| Background:IgA nephropathy (IgAN) is recognized as the most common form of primary glomerulonephritis worldwide.Approximately20%to40%of patients with IgAN progress to end-stage renal failure within20years since diagnosis.Although multiple mechanisms have been suggested, the pathogenesis and mechanisms of IgAN remain to be elucidated.B-cell-activating factor belonging to the tumor necrosis factor family (BAFF)has been found to have the function of activating B cells and participating in the class switching of B cells.It is critical for the maintenance of normal B cell development and homeostasis.However, its clinical application needs further study.Increasing evidence suggests that IgAN is an immune-complex (IC) disease in which circulating immune complexes (CICs)are deposited in the glomerular mesangium.The composition of CICs and that of ICs in the mesangium of IgAN patients display a remarkable parallelism:IgA1is the exclusive IgA subclass and C3is commonly present. Comparative structural studies of the amino-acid sequences of IgA1and IgA2H chains revealed major differences in their hinge region. IgA1contains an extended polyproline peptide bearing multiple serine and threonine residues and distinctive O-glycans.Considerable evidence has revealed that abnormalities of IgA1O-glycosylation may be one of the key pathogeneses of IgAN over the past10years. The IgA1O-glycans proceeds by the addition of Gal to GalNAc at position β1,3,which is catalyzed by a single UDP-Gal:GalNAc-α-Ser/Thr β1,3-galactosyltransferase (ClGalTl)working with its chaperone Cosmc (core I β3-Gal-T-specific molecular chaperone). This process may be blocked through the attachment of SA to the GalNAc residues forming gal-deficient IgA1(Gd-IgA1),which is catalyzed by an a2,6-sialyltransferase (ST6GalNAc).And ST6GalNAc-II may participate in the synthesis of Gd-IgA1through the studies of IgA1-producing cells. C1GALT1activity was remarkably lower in peripheral B lymphocyte of IgAN patients when compared with that of controls, and the Cosmc mRNA expression level in IgAN patients was significantly decreased and correlated with IgA glycosylation abnormality degree as well as clinical manifestations.Although the pathogenesis of this disease is unclear, some researchers suggested that it might not be genetic disorders but external suppression that causes the high ST6GalNAc-II and low COSMC and C1GALT1C1mRNA expression in IgAN.It is known that many IgAN patients manifest episodic macrohematuria, which coincides with mucosal infection especially in the upper respiratory tract or gastrointestinal tract. Previous studies show that a transient deterioration of urinal findings and the high level of serum IgA and IgAl after tonsillectomy. That suggests infection of tonsil can be simulated by tonsillectomy. This study focus on the changes of BAFF and the factors of aberrant IgA1glycosylation in peripheral blood before and after tonsillectomy, mimicking the infection of tonsil, compared the difference of the patients with IgA nephropathy, patients without IgA nephropathy and healthy people who didn’t undergo tonsillectomy, to study the activation of B cells, expression of IgA1,aberrant IgAl glycosylation and the relationship between them. Chapter I. Comparison of BAFF and IgAl in peripheral blood before and after tonsillectomy in the patients with IgA nephropathyObjective:To detect the different expression levels of BAFF in peripheral blood mononuclear cells(PBMCs)and IgAl in plasma before and after tonsillectomy in the patients with IgA nephropathy, analyze the relationship between B cells activation, IgAl expression and tonsil.Methods:Peripheral blood samples were obtained from16patients with IgAN before(24-48h)and after(36-60h) tonsillectomy. Diagnosis of IgAN based on immunofluorescent and light microscopic findings from percutaneous renal biopsy tissue. Peripheral blood samples from patients without renal diseases (non-IgAN) before and after tonsillectomy and16healthy people (healthy control) were obtained as controls. The expression of BAFF mRNA in PBMCs was tested by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). IgAl content in plasma was measured in duplicate using ELISA.Results:1.The expression of BAFF mRNA in the patients with IgAN before tonsillectomy was significantly higher than healthy people,(P<0.01); BAFF mRNA expression in IgAN patients post-tonsillectomy was significantly higher than that of pre-tonsillectomy,(P<0.01),and in non-IgAN patients post-tonsillectomy (P<0.05). 2.The concentration of IgAl in IgAN patients’ plasma pre-tonsillectomy was higher than healthy people (P<0.05).The concentration of IgAl in post-tonsillectomy IgAN patients’plasma was higher than pre-tonsillectomy (P<0.01),than post-tonsillectomy non-IgAN patients (P<0.05).3.The level of BAFF mRNA expression significantly positively correlated with IgA1concentration (r=0.604, P<0.01).Conclusion:Through stimulating tonsil, we observed increase gene expression of BAFF in PBMCs from the patients with IgAN, elevated concentration levels of IgAl in plasma. And it seems that BAFF plays a key role in IgAl secretion in peripheral blood. BAFF and its gene expression are also expected to be a biomarker in assessment of the change of IgAN. Chapter Ⅱ.The expression of ST6GalNAc Ⅱ, C1GALT1, COSMC in peripheral blood before and after tonsillectomy and its relationship with aberrant IgAl glycosylation in patients with IgA nephropathyObjective:To detect the different expression levels of ST6GalNAc II, C1GALT1and COSMC in PBMCs and aberrant O-glycosylation IgA1in plasma before and after tonsillectomy in the patients with IgA nephropathy, analyze the correlation between tonsil and aberrant O-glycosylation IgA1.Methods:Immunohistochemical stains were performed to detect the expression of ST6GalNAc Ⅱ in tonsil components.The expression of ST6GalNAc Ⅱ, C1GALT1and COSMC mRNA in PBMCs was tested by qRT-PCR. The levels of IgAl O-glycosylation were determined by Vicia villosa (VV) lectin-binding assay.Results:1.The expression of ST6GalNAc Ⅱ was higher in IgAN patient’s tonsil compared to non-IgAN patient. They localized in all tonsil components (including germinal center and tonsillar crypt epithelium) of IgAN patient.2.The expression of ST6GalNAc Ⅱ mRNA in the patients with IgAN before tonsillectomy was significantly higher than healthy people,(P<0.01);ST6GalNAc II mRNA expression in IgAN patients post-tonsillectomy was significantly higher than pre-tonsillectomy,(P<0.01), and in non-IgAN patients post-tonsillectomy (P<0.05).3.In IgAN patients,the expression of ST6GalNAc II mRNA36to48hours post-tonsillectomy was significantly higher than pre- tonsillectomy,about1.95times (P<0.01),and the expression of ST6GalNAc II mRNA48to60hours post-tonsillectomy was significantly higher than pre-tonsillectomy,about1.38times (P<0.01).4. In IgAN patients, the expression of C1GALT1and COSMC mRNA post-tonsillectomy was significantly lower than pre-tonsillectomy (P<0.05).5.The OD value of VV lectin binding ELISA of IgAl in IgAN patients’plasma was significantly higher than that of healthy people (P<0.01).There was no significant difference between pre-and post-tosillectomy.6. The level of ST6GalNAc II mRNA expression significantly positively correlated with VVL-binding ELISA OD value (r=0.604, P<0.01).Conclusion:Through stimulating tonsil, we observed transient increase gene expression of ST6GalNAc II in PBMCs from the patients with IgAN, and decrease gene expression of C1GALT1and COSMC. The concentration level of Gd-IgAl in IgAN patients’plasma was higher than in healthy people. According to previous studies and our studies, it suggested that increase gene expression of ST6GalNAc II and decrease gene expression of C1GALT1and COSMC in PBMCs leaded to increase Gd-IgAl secretion in peripheral blood. |
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