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