Activation of fibroblast growth factor-inducible 14 in the early phase of childhood IgA nephropathy

Abstract

IgA nephropathy (IgAN) is the most common form of glomerulonephritis worldwide. Pediatric patients in Japan are diagnosed with IgAN at an early stage of the disease through annual urinary examinations. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible 14 (Fn14) have various roles, including proinflammatory effects, and modulation of several kidney diseases; however, no reports have described their roles in pediatric IgAN. In this study, we performed pathological and immunohistochemical analyses of samples from 14 pediatric IgAN patients. Additionally, gene expression arrays of glomeruli by laser-captured microdissection were performed in hemi-nephrectomized high serum IgA (HIGA) mice, a model of IgA nephropathy, to determine the role of Fn14. Glomeruli with intense Fn14 deposition were observed in 80% of mild IgAN cases; however, most severe cases showed glomeruli with little or no Fn14 deposition. Fn14 deposition was not observed in obvious mesangial proliferation or the crescent region of glomeruli, but was detected strongly in the glomerular tuft, with an intact appearance. In HIGA mice, Fn14 deposition was observed mildly beginning at 11 weeks of age, and stronger Fn14 deposition was detected at 14 weeks of age. Expression array analysis indicated that Fn14 expression was higher in HIGA mice at 6 weeks of age, increased slightly at 11 weeks, and then decreased at 26 weeks when compared with controls at equivalent ages. These findings suggest that Fn14 signaling affects early lesions but not advanced lesions in patients with IgAN. Further study of the TWEAK/Fn14 pathway will contribute to our understanding of the progression of IgAN.

Fig 1. Fn14 expression in glomeruli from biopsied specimens obtained from patients with IgAN.

(A) Fn14 expression was evaluated semiquantitatively in each patient. Ten patients with mild disease (cases 1–10) and four patients with severe disease (cases 11–14) were analyzed. Semiquantitative scoring of Fn14 positivity was categorized as follows: 0, no staining of tufts; 1, staining within 25% of tufts; 2, staining in 25–50% of tufts; 3, staining in 50–75% of tufts; and 4, staining in more than 75% of tufts. (B) Fn14 staining was frequently detected in glomeruli with an intact appearance. Double PAS staining and Fn14 immunostaining in a case with mild IgAN (case 5 in Table 1). Two representative glomeruli are shown. Fn14 deposits existed segmentally in one glomerulus and were detected in glomeruli showing an intact appearance without any mesangial proliferation in a patient with mild disease. This patient had mild proteinuria and repeated macrohematuria during upper respiratory infection and enterocolitis.

Fig 2. Development of IgA nephropathy in HIGA mice with hemi-nephrectomy.

(A) Time course of IgA nephropathy HIGA mice. Left hemi-nephrectomy under inspiratory anesthesia was performed to accelerate glomerular injury at 6 weeks of age. After hemi-nephrectomy, the mice were sacrificed at 9, 11, 14, 26, or 40 weeks of age, and the right residual kidney was collected for further analysis. (B) Urinary protein excretion of hemi-nephrectomized HIGA mice. The mean U-pro/Cr ratios in HIGA (n = 4) and control mice (n = 3) are shown. U-pro/Cr tended to be increased in HIGA mice from 14 weeks of age, with a peak at 26 weeks of age. (C) Pathological observations in HIGA mice after hemi-nephrectomy. Light microscopy findings (PAS staining) in HIGA mice are shown. Mesangial proliferative lesions were not obvious at 6 (a), 9 (b), and 11 weeks (c). After 14 weeks, the proliferation of mesangial matrix was more evident than the mesangial cell proliferation, particularly at 40 weeks (f), apparent proliferation of the mesangial matrix was observed. 200× magnification.

Fig 3. Pathological observations in HIGA mice after hemi-nephrectomy.

(A) Immunohistochemical staining with IgA. IgA deposition was not observed at 6 or 9 weeks (a, b). Slightly IgA-positive glomeruli were detected at 11 weeks (c). IgA-positive glomeruli were frequently detectable after 14 weeks (d–f). 200× magnification. (B) Immunohistochemical staining for Fn14. Positive Fn14 staining in glomeruli was observable at 6 and 9 weeks (a, b). Fn14 deposition became more obvious at 14 weeks (d). Fn14 deposition was not evident at 26 or 40 weeks (e, f). Negative Fn14 staining in glomeruli of a control mouse (g). Arrows indicate positive Fn14 staining in glomeruli. (C) The proportion of the Fn14 stained area in the glomeruli at different time points. The average values of 10 glomeruli are shown at 6, 9, 11, 14, 26, and 40-week-old HIGA mice. *: p<0.001(Mann-Whitney U test).

Fig 4. Expression patterns of Fn14, Tweak and their downstream targets in the glomeruli of HIGA mice.

(A) Expression levels of Fn14 and Tweak in glomeruli obtained from control and HIGA mice at each time point. Normalized counts of each gene are used to indicate the expression level. (B) Comparison of the expressions of genes reported to be regulated by the Tweak/Fn14 pathway. (C, D) Representative pattern of gene expression is shown.

Fig 5. Expression pattern of differentially expressed genes in glomeruli obtained from HIGA mice.

(A) Differentially expressed genes in the glomeruli of HIGA mice at each time point. (B) Several biological pathways were identified from these differentially expressed gene sets. Representative pathways and expression patterns of the involved genes are shown. Ratio of the normalized count of HIGA mice to control mice was used as a relative normalized count.