Eishin Oki

Retinoic acid-inducible gene-I is induced by double-stranded RNA and regulates the expression of CC chemokine ligand (CCL) 5 in human mesangial cells

BACKGROUND Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase involved in immune reactions against RNA viruses and various inflammatory and autoimmune diseases. The purpose of the present study was to investigate the role of RIG-I in glomerular diseases. METHODS We treated human mesangial cells in culture with polyinosinic-polycytidylic acid (poly IC), which is an authentic double-stranded RNA, and analysed the expression of RIG-I, CC chemokine ligand 5 (CCL5) and interferon (IFN)-β by western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbent assay (ELISA). To elucidate the poly IC-signalling pathway, we subjected the cells to RNA interference (RNAi) against RIG-I, IFN-β or Toll-like receptor (TLR) 3. Furthermore, we studied the effects of IFN-β receptor blocking and IFN-β overexpression. RESULTS Poly IC induced the expression of RIG-I and CCL5 in human mesangial cells, and RNAi against RIG-I inhibited this poly IC-induced CCL5 expression. Poly IC-induced RIG-I expression was also inhibited by RNAi against IFN-β and by an antibody against the IFN-β receptor. IFN-β overexpression induced the expression of both RIG-I and CCL5. The knockdown of TLR3 abolished poly IC-induced RIG-I expression. CONCLUSIONS The TLR3/IFN-β/RIG-I/CCL5 signalling pathway may mediate immune and inflammatory responses against viral infection in mesangial cells, suggesting the role of this pathway in the aggravation of glomerulonephritis due to viral infection.

Retinoic acid-inducible gene-I (RIG-I) is induced by IFN-γ in human mesangial cells in culture: possible involvement of RIG-I in the inflammation in lupus nephritis

Interferon-γ is a potent Th1-type cytokine and a key molecule in the pathogenesis of autoimmune diseases including lupus nephritis. Retinoic acid-inducible gene-I is a putative RNA helicase that plays an important role in immune and inflammatory reactions. We previously demonstrated the increased expression of the retinoic acid-inducible gene-I protein in the kidney tissue of patients with lupus nephritis, and the presence of a significant amount of retinoic acid-inducible gene-I mRNA in the urinary sediment of patients with this inflammatory renal disease. In the present study, interferon-γ was found to induce the expression of retinoic acid-inducible gene-I in human mesangial cells in culture. Knockdown of retinoic acid-inducible gene-I inhibited the interferon-γ-induced upregulation of interferon regulatory factor 7, a transcriptional factor involved in immune and inflammatory reactions. These findings suggest that retinoic acid-inducible gene-I produced by mesangial cells may be involved in the pathogenesis of lupus nephritis.

Mizoribine Treatment of Young Patients with Severe Lupus Nephritis: A Clinicopathologic Study by the Tohoku Pediatric Study Group

Background: A novel purine synthesis inhibitor, mizoribine (MZR), with a similar activity to that of mycophenolate mofetil, was developed in Japan. We suspected that long-term oral MZR intermittent pulse therapy (MZR-P) might be more effective than the conventional daily MZR regimen due to the higher peak serum MZR levels that are achieved. Here, we examined the clinicopathologic efficacy of MZR-P treatment in 10 young patients with diffuse proliferative lupus nephritis (DPLN), including 3 patients who received MZR-P as their primary cytotoxic therapy. Methods: After their most recent renal flare-ups, all the patients were treated using MZR-P combined with oral prednisolone (PDN). MZR was administered as a single daily dose of 6–10 mg/kg per day (maximum dose of 500 mg) on 2 days of the week (Monday and Thursday) for at least 12 months or longer. The concomitantly administered PDN dose was gradually reduced. Results: The baseline characteristics of the patients were as follows: mean age 15 years; urinary protein/creatinine (Up/cr) ratio 1.57 ± 1.05 mg/mg; serum C3 level 50.3 ± 19.7 mg/dl; serum complement hemolytic activity (CH50) 18.1 ± 9.9 U/ml; serum anti-dsDNA antibody titer 177.5 ± 152.7 IU/ml; serum creatinine 0.6 ± 0.1 mg/dl, and European Consensus Lupus Activity Measurement (ECLAM) index 4.9 ± 2.6. Despite the gradual tapering of the PDN dose, marked improvements compared with the baseline values were observed at the last observation, mean interval of 29 months after the start of treatment: Up/cr ratio 0.19 ± 0.14; ECLAM index 1.3 ± 0.7 (p < 0.01); serum C3 level 76.5 ± 22.1 mg/dl; serum CH50 value 31.9 ± 8.7 U/ml, and anti-dsDNA antibody titer 34.2 ± 20.5 IU/ml (p < 0.05). The serum creatinine level remained within the normal range in all study participants. Post-treatment renal biopsies were performed in 5 of the patients; histology showed a marked attenuation of lesion progression. No serious adverse effects were observed. Conclusion: We believe that long-term MZR-P may prove to be a treatment of choice for young patients with DPLN. However, confirmation is needed as this preliminary study is limited by the small number of subjects, lack of controls, and its retrospective nature.

Mizoribine attenuates renal injury and macrophage infiltration in patients with severe lupus nephritis.

The purine synthesis inhibitor mizoribine (MZR) has been successfully used without serious adverse effects in the treatment of several renal diseases including lupus nephritis. Besides its immunosuppressive effects, MZR has recently been reported to ameliorate tubulointerstitial fibrosis in rats via suppression of macrophage infiltration. However, there has been little information regarding the beneficial effects of MZR from the histologic standpoint in human lupus nephritis. Pre- and posttreatment renal biopsy specimens obtained from nine patients with diffuse proliferative lupus nephritis (DPLN) were divided into two groups (group A, five patients who received immunosuppressive treatment with MZR and group B, four patients who received immunosuppressive treatment without MZR) and histologically evaluated. Grading was done according to the 2003 classification system for lupus nephritis developed by the International Society of Nephrology/Renal Pathology Society, which considers the activity and chronicity indices, an immunohistologic study to assess intraglomerular and interstitial infiltration by macrophages, and the expression of osteopontin. Although in all the patients the posttreatment renal biopsy showed improvement of histologic grading and activity indices, group A patients showed a significant decrease of the chronicity indices and of intraglomerular infiltration by macrophages when compared to group B patients (2.6 ± 0.5 vs 4.0 ± 1.4 and 0.5 ± 0.2 vs 2.4 ± 1.9 cells per glomerulus, respectively; p < 0.05). Although this was a preliminary study in a small number of subjects, these histological observations may further confirm the beneficial effects of MZR for selected patients with DPLN.

Expression of mRNA for functional molecules in urinary sediment in glomerulonephritis

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 method for clinical monitoring and studying of pathogenesis in glomerulonephritis.

Long-Term Tacrolimus-Based Immunosuppressive Treatment for Young Patients with Lupus Nephritis: A Prospective Study in Daily Clinical Practice

Background: The optimal long-term treatment for lupus nephritis (LN) in pubertal patients remains to be determined. Tacrolimus (Tac) inhibits T cell activation, and is therefore expected to be effective in patients with LN. However, little has been published about the long-term efficacy and safety of Tac-based immunosuppressive treatment of young patients with LN in daily clinical practice. Methods: Nineteen consecutive patients with biopsy-proven LN were recruited for an open-label, prospective, long-term Tac-based treatment regimen. Tac was administered once daily at a dose of 3 mg as induction- or reinduction-maintenance treatment. Four patients (21%) with new-onset LN received mizoribine at a dose of 150 mg once daily in addition to Tac. Treatment outcomes were defined by the European Consensus Lupus Activity Measurement (ECLAM) index, urinary protein/creatinine ratio (Up/cr), serum creatinine and serological lupus markers (complement C3, complement hemolytic activity, CH50, and anti-dsDNA antibody titer). Data on these parameters were collected prospectively. The median follow-up was 42 months. Results: Baseline characteristics of the patients were as follows: mean age, 18 years; Up/cr, 0.89 ± 1.17; serum C3, 68.1 ± 23.2 mg/dl (normal, 79–152 mg/dl); serum CH50, 26.4 ± 10.5 U/ml (normal, 23–46 U/ml); serum anti-dsDNA antibody titer, 69.3 ± 67.5 IU/ml (normal, <12.0 IU/ml); serum creatinine, 0.55 ± 0.18 mg/dl, and ECLAM index, 4.6 ± 1.9. Despite gradually tapering the dose of concomitantly administered prednisolone, a marked improvement compared with baseline values was observed in all outcome measures as early as 3 months after the initiation of treatment, and the favorable changes persisted throughout the treatment period in most of the patients. Sustained improvements in the outcome measures compared with the baseline values were confirmed after a mean of 42 months of treatment: ECLAM index, 1.1 ± 1.1; serum CH50, 36.0 ± 12.8 U/ml, anti-dsDNA antibody titer, 22.5 ± 26.5 IU/ml (all p < 0.01); Up/cr ratio, 0.35 ± 0.58, and serum C3 level, 79.7 ± 17.6 mg/dl (both p < 0.05). Serum creatinine level remained within the normal range in all the study participants. Complete response was achieved in 12 patients (63%), and a partial response was achieved in 5 patients (26%). The remaining 2 patients showed no response. No serious adverse effects were observed. Conclusion: The data suggest that long-term, relatively low-dose Tac-based immunosuppressive treatment is beneficial and has low cytotoxicity, and therefore represents an attractive option for the treatment of young patients with LN in daily clinical practice. Further studies involving a larger number of patients are needed to confirm these results.

Treatment of difficult cases of systemic-onset juvenile idiopathic arthritis with tacrolimus

Since a proportion of systemic-onset juvenile idiopathic arthritis (SOJIA) patients continue to require long-term corticosteroid therapy for disease control, an effective and safe therapeutic strategy for controlling the activity of refractory SOJIA remains to be established. We report the efficacy of tacrolimus for the treatment of SOJIA in two patients with refractory SOJIA, one of them showing poor response to cyclosporine A. Tacrolimus might be the treatment of choice in selected patients with refractory systemic-onset juvenile idiopathic arthritis. Further studies to confirm the long-term efficacy and safety of tacrolimus in larger numbers of patients are, however, needed.

Efficacy of mizoribine-tacrolimus-based induction therapy for pediatric lupus nephritis:

Background Recent advances in the management of lupus nephritis (LN) have also contributed to a favorable outcome in patients with pediatric-onset LN. Nevertheless, we believe that a more effective and less toxic treatment is needed to attain optimal control of pediatric-onset LN. Methods Seven consecutive children with biopsy-proven LN (four with class III/IV and three with class V) received multitarget induction therapy consisting of mizoribine (MZR), tacrolimus (Tac), and prednisolone (PDN). They were prospectively evaluated at three, six, and 12 months, and at the latest observation point after a mean period of 32 months. Post-treatment renal biopsy was performed in two patients with class III/IV. Results Despite gradually tapering the dose of concomitantly administered PDN, a significant improvement compared with baseline values was observed in the urinary, serological, and clinical assessment measures even at three months of treatment, and the favorable changes persisted throughout the treatment period in most of the study participants except for one. In two patients who underwent post-treatment renal biopsy, a marked histologic improvement was confirmed. No serious adverse events were observed. Conclusions Multitarget therapy may be an attractive option for the treatment of pediatric-onset LN. Further studies involving a larger number of patients are needed.

Imbalance towards Th1 pathway predominance in purpura nephritis with proteinuria

Although recent reports suggest a possible role for an imbalance in Th1 and Th2 proinflammatory cytokines in the development and progression of glomerulonephritis, there is little information on Th1 or Th2 predominance in Henoch–Schönlein purpura nephritis (HSPN). Since T-bet and GATA-3 are transcriptional factors that regulate the differentiation of helper T lymphocytes into Th1 and Th2, we examined the relative mRNA expression of T-bet and GATA-3 in the urinary sediment of children with proteinuric HSPN by real-time quantitative PCR. Eight consecutive patients with proteinuric HSPN (4 with grade IIIa and 4 with grade IIIb according to the International Study of Kidney Disease in Children criteria) and 20 healthy subjects were enrolled in this study. The relative expression level of T-bet was significantly higher in the urinary sediment of patients with HSPN at presentation than in that of the healthy controls (p = 0.021), while the relative expression of GATA-3 was significantly lower in the urinary sediment of patients than in that of controls (p = 0.002). Urinary mRNA expression of T-bet correlated with the urinary protein/creatinine ratio (r = 0.608, p = 0.013), and correlated inversely with serum level of total protein (r = −0.574, p = 0.020). Moreover, a significantly increased intensity of T-bet immunostaining was observed in the glomeruli in the biopsy specimen of all study patients. All patients received immunosuppressive therapy. Repeat measurements of urinary mRNA expression of T-bet and GATA-3 after treatment revealed that the expression of T-bet in patients had significantly decreased relative to the baseline (p = 0.003), while the expression of GATA-3 remained static. In a patient subjected to a post-treatment renal biopsy, the increased intensity of immunostaining of T-bet had clearly diminished following immunosuppressive treatment, in accordance with a significant decrease in urinary mRNA expression of T-bet. These observations suggest that patients with proteinuric HSPN demonstrate increased T-bet and depressed GATA-3 expression in the urinary sediment, indicating a possible shift in Th1/Th2 balance towards Th1 predominance.

Potential Th1/Th2 predominance in children with newly diagnosed IgA nephropathy

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis in children and adults. Although its pathogenesis remains to be elucidated, recent reports have suggested a possible role of imbalance of Th1 ⁄ Th2 proinflammatory cytokines in the development and progression of IgAN (1–3). Because T-bet and GATA3 are the transcriptional factors for the differentiation of helper T lymphocytes toward Th1 and Th2 (4), we examined mRNA expression of T-bet and GATA3 in urinary sediment of children with newly diagnosed IgAN and correlated their intra-renal protein expression with pathological findings assessed using a semiquantitative histologic grading system. Seven consecutive children who were newly diagnosed with IgAN at our hospital from January 2008 to April 2009 were enrolled in this study. The patients were four boys and three girls with a median age of 12 years (range, 7–15 years) at entry. All the patients had normal blood pressure and normal renal function at presentation. A diagnostic renal biopsy was performed within 3 months from the initial detection of urinary abnormalities in all of them. The biopsy specimens were scored semiquantitatively in a blinded fashion by one of the authors, using the scoring system for childhood IgAN described by Andreoli and Bergstein (5). The activity index (AI) was determined by grading mesangial proliferation, interstitial mononuclear cell infiltration and cellular crescent formation using a 4-grade scale (0, none; 1, mild; 2, moderate; 3, severe), on the basis of the percentage of glomeruli involved (0, 0%; 1, 1–20%; 2, 21–50%; 3, >50%). The chronicity index was determined by counting the number of glomeruli demonstrating fibrous crescents and segmental or global sclerosis, each being scored on a scale of 0–3, on the basis of the percentage of glomeruli involved as indicated. Tubular atrophy and interstitial fibrosis were each scored on a scale of 0–3. The sum of these numbers comprised the AI (maximum = 9) and the chronicity index (maximum = 12), respectively. A whole-stream, early morning, urine sample was collected for the gene expression study (4,6). Briefly, the urine samples were centrifuged at 1650 g for 30 min. Total RNA was extracted using an RNeasy Mini Kit (Qiagen Science, Germantown, MD, USA), according to the manufacturer’s instructions. All the specimens were stored at )80 C. The purity of RNA was confirmed by the relative absorbance at 260 ⁄ 280 nm ratio. We quantified the mRNA expression of T-bet and GATA3 in the urinary sediment. For each reaction, approximately 0.1 lg of total RNA was reverse transcribed to cDNA using an iScript cDNA synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). The resulting cDNAs were stored at )80 C until required. The relative mRNA abundance was quantified using Chromo 4 (BioRad Laboratories). The primer sequences were as follows: T-bet: -forward, 5¢-CTG CCT ACC AGA ATG CCG AGA3¢, reverse, 5¢-AAG CGG CTG GGA ACA GGA TAC-3¢-; GATA3: -forward, 5¢-GCA GGA GCA GTA TCA TGA AGC CTA A-3¢, reverse, 5¢-TTG GAA CAC AGA CAC CAC AGT GAG-3¢-; and 18S rRNA: -forward, 5¢-ACT CAA CAC GGG AAA CCT CA-3¢, reverse, 5¢-AAC CAG ACA AAT CGC TCC AC-3¢(Takara Bio Inc, Otsu, Japan). The mRNA expression level of each target was normalized to that of the housekeeping gene, 18S rRNA. PCR amplifications were performed in 20 lL at 95 C for 3 min, followed by 40 cycles of incubation at 95 C for 10 sec and at 60 C for 30 sec. Each sample was run in triplicate. The results were analysed 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 patients was calculated by the 2 method (4,6). Urine Acta Pædiatrica ISSN 0803–5253