1 Department of Pathology, University of Cologne and 2 Department of Internal Medicine I, Cologne General Hospital, Merheim Medical Center, Cologne, Germany
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Abstract |
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Methods. By laser-microdissection, 10 glomeruli/case were isolated from 5 µm thick tissue slices in a total of 15 cases of mesangial proliferation with different renal diseases (IgA nephropathy, lupus nephritis and mesangial proliferative lesions of unknown aetiology) vs transplant biopsies as negative and TNF -treated cultured human mesangial cells as positive controls. After reverse transcription of isolated RNA, cDNA aliquots were quantified for VCAM-1 expression by real-time PCR using the threshold cycle (Ct) method, normalized for the housekeeping gene ß-actin, and compared with qualitative RTPCR results.
Results. Unsuspected VCAM-1 transcript steady-state levels could be detected by real-time PCR in agreement with qualitative PCR, while morphologic and immunhistologic analyses were unrevealing. As yields of RNA extraction in femtogram quantities cannot be measured spectrophotometrically, a Ct-ratio was formed between ß-actin and VCAM-1 per case showing high VCAM-1 expression in lupus nephritis (1.39), and moderate expression in IgA nephropathy (1.081.23) vs TNF -treated mesangial cells (0.971.23) and negative control cases (0.660.68).
Conclusions. This is the first reported gene expression analysis method for routinely paraffininzed human renal biopsies, demonstrating the power of combined laser-microdissection and PCR quantification as novel methods for the evaluation of minimal tissue beyond purely descriptive morphologic analysis.
Keywords: human; kidney; laser-microdissected glomeruli; paraffinined biopsies; quantitative RTPCR; VCAM-1
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Introduction |
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Recently, laser-microdissection has been established as a reliable method for procuring morphologically distinct tissue regions of interest in a large variety of tissues. For the kidney, studies have used unfixed, frozen kidney slices from murine models [13] providing sufficient tissue to analyse. So far, only a single report exists on the use of one frozen human biopsy [3]. However, routine evaluation of renal biopsies in Europe has been performed for many years on formalin-fixed, paraffinized needle core biopsies because of their vast superior preservation of morphologic details compared with frozen material. Thus, presently no study exists describing the specific isolation and RNA extraction from small tissue targets such as glomeruli in routinely fixed and paraffin-embedded needle core biopsies.
As for the second prerequisite, the determination of different levels of gene expression, quantitative PCR methods have been developed. They have been used to study intrarenal gene expression in native kidneys from murine models [4,5], whole organ nephrectomies [6] and larger fractions of frozen human renal core biopsies from transplant kidneys [7]. However, no data presently exist applying this method to minute targets such as glomerular slices after laser-microdissection of formalin-fixed, paraffinized renal biopsies.
Experimental reports have demonstrated the progression of an existing mesangial proliferation due to an immunologic or non-immunologic activation of nuclear-factor kappa B (NF-B)-mediated inflammatory signal pathway. Our group has shown that activation of this pathway in vitro [8] and in vivo [9] leads to a mesangial surface expression of vascular cell adhesion molecule-1 (VCAM-1), a member of the immunoglobulin supergene family [10].
As intrarenal upregulation of gene expression may proceed structurally detectable alterations by days [11], quantification of VCAM-1 as marker gene for mesangial cell (MC) stimulation may be an important alarm signal indicating further progression of MC lesions. Thus, we describe here a novel evaluation method applicable for routine renal biopsies. It consists of combining laser-microdissection of glomeruli from formalin-fixed, paraffinized renal needle core biopsies with so-called real-time, quantitative RTPCR, using as study objective the analysis of VCAM-1 expression in mesangial proliferative lesions. As VCAM-1 expression could be detected in mesangial lesions unsuspected of inflammatory activation by standard routine analysis, the specific evaluation of VCAM-1 levels may be important for further diagnostic considerations and possible therapeutic interventions.
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Subjects and methods |
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From a freshly explanted human kidney (with a small urothelial carcinoma and no other detectable parenchymal disease) sent for pathological evaluation by frozen section, a small slice of cortex was immediately frozen in OCT compound (Sakura Finetek Europe B.V., The Netherlands [9]).
Laser-manipulated microdissection, and laser pressure catapulting
Ten to 25 glomeruli were individually microdissected from 5 µm thick sections of either mouse kidney or human renal biopsy cores using a laser microbeam (P.A.L.M., Wolfrathshausen, Germany). Contamination by parietal epithelial cells, expressing VCAM-1 constitutively [12], was carefully avoided. Subsequently to dissection, each glomerulus was laser-catapulted into a microfuge tube containing RNase-free water, as well as RNAsin (2040 U/ml, Promega, Mannheim, Germany), and stored frozen at -70°C until extraction.
