IL-4/STAT6、HIPK2对IgA肾病人腭扁桃体低糖基化IgA1表达的调控机制研究
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摘要
研究背景：
IgA肾病是世界范围内最常见的原发性肾小球疾病,大约20%的患者在20年内会进展至终末期肾功能衰竭,是导致尿毒症最常见原因,然而其发病机制尚不明确。IgA肾病以IgAl/低糖基化IgA1沉积于肾小球系膜区及毛细血管袢为病理特征,以血尿蛋白尿、高血压及肾功能损害为主要临床表现。IgAl与IgA2的主要区别点在与IgAl存在铰链区。在IgAl铰链区,β1,3半乳糖转移酶(C1GALT1)在其特异性分子伴侣COSMC的辅助下,通过UDP-半乳糖载体将半乳糖转移至IgA1的β1,3位乙酰半乳糖胺残基上,完成IgA1的糖基化修饰。近年的研究证实低糖基化IgA1是IgA肾病的关键致病因子。IgA肾病外周血B淋巴细胞β1,3半乳糖转移酶及分子伴侣活性COSMC明显下降,并且与IgAl病理损伤严重程度呈正相关,但是其活性下降的具体机制尚未阐明。
有学者认为外界环境因素的刺激,而非基因遗传因素,导致IgA肾病IgA1铰链区异常糖基化。脂多糖能抑制C1GALT1及COSMC活性；体外实验证实Th2细胞因子IL-4能下调C1GALT1及COSMC的表达,影响IgAl铰链区糖基化；IL-4及其下游转录因子STAT6,能够调节Th2细胞分化,促进炎症反应的发生。然而目前尚未有研究涉及IL-4/STAT6信号通路与IgA肾病IgA1及其糖基化之间的关系。
同源异型结构域相互作用蛋白激酶-2(homeodomain-interactiing protein kinase-2HIPK2)是丝/苏氨酸激酶,包括HIPK1、HIPK2与HIPK3。HIPK2通过相互作用蛋白对靶点进行磷酸化修饰,调控相关蛋白酶的活性及基因转录表达。有学者在对IL-4刺激STAT6-/-与STAT6+/+小鼠B细胞基因表达研究中,发现HIPK2表达尤为活跃,提示HIPK2在IL-4(JAK/STAT6)信号通路中具有重要作用。在IL-4诱导JAK/STAT6信号通路中,HIPK2表达明显升高,提示HIPK2可能参与IL-4介导JAK/STAT6信号通路中相关蛋白酶的磷酸化修饰及转录表达调控。已证实,IL-4介导C1GALT1的活性及分子伴侣COSMC表达调控,这让我们思考,HIPK2是否通过调控C1GALT1的活性及分子伴侣COSMC表达,参与IL-4(JAK/STAT6)信号通路介导IgA肾病腭扁桃体淋巴细胞低糖基化IgA1形成的作用机制。
我们知道IgA肾病患者在黏膜组织,特别是上呼吸道和肠道黏膜感染后常出现阵发性肉眼血尿。许多回顾性或前瞻性临床对照研究证实：IgA肾病患者摘除腭扁桃体后尿检正常率、肾功能稳定率和肾脏生存率均高于对照组；循环中lgA水平明显下降。目前很多学者认为IgA肾病的发生与黏膜组织对外来抗原免疫应答紊乱有关。我们前期的研究证实,革兰氏阳性菌的代表甲型溶血性链球菌是慢性扁桃体炎最常见的致病菌,而脂多糖是革兰氏阴性菌细胞壁的主要抗原成分,本课题中我们采用灭活的甲型溶血性链球菌和脂多糖作为干预手段,模拟扁桃体感染,观察腭扁桃体单个核细胞IL-4/STAT6、HIPK2表达的变化,分析其与IgA1及其糖基化之间的关系(学术思想见下图),为IgA肾病的防治提供新的理论依据和治疗靶点。
第一章IL-4/STAT6、HIPK2、C1GALT1及COSMC在腭扁桃体中的表达及其与IgA1糖基化的相关性研究
目的：检测腭扁桃体IL-4、STAT6、HIPK2、(31,3半乳糖苷转移酶(C1GALT1)及分子伴侣COSMC的表达,分析IL-4/STAT6、HIPK2与IgA1及其铰链区糖基化之间的关系。
方法：收集经临床和肾脏病理确诊为IgA肾病患者腭扁桃体22例做为观察组(IgAN组),慢性扁桃体炎非肾炎患者腭扁桃体24例作为对照组(CT组)。免疫组织化学检测腭扁桃体组织IL-4、STAT6、HIPK2的表达水平；免疫荧光检测C1GALT1及COSMC;密度梯度离心法分离腭扁桃体单个核细胞,采用RT-PCR和Western blot技术分别检测IL-4、STAT6、HIPK2、C1GALT1及COSMC mRNA和蛋白表达；ELISA检测培养液上清中IgA1含量,蚕豆凝集素(VVL)亲和ELISA法测定VVL与IgA1结合力,间接检测IgA1铰链区糖基化水平。
结果：
1.免疫组化结果显示与CT组比较,IgAN组IL-4/STAT6、 HIPK2在网状上皮及淋巴小结生发中心分布明显增加；免疫荧光提示IgAN组C1GALT1及COSMC在腭扁桃体组织中表达较CT组减少；
2.RT-PCR及Western blot结果均提示IgAN组IL-4/STAT6、 HIPK2表达较CT组明显升高,C1GALT1、COSMC表达较CT组下降；
3.IgAN组腭扁桃体单个核细胞培养上清IgA1含量较CT组明显升高；VVL-ELISA结果提示IgAN组IgA1与蚕豆凝集素结合力明显高于CT组,说明IgAN组腭扁桃体单个核细胞分泌的IgA1存在明显的低糖基化；
4.相关性分析提示IgAN腭扁桃体单个核细胞HIPK2mRN表达与eGFR呈负相关,与蛋白尿、IgA1及VVL-ELISA OD值呈正相关。
结论：IL-4/STAT6信号通路在IgAN患者腭扁桃体中处于激活状态；HIPK2在IgAN腭扁桃体单个核细胞中高表达,HIPK2与IgAN主要临床指标、IgA1及其糖基化水平关系密切,提示HIPK2是IgAN的重要致病因子。
第二章脂多糖、甲型溶血性链球菌对腭扁桃体Th1.Th2及Th1/Th2的影响
目的：研究IgAN腭扁桃体Th1/Th2极化是否存在异常；观察脂多糖、甲型溶血性链球菌刺激对腭扁桃体Th1、Th2及Th1/Th2的影响。
方法：将IgAN和CT患者腭扁桃体单个核细胞各分成三组,第一组为空白对照组；第二组加入脂多糖LPS(浓度为10ug/ml)；第三组加入灭活的甲型溶血性链球菌HS(浓度为1×108cfu/ml).培养72小时后加入PMA/Ionomycin mixture及BFA/Monensin mixture作为淋巴细胞刺激剂孵育6小时,CD3+CD8-设门,流式细胞术检测IL-4、IFN-γ含量,Thl为CD3+/CD8-/IFN-γ+细胞,Th2为CD3+/CD8-/IL-4+细胞。
结果：
1.IgAN组Th1:5.73±2.14%；Th2:1.24±0.55%；Th1/Th2:4.62±2.12。C组Th1:4.71±1.98%；Th2:0.37±O.22%;Th1/Th2:12.73±5.78。
