help button home button ClinMed NetPrints
HOME HELP FEEDBACK BROWSE ARTICLES BROWSE BY AUTHOR
Warning: This article has not yet been accepted for publication by a peer reviewed journal. It is presented here mainly for the benefit of fellow researchers. Casual readers should not act on its findings, and journalists should be wary of reporting them.

This Article
Right arrow Abstract Freely available
Services
Right arrow Similar articles in this netprints
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by BANERJEE, H.
Right arrow Search for Related Content
PubMed
Right arrow Articles by BANERJEE, H.
Related Collections
Right arrow Pathology:
Histopathology

Right arrow CLINICAL:
Renal Medicine

clinmed/2002040002v1 (April 24, 2002)
Contact author(s) for copyright information

Detection of differentially expressed genes in LPS treated human renal tubular epithelial cells using c DNA arrays.

Hirendra Banerjee M.D./Ph.D.

Medical University of South Carolina,Charleston,SC,USA.

ABSTRACT

This study reports the use of gene array technology for the identification of differentially

expressed genes in Lipopolysaccharide (LPS) treated human renal tubular epithelial cells. The tubular epithelial

response to bacterial infection has not been well studied at a molecular level. In order to study differential

Gene expression in response to LPS stimulation, cultured tubular epithelial cells were treated with 1mg/ml

LPS for 24 hours. RNA was isolated from the treated and control cells and cDNA synthesized by Reverse

Transriptase reaction. Radio labeled cDNA was hybridized to a cDNA human expression array with

several known genes.The results showed upregulation of several genes in response to LPS including ubiquitin, zinc finger protein 91 (ZNF92), G2 mitotic specific cyclin (CCNA) etc being the novel and the most notable ones.

This study illustrates the enormous potential of gene array technologies for understanding the molecular mechanisms involved in kidney diseases caused by bacterial infection.

INTRODUCTION

During the last half of the twentieth century analysis of the regulation and for function of genes has been largely driven by step by step studies and individual analysis of genes and proteins. In the past decade a paradigm shift has emerged due to which we are now able to produce large amount of data about large number of genes in a largely parallel and highly socialized manner. An important tool in this process has been the development of DNA microarrays. These arrays consist of matrix of thousands of DNA sequences, which can be used to measure DNA and RNA variations in application. Most include gene expression, profiling, comparative genomics and genotyping (1-4). LPS induces glomerulonephritis, and create renal failure (4-6). Bacterial LPS has been shown to induce apoptosis in endothelial cells (7). LPS also causes hemolytic uremic syndrome as determined by animal model. LPS are endotoxin, which in the main component of the outersurface of gram negative bacteria. LPS are potent activators of cells of immune inflammatory system including macrophages, monocytes and endothelial cells. LPS contribute to systemic changes seen in septic shock ( 8). Septic and septic shock are increasing cause of mortality and morbidity causing approximately 200,000 deaths in USA. The basic paradigm of septic shock is not microbial antigen such as LPS, bacterial cell wall component such as lipotechoic acid, and bacterial lipoprotein which initiate an uncontrollable network of proinflammatory mediators which can lead to cardiovascular shock and death (9). Vertebrate and invertebrates initiate a series of defense mechanism after being infected by various microorganisms by sensing the conserved pathogen associated patterns such as bacterial LPS. Most organisms have developed a set of receptors that can specifically recognize pathogen associated molecular patterns are referred to as pattern recognition receptors. There are receptors that LPS induces signal transduction pathway molecules like NFKb, IL 1 receptor, TNF receptor, MAP kinase receptor etc. (10).

An important step in understanding the disease process in septic shock or LPS induced septicimea in renal disease is to eliminate the molecular mechanism that operate in the kidney specifically the tubular epithelium during the initial colonization of the bacteria. It has been previously shown that bacteria can attach to tubular epithelial cells and produce LPS, which stimulates immune host mechanism. But it is very difficult to determine the total number of signalling molecules elaborated by the bacteria and concomitantly the number of signalling pathways that lead to the initial response.

In this report, interaction of kidney tubule with LPS is studied. RNA isolated from cultured human tubular epithelial cells were used to label high density DNA arrays consisting of several cDNA clones. Differences in gene expression were identified by pair wise hybridization of probes generated from human tubular epithelial cells before and after treatment with LPS. Overall results show that LPS treatment to human tubular epithelial cells causes an increase in transcript level of several genes present on the array. Although the exact function of majority of these LPS responsive genes are unknown, several known signalling molecules were activated many of which were not previously shown to be activated in bacterial LPS induced renal infections.