RNA extraction and reverse transcription
RNA was extracted using the Purescript RNA isolation kit (Gentra, Minneapolis, MN) according to manufacturer's instructions with the following modifications and volume adjustments for human renal biopsies. For each microdissected sample of 20 glomeruli collected from 5 µm thick formalin-fixed and paraffinized sections, only 30 µl of lysis buffer were used and the tube gently shaken. One microgram of poly A-RNA (Qiagen, Hilden) and 10 µl DNAprotein-precipitation solution were added for 5 min on ice, and centrifuged to form a tight pellet as advised by the manufacturer. The supernate was transfered to a new tube and mixed with 30 µl of 100% ice-cold isopropanol and 1 µl of 2 µg/ml glycogen (Boehringer Ingelheim; no 901393) by inverting 50 times. After centrifugation and washes with 70% ice-cold ethanol a small pellet should be visible which was air dried and resuspended in RNase-free water. Subsequently, an additional DNase (Sigma-Aldrich, Taufkirchen) treatment according to manufacturer's recommendation was performed to prevent amplification from contaminating DNA. These optimized and adjusted volume requirements were tested using batches of microdissected, formalin-fixed and paraffinized mouse glomeruli. Extraction and reverse transcription procedures were also optimized with cDNA from microdissected mouse glomeruli. To evaluate the efficiency of this extraction procedure, one of the most recent commercially available extraction kits, called PicoPure RNA isolation kit (Arcturus, Mountain View, CA) was used. According to the manufacturer, this extraction procedure is fast (total extraction time 2 h) and optimized to work on minute tissue samples with as little as 10 microdissected cells. The provided protocol was followed strictly without any additional volume or procedure adjustment. Mouse glomeruli (in separate sets of 20) were microdissected from frozen and formalin-fixed, paraffinized kidneys and extracted using this protocol. For quantitative analysis, ß-actin was measured using the QuantiTect SYBR Green PCR kit (Qiagen, Hilden) with the ABI PRISM 7700 PCR cycler (PE Biosystems, Weiterstadt) in a two-step RTPCR procedure according to a protocol provided by the manufacturer.
The entire amount of extracted RNA was subsequently used in a reverse transcription reaction (TaqMan reverese transcription reagent kit; PE Biosystems) using random hexamers. cDNA was stored at 4°C until further use.
Qualitative semi-nested PCR
For qualitative assessment of VCAM-1 expression, the cDNA material of one to five glomeruli per case was used in a so-called nested PCR reaction. Reverse transcribed RNA from TNF -stimulated human mesangial cells served as control. First and second round PCR primers were used as described previously [13]; their products were checked by sequence analysis [13]. Primers for ß-actin semi-nested PCR were manufactured by MWG Biotech (Ebersberg, Germany; forward primer: 5'-TTGCAATGAGCGGTTCCGCT-3'; reverse primer 1: 5'-TACAGCTGTTTGCGGATGTCC-3'; reverse primer 2: 5'-CACGTCACACTTCATGATGGAG-3'). Each PCR was performed in a total volume of 50 µl at an annealing temperature of 55°C with each run consisting of 30 cycles [13]. All PCR products were analysed using ethidium brominde stained, 1.8% agarose gels.
Quantitative real-time TaqMan PCR
For VCAM-1, all primers and the probe were obtained from PE Biosystems. Quantification was performed using the same forward and reverse primers (no. 2) as in the qualitative semi-nested PCR. The probe was 5'-TTCTCCTGAGCTTCTCGTGCTCTATTTTGC-3' with its 5' nucleotide labelled with a quencher dye (TAMRA=6-carboxy-tetramethylrhodamine) and its 3' nucleotide labelled with the fluorescent 6-FAM. For ß-actin detection, primers and probe were obtained from Eurogentics (Seraing, Belgium). Universal master mix (PE Biosystems) contained all reagents including Taq-Polymerase apart from specific primers and probes. Volume equivalents of one, three or five glomeruli were tested in a total reaction volume of 50 µl. PCR conditions were as follows: 2 min at 50°C for uracil-N-glycosylase incubation, 10 min at 95°C for the activation of AmpliTaq Gold DNA polymerase and then 60 cycles of 95°C for 15 s and 60°C for 1 min. The expression of VCAM-1 and ß-actin in each sample were quantified in separate tubes with the respective primers. The amplification was performed using the ABI 7700 PRISM PCR cycler (PE Biosystems) in a two-step RTPCR procedure. Gels of the final products were analysed as described before.
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Results |
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In order to validate our Purescript extraction method further, we performed a qualitative RTPCR using the mRNA equivalent of one glomerular slice (Figure 2). For VCAM-1 a 103 bp amplification product, for ß-actin as housekeeping gene, a 110 bp product was obtained.
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Discussion |
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As the minimal amount of RNA obtained from a few glomerular slices cannot be quantified by a spectrophotometer, we used a ratio between the threshold of ß-actin and VCAM-1 to calculate the expression level of the gene of interest, thus correcting for possible variations through the extraction procedure and enabling a more meaningful comparison between cases and different studies than absolute expression values would have done. Thus, we observed in our small biopsy series a clear distinction between cases positive or negative for increased VCAM-1 expression.