2.LPS刺激后IgAN组Thl:13.30±4.15%；Th2:1.20±0.78; Th1/Th2:11.08±4.33。CT组Th1:7.39±3.78%；Th2:0.58±0.33%; Th1/Th2:12.74±5.01.
3.予以HS刺激后IgAN组Th1:7.03±3.14%;Th2:8.11±3.98%Th1/Th2:0.87±0.25。CT组Th1:3.52±2.55%；Th2:0.77±0.41%; Th1/Th2:4.57±1.92.
4.采用Pearson相关分析及直线回归,发现IgAN组Th1/Th2匕值与蛋白尿呈负相关,r=-0.785。
结论：IgAN腭扁桃体中Th2细胞免疫占优势地位,并且与蛋白尿等临床指标的严重程度呈正相关；LPS、HS能促使Th1/Th2极化失衡,其中LPS促进Thl分化为主,HS促进Th2分化更明显。
第三章白介素-4、脂多糖、甲型溶血性链球菌对腭扁桃体单个核细胞STAT6、HIPK2、C1GALT1/COSMC、IgAl及其糖基化的影响
目的：采用IL-4、LPS、HS作为干预刺激,探讨IL-4/STAT6信号通路、HIPK2与低糖基化IgA1表达的关系。
方法：腭扁桃体单个核细胞培养体系中,分别加入用IL-4(浓度为10ng/ml)、LPS (终浓度为10ug/ml)、灭活的HS(浓度为1×108cfu/ml)刺激72小时,RT-PCR, Western blot技术,分别测定STAT6、HIPK2、C1GALT1、COSMC mRNA和蛋白表达,ELISA技术测定培养液上清中IgA1及其糖基化水平的变化。
结果：
1.IL-4、LPS、HS均能上调IgAN组STAT6、HIPK2mRNA和蛋白表达,IL-4、HS较LPS上调作用更为明显；同时IL-4、LPS、HS均能下调C1GALT1及COSMC mRNA和蛋白表达,HS较IL-4、LPS下调作用更明显。
2.CT组在接受IL-4、LPS、HS刺激后,STAT6、HIPK2mRNA和蛋白表达上调,但升高程度较IgAN组低；C1GALT1、COSMC mRNA和蛋白表达较空白对照组无明显变化。
3.IgAN组及CT组腭扁桃体单个核细胞IgA1分泌较空白对照组明显增加,其中IL-4、HS较LPS刺激单个核细胞分泌IgA1效果更为明显；蚕豆凝集素亲和ELISA测定IgA1半乳糖基水平,IgAN刺激组较空白对照组VVL-ELISAOD值明显增加,HS刺激对OD值影响较IL-4、LPS大；IL-4、LPS、HS刺激后,CT组VVL-ELISA检测所得OD值无明显变化。
结论：IL-4、LPS、HS能激活IgA肾病腭扁桃体IL-4/STAT6信号通路,促进HIPK2表达,从而下调C1GALT1/COSMC活性,导致IgA1分泌增多,IgA1糖基化水平下降。
第四章沉默HIPK2基因对IgAl及其糖基化的影响
目的：HIPK2在IgA肾病腭扁桃体中高表达,且与低糖基化IgAl分泌关系密切。我们拟采用HIPK2-siRNA沉默HIPK2基因表达,观察其对腭扁桃体细胞IgAl及糖基化的影响。
方法：分离腭扁桃体单个核细胞,转染HIPK2-siRNA, Annexin V-FITC法检测转染后细胞凋亡率；RT-PCR、Western blot分别检测HIPK2-siRNA转染后,C1GALT1及COSMC mRNA和蛋白表达；ELISA测定培养液上清中IgAl及其糖基化水平的变化。
结果：
1.HIPK2-siRNA转染单个核细胞后24小时,流式细胞术测得早期凋亡率百分比为(13.801±4.371)%,晚期凋亡率为(10.204±3.441)%；细胞凋亡率较对空白照组明显升高。
2.转染HIPK2-siRNA后,IgAN组C1GALT1、COSMC mRNA和蛋白水平明显升高,CT组C1GALT1、COSMC mRNA和蛋白表达无明显变化
3.转染72小时后检测腭扁桃体单个核细胞培养上清中IgA1及其糖基化水平：IgAN组腭扁桃体单个核细胞IgA1分泌较空白对照组明显减少；CT组IgA1含量无明显变化；VVL-ELISA测定IgA1糖基化水平,IgAN转染组较空白对照组OD值明显降低,CT组转染后OD值无明显变化。
结论：IgA肾病腭扁桃体单个核细胞HIPK2基因沉默后,C1GALT1及COSMC表达增多,IgAl分泌减少,铰链区糖基化水平增高,提示HIPK2是IgA肾病异常糖基化IgA1表达的关键致病因子,沉默HIPK2基因可以减少低糖基化IgA1的产生。
Background:
IgA nephropathy (IgAN) is recognized as the most common immune complex related to the cause of glomerulonephritis worldwide. Approximately20%of patients progress to end-stage renal disease within20years. However, the pathogenesis and mechanisms of IgAN remain to be elucidated. The disease is characterized by the predominant deposition of IgA1in the mesangial area of glomeruli. IgAl differs from IgA2, particularly at the 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 IgAl O-glycans are based on a core N-acetyl galactosamine (GalNAc) usually extended with galactose (Gal) under the effect of a j31,3Gal transferase (C1GALT1) working with its chaperone Cosmc (core I β3-Gal-T-specific molecular chaperone). β1,3-galacto-syltransferase (β1,3GT) synthesis activity 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. Yet, no study has been carried out to clarify the underlying basis of the expression suppression.