MATERIALS AND METHODS

Cell lines and LPS Treatment - Human tubular epithelial cells were kind gift from the nephrology division of the Medical University of South Carolina. The cells were maintained in DMEM-F12 medium at 370C in a CO2 incubator.

RNA Extraction: 1mg/ml of LPS was added to about 8x106 cells - Total RNA from the LPS treated and control cells was extracted using TRI 201 reagent (Life Technologies, Grand Island, NY, USA). Poly (A)+ RNA was separated from the total RNA using Oligotex resin (Origine, USA).

cDNA Library Arrays: The Atlas cDNA array was purchased from Clontech Corporation, (Palo Alto, CA). The array contained several important genes like C-myc, p53, MAP Kinane Kinase etc.

Probe preparation: For each total cDNA probe preparation, 500 ug of poly (A)+ RNA was used from LPS treated and control cells. Radioactively labeled first strand cDNA was generated by incorporating [ 32P] dCTP nucleotides using Oligo (DT) primer and Super Script II RNAse H - Reverse Transcriptase (Life Technologies, Rockville, MD, USA) according to manufacturers instructions. Unincorporated nucleotides were removed using NucTrap Probe purification columns, (Stratagene, Lajolla, CA, USA). The whole-labelled cDNA preparation was used as the probe in the array hybridization.

Hybridization of the arrayed membranes:

The arrayed membranes were prehybridized at + 42oC for 3 hours in hybridization solution (50% fomamide, 6 x SSC; 5X Denhardt's solution, 1/-SDS, 0.5 mg/ml poly-dA and 0.5 mg/ml cot-1-DNA), after which the denatured probe was added to the solution and hybridized to the membranes at +42oC overnight. Following hybridization, the membranes were washed twice in 2 x SSC /1%. SDS at room temperature for 15 minutes, twice in .1X SSC/0.5% SDS at +65oC for 20 minutes and once in 0.1XSSC/0.5%. SDS at + 68oC for 15 minutes. After the final wash, the membranes were exposed to Kodak XOMAT films for 24 hours and then developed.

Data Analysis:

Hybridization signals were detected and analyzed using a scanner and Image OvaNT software program (Molecular Dynamics).

RT-PCR

Three microgram of total RNA from the control and LPS treated cells were incubated with 90 units of DNAseI and then reverse transcribed into cDNA using 800 ng of hexamers (PDNG, Boehringer - Mannheim, USA) and 200U of Superscript-reverse transcriptase (GIBCO-BRL). Beta Actin primers were used to do the PCR reaction. For each reaction, one tenth of the cDNA was amplified on an automated thermal cycle (Perkin-Elmer) in a final volume of 50 ul with 1.5 U of Amplitaq Gold (Perkin Elmer, CT, USA), 1X buffer, 2.5 mmol/l Mgcl2, 200 mmol/l of each deoxynucleotide triphosphate, and 50 pmol of each primer. After an initial step at 94oC for 15 minutes, 30 cycles were performed, each cycle consisting of denaturation at 94oC for 1 minute, annealing at 60oC for 1 minute, and elongation at 72oC for 1 minute. RT-PCR amplicans (10 ul) were electrophoesed on a .8% agarose gel and photographed under ultraviolet light. The housekeeping gene beta actin thus served as internal control in normalizing the differences in separate hybridization.

RESULTS

To test the genes induced by Lipopolysaccharide stimulation, two identical membranes were hybridized with cDNA probes made from 500 ng of poly (A)+ RNA from control and LPS stimulated human tubular epithelial cells. The hybridization signals were normalized using the house keeping gene beta actin by RT-PCR. The results were repeated three times. No genes were found to be induced in the control array membrane while several genes were induced in the array probed by LPS treated cDNA. The following genes were induced:

1) Ribosomal protein S9

2) Myb related protein B

3) V-ERB-A related protein ERB-2

4) MAP kinase Kinase G

5) Gadd 45

6) Zinc finger protein 91

7) G 1 Mitotic specific cyclin A.



Discussion: Partial analysis of gene expression profile is a new tool for studying the interplay of signals and transcriptional molecules in complex biological system. We showed the expression of several genes in response to LPS treatment to human tubular epithelial cells by using a cDNA expression array. Although, this is only a small fraction but important information for a particular pathway can be obtained because differential transcription of a gene also plays a role in alteration of the pathway in which that gene is involved. One of the outcomes of this microarray analysis is the discovery of expression of MAP kinase Kinase 6, which is an important molecule in several important signal transduction pathways.



