Department of Nephrology, Medizinische Poliklinik, University of Munich, Germany
Correspondence and offprint requests to: Matthias Kretzler MD, Med. Poliklinik, Innenstadt, Ludwig Maximilians University of Munich, Pettenkoferstrasse 8a, D-80336 München, Germany. Email: kretzler{at}med-poli.med.uni-muenchen.de
Keywords: cDNA array; gene expression; kidney; microdissection; renal biopsy; RTPCR
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Introduction |
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The human genomic information now available opens a wide range of new opportunities in biomedicine [3,4]. A comprehensive description of mRNA levels encoding functionally relevant molecules could play an important role in molecular analysis and understanding of human disease [5]. Identification of specific mRNA expression pattern in diseased tissues and their correlation with diagnosis, prognosis and response to the different treatments available could be a promising immediate clinical application. In addition, the study of gene expression analysis should provide valuable information about the nature and prognosis of disease and should lead to the identification of novel therapeutic targets.
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The ERCB project: goals and principles |
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Sample collection, preparation and expression profiling
For acquisition of biopsy samples a robust and reproducible protocol facilitating the participation of clinical centres was established in the framework of the ERCB [6].
After fine needle biopsy, 10% of the biopsy cylinder is transferred directly into RNase inhibitor solution. To standardize storage conditions and to address the loss or degradation of RNA a commercially available aqueous RNase inhibitor (RNAlater, Ambion, USA) was used to store and ship the biopsies.
The complex architecture of the kidney and the segment-specific expression regulation in renal disease makes it necessary to dissect nephrons prior to gene expression. Manual microdissection under a stereomicroscope is an effective approach that has allowed evaluation of nephron segment-specific gene expression in animal models [7,8]. In human biopsy studies, manual microdissection and PCR-based gene expression analysis of nephron segments has also been reported [912]. This approach is also applied in the processing of the kidney biopsy tissue of the ERCB. The renal biopsies are centrally microdissected under a stereomicroscope into nephron segments (glomeruli and tubular interstitial compartment). This offers a reliable and fast dissection, as evaluated by high power phase contrast microscopy and analysis for nephron segment-specific markers by real-time RTPCR. Samples containing only glomerular structures gave a 300-fold higher signal for WT-1, a marker for glomerular epithelial cells, than tubulo-interstitial samples.
For optimal tissue recovery, several protocols (including lysis in 2% Triton-X 100 combined with freezethaw cycles in liquid nitrogen, denaturing lysis buffer, ultrasound treatment or shredder columns) were systematically compared. The highest cDNA yield was achieved by denaturation of the sample with ß-mercaptoethanol and guanidine thiocyanate containing lysis buffer for 10 min at room temperature. Alternative tissue fragmentation protocols with ultrasound or shredder columns did not improve the yield.
A silica gel-based RNA isolation procedure (RNeasy-Mini, Qiagen, Germany) and reverse transcription revealed a 75-fold higher cDNA yield compared with an in situ reverse transcription protocol. In more than 1200 RNA isolations on minimal tissue samples this technique gave reproducible RNA yields.
The low amount of mRNA templates from small tissue compartments, such as microdissected nephron segments, requires the application of detection techniques with extreme sensitivity.
To evaluate gene expression in renal biopsies, two approaches are currently followed.
Real-time RTPCR
Real time RTPCR combines excellent sensitivity with quantification by the direct determination of amplicon numbers after each PCR. This method even allows us to scale the assay down up to the level of a single microdissected cell [13,14]. Real-time RTPCR is applied for mRNA expression of selected candidate marker genes. The expression of the mRNAs is standardized for housekeeper gene expression and compared with expression levels in control tissue.
Oligonucleotide or cDNA arrays
DNA arrays offer parallel quantitative mRNA investigation of hundreds or thousands of genes. However, the minimal amount of starting material required is currently 1 µg total RNA. Using microdissected glomeruli from renal biopsies an amplification procedure prior to array analysis is required. Linear amplification of mRNA can show a >1000-fold amplification efficiency [15,16]. However, for quantitative analysis, the linearity of the amplification, i.e. maintaining the different expression ratios between the diverse mRNA populations, is crucial. Careful evaluation of the resulting expression profiles by independent techniques remains an important save guard of these approaches [17].
Laser-microdissection and mRNA quantification of routine processed renal biopsies
In recent years the use of laser microdissection has enabled the separation of nephron segments from frozen and fixed sections [1821]. Using renal tissue sections, areas of interest can be harvested for expression analysis via laser capture microdissection, manual harvesting with micro-manipulators or laser beam catapulting.
Recently, mRNA quantification of laser capture microdissected renal tissue obtained from formaldehyde-fixed and paraffin-embedded sections have been demonstrated [18]. This method allows the mRNA analysis of archival material even after prolonged storage times. A caveat of this approach is a significant reduction in mRNA yield over one to two orders of magnitude compared with manual microdissection of intact nephron segments [22].
To evaluate this alternative approach, mRNA expression of the chemokines IP-10 and RANTES as important inflammatory mediators in transplant rejection [23] was analysed in renal allografts and control kidneys.
A parallel processing of the same tissue allowed a direct comparison of formaldehyde-fixation and cryo-preservation. Formaldehyde-fixed biopsies were used to examine the performance of the protocol on routine processed archival renal biopsies. To demonstrate the reproducibility of the microdissection and the gene expression analysis, the ratio of IP-10 and RANTES to the housekeeper gene GAP-DH, was determined on formaldehyde-fixed and paraffin-embedded samples on serial sections by real-time RTPCR [18].
Microdissected tissue samples from cryo-sections, formaldehyde-fixed tissue and routine formaldehyde-fixed biopsies showed comparable expression pattern with a pronounced increase in the IP-10 and RANTES expression in allograft rejection.
This protocol enables the analysis of a specific mRNA on routine diagnostic material and opens archival tissue samples for retrospective expression analysis.
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Application of molecular diagnostics and gene expression analysis |
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In the renal field, we envision two potential molecular-diagnostic strategies using gene expression quantification [25].
The first approach will use molecular-diagnostic tools for specific differential diagnostic problems, which cannot be resolved by conventional diagnostic procedures. A limited number of cDNAs with a high predictive value could be determined using the laser microdissection protocol described above. This strategy is suited ideally to be used as an add-on procedure after completion of the routine diagnostics.
The diagnostic markers will have to be identified by comprehensive expression profiling of the diseases under consideration and selected on the basis of their predictive strength concerning identification of defined diagnoses and predicting optimal therapeutic strategies.
Following the above strategy, a panel of one to five markers could be established for standard differential-diagnostic problems in nephrology. This flexible approach can be added to the biopsy analysis, does not require special tissue processing and uses consecutive sections of the same material evaluated by histopathology.
The second approach would use gene expression profiles as a separate information source in the diagnostic process.
First studies defining inflammatory vs fibrotic gene expression profiles in human kidneys suggest [25] that cDNA array based techniques can be used to determine specific gene expression patterns within subsets of a renal disease. Clearly a careful verification of the different marker sets will be required in comprehensive sample populations to assess their predictive strength.
Identification of regulatory pathways in renal disease using gene expression strategies is currently under way in animal models and human diseases.
The complex expression profiles obtained will require an in-depth bioinformatic analysis followed by careful characterization of the identified molecular pathways in cell culture and animal models. A schematic of a potential approach is given in Figure 1.
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Outlook and conclusion |
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As more expression data from different diseases are generated, molecular markers may be able to identify disease-specific fingerprints and importantly, potential mechanisms underlying the pathology of specific diseases.
The introduction of molecular diagnostics to renal biopsies could result in functional-defined diagnostic categories, improved prognostic information and evidence-based selection of available therapeutic strategies.
Conflict of interest statement. None declared.
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Acknowledgments |
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References |
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