Although the pathogenesis of this disease is unclear, some researchers suggested that it might not be genetic disorders but external suppression that causes the low COSMC and C1GALT1C1mRNA expression in IgAN. LPS could significantly inhibit COSMC and C1GALT1C1expression. Recent work using a surface IgA1-positive human B lymphoma cell line has shown that the T-helper2cytokine interleukin-4may play a key role in controlling O-glycosylation of the IgAl hinge region. Interleukin-4stimulation significantly decreased the mRNA levels of both and Cosmc. In parallel, C1GALT1C1activity was reduced and the synthesized IgAl O-glycoforms were poorly galactosylated. Moreover, IgAN patients with severe renal dysfunction are more likely to hyperproduce Th2cytokine and synthesize more IL-4compared to patients with mild disease. These findings suggest that Th2polarity in systemic immune response may induce dysregulation of systemic tolerance, followed by glomerular IgA deposition and injury. The T cell-derived cytokines IL-4specifically activate STAT6, which plays important roles in Th2differentiation and inflammation. Yet, no study has been carried out to clarify the underlying relationship between the expression suppression of C1GALT1and COSMC with IL-4/STAT6signaling in IgA nephropathy.
More recently, high-throughput sequencing of chromatin immunoprecipitated DNA has identified genes bound by STAT6. One study compared genes bound by STAT6in wild-type and Stat6-/-Th2cells and found that some of the STAT6-bound regions coincided with various permissive epigenetic marks, and the corresponding genes include IL-4and home-odomain-interacting protein kinase2(HIPK2). IL-4induced STAT6-dependent expression of a selection of genes identified by GeneChip analysis, including HIPK2was confirmed by Northern blot. STAT6deficiency clearly inhibited the IL-4-induced up-regulation of HIPK2which is most likely a primary target gene of STAT6. HIPK2is a nuclear serine-threonine kinase participating in transcriptional regulation and growth control.While O-glycans of IgAl are synthesized in a step-wise manner, beginning with attachment of GalNAc to Ser or Thr catalyzed by a member of the UDP-GalNAc-transferase enzyme family. We hypothesized and experimentally validated that HIPK2, a previously unrecognized kinase in the context of IgA nephropathy, contributes to increased synthesis of aberrantly galactosylated IgAl.
We know that many IgAN patients show episodic macrohematuria, which coincides with mucosal infection especially in the upper respiratory tract or gastrointestinal tract. Two groups have reported tonsillectomy to be an effective treatment in retrospective and prospective studies. The results of immunization studies in IgAN patients support the notion that mucosal immunization with neoantigen results in impaired mucosal and systemic IgA responses but normal IgG and IgM responses. There is a growing body of evidence to suggest that impaired mucosal IgA response may result in impaired elimination of mucosal antigens. In our previous study, we identified that a-hemolytic streptococcus (HS) was the most common Gram-positive bacteria isolated from tonsils of patients with IgAN or chronic tonsillitis. Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, so we used both HS and LPS to stimulate the tonsil mononuclear cells (TMCs) to imitate mucosal infection in IgAN.
Chapter I. The expression of IL-4/STAT6, HIPK2, C1GALT1, COSMC and its relationship with IgAl secretion and O-Glycosylation in tonsil from patients with IgA nephropathy
Obejective:To detect the expression levels of IL-4, STAT6, C1GALT1C1, COSMC, HIPK2in tonsil components and mononuclear cells, and analyze the correlation between IL-4/STAT6pathway and HIPK2with aberrantly glycosylated IgA1in IgA nephropathy.
Methods:Tonsillar tissue specimens were obtained from22patients with IgA nephropathy (IgAN) who had been diagnosed based on immunofluorescent and light microscopic findings from percutaneous renal biopsy. The tonsils from24patients with chronic tonsillitis (CT) lacking renal diseases, were used as controls. Immunofluorescence and immunohistochemical stains were performed to detect the expression of IL-4, STAT6, HIPK2, C1GALT1C1, COSMC in tonsil components; Mononuclear cells were separated by density-gradient centrifugation using Lymphocyte Separation Medium, and the expression of IL-4, STAT6, C1GALT1C1, Cosmc, HIPK2in tonsil mononuclear cells (TMCs) were tested by RT-PCR and Western-blot. IgAl content in the supernatant was measured in duplicate using ELISA. The levels of IgAl O-glycosylation were determined by Vicia villosa (VV) lectin-binding assay.
