NDT Advance Access originally published online on October 4, 2005
Nephrology Dialysis Transplantation 2005 20(12):2598-2601; doi:10.1093/ndt/gfi176
© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Editorial Comment
Messenger RNA assessment in clinical nephrology: perspectives and progress of methodology
Michael Eikmans,
Hans J. Baelde,
Emile de Heer and
Jan A. Bruijn
Department of Pathology, Leiden University Medical Center, The Netherlands
Correspondence and offprint requests to: Michael Eikmans, Department of Pathology, Leiden University Medical Center, The Netherlands. Email: m.eikmans{at}lumc.nl
Keywords: kidney disease; laser-capture microdissection; messenger RNA; prognosis; real-time PCR; RNA extraction
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Introduction
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Molecular biology offers new opportunities for clinical medicine.
A promising clinical application of molecular biology is the
identification of mRNA expression patterns in diseased organs.
These mRNA expression patterns may provide information regarding
diagnosis, prognosis and responsiveness to treatment. The development
of technologies such as microarray analysis and real-time PCR
enables the study of gene expression networks in renal biopsies.
This fact, in combination with the usage of laser-capture microdissection,
enables gene expression analysis in a nephron segment-specific
way. We will elaborate on the potential applications of mRNA
assessment in clinical nephrology. Progress in the optimization
of protocols for acquiring adequate renal tissue and intact
RNA will be discussed.
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Perspectives of mRNA assessment in clinical practice
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The question of why it is useful to analyse levels of mRNA in
renal biopsies must be answered. Firstly, mRNA quantitation
could be used as a diagnostic tool. In renal pathology, it is
sometimes difficult to formulate a diagnosis on the sole basis
of clinical and histological findings. Molecular analysis of
the tissue with RNA quantitation may help improve diagnostic
accuracy. Secondly, mRNA levels could be used as prognostic
tools. Studies in animal models [
1–3] and, more recently,
in biopsy specimens from patients [
4–6] have shown that
alterations in mRNA levels for extracellular matrix (ECM) molecules
and ECM-regulating molecules predict the extent of scarring
in later phases of the disease. Furthermore, recent studies
in kidney transplantation [
7–9] and in native kidney diseases
[
10,
11] show that mRNA levels of transcripts identified by microarray
analysis may serve as a complement to histological findings
for the formulation of the patient's prognosis. Thirdly, mRNA
assessment may be used as a tool to predict the response of
the patient to therapy. Support for this concept has been provided
by Sarwal
et al. They showed that a relatively high expression
of CD20, a marker for B cells, during acute rejection is associated
with resistance to anti-rejection therapy [
8]. In another report,
Fas ligand mRNA levels predicted unresponsiveness to anti-rejection
therapy [
12]. Fourthly, mRNA assessment may be used as a tool
to monitor the extent of a therapy's negative side effects over
time. For example, mRNA levels of TGF-ß and collagens
I and III have been used to compare the fibrogenic effects of
different calcineurin inhibitors on kidney grafts [
6,
13,
14].
Usage of sequential protocol biopsies in mRNA assessment makes
it possible to accurately monitor changes over time in the activity
rate of profibrotic pathways under the influence of maintenance
immunosuppressive medications. Fifthly, investigation of gene
expression levels in renal tissue samples will elucidate the
pathogenesis of kidney diseases.
Overall, we envision that the use of molecular markers in addition to conventional parameters including morphology will lead to a more accurate prognosis and will therefore enable the application of more individualized therapeutic regimens. The response of patients to anti-rejection medication and maintenance therapy may be predicted more accurately on the basis of molecular markers.
Messenger RNA assessment in clinical biopsies for the applications mentioned above requires optimal protocols for tissue acquirement and RNA extraction. In the next chapter, we will discuss the progress that has been made in techniques for acquiring glomeruli from renal biopsies and extraction of intact RNA.
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Toward an optimal protocol for the acquisition of intact RNA from renal tissue
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During the course of renal disease, different compartments of
the kidney are affected in different ways. Molecular mechanisms
that pertain to the development of glomerular lesions may differ
from those that pertain to the development of tubulointerstitial
lesions. Therefore, separate analysis of glomerular tissue from
renal biopsies at the mRNA level provides useful information,
which complements the information gathered through mRNA analysis
of whole cortical renal tissue alone. In some renal disease
entities including minimal change disease, morphological alterations
are generally observed only in the glomeruli. Messenger RNA
levels determined in glomerular tissue, rather than in the whole
renal cortex, would in that case more directly and adequately
reflect the molecular composition of the glomeruli.