ACKNOWLEDGEMENT

The author is grateful to Dr.John Raymond of the Nephrology Department and Dr.I.Singh of Pediatrics Department

of Medical University of South Carolina for their help and support.This research was supported by a NIH (NRSA)

Fellowship T32 DK07752 to Dr.H.Banerjee.



REFERENCES

1) Yokoo T, Ohashi T, Utsunomiya Y, Shiba H, Shen JS, Hisada Y, Eto Y, Kawamura

Hosoya T. Inflamed glomeruli-specific gene activation that uses recombinant adenovirus with the Cre/lox system. J Am Soc Nephrol. 2001 Nov;12(11):2330-7.

2) Imai E, Takenaka M, Nagasawa Y, Kaimori J, Hori M. Application of microarray assay to nephrology.

Nephrol Dial Transplant. 2000;15 Suppl 6:78-80.

3) Messmer UK, Briner VA, Pfeilschifter. Basic fibroblast growth factor selectively enhances TNF-alpha-induced apoptotic cell death in glomerular endothelial cells: effects on apoptotic signaling pathways. J Am Soc Nephrol. 2000 Dec;11(12):2199-211.

4) Baran D, Vendeville B, Ogborn M, Katz N. Cell adhesion molecule expression in murine lupus-like nephritis induced by lipopolysaccharide. Nephron. 2000 Feb;84(2):167-76.

5) Moll S, Menoud PA, French L, Sappino AP, Pastore Y, Schifferli JA, Izui S.Tubular up-regulation of clusterin mRNA in murine lupus-like nephritis. Am J Pathol. 1998 Apr;152(4):953-62.

6) Rigolet M, Faussillon M, Baudry D, Junien C, Jeanpierre C. Profiling of differential gene expression in Wilms tumor by cDNA expression array. Pediatr Nephrol. 2001 Dec;16(12):1113-21.

7) Boer JM, Huber WK, Sultmann H, Wilmer F, von Heydebreck A, Haas S, Korn B, Gunawa, B, Vente A, Fuzesi L, Vingron M, Poustka A.Identification and classification of differentially expressed genes in renal cell carcinoma by expression profiling on a global human 31,500-element cDNA array . Genome Res. 2001 Nov;11(11):1861-70.

8) Vasselon T, Hanlon WA, Wright SD, Detmers PA . Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38..J Leukoc Biol. 2002 Mar;71(3):503-10.

9) Gohda T, Makita Y, Shike T, Funabiki K, Shirato I, Tomino Y. Dilazep hydrochloride, an antiplatelet drug, inhibits lipopolysaccharide-induced mouse mesangial cell IL-6 secretion and proliferation. Kidney Blood Press Res. 2001;24(1):33-8.

10) Dadgostar H, Zarnegar B, Hoffmann A, Qin XF, Truong U, Rao G, Baltimore D, Cheng G. Cooperation of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes. Proc Natl Acad Sci U S A. 2002 ; Feb 5;99(3):1497-502.

11) Kim J, Kim HR, Lee JC, Jang YS. Involvement of ERK, p38 MAP kinase, and PKC in MHC class II-mediated signal transduction in a resting B cell line .Biochem Biophys Res Commun. 2002 Feb 15;291(1):139-45.

12) Diks SH, van Deventer SJ, Peppelenbosch MP. Lipopolysaccharide recognition, internalisation, signalling and other cellular effects. J Endotoxin Res. 2001;7(5):335-48.



13 )Hofer S, Rescigno M, Granucci F, Citterio S, Francolini M, Ricciardi-Castagnoli P. Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide.

Microbes Infect. 2001 Apr;3(4):259-65.



14) Beutler B, Poltorak A. The sole gateway to endotoxin response: how LPS was identified as Tlr4, and its role in innate immunity. Drug Metab Dispos. 2001 Apr;29(4 Pt 2):474-8.



15) Wyllie DH, Kiss-Toth E, Visintin A, Smith SC, Boussouf S, Segal DM, Duff GW, Dower SK.Evidence for an accessory protein function for Toll-like receptor 1 in anti-bacterial responses. J Immunol. 2000 Dec 15;165(12):7125-32.





































































































.



.

































This Article
Right arrow Abstract Freely available
Services
Right arrow Similar articles in this netprints
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by BANERJEE, H.
Right arrow Search for Related Content
PubMed
Right arrow Articles by BANERJEE, H.
Related Collections
Right arrow Pathology:
Histopathology

Right arrow CLINICAL:
Renal Medicine


HOME HELP FEEDBACK BROWSE ARTICLES BROWSE BY AUTHOR