was extracted. RNA isolation. Total RNAs from cultured podocytes were isolated using the RNeasy mini kit (QIAGEN GmbH, Hilden, Germany). Total RNA quantity and purity were assessed by measurement of OD260/280 using a NanoDrop spectrophotome- ter. RNA with an A 260 /A 280 ratio of >1.8 was considered acceptable for microarray experiments. Mouse oligonucleotide microarray. Mouse oligonucleotide microarrays (Micro- arrays, Inc., Nashville, TN) spotted with a 38,500 Illumina MEEBO set (Mouse Exonic Evidence-Based Oligonucleotide) were used for the experiments. Fluores- cently labeled cRNA for micorarrays was prepared by amplification of total RNA in the presence of aminoallyl-UTP, followed by the coupling of Cy3 or Cy5 dye (AmershamPharmacia, Uppsala, Sweden). cRNAs from LG-treated podocytes (Cy3) were competitively hybridized with those from HG-treated podocytes (Cy5) in each hybridization. Triplicate hybridizations were performed for each time point. To assess the variability of the method, an identical batch of cRNAs was used in two independent hybridizations, resulting in a Pearson correlation coefficient of 0.9. Microarrays were incubated at 60 °C for 16 h, washed, and sub- sequently scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA), and the results were ultimately saved in the GenePix Result (GPR) format. Real-time PCR. The primers used in the experiments are summarized in Table 1. Using the PRISM Ò 7700 Sequence Detection System (Applied Biosystems, Foster City, CA), PCR was performed in a 20 ll reaction, containing 10 ll of SYBR Green Ò PCR Master Mix (Applied Biosystems), 5 ll of cDNA corresponding to 25 ng of RNA, and 5 pmol sense and antisense primers according to the manufacturer’s instructions. The cDNA content of each specimen was determined using a 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.03.121 q Seung Hyeok Han and Sanghwa Yang equally contributed to this work. * Corresponding author. Fax: +82 2 393 6884. E-mail address: kswkidney@yuhs.ac (S.-W. Kang). Biochemical and Biophysical Research Communications 370 (2008) 514–518 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc