NDT Advance Access originally published online on April 1, 2007
Nephrology Dialysis Transplantation 2007 22(8):2407-2409; doi:10.1093/ndt/gfm175
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Expression of retinoic acid-inducible gene-I in lupus nephritis
Correspondence and offprint requests to: Email: skoichi{at}infoaomori.ne.jpSir,
Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH-box family of proteins, which is involved in inflammatory reactions related to RNA metabolisms, although the bioactivities of RIG-I still remain to be clarified in detail [1]. Previous studies have reported that expression of RIG-I is induced in various inflammatory diseases, such as viral infections, leukaemia and bladder carcinoma [1]. However, there are no reports on the expression of RIG-I in the kidneys of patients with lupus nephritis. In the present study, we examined the expression of RIG-I in kidney tissue specimens obtained from cases of human lupus nephritis, and evaluated the correlation between its expression and the histological activity of the renal disease in these patients.
From January 2000 to August 2006, lupus nephritis was diagnosed in 15 children and adolescents at Hirosaki University Hospital. All met the International Society of Nephrology/Renal Pathology Society (ISN/RPS) criteria for the histological diagnosis of lupus nephritis. Tissue cylinders obtained by renal biopsy were divided into three portions and processed for routine light-microscopic, electron-microscopic and immunofluorescence examinations. After the renal tissue samples for the immunofluorescence study were embedded in optimal cutting temperature (OCT), routine studies to determine the deposition of IgG, IgA, IgM, C3 and C1q were performed, and the samples were stored at –30°C until further use. Of these OCT-embedded kidney tissue specimens, 10 specimens obtained from eight patients, in good condition, were selected for this study. Repeat renal biopsy was performed in two of the eight patients (cases 3 and 6) who exhibited changes of proliferative lupus nephritis at presentation. Eight kidney tissue specimens obtained from patients with minimal-change (MC) disease were used as controls.
Each of the kidney tissue specimens from the patients with lupus nephritis was examined by light microscopy in a blinded fashion by one of the examiners and the histological findings were scored. The activity index was evaluated semi-quantitatively using the scoring system described by Austin et al. [2].
The OCT-embedded tissue specimens were cut into 5 µm-thick sections in a cryostat, briefly fixed in cold acetone and then air-dried; the slides were then washed in PBS immediately before the immunohistochemical procedure. After blocking by incubating with 1% goat serum, the slides were incubated with anti-RIG-I antibody (1 : 1000), as described by Imaizumi et al. [1]. The samples were then incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Sigma, Saint Louis, USA).
According to ISN/RPS criteria for light-microscopic histological classification, two patients (cases 2 and 8) were classified as class II nephritis, four (cases 1, 3, 4 and 6) as class IV nephritis, and two (cases 5 and 7) as class V nephritis. The mean activity index was 6.3, and two patients (cases 3 and 6) had a very high activity index of more than 10 (Table 1). Significant histological improvement was observed at the second renal biopsy in both cases 3 and 6. According to the ISN/RPS criteria, case 3 was classified as class II and case 6 as class III. Indeed, the activity index was also significantly decreased in both the cases (to three in case 3 and seven in case 6). None of the patients with MC disease showed any more than minor glomerular abnormalities on light-microscopic examination.
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Glomerular immunoreactivity for RIG-I was detectable in all the cases of lupus nephritis and an intense granular pattern of immunofluorescence was observed in a mesangial area and capillary loop distribution. Immunoreactivity of variable intensity was also present in the interstitial lesions from the patients with lupus nephritis. The intensity of the immunostaining correlated well with the renal histological activity index, but not with the findings of routine immunofluorescence examination. Especially, cases with high fluorescence intensity signals for RIG-I expression showed severe leucocyte exudation in the glomeruli on light-microscopic examination (Table 1). In cases 3 and 6 with a high index of histological activity, at the first biopsy in particular, a significantly increased intensity of immunostaining for RIG-I was observed (Figure 1). Furthermore, the immunostaining intensity was decreased at the second biopsy in both the cases. In the cases with MC disease, immunofluorescence staining was either absent or only trace-like in scattered areas of the glomeruli.
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It has been reported that Th1-derived cytokines play an important role in the progression of lupus-associated renal injury [3]. Interferon
(IFN-) has been shown to induce the expression of various factors, including adhesion molecules, chemokines, growth factors and enzymes in both animal models and human lupus nephritis, and is believed to play an important role in the inflammatory and immune processes involved in the pathogenesis of lupus nephritis [3]. Patole et al. [3] reported that the expression of toll-like receptors (TLRs) was up-regulated in lupus-like immune complex glomerulonephritis of MRL-Fas(lpr) mice, and the regulation of TLRs was involved in infection-associated exacerbation. In contrast, it has been reported that up-regulation of interleukin-4, a Th2-derived cytokine, may represent the T cell dysfunction involved in MC disease [4]. In the present study, our results revealed strong immunostaining for the RIG-I protein in the kidney specimens obtained from the cases with lupus nephritis, but negative or only very weak staining in the specimens obtained from the cases with MC disease. Imaizumi et al. [1] reported that RIG-I expression may mainly contribute to the Th1 type reaction, because its expression is induced in human endothelial cells stimulated by IFN-. Also, Paladino et al. [5] reported that TLRs and RIG-I would serve an important role during the type I IFN-dependent antiviral response in epithelial and fibroblast cell layers. Considering the reports in the literature and our own results, we suggest that the Th1 type reaction may play an important role in the expression of RIG-I in the human kidney. To the best of our knowledge, this is the first report showing the expression of RIG-I in diseased human kidneys by an immunohistochemical technique. In this study, it appeared that the amount of immunohistochemically detectable RIG-I was related to the severity of the glomerular lesions, such as that of leucocyte exudation into the glomeruli, in the specimens obtained from cases with active lupus nephritis. Two cases, with severe histological changes and a very high histological activity index, showed significantly more intense immunostaining for RIG-I. Furthermore, the staining intensity decreased in parallel with the improvement of the histological activity index at the second biopsy. These observations suggest that the expression of RIG-I in lupus nephritis could be useful as a parameter for reflecting the histological activity and severity of the renal disease.
In conclusion, we demonstrated that RIG-I expression occurs at levels detectable by indirect immunofluorescence examination, and that the intensity of its expression is correlated with the histological activity index in cases of lupus nephritis. The mechanisms by which RIG-I mediates the inflammatory and immunological processes involved in lupus nephritis still remain to be determined, and must be addressed in future studies.
Conflict of interest statement. None declared.
1Department of Pediatrics
2Department of Vascular Biology
Institute of Brain Science
Hirosaki University School of Medicine
Hirosaki 036-8562, Japan
References
- Imaizumi T, Hatakeyama M, Yamashita K, et al. Interferon-gamma induces retinoic acid-inducible gene-I in endothelial cells. Endothelium (2004) 11:169–173.[CrossRef][Web of Science][Medline]
- Austin HA, Muenz LR, Joyce KM, Antonnovych TT, Balow JE. Diffuse proliferative lupus nephritis: identification of specific pathologic features affecting renal outcome. Kidney Int (1984) 25:689–695.[Web of Science][Medline]
- Patole PS, Pawar RD, Lech M, et al. Expression of toll-like receptors in lupus-like immune complex glomerulonephritis of MRL-Fas(lpr) mice. Nephrol Dial Transplant (2006) 21:3062–3073.
[Abstract/Free Full Text] - Cho BS, Yoon SR, Jang JY, Pyun KH, Lee CE. Up-regulation of interleukin-4 and CD23/Fc
RII in minimal change nephritic syndrome. Pediatr Nephrol (1999) 13:199–204.[CrossRef][Web of Science][Medline] - Paladino P, Cummings DT, Noyce RS, Mossman KL. The IFN-independent response to virus particle entry provides a first line of antiviral defense that is independent of TLRs and retinoic acid-inducible gene I. J Immunol (2006) 177:8008–8016.
[Abstract/Free Full Text]
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