Glomeruli were initially separated from the tubulointerstitium through manual microdissection of fresh renal biopsies [15–19,21]. Only when a biopsy yielded an amount of tissue that was sufficient for diagnostic purposes, a small piece of cortex (usually 1–2 mm in length) was cut off for microdissection. The cortical tissue was disrupted with the aid of two small needles, and at that point the glomeruli became visible under a stereomicroscope as separate structures among the tubulointerstitial tissue (Figure 1A). The glomeruli were removed from the surrounding tissue with a pipette and washed to remove tissue debris. Glomeruli were then stored at –80°C until RNA extraction.
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Fig. 1. (A) Glomeruli obtained with manual microdissection of a fresh renal biopsy under stereomicroscopy. (B,C) Glomerular tissue obtained with laser-capture microdissection from a frozen renal biopsy specimen.
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Several methods can be used to extract RNA from small amounts
of glomerular tissue. With mild soap solutions such as Nonidet
P-40 and Triton X-100 glomeruli are permeabilized, and subsequently
cDNA can be synthesized
in situ. This method has often been
applied to freshly obtained, microdissected glomeruli [
15,
17,
19].
For several years, an RNA-extraction method which makes use
of Trizol® (Invitrogen) has been available. This method
is based on phenol/chloroform-mediated extraction of RNA. More
recently, silica-gel spin columns have been developed (e.g.
RNeasy mini columns, Qiagen), which can be used in combination
with the stringency of guanidine-isothiocyanate lysis. Silica-gel
based spin columns offer a rapid method to isolate intact RNA
from renal biopsy tissue, derived from either the whole cortex
or the glomeruli.
One of the principal problems with RNA isolation is that mRNA molecules are sensitive to degradation by endonucleases and exonucleases. Thawing of frozen archival biopsy material leads to RNA degradation [19,20]. The RNA-preserving compound RNAlater has been proven to conserve the integrity of the RNA in whole renal cortical tissue that is stored at 4°C or lower temperatures for a prolonged time period. It will also minimize RNA degradation in frozen tissue during thawing [20,21]. However, treatment of the whole renal cortical tissue with RNAlater is detrimental for glomerular morphology and complicates manual glomerular microdissection [20]. For several years, laser-capture microdissection has been used to obtain specific structures, such as glomeruli, from frozen [22,23] and formalin-fixed [24] renal biopsies. With this technique, glomeruli can be selected from a renal tissue slide, captured by a laser beam (Figure 1B), and catapulted into a reaction tube for mRNA assessment; RNA-preserving compounds are not necessary. Laser-capture microdissection has made archival material available for research purposes. Novel data – obtained through real-time PCR of a household gene on the corresponding cDNA – show that yields of intact RNA from laser-captured glomeruli of frozen renal biopsies are significantly higher than those from manually microdissected glomeruli of fresh renal biopsies (Figure 2).
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Fig. 2. Significantly higher yields of intact mRNA are obtained from laser-captured glomeruli than from manually microdissected glomeruli. For assessment of mRNA yield, real-time PCR of the household gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed on cDNA from laser-captured glomeruli of 35 frozen renal biopsies (mean Ct value 27.0±1.3) and on cDNA from manually microdissected glomeruli of 33 freshly obtained renal biopsies (mean Ct value 32.5±3.8). The figure depicts Ct values, which represent the number of PCR cycles needed to obtain an amplification product above the minimum threshold level: the lower the Ct value, the higher the yield of GAPDH mRNA from a certain sample. *P<0.001.
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The combination of laser-capture microdissection and novel RNA-amplification
protocols with a 1000-fold linear amplification efficiency [
25]
generates nephron segment specific gene profiles in patient
material. Recently, gene expression profiling has been performed
on individual glomeruli obtained from biopsies with lupus nephritis
[
26]. These glomeruli had been obtained with laser-capture microdissection.
These experiments have provided insight into the extent of interglomerular
molecular variations within one patient and into variations
in the glomerular expression profile between different patients
with a similar renal disease entity. Additionally, nanotechnology-based
biochips will definitely gain importance as tools for studying
molecular mechanisms at the single cell level [
27].
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Conclusion
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Measurement of mRNA transcripts may be a sensitive and efficient
means to predict outcome in kidney transplantation and native
kidney diseases. Messenger RNA assessment may be instrumental
in predicting the patient's response to therapy and in monitoring
potential long-term side effects of maintenance therapy. Any
of these applications in clinical practice requires optimized
protocols for extraction of intact RNA from renal biopsy specimens.
Research in recent years has generated such protocols. Laser-capture
microdissection enables analysis of gene expression in specific
renal tissue compartments of archival biopsy tissue. The combination
of molecular biological applications, including laser-capture
microdissection and differential gene expression analysis, renders
a powerful instrument to investigate the mechanisms and pathogenesis
of renal disease.
Conflict of interest statement. None declared.
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Introduction
Perspectives of mRNA assessment...