Results:
1. The expression of IL-4, STAT6and HIPK2were significantly higher compared to CT group. They localized in all tonsil components (including germinal center and tonsillar crypt epithelium) of IgAN patients, but the expression of C1GALT1and COSMC decreased significantly in patients with IgAN in comparison with those of CT.
2. The mRNA and protein amount of IL-4, STAT6and HIPK2in the tonsil derived from IgAN patients was significantly higher than that of CT patients. While the expression of C1GALT1and COSMC decreased significantly in patients with IgAN
3. The concentration of IgA1in supernatants from cells cultured was higher than control cells. The OD value of VV lectin binding ELISA was significantly higher than that of CT patients.
4. The level of HIPK2mRNA expression significantly negatively correlated with renal function as expressed by eGFR, and also significantly positively correlated with daily proteinuria, IgAl concentration and its VVL-binding ELISA OD value.
Conclusion:IL-4/STAT6signaling pathway and HIPK2was highly activated in IgAN group; the baseline levels of O-glycosylation of patients with IgAN were significantly lower than CT group. It seems that HIPK2plays a key role in in IgAl secretion and O-Glycosylation.
Chapter Ⅱ. Effects of LPS and HS stimulation on TH1, TH2and TH1/TH2ratio in tonsillar lymphocyte
Objective:To detect Th1/Th2polarization in palatine tonsil of IgA nephropathy; observe the effects of LPS and HS stimulation on TH1, TH2and TH1/TH2ratio in tonsillar lymphocyte
Methods:Tonsillar lymphocyte were cultured with or without LPS (lOug/ml) or HS (1*108/ml) for72hours. PMA/Ionomycin mixture and BFA/Monensin mixture were added as lymphocyte stimulating agent before Flow cytometry test.The determination of CD4+cells was inferred by differential gatering in a flow cytometer, considering CD3+CD8-T cells. Cell populations were defined as follows, TH1: CD3-positive, CD8-negative, IFN-y-positive and IL-4-negative; TH2: CD3-positive, CD8-negative, IFN-y-negative and IL-4-positive.
Results:
1. Flow cytometry analyses showed percentage of TH1population in IgAN group vs CT group were5.73±2.14%vs4.71±1.98%without stimulation,1.24±0.55%vs0.37±0.22%, in the TH2population. The mean±SD Th1:Th2ratio for the IgAN group without situmlation was4.62±2.12, and in CT group was12.73±5.78.
2. Percentage of TH1population in IgAN group vs. CT group were13.30±4.15%vs7.39±3.78%after LPS stimulation,1.20±0.78%vs0.58±0.33%, in the TH2population. The mean±SD values for LPS stimulation group were11.08±4.33vs12.74±5.01. The value in CT group was significantly higher than that in IgAN group without or with stimulation.
3. Percentage of TH1population in IgAN group vs CT group were7.03±3.14%vs3.52±2.55%after HS stimulation,8.11±3.98%vs0.77±0.41%, in the TH2population. The mean±SD values for HS stimulation group in IgAN group vs CT group were0.87±0.25vs4.57±1.92.
4. The values of the Th1:Th2ratio varied widely among the patients in IgAN group, and we selected those patients who had undergone proteinuria. The Th1:Th2ratio significantly negatively correlated with proteinuria.
Conclusion:The value of Thl/Th2in CT group was significantly higher than that in IgAN group without or with stimulation. We hypothesized that a polarization toward TH2response at the stimulated lymphocyte level may lead to immune abnormalities in IgAN. THO cells are differentially mobilized during contact sensitization and by adjuvants such as LPS that induce T helper typel (Th1) responses, or HS that induces T helper type2(Th2) responses.
Chapter Ⅲ. Effects of IL-4, LPS and HS on STAT6, HIPK2, C1GALT1and COSMC expression, IgAl secretion and O-Glycosylation
Objective:To detect the Effects of IL-4, LPS and HS on IL-4/STAT6, HIPK2, C1GALT1and COSMC expression, IgAl secretion and O-Glycosylation.
Methods:Tonsillar lymphocyte were cultured with or without10ng/mL of recombinant human IL-4or HS (1*108/ml) or LPS (10ug/ml) for72hours. The expression of STAT6, HIPK2, C1GALT1and COSMC were examined by RT-PCR and Western blot. IgAl content in the supernatant was measured in duplicate using ELISA. The levels of IgA1O-glycosylation were determined by Vicia villosa (VV) lectin-binding assay
Results:
1. The levels of mRNA and protein encoding IL-4, STAT6and HIPK2in cells coincubated with IL-4, LPS, HS were significantly higher than that in control without stimulation after72h in IgAN group. Whereas the levels of mRNA and protein encoding C1GALT1and COSMC was significantly lower compared to that without stimulation in IgAN group. The C1GALT1and COSMC mRNA and protein expression from cells cultured with HS was significantly lower than cells cultured with IL-4or LPS in IgAN group (P<0.05). Moreover, the C1GALT1and COSMC expression of TMCs in IgAN patients after stimulation was significantly lower than that in CT patients.
2. The levels of mRNA and protein encoding IL-4, STAT6and HIPK2in cells coincubated with IL-4, LPS, HS were significantly higher than that in control in CT group. While no significantly difference could be seen in C1GALT1and COSMC expression of CT group among different stimulations compared to control group.
