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 [13] and, more recently, in biopsy specimens from patients [46] 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 [79] 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 [1519,21]. Only when a biopsy yielded an amount of tissue that was sufficient for diagnostic purposes, a small piece of cortex (usually 12 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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References
|
---|
- Lee SK, Goyal M, De Miguel M et al. Renal biopsy collagen I mRNA predicts scarring in rabbit anti-GBM disease: comparison with conventional measures. Kidney Int 1997; 52: 10001015[ISI][Medline]
- Munaut C, Bergijk EC, Baelde JJ et al. A molecular biological study of extracellular matrix components during the development of glomerulosclerosis in murine chronic graft-versus-host disease (GvHD). Lab Invest 1992; 67: 580587[ISI][Medline]
- He C-J, Yang C-W, Peten EP et al. Collagen and collagenase mRNAs in normal and sclerotic glomeruli: Predictors of progression and response to therapy. Kidney Int 1995; 47 [Suppl 49]: S39S43
- Delarue F, Hertig A, Alberti C et al. Prognostic value of plasminogen activator inhibitor type 1 mRNA in microdissected glomeruli from transplanted kidneys. Transplantation 2001; 72: 12561261[CrossRef][ISI][Medline]
- Eikmans M, Sijpkens YW, Baelde HJ et al. High transforming growth factor-beta and extracellular matrix mRNA response in renal allografts during early acute rejection is associated with absence of chronic rejection. Transplantation 2002; 73: 573579[ISI][Medline]
- Baboolal K, Jones GA, Janezic A, Griffiths DR, Jurewicz WA. Molecular and structural consequences of early renal allograft injury. Kidney Int 2002; 61: 686696[CrossRef][ISI][Medline]
- Scherer A, Krause A, Walker JR et al. Early prognosis of the development of renal chronic allograft rejection by gene expression profiling of human protocol biopsies. Transplantation 2003; 75: 13231330[CrossRef][ISI][Medline]
- Sarwal M, Chua MS, Kambham N et al. Molecular heterogeneity in acute renal allograft rejection identified by DNA microarray profiling. N Engl J Med 2003; 349: 125138[Abstract/Free Full Text]
- Eikmans M, Van der Wal A, Baelde JJ et al. Renal mRNA expression of array-selected molecular markers is an additive prognostic tool to morphologic and clinical parameters during acute rejection of kidney allografts. J Am Soc Nephrol 2004; 15: 21A22A
- Eikmans M, Baelde HJ, Hagen EC et al. Renal mRNA levels as prognostic tools in kidney diseases. J Am Soc Nephrol 2003; 14: 899907[Abstract/Free Full Text]
- Henger A, Kretzler M, Doran P et al. Gene expression fingerprints in human tubulointerstitial inflammation and fibrosis as prognostic markers of disease progression. Kidney Int 2004; 65: 904917[CrossRef][ISI][Medline]
- Desvaux D, Schwarzinger M, Pastural M et al. Molecular diagnosis of renal-allograft rejection: Correlation with histopathologic evaluation and antirejection-therapy resistance. Transplantation 2004; 78: 647653[ISI][Medline]
- Bicknell GR, Williams ST, Shaw JA et al. Differential effects of cyclosporin and tacrolimus on the expression of fibrosis-associated genes in isolated glomeruli from renal transplants. Br J Surg 2000; 87: 15691575[CrossRef][ISI][Medline]
- Miyagi M, Muramatsu M, Ishikawa Y et al. Comparison of the therapeutic effects between CsA and FK506 on chronic renal allograft injury and TGF-beta expression. Transplant Proc 2002; 34: 15891590[CrossRef][ISI][Medline]
- Carome MA, Striker LJ, Peten EP et al. Human glomeruli express TIMP-1 mRNA and TIMP-2 protein and mRNA. Am J Physiol Renal Fluid Electrolyte Physiol 1993; 264: F923F929[Abstract/Free Full Text]
- Peten E, Striker LJ, Carome MA et al. The contribution of increased collagen synthesis to human glomerulosclerosis: A quantitative analysis of a2IV collagen mRNA expression by competitive polymerase chain reaction. J Exp Med 1992; 176: 15711576[Abstract/Free Full Text]
- Striker LJ, Esposito C, Striker GE. Molecular biology of human glomerular diseases. Kidney Int 1997; 51 [Suppl 58]: S62S65
- Del Prete D, Forino M, Gambaro G et al. A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens. Exp Nephrol 1998; 6: 563567[CrossRef][ISI][Medline]
- Eikmans M, Baelde HJ, De Heer E, Bruijn JA. Processing renal biopsies for diagnostic mRNA quantification: improvement of RNA extraction and storage conditions. J Am Soc Nephrol 2000; 11: 868873[Abstract/Free Full Text]
- Roos-Van Groningen MC, Eikmans M, Baelde HJ, De Heer E, Bruijn JA. Improvement of extraction and processing of RNA from renal biopsies. Kidney Int 2004; 65: 97105[CrossRef][ISI][Medline]
- Cohen CD, Frach K, Schlondorff D, Kretzler M. Quantitative gene expression analysis in renal biopsies: A novel protocol for a high-throughput multicenter application. Kidney Int 2002; 61: 133140[CrossRef][ISI][Medline]
- Kohda Y, Murakami H, Moe OW, Star RA. Analysis of segmental renal gene expression by laser capture microdissection. Kidney Int 2000; 57: 321331[CrossRef][ISI][Medline]
- Nagasawa Y, Takenaka M, Matsuoka Y, Imai E, Hori M. Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting. Kidney Int 2000; 57: 717723[CrossRef][ISI][Medline]
- Cohen CD, Grone HJ, Grone EF et al. Laser microdissection and gene expression analysis on formaldehyde-fixed archival tissue. Kidney Int 2002; 61: 125132[ISI]
- Upson JJ, Stoyanova R, Cooper HS et al. Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection. J Cell Physiol 2004; 201: 366373[CrossRef][ISI][Medline]
- Peterson KS, Huang JF, Zhu J et al. Characterization of heterogeneity in the molecular pathogenesis of lupus nephritis from transcriptional profiles of laser-captured glomeruli. J Clin Invest 2004; 113: 17221733[Abstract/Free Full Text]
- Jain KK. Nanodiagnostics: application of nanotechnology in molecular diagnostics. Expert Rev Mol Diagn 2003; 3: 153161[CrossRef][ISI][Medline]