Third Department of Internal Medicine, Akita University School of Medicine, Akita, Japan
Correspondence and offprint requests to: Atsushi Komatsuda, MD, Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan. E-mail: komatsud{at}med.akita-u.ac.jp
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Abstract |
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Methods. To identify up-regulated genes during the nephrosis phase, we undertook serial analyses of gene expression (SAGE) in peripheral blood mononuclear cells (PBMC) from a patient with MCNS sampled during the nephrosis and remission phases. To confirm the SAGE results, we performed quantitative real-time reverse transcriptionpolymerase chain reaction (RTPCR) analyses. We also measured the serum levels of the identified gene product in nephrosis and remission samples from 29 MCNS patients, 57 patients with nephrotic syndrome due to other types of glomerular diseases and 30 healthy individuals.
Results. Using more than 20 000 SAGE tags, we identified 15 functionally known genes that were up-regulated (4-fold) in PBMC from the MCNS patient during the nephrosis phase. For further examination, we selected two genes encoding secretory proteins, namely tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and tissue inhibitor of metalloprotease 1. Real-time RTPCR analysis confirmed a higher expression of TRAIL mRNA in PBMC during nephrosis than during remission in eight MCNS patients. The expression pattern of TRAIL mRNA, however, was variable among four patients with membranous nephropathy. There was no significant increase of serum levels of a soluble form of TRAIL in MCNS patients during the nephrosis phase.
Conclusions. These results suggest that the intracellular TRAIL mRNA expression in PBMC is up-regulated in MCNS patients during the nephrosis phase. This change may represent either an epiphenomenon or it may provide a potential explanation for the altered T-cell function in MCNS.
Keywords: gene expression profile; minimal-change nephrotic syndrome; serial analysis of gene expression; tumour necrosis factor-related apoptosis-inducing ligand
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Introduction |
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Recently, Sahali et al. [6], screening a library of subtracted cDNA, reported on a novel approach for the identification of PBMC genes that are potentially involved MCNS relapse. Their preliminary screening of the library, with relapse and remission unsubtracted cDNA probes, identified approximately 1000 clones. They finally found 42 up-regulated known transcripts in MCNS relapse. These transcripts included genes encoding proteins associated with signalling pathways and the cytoskeletal scaffold.
Serial analysis of gene expression (SAGE), a recently developed functional genomic approach, has enabled us to identify simultaneously the expression pattern of thousands of genes [7]. Therefore, SAGE can be applied for comparing differences of gene expression between PBMC from MCNS patients during the nephrosis and remission phases. In the present study, we capitalized on an advantage of SAGE for screening of mRNA expression in PBMC from a MCNS patient during the two phases. Among the up-regulated genes in the nephrosis sample, two were for secretory proteins, namely tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (approved gene symbol TNFSF10) [8,9] and tissue inhibitor of metalloprotease 1 (TIMP-1). We selected these genes for further examinations.
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Subjects and methods |
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Based on these findings, she was diagnosed to have MCNS and was treated with prednisolone (40 mg/day). With the treatment, she reached complete remission within 2 weeks. While her dosage of prednisolone was being tapered, her MCNS relapsed. Blood samples were obtained from her during the relapse in August 2002 (while the patient was receiving 15 mg/day prednisolone) and 3 months after the remission of this episode of relapse (while the patient was receiving 15 mg/day prednisolone therapy).
In this study, we also examined blood samples from 29 patients during the nephrosis and remission phases with biopsy-proven MCNS, 30 healthy subjects and 57 patients with nephrotic syndrome [biopsy-proven; 27 with FSGS and 30 with membranous nephropathy (MN)]. The protocol of this study was approved by the ethics committee of the institution involved and informed consent for genetic studies was obtained from all subjects.