Importantly, positive VCAM-1 quantification could be revealed in several positive biopsy cases in which microscopically an inflammatory response was not readily visible, such as in IgA nephropathy and in mesangial proliferative lesions of unclear etiology. Surprisingly, all IgA cases studied showed a positive signal for VCAM-1. As cases of other entities analysed in the same quantitative PCR run resulted in positive as well as negative amplifications and all controls displayed correct negative results, this observation cannot easily be interpreted as false positive. As aggregated IgA may lead to a NF-B-mediated stimulation of cultured human mesangial cells [14], our observation from human renal biopsies supports the idea that IgA nephropathy does not only represent a proliferative lesion but may well have an inflammatory component at least in some IgA cases. Currently, the role of VCAM-1 in IgA nephropathy remains speculative, and requires further investigation to evaluate whether this expression is of major functional importance or represents a non-specific bystander reaction.
In contrast to IgA nephropathy, VCAM-1 levels in one of the cases of lupus nephritis provided evidence for activation of the inflammatory pathway while another case did not, despite of their common histomorphologic classification (as diffuse proliferative). As activation of inflammatory signal transduction may contribute to the progression of a pre-existing mesangial proliferative lesion, these results clearly indicate the potential usefulness of quantitative, real-time PCR in future diagnostics and possible therapeutic implications.
Thus, in the near future, quantitative PCR analysis may enhance our understanding of pathophysiologic aspects of different renal diseases, once sufficient cases have been studied per disease entity and the range of expression levels for a specific gene have been defined.
However, there are also distinct methological limits using formalin-fixed, paraffin-embedded renal biopsy cores. These are predominantly caused by the degree of degradation of RNA due to handling time before fixation, the use of a specific fixative and finally the paraffin embedding [15]. Four per cent of buffered formalin as fixative is essential, while a fixation duration of 26 h seems optimal. Unfortunately, the quality of formalin fixation as well as its duration cannot be determined from morphologic analysis alone. Whenever this information seems critical as well as for prospective cases, a close interaction with the respective nephrologist performing the biopsy is essential. The problem using archival paraffinized tissues is well known [16], and has been confirmed again for quantitative PCR by reports studying the influence of different fixatives on RNA quality [1]. Thus, a negative result for the quantification of the gene of interest can only be accepted as truly negative, if one has a positive control gene, indicating that the RNA is not completely degraded and if all positive and negative controls give the expected results. In this aspect, quantitative PCR is really not different as its older sister, qualitative PCR.
The success of our amplification is in part also due to avoiding known RNase destroyers and inhibitors of Taq polymerase such as diethyl pyrocarbonate [17]. Furthermore, and in contrast to many studies, our glomerular sections are representing very thin slices of non-solid tissue pieces more similar to the microdissection of single cells [18]. In currently published procedures for solid tissue blocks of 20 µm or more in thickness, full digestion with proteinase K is crucial before cell lysis [19]. The extraction protocols from both kits used in this study do not list this additional digestion step, while the composition of their lysis buffer as part of their patent remains a non-disclosed secret. Thus, the success of our extraction protocol may be in part due to properties of our special material such as delicate structures and a thickness of about one fourth of a cell, in part due to the special combination of ingredients in the lysis buffer itself. The lack of an additional digestion step is advantageous, since it allows us to reduce the necessary handling time by 2 h.
Another important problem may arise from the limitations of the material available, particularly if thin needles are used to obtain the biopsy core. Most often, after sections for the routine analysis have been cut, there is not enough material, particularly glomeruli, left for evaluation. One has to postulate that a suitable biopsy should contain at least five, better 10 glomerular cross sections with completely intact Bowman capsule.
The decision to employ this novel technology is facilitated considering its rather limited time requirement. In our experience, one to three sections of 5 µm thickness can be cut and deparaffinized in 1 h. The microdissection of a minimum of 10 glomeruli takes
30 min each, while extraction and reverse transcription each need 1 h to complete. Thus, cDNA for analysis can be obtained within 1 working day. If a convenient overnight run is not possible, quantitative PCR for the transcript of interest and its control can be achieved the next morning simultaneously (provided the probes have different fluorescent labels) within 23 h depending on the number of cycles. Thus, compared with a routine overnight immunhistologic analysis, quantitative PCR is at least as fast.
For future diagnostic considerations, an additional, second (frozen) biopsy core could be advantageous, particularly regarding the improved results for RNA extraction from frozen vs formalin-fixed and paraffinized glomeruli. Besides an improvement in our daily immunhistologic analysis (i.e. particularly for IgG), its superior RNA yield could enable the study of many genes of interest, which is presently impossible by conventional immunhistology. In summary, quantitative PCR of microdissected glomeruli from formalin-fixed, paraffin-embedded needle core renal biopsies seems to bewithin the limits set by RNA degradationa useful and reliable method for evaluation of gene expression, being superior in its diagnostic value to any immunhistologic or simple morphologic evaluation. Its demonstrated potential to detect an activation of an inflammatory pathway as a possible progression factor of renal lesions could make it even a very promising method to obtain data useful for future therapeutic considerations.
Conflict of interest statement. None declared.
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Acknowledgments |
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Notes |
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References |
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