Activation of the interleukin-4/signal transducer and activator of transcription 6 signaling pathway and homeodomain-interacting protein kinase 2 production by tonsillar mononuclear cells in IgA nephropathy.

BACKGROUND/AIM: Clinical development and exacerbation of IgA nephropathy (IgAN) are frequently preceded by episodes of upper respiratory tract infection such as tonsillitis. This study aimed to determine the role of the interleukin-4 (IL-4)/signal transducer and activator of transcription 6 (STAT6) signaling pathway and homeodomain-interacting protein kinase 2 (HIPK2) in aberrant IgA1 O-glycosylation production, and identify potential therapeutic targets in IgAN.

METHODS: Expression levels of IL-4, STAT6, core1β1,3-galactosyltransferase (C1GALT1C1), core1β3GalT-specific molecular chaperone (Cosmc) and HIPK2 in tonsil components were examined by immunohistochemical and immunofluorescence staining. Lymphocytes isolated from 22 patients with IgAN and 24 patients with chronic tonsillitis (CT) as controls were cultured for 72 h with or without IL-4, lipopolysaccharide (LPS) and α-hemolytic streptococcus (HS) stimulation. Expression levels of STAT6, C1GALT1C1, Cosmc, HIPK2-mRNA and protein were measured by real-time PCR and Western blot analysis, respectively. The concentration of IgA1 and level of O-glycosylation were determined by ELISA and Vicia villosa (VV) lectin-binding assay. To determine the contribution of HIPK2 in IgA secretion and O-glycosylation, cells were subjected to experiments for evaluation of HIPK2 silencing by Hipk2-siRNA transfection.

RESULTS: The IL-4/STAT6 signaling pathway was highly activated in all tonsil tissues (including the germinal center and tonsillar crypt epithelium) of IgAN patients, but the gene or protein expression of β1,3-Gal transferase (C1GALT1) and COSMC decreased significantly in patients with IgAN in comparison with those with CT. Hipk2 production in the tonsils derived from IgAN patients was significantly higher than that of CT patients. HIPK2-mRNA expression significantly negatively correlated with renal function as expressed by the estimated glomerular filtration rate, and also significantly positively correlated with daily proteinuria. The level of IL-4, STAT6 and HIPK2 were closely related with Lee's pathological grading system. The levels of mRNA and protein encoding STAT6 and Hipk2 in cells coincubated with IL-4, LPS and HS were significantly higher than those in the controls without stimulation; however, in the IgAN group the levels of mRNA and protein encoding C1GALT1 and Cosmc were significantly lower compared to the controls. IgA1 concentrations of supernatants in IgAN patients were remarkably higher under conditions of external stimulation. As expected, the optical density value of VV lectin binding to IgA1 increased after external stimulation in the IgAN group. By siRNA transfection, our results clearly indicate that Hipk2 negatively regulates C1GALT1 and Cosmc expression. Importantly, HIPK2-siRNA attenuates the aberrant glycosylation of IgA1 secretion.

CONCLUSION: We identified and confirmed that activation of the IL-4/STAT6 signaling pathway has a crucial role in aberrant glycosylation of IgA1 secretion. HIPK2, a protein kinase previously unrecognized in kidney disease, may mediate the glycosylation of IgA1. We believe that HIPK2 could be a new therapeutic target for IgAN, especially as protein kinases are 'drugable' targets.