SAGE protocol
The libraries for SAGE of PBMC from Patient 1 during nephrosis and remission phases were generated as described previously [10]. Total RNA was prepared using a Trizol reagent (Gibco-BRL, Gaithersburg, MD, USA). PolyA+ RNA was obtained using a Messagemaker kit (Gibco-BRL) according to the manufacturer's instruction and was converted to cDNA with a SuperScript Choice System (Gibco-BRL) with a 5'-biotinylated oligo(dT). Biotinylated double-strand cDNA was cleaved with the restriction enzyme NlaIII (New England Biolaboratories, Beverly, MA, USA) and the 3'-terminal fragments were bound to streptoavidin-coated magnetic beads (Dynal, Oslo, Norway). Captured 3' cDNA fragments were divided into two pools and each pool was ligated to one of the linkers containing recognition sites for BsmFI.
Nucleotide sequences of the linkers were as follows:
PCR products were analysed by polyacrylamide gel electrophoresis (PAGE) and the band containing the ditags was excised. After digestion with NlaIII, the ditags were ligated together to produce concatemers. The concatemers were separated by PAGE, and the products that consisted of between 400 and 1200 base pairs (bp) were excised. These products were cloned into pZero-1 (Invitrogen, Carlsbad, CA, USA), digested with SphI (New England Biolaboratories). PCR for insert screening of colonies was performed with the M13 forward and reverse sequences as primers. PCR products longer than 600 bp were sequenced using the BigDye terminator cycle sequence kit (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Sequencing was performed with an ABI 377 automated DNA sequencer (Perkin-Elmer Applied Biosystems).
SAGE data analysis
Concatemer sequences were analysed as described previously [10] with the SAGE 2000 software, version 4.12, which was kindly provided by Dr Kinzler (Johns Hopkins University). To identify the individual genes, the search for the expressed genes was conducted at the Serial Analysis of Gene Expression Tag of the Gene Mapping home page (http://www.ncbi.nlm.gov/SAGE). The detailed SAGE protocol can be seen at the SAGE home page (http://www.sagenet.org).
Quantitative real-time RTPCR
We quantified TRAIL and TIMP-1 mRNA expressions in the nephrosis and remission PBMC samples from four MCNS patients (including Patient 1) and eight MN patients. Table 1 summarizes the medical regimens of these patients at the time blood samples were obtained.
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Real-time reverse transcription (RT)PCR reaction was carried out according to the manufacturer's instructions in a final 20 µl volume containing 10 µl DNA Master Hybridization Probe 2X (Qiagen), 1 µl of 10 pmol forward and reverse primers, 1 µl cDNA and 7 µl water. After an initial denaturation step at 95°C for 900 s, temperature cycling was initiated. Each cycle consisted of denaturation at 95°C for 15 s, hybridization at 56°C (for TRAIL and ß-actin) or at 60°C (for TIMP-1) for 20 s and elongation at 72°C for 20 s, using a LightCycler (Roche Diagnostics, Mannheim, Germany). Forty-five cycles were performed and each sample was run in triplicate.
Quantitative real-time RTPCR curves were analysed by the LightCycler 3.5 software (Roche Diagnostics). For the relative quantification of TRAIL and TIMP-1 mRNA expressions, the mRNA expression of ß-actin was used as a control.
ELISA for a soluble form of TRAIL
Serum levels of a soluble form of TRAIL (sTRAIL) were measured in duplicate for each sample, using an enzyme-linked immunosorbent assay (ELISA) kit for sTRAIL (Active Motif North America, Carlsbad, CA, USA). Optical density was measured in an ELISA reader at 405 nm. Concentrations of sTRAIL were determined by a standard curve, according to the manufacturer's instructions.
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Results |
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Quantification of TRAIL and TIMP-1 mRNA expressions in PBMC from MCNS patients during nephrosis and remission phases by real-time RTPCR
SAGE showed that two genes for secretory proteins (TRAIL and TIMP-1) were up-regulated in the PBMC from Patient 1 during the nephrosis phase of MCNS (Table 2). To confirm this result, we performed quantitative real-time RTPCR analyses for TRAIL and TIMP-1 mRNA expressions in nephrosis and remission PBMC samples from seven additional MCNS patients. We confirmed that TRAIL mRNA expressions were higher in the nephrosis samples than in the remission samples from all the MCNS patients analysed (patients 18 in Table 1). On the other hand, we could not confirm that the expressions of TIMP-1 mRNA in the nephrosis PBMC samples were higher than in the remission samples from all the MCNS patients (Table 1).
