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clinmed/2002040002v1 (April 24, 2002)
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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.
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.
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.
Hybridization signals were detected and analyzed using a scanner and Image OvaNT software program (Molecular Dynamics).
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.
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.
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.
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