3. The IgAl production of TMCs stimulated with IL-4, LPS or HS was significantly higher compared to that without stimulation in both groups. Mean IgAl levels of supernatants were remarkable higher after IL-4or HS stimulation compared with cells cultured with LPS. As expected, VV lectin binding to IgAl derived from cells stimulated by HS, IL-4and LPS was significantly higher than that from unstimulated cells in IgAN group. No significant increase in lectin binding was observed for IgA stimulated with IL-4, LPS or HS relative to the unstimulated cultures in CT group.
Conclusion:IL-4/STAT6signaling pathway in tonsil of IgA nephropathy can be activated by IL-4, LPS and HS, which can together promoting the production of HIPK2, and downregulation the expression of C1GALT1and COSMC. All the above lead to the production of Under-O-glycosylation IgAl
Chapter IV. The effect of silencing HIPK2gene expression in IgAl secretion and O-Glycosylation
Objective:HIPK2was highly activated in tonsil of IgAN group, and it has a close relationship with IgAl secretion and O-Glycosylation. In this chapter, we silenced the gene expression of HIPK2to observe the influence in IgAl secretion and O-Glycosylation by HIPK2-siRNA.
Methods:To assess the percentage of apoptotic and viable cell fractions after transfection of HIPK2siRNA, a human Annexin V-FITC/PI staining kit was used. The expression of C1GALT1C1and COSMC in tonsil mononuclear cells (TMCs) were tested by RT-PCR and Western-blot. IgAl content and the levels of O-glycosylation in the supernatant were measured in duplicate using ELISA.
Results:
1. We detected the apoptosis of mononuclear cells after24hours since transfection of HIPK2siRNA. The early stage of apoptosis is (13.801±4.371)%, while the late stage of apoptosis is (10.204±3.441)%. The apoptosis of mononuclear cells increased after HIPK2-siRNA transfection.
2. Our results showed that the expression of C1GALT1and COSMC protein and mRNA was increased significantly in IgAN-HIPK2siRNA-treated group.While in CT group the expression of C1GALT1and COSMC protein and mRNA remains stable after transfection of HIPK2siRNA.
3. IgAl protein detected by ELISA decreased after HIPK2-siRNA transfection, while in CT group, IgAl level maintain stable. In IgAN-HIPK2siRNA-treated group, VV lectin binding to IgAl was significantly lower than that from normal controls. While in CT group the OD level of VV lectin binding ELISA remains stable.
Conclusion:HIPK2-siRNA negatively regulates IL-4/STAT6pathway-induced IgA secretion. Importantly, HIPK2-siRNA attenuates the aberrant glycosylation of IgAl secretion. Thus, these results suggest that HIPK2is indispensable for IgA production and may play a key role in IgA glycosylation.
