Pediatr Nephrol (2008) 23:395–401 DOI 10.1007/s00467-007-0683-z
ORIGINAL ARTICLE
Expression of mRNA for functional molecules in urinary sediment in glomerulonephritis Koji Tsugawa & Eishin Oki & Koichi Suzuki & Tadaatsu Imaizumi & Etsuro Ito & Hiroshi Tanaka
Received: 25 September 2007 / Revised: 17 October 2007 / Accepted: 17 October 2007 / Published online: 19 December 2007 # IPNA 2007
Abstract Recent studies have suggested that gene expression studies using urinary sediment might be a non-invasive approach to assessing activity and pathogenesis in glomerulonephritis. However, little information is available regarding the mRNA expression patterns of functional molecules, such as T-bet, GATA-3, FOXP3, and retinoic acid-inducible gene-I (RIG-I), in urinary sediment, from patients with immunocomplex-mediated glomerulonephritis. Fourteen lupus nephritis (LN) patients, 13 IgA nephropathy (IgAN) patients, and 12 healthy controls were enrolled in the study. The mRNA expressions of T-bet, GATA-3, FOXP3 and RIG-I in urinary sediment were measured using real time quantitative polymerase chain reaction. We also studied the expression of RIG-I in kidney tissue specimens obtained from LN and IgAN patients. Significant differences in the expression patterns of GATA-3, FOXP3 and RIG-I, and marginal differences in T-bet expression, were observed between the three study groups. Immunofluorescent staining for RIG-I was observed in the tissue specimens from the LN patients, but not in those from the IgAN patients. The mRNA expression patterns of T-bet, GATA-3, FOXP3 and RIG-I in urinary sediment differ according to diagnostic category. These results suggest that the measurement of these target gene expressions might be a useful, non-invasive
K. Tsugawa (*) : E. Oki : K. Suzuki : E. Ito : H. Tanaka Department of Pediatrics, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan e-mail:
[email protected] T. Imaizumi Department of Vascular Biology, Hirosaki University School of Medicine, Hirosaki, Japan
method for clinical monitoring and studying of pathogenesis in glomerulonephritis. Keywords Th1/Th2 balance . T-bet . GATA-3 . FOXP3 . RIG-I . Urinary sediment . Lupus nephritis . IgA nephropathy
Introduction Although the pathogenesis of immunocomplex-mediated glomerulonephritis, such as lupus nephritis (LN) and IgA nephropathy (IgAN), is not fully understood, the release of pro-inflammatory cytokines from activated renal cells has been reported to play an important role [1]. Recent reports have suggest that an imbalance between Th1 and Th2 cells might be associated with the development and progression of several forms of glomerulonephritis [2, 3]. Most patients with LN or IgAN are thought to exhibit Th1/Th2 heterogeneity; however, no clear evidence of this exists [4–6]. Rengarajan et al. reported that the relative predominance of T-bet or GATA-3 may determine Th1/Th2 polarization [7]. T-bet is the principal Th1 transcription factor, while GATA-3 is the principal Th2 transcription factor. Thus, the relative expressions of T-bet and GATA-3, which can lead to Th1/Th2 imbalances, has been implicated in a number of immunological diseases. Recent studies have also highlighted the importance of a specialized subgroup of CD4+CD25+ T lymphocytes, termed regulatory T lymphocytes (Tregs), in the suppression of autoimmunity and the maintenance of self-tolerance. In this context, the transcription factor FOXP3 plays a key role in the development and function of Tregs [8, 9]. Although the mechanisms deployed by FOXP3-expressing Tregs in humans are not clear, they are thought to include direct cell contact, cytokine signaling, and the inhibition of gene
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transcription that is central to effector functions [10]. FOXP3-expressing Tregs, which are specialized for the suppression of Th1 pathogenic immune responses, may participate in the development of glomerular diseases. Recently, we reported the expression of retinoic acid inducible gene-I (RIG-I) in renal biopsy specimens from patients with LN [11]. RIG-I is a member of the DExH-box family of proteins, and is thought to be a putative helicase, based on its amino acid sequence [12]. Although the details of the biological functions of RIG-I are not yet known, previous studies have indicated that the expression of RIG-I is induced in various inflammatory diseases, such as viral infections, leukemia and bladder carcinoma [13–16]. The expression of RIG-I is thought to contribute mainly to Th1-type reactions, because its expression is induced in human endothelial cells stimulated by interferon-gamma (IFN-γ) [17]. These studies suggest that an evaluation of the expression of RIG-I could be useful for elucidating the pathogenic mechanisms in immunocomplex-mediated glomerulonephritis. In clinical settings, the examination of renal biopsies is an accurate method for determining the histologic profile and activity of glomerular diseases. However, complications can occur from this procedure, and serial monitoring would be technically difficult. In this context, the measurement of mRNA expression in urinary sediment, which might directly reflect some conditions of glomerulonephritis, using reverse transcription and real-time quantitative polymerase chain reaction assays (RT-QPCR) has recently become possible [18–23]. In the study described here, we examined the relative mRNA expressions of four functional molecules, Tbet, GATA-3, FOXP3 and RIG-I, in urinary sediment obtained from young patients with LN or IgAN, to determine whether the expression patterns of these factors differed according to the diagnostic category.
Patients and methods Patients We studied 14 patients with LN. All patients fulfilled the American College of Rheumatology diagnostic criteria for systemic lupus erythematosus (SLE). The disease activity of SLE was assessed clinically with the European Consensus Lupus Activity Measurement (ECLAM) index [24]. Thirteen patients underwent kidney biopsies; a biopsy was not performed in the remaining one patient because of concomitant thrombocytopenia. We also studied 13 patients with early onset, biopsy-proven IgA nephropathy and 12 healthy subjects as controls. The histological grading of IgAN was assessed using a semi-quantitative scoring system: the activity and chronicity scores described by Andreoli and Bergstein [25] and the tubulointerstitial scores
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described by Foster et al. [26]. Informed consent was obtained from all the study subjects. Messenger RNA from urinary sediment A whole-stream, early morning, urine specimen was collected from all the study subjects for use in the gene expression study. The method of mRNA isolation has been described by Li et al. [18]. Briefly, the urine samples were centrifuged at 3,000 r.p.m. for 30 min at 4°C. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, MD, USA), according to the manufacturer’s instructions. All the specimens were stored at −70°C. The purity of the RNA was confirmed by the relative absorbance at the 260/ 280 nm ratio, as determined by spectrometry. Real-time polymerase chain reaction In our study, we quantified the mRNA expression of T-bet, GATA-3, FOXP3 and RIG-I in the urinary sediment. For each reaction, approximately 0.1 μg of total RNA was reverse transcribed to cDNA with an iScript cDNA synthesis Kit (Bio-Rad Laboratories). The resulting cDNAs were stored at −70°C until required. The relative mRNA abundances were quantified using Chromo 4 (Bio-Rad Laboratories). The primer sequences were as follows: T-bet (forward, 5′-CTG CCT ACC AGA ATG CCG AGA-3′, reverse, 5′-AAG CGG CTG GGA ACA GGA TAC3′); GATA-3 (forward, 5′-GCA GGA GCA GTA TCA TGA AGC CTA A-3′, reverse, 5′-TTG GAA CAC AGA CAC CAC AGT GAG-3′); FOXP3 (Qiagen Hs_FOXP3_1_SG QuantiTect Primer Assay); RIG-I (forward, 5′-GCA TAT TGA CTG GAC GTG GCA-3′, reverse, 5′-CAG TCA TGG CTG CAG TTC TGT C-3′); and 18S rRNA (forward, 5′-ACT CAA CAC GGG AAA CCT CA-3′, reverse, 5′-AAC CAG ACA AAT CGC TCC AC-3′). The mRNA expression level of each target was normalized to that of a housekeeping gene, 18S rRNA. PCR amplifications were performed in a 20-μl volume at 95°C for 3 min, followed by 40 cycles at 95°C for 5 s and at 60°C for 20 s. Each sample was run in triplicate. The results were analyzed with an Opticon Monitor 3 (Bio-Rad Laboratories). To quantify the abundance of the target mRNAs, we calculated the differences in the threshold cycles between the target genes and 18S rRNA. The relative mRNA abundance in the patient groups was calculated by the 2 ΔΔCt method [27]. Immunofluorescence staining Embedded tissue specimens were cut into 5 μm-thick sections with a cryostat at the optimal cutting temperature; the sections were then briefly fixed in cold acetone and air-dried. The slides were washed in phosphate-buffered saline (PBS)
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solution immediately before the immunohistochemical procedure. After blocking by incubation with 1% goat serum, the slides were incubated with anti-RIG-I antibody (1:1,000), as described by Imaizumi et al. [17]. The samples were then incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:40) (Sigma, Saint Louis, USA). Statistical analysis The statistical analysis was performed with SPSS for Windows, version 11.0.1 (SPSS, Chicago, IL, USA). The results were presented as mean ± SD, unless otherwise specified. Because the distributions of the gene expression levels were highly skewed, they were compared between groups, with a Kruskal– Wallis test or a Mann–Whitney U test, as appropriate. Correlations between mRNA expression levels and clinical parameters were determined with the Spearman rank correlation coefficient. A P value of < 0.05 was considered statistically significant. All probabilities were two-tailed.
Results We studied a total of 39 subjects. The baseline demographic and clinical data of the patients are summarized in Table 1. Table 1 Baseline demographic and clinical data
Significant differences in age, urine protein excretion, occult blood in the urine and prednisolone (PDN) doses were observed among the diagnostic groups. A post-hoc analysis showed that the control group was significantly older than the other groups and that the IgAN group had a more severe level of occult blood in the urine than the LN group, while the LN group had a significantly higher proteinuria level and a higher PDN dose than the IgAN group. Urinary mRNA expression between disease groups and control We first compared the levels of gene expression between the two disease groups and the healthy controls. Figure 1 summarizes the expressions of the target genes T-bet, GATA-3, FOXP3 and RIG-I in urinary sediment from all the study subjects. The expression of T-bet in the urinary sediment was marginally higher in the IgAN group than in the other two groups (P