We also examined TRAIL mRNA expressions in the nephrosis and remission PBMC samples from four MN patients. The results showed that expression patterns were variable among these patients (patients 912 in Table 1).
Serum sTRAIL levels in MCNS patients during nephrosis and remission phases
Based on the results of SAGE and real-time RTPCR analyses, we used ELISA to measure serum sTRAIL levels in 29 MCNS patients during the nephrosis and remission phases. We also measured serum sTRAIL levels in 30 healthy subjects and 57 patients with nephrotic syndrome due to other types of glomerular diseases (27 FSGS and 30 MN). The results showed that there was no significant increase in the serum sTRAIL level in MCNS patients during the nephrosis phase (Figure 1).
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Discussion |
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Sahali et al. [6] recently reported their findings of a subtracted cDNA library screening, using cDNA from PBMC from the same MCNS patient and comparing the relapse and remission phases. They found various up-regulated PBMC transcripts for signalling pathways, including the mitogen-activated protein kinase cascade. Our SAGE results also showed considerable differences in the expression profiles between nephrosis and relapse PBMC samples from the same MCNS patient. During the nephrosis phase, there were several up-regulated genes for various proteins involved in signalling pathways, such as calcium/calmodulin-dependent protein kinase kinase 1, mitogen-activated protein kinase-activated protein kinase, FK506 binding protein 1A and cyclophilin F, in addition to the gene for TRAIL. These observations suggest that multiple signalling pathways might be activated in PBMC during the development of MCNS.
TRAIL (also known as Apo-2 ligand) has been identified as a member of the TNF ligand family that induces apoptosis in a wide variety of tumour cells [8,9]. TRAIL can selectively induce apoptosis in tumorigenic cells, but not in normal cells, highlighting its potential therapeutic application in cancer treatment. In contrast to other members of the TNF ligand family, which are often only transiently expressed on activated cells, TRAIL mRNA is expressed constitutively in a wide range of tissues [8]. Although TRAIL is primarily expressed as a type II transmembrane protein, bioactive sTRAIL might be released from activated T-cells in association with microvesicles or cleaved from the cell surface by proteases, or both [11,12]. Thus, the serum levels of sTRAIL are likely to be regulated not only by mRNA TRAIL expressions in PBMC but also by other factors. This might be the reason why in our study there was no correlation between the levels of mRNA TRAIL expression in PBMC and serum sTRAIL in MCNS patients during nephrosis.
Several studies have demonstrated that TRAIL is associated with the pathogenesis of some autoimmune diseases, such as neutropenia of systemic lupus erythematosus [13 and references therein]. However, there have been no reports suggesting the possibility of TRAIL being associated with the pathogenesis of MCNS. Our results suggest that the intracellular TRAIL mRNA expression is up-regulated in PBMC during the nephrosis phase of MCNS, although the bulk of the investigations were based on preliminary SAGE findings from a single patient and real-time RTPCR findings from a total of eight patients. Our observations could represent an epiphenomenon or they could provide a potential explanation for the altered T-cell function seen in MCNS.
It is known that therapeutic concentrations of IFN-ß and IFN- stimulate the expression of high levels of TRAIL mRNA in PBMC and neutrophils, and the release of elevated amounts of sTRAIL [14,15]. In fact, there are several case reports that describe an acute onset or a relapse of MCNS during the therapy of malignancies, or chronic viral hepatitis with IFN-ß or IFN-
[1619]. It is therefore possible that the up-regulation of a TRAIL induced by IFN-ß or IFN-
might be involved in the development of MCNS in these cases. In particular, nephrotic-range proteinuria regressed without steroid treatment after the cessation of IFN-ß injections in the case reported by Nakao et al. [17]. Although TRAIL is now considered to have a promising therapeutic potential in cancer treatment [20], careful patient monitoring, including urinalysis, will be necessary when using it.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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