IgA肾病是世界范围内最常见的原发性肾小球疾病,大约20%的患者在20年内会进展至终末期肾功能衰竭,是导致尿毒症最常见原因,然而其发病机制尚不明确。IgA肾病以IgAl/低糖基化IgA1沉积于肾小球系膜区及毛细血管袢为病理特征,以血尿蛋白尿、高血压及肾功能损害为主要临床表现。IgAl与IgA2的主要区别点在与IgAl存在铰链区。在IgAl铰链区,β1,3半乳糖转移酶(C1GALT1)在其特异性分子伴侣COSMC的辅助下,通过UDP-半乳糖载体将半乳糖转移至IgA1的β1,3位乙酰半乳糖胺残基上,完成IgA1的糖基化修饰。近年的研究证实低糖基化IgA1是IgA肾病的关键致病因子。IgA肾病外周血B淋巴细胞β1,3半乳糖转移酶及分子伴侣活性COSMC明显下降,并且与IgAl病理损伤严重程度呈正相关,但是其活性下降的具体机制尚未阐明。
有学者认为外界环境因素的刺激,而非基因遗传因素,导致IgA肾病IgA1铰链区异常糖基化。脂多糖能抑制C1GALT1及COSMC活性；体外实验证实Th2细胞因子IL-4能下调C1GALT1及COSMC的表达,影响IgAl铰链区糖基化；IL-4及其下游转录因子STAT6,能够调节Th2细胞分化,促进炎症反应的发生。然而目前尚未有研究涉及IL-4/STAT6信号通路与IgA肾病IgA1及其糖基化之间的关系。
同源异型结构域相互作用蛋白激酶-2(homeodomain-interactiing protein kinase-2HIPK2)是丝/苏氨酸激酶,包括HIPK1、HIPK2与HIPK3。HIPK2通过相互作用蛋白对靶点进行磷酸化修饰,调控相关蛋白酶的活性及基因转录表达。有学者在对IL-4刺激STAT6-/-与STAT6+/+小鼠B细胞基因表达研究中,发现HIPK2表达尤为活跃,提示HIPK2在IL-4(JAK/STAT6)信号通路中具有重要作用。在IL-4诱导JAK/STAT6信号通路中,HIPK2表达明显升高,提示HIPK2可能参与IL-4介导JAK/STAT6信号通路中相关蛋白酶的磷酸化修饰及转录表达调控。已证实,IL-4介导C1GALT1的活性及分子伴侣COSMC表达调控,这让我们思考,HIPK2是否通过调控C1GALT1的活性及分子伴侣COSMC表达,参与IL-4(JAK/STAT6)信号通路介导IgA肾病腭扁桃体淋巴细胞低糖基化IgA1形成的作用机制。
我们知道IgA肾病患者在黏膜组织,特别是上呼吸道和肠道黏膜感染后常出现阵发性肉眼血尿。许多回顾性或前瞻性临床对照研究证实：IgA肾病患者摘除腭扁桃体后尿检正常率、肾功能稳定率和肾脏生存率均高于对照组；循环中lgA水平明显下降。目前很多学者认为IgA肾病的发生与黏膜组织对外来抗原免疫应答紊乱有关。我们前期的研究证实,革兰氏阳性菌的代表甲型溶血性链球菌是慢性扁桃体炎最常见的致病菌,而脂多糖是革兰氏阴性菌细胞壁的主要抗原成分,本课题中我们采用灭活的甲型溶血性链球菌和脂多糖作为干预手段,模拟扁桃体感染,观察腭扁桃体单个核细胞IL-4/STAT6、HIPK2表达的变化,分析其与IgA1及其糖基化之间的关系(学术思想见下图),为IgA肾病的防治提供新的理论依据和治疗靶点。
第一章IL-4/STAT6、HIPK2、C1GALT1及COSMC在腭扁桃体中的表达及其与IgA1糖基化的相关性研究
目的：检测腭扁桃体IL-4、STAT6、HIPK2、(31,3半乳糖苷转移酶(C1GALT1)及分子伴侣COSMC的表达,分析IL-4/STAT6、HIPK2与IgA1及其铰链区糖基化之间的关系。
方法：收集经临床和肾脏病理确诊为IgA肾病患者腭扁桃体22例做为观察组(IgAN组),慢性扁桃体炎非肾炎患者腭扁桃体24例作为对照组(CT组)。免疫组织化学检测腭扁桃体组织IL-4、STAT6、HIPK2的表达水平；免疫荧光检测C1GALT1及COSMC;密度梯度离心法分离腭扁桃体单个核细胞,采用RT-PCR和Western blot技术分别检测IL-4、STAT6、HIPK2、C1GALT1及COSMC mRNA和蛋白表达；ELISA检测培养液上清中IgA1含量,蚕豆凝集素(VVL)亲和ELISA法测定VVL与IgA1结合力,间接检测IgA1铰链区糖基化水平。
结果：
1.免疫组化结果显示与CT组比较,IgAN组IL-4/STAT6、 HIPK2在网状上皮及淋巴小结生发中心分布明显增加；免疫荧光提示IgAN组C1GALT1及COSMC在腭扁桃体组织中表达较CT组减少；
2.RT-PCR及Western blot结果均提示IgAN组IL-4/STAT6、 HIPK2表达较CT组明显升高,C1GALT1、COSMC表达较CT组下降；
3.IgAN组腭扁桃体单个核细胞培养上清IgA1含量较CT组明显升高；VVL-ELISA结果提示IgAN组IgA1与蚕豆凝集素结合力明显高于CT组,说明IgAN组腭扁桃体单个核细胞分泌的IgA1存在明显的低糖基化；
4.相关性分析提示IgAN腭扁桃体单个核细胞HIPK2mRN表达与eGFR呈负相关,与蛋白尿、IgA1及VVL-ELISA OD值呈正相关。
结论：IL-4/STAT6信号通路在IgAN患者腭扁桃体中处于激活状态；HIPK2在IgAN腭扁桃体单个核细胞中高表达,HIPK2与IgAN主要临床指标、IgA1及其糖基化水平关系密切,提示HIPK2是IgAN的重要致病因子。
第二章脂多糖、甲型溶血性链球菌对腭扁桃体Th1.Th2及Th1/Th2的影响
目的：研究IgAN腭扁桃体Th1/Th2极化是否存在异常；观察脂多糖、甲型溶血性链球菌刺激对腭扁桃体Th1、Th2及Th1/Th2的影响。
方法：将IgAN和CT患者腭扁桃体单个核细胞各分成三组,第一组为空白对照组；第二组加入脂多糖LPS(浓度为10ug/ml)；第三组加入灭活的甲型溶血性链球菌HS(浓度为1×108cfu/ml).培养72小时后加入PMA/Ionomycin mixture及BFA/Monensin mixture作为淋巴细胞刺激剂孵育6小时,CD3+CD8-设门,流式细胞术检测IL-4、IFN-γ含量,Thl为CD3+/CD8-/IFN-γ+细胞,Th2为CD3+/CD8-/IL-4+细胞。
结果：
1.IgAN组Th1:5.73±2.14%；Th2:1.24±0.55%；Th1/Th2:4.62±2.12。C组Th1:4.71±1.98%；Th2:0.37±O.22%;Th1/Th2:12.73±5.78。
2.LPS刺激后IgAN组Thl:13.30±4.15%；Th2:1.20±0.78; Th1/Th2:11.08±4.33。CT组Th1:7.39±3.78%；Th2:0.58±0.33%; Th1/Th2:12.74±5.01.
3.予以HS刺激后IgAN组Th1:7.03±3.14%;Th2:8.11±3.98%Th1/Th2:0.87±0.25。CT组Th1:3.52±2.55%；Th2:0.77±0.41%; Th1/Th2:4.57±1.92.
4.采用Pearson相关分析及直线回归,发现IgAN组Th1/Th2匕值与蛋白尿呈负相关,r=-0.785。
结论：IgAN腭扁桃体中Th2细胞免疫占优势地位,并且与蛋白尿等临床指标的严重程度呈正相关；LPS、HS能促使Th1/Th2极化失衡,其中LPS促进Thl分化为主,HS促进Th2分化更明显。
第三章白介素-4、脂多糖、甲型溶血性链球菌对腭扁桃体单个核细胞STAT6、HIPK2、C1GALT1/COSMC、IgAl及其糖基化的影响
目的：采用IL-4、LPS、HS作为干预刺激,探讨IL-4/STAT6信号通路、HIPK2与低糖基化IgA1表达的关系。
方法：腭扁桃体单个核细胞培养体系中,分别加入用IL-4(浓度为10ng/ml)、LPS (终浓度为10ug/ml)、灭活的HS(浓度为1×108cfu/ml)刺激72小时,RT-PCR, Western blot技术,分别测定STAT6、HIPK2、C1GALT1、COSMC mRNA和蛋白表达,ELISA技术测定培养液上清中IgA1及其糖基化水平的变化。
结果：
1.IL-4、LPS、HS均能上调IgAN组STAT6、HIPK2mRNA和蛋白表达,IL-4、HS较LPS上调作用更为明显；同时IL-4、LPS、HS均能下调C1GALT1及COSMC mRNA和蛋白表达,HS较IL-4、LPS下调作用更明显。
2.CT组在接受IL-4、LPS、HS刺激后,STAT6、HIPK2mRNA和蛋白表达上调,但升高程度较IgAN组低；C1GALT1、COSMC mRNA和蛋白表达较空白对照组无明显变化。
3.IgAN组及CT组腭扁桃体单个核细胞IgA1分泌较空白对照组明显增加,其中IL-4、HS较LPS刺激单个核细胞分泌IgA1效果更为明显；蚕豆凝集素亲和ELISA测定IgA1半乳糖基水平,IgAN刺激组较空白对照组VVL-ELISAOD值明显增加,HS刺激对OD值影响较IL-4、LPS大；IL-4、LPS、HS刺激后,CT组VVL-ELISA检测所得OD值无明显变化。
结论：IL-4、LPS、HS能激活IgA肾病腭扁桃体IL-4/STAT6信号通路,促进HIPK2表达,从而下调C1GALT1/COSMC活性,导致IgA1分泌增多,IgA1糖基化水平下降。
第四章沉默HIPK2基因对IgAl及其糖基化的影响
目的：HIPK2在IgA肾病腭扁桃体中高表达,且与低糖基化IgAl分泌关系密切。我们拟采用HIPK2-siRNA沉默HIPK2基因表达,观察其对腭扁桃体细胞IgAl及糖基化的影响。
方法：分离腭扁桃体单个核细胞,转染HIPK2-siRNA, Annexin V-FITC法检测转染后细胞凋亡率；RT-PCR、Western blot分别检测HIPK2-siRNA转染后,C1GALT1及COSMC mRNA和蛋白表达；ELISA测定培养液上清中IgAl及其糖基化水平的变化。
结果：
1.HIPK2-siRNA转染单个核细胞后24小时,流式细胞术测得早期凋亡率百分比为(13.801±4.371)%,晚期凋亡率为(10.204±3.441)%；细胞凋亡率较对空白照组明显升高。
2.转染HIPK2-siRNA后,IgAN组C1GALT1、COSMC mRNA和蛋白水平明显升高,CT组C1GALT1、COSMC mRNA和蛋白表达无明显变化
3.转染72小时后检测腭扁桃体单个核细胞培养上清中IgA1及其糖基化水平：IgAN组腭扁桃体单个核细胞IgA1分泌较空白对照组明显减少；CT组IgA1含量无明显变化；VVL-ELISA测定IgA1糖基化水平,IgAN转染组较空白对照组OD值明显降低,CT组转染后OD值无明显变化。
结论：IgA肾病腭扁桃体单个核细胞HIPK2基因沉默后,C1GALT1及COSMC表达增多,IgAl分泌减少,铰链区糖基化水平增高,提示HIPK2是IgA肾病异常糖基化IgA1表达的关键致病因子,沉默HIPK2基因可以减少低糖基化IgA1的产生。
Background:
IgA nephropathy (IgAN) is recognized as the most common immune complex related to the cause of glomerulonephritis worldwide. Approximately20%of patients progress to end-stage renal disease within20years. However, the pathogenesis and mechanisms of IgAN remain to be elucidated. The disease is characterized by the predominant deposition of IgA1in the mesangial area of glomeruli. IgAl differs from IgA2, particularly at the 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 IgAl O-glycans are based on a core N-acetyl galactosamine (GalNAc) usually extended with galactose (Gal) under the effect of a j31,3Gal transferase (C1GALT1) working with its chaperone Cosmc (core I β3-Gal-T-specific molecular chaperone). β1,3-galacto-syltransferase (β1,3GT) synthesis activity 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. Yet, no study has been carried out to clarify the underlying basis of the expression suppression.
Although the pathogenesis of this disease is unclear, some researchers suggested that it might not be genetic disorders but external suppression that causes the low COSMC and C1GALT1C1mRNA expression in IgAN. LPS could significantly inhibit COSMC and C1GALT1C1expression. Recent work using a surface IgA1-positive human B lymphoma cell line has shown that the T-helper2cytokine interleukin-4may play a key role in controlling O-glycosylation of the IgAl hinge region. Interleukin-4stimulation significantly decreased the mRNA levels of both and Cosmc. In parallel, C1GALT1C1activity was reduced and the synthesized IgAl O-glycoforms were poorly galactosylated. Moreover, IgAN patients with severe renal dysfunction are more likely to hyperproduce Th2cytokine and synthesize more IL-4compared to patients with mild disease. These findings suggest that Th2polarity in systemic immune response may induce dysregulation of systemic tolerance, followed by glomerular IgA deposition and injury. The T cell-derived cytokines IL-4specifically activate STAT6, which plays important roles in Th2differentiation and inflammation. Yet, no study has been carried out to clarify the underlying relationship between the expression suppression of C1GALT1and COSMC with IL-4/STAT6signaling in IgA nephropathy.
More recently, high-throughput sequencing of chromatin immunoprecipitated DNA has identified genes bound by STAT6. One study compared genes bound by STAT6in wild-type and Stat6-/-Th2cells and found that some of the STAT6-bound regions coincided with various permissive epigenetic marks, and the corresponding genes include IL-4and home-odomain-interacting protein kinase2(HIPK2). IL-4induced STAT6-dependent expression of a selection of genes identified by GeneChip analysis, including HIPK2was confirmed by Northern blot. STAT6deficiency clearly inhibited the IL-4-induced up-regulation of HIPK2which is most likely a primary target gene of STAT6. HIPK2is a nuclear serine-threonine kinase participating in transcriptional regulation and growth control.While O-glycans of IgAl are synthesized in a step-wise manner, beginning with attachment of GalNAc to Ser or Thr catalyzed by a member of the UDP-GalNAc-transferase enzyme family. We hypothesized and experimentally validated that HIPK2, a previously unrecognized kinase in the context of IgA nephropathy, contributes to increased synthesis of aberrantly galactosylated IgAl.
We know that many IgAN patients show episodic macrohematuria, which coincides with mucosal infection especially in the upper respiratory tract or gastrointestinal tract. Two groups have reported tonsillectomy to be an effective treatment in retrospective and prospective studies. The results of immunization studies in IgAN patients support the notion that mucosal immunization with neoantigen results in impaired mucosal and systemic IgA responses but normal IgG and IgM responses. There is a growing body of evidence to suggest that impaired mucosal IgA response may result in impaired elimination of mucosal antigens. In our previous study, we identified that a-hemolytic streptococcus (HS) was the most common Gram-positive bacteria isolated from tonsils of patients with IgAN or chronic tonsillitis. Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, so we used both HS and LPS to stimulate the tonsil mononuclear cells (TMCs) to imitate mucosal infection in IgAN.
Chapter I. The expression of IL-4/STAT6, HIPK2, C1GALT1, COSMC and its relationship with IgAl secretion and O-Glycosylation in tonsil from patients with IgA nephropathy
Obejective:To detect the expression levels of IL-4, STAT6, C1GALT1C1, COSMC, HIPK2in tonsil components and mononuclear cells, and analyze the correlation between IL-4/STAT6pathway and HIPK2with aberrantly glycosylated IgA1in IgA nephropathy.
Methods:Tonsillar tissue specimens were obtained from22patients with IgA nephropathy (IgAN) who had been diagnosed based on immunofluorescent and light microscopic findings from percutaneous renal biopsy. The tonsils from24patients with chronic tonsillitis (CT) lacking renal diseases, were used as controls. Immunofluorescence and immunohistochemical stains were performed to detect the expression of IL-4, STAT6, HIPK2, C1GALT1C1, COSMC in tonsil components; Mononuclear cells were separated by density-gradient centrifugation using Lymphocyte Separation Medium, and the expression of IL-4, STAT6, C1GALT1C1, Cosmc, HIPK2in tonsil mononuclear cells (TMCs) were tested by RT-PCR and Western-blot. IgAl content in the supernatant was measured in duplicate using ELISA. The levels of IgAl O-glycosylation were determined by Vicia villosa (VV) lectin-binding assay.
Results:
1. The expression of IL-4, STAT6and HIPK2were significantly higher compared to CT group. They localized in all tonsil components (including germinal center and tonsillar crypt epithelium) of IgAN patients, but the expression of C1GALT1and COSMC decreased significantly in patients with IgAN in comparison with those of CT.
2. The mRNA and protein amount of IL-4, STAT6and HIPK2in the tonsil derived from IgAN patients was significantly higher than that of CT patients. While the expression of C1GALT1and COSMC decreased significantly in patients with IgAN
3. The concentration of IgA1in supernatants from cells cultured was higher than control cells. The OD value of VV lectin binding ELISA was significantly higher than that of CT patients.
4. The level of HIPK2mRNA expression significantly negatively correlated with renal function as expressed by eGFR, and also significantly positively correlated with daily proteinuria, IgAl concentration and its VVL-binding ELISA OD value.
Conclusion:IL-4/STAT6signaling pathway and HIPK2was highly activated in IgAN group; the baseline levels of O-glycosylation of patients with IgAN were significantly lower than CT group. It seems that HIPK2plays a key role in in IgAl secretion and O-Glycosylation.
Chapter Ⅱ. Effects of LPS and HS stimulation on TH1, TH2and TH1/TH2ratio in tonsillar lymphocyte
Objective:To detect Th1/Th2polarization in palatine tonsil of IgA nephropathy; observe the effects of LPS and HS stimulation on TH1, TH2and TH1/TH2ratio in tonsillar lymphocyte
Methods:Tonsillar lymphocyte were cultured with or without LPS (lOug/ml) or HS (1*108/ml) for72hours. PMA/Ionomycin mixture and BFA/Monensin mixture were added as lymphocyte stimulating agent before Flow cytometry test.The determination of CD4+cells was inferred by differential gatering in a flow cytometer, considering CD3+CD8-T cells. Cell populations were defined as follows, TH1: CD3-positive, CD8-negative, IFN-y-positive and IL-4-negative; TH2: CD3-positive, CD8-negative, IFN-y-negative and IL-4-positive.
Results:
1. Flow cytometry analyses showed percentage of TH1population in IgAN group vs CT group were5.73±2.14%vs4.71±1.98%without stimulation,1.24±0.55%vs0.37±0.22%, in the TH2population. The mean±SD Th1:Th2ratio for the IgAN group without situmlation was4.62±2.12, and in CT group was12.73±5.78.
2. Percentage of TH1population in IgAN group vs. CT group were13.30±4.15%vs7.39±3.78%after LPS stimulation,1.20±0.78%vs0.58±0.33%, in the TH2population. The mean±SD values for LPS stimulation group were11.08±4.33vs12.74±5.01. The value in CT group was significantly higher than that in IgAN group without or with stimulation.
3. Percentage of TH1population in IgAN group vs CT group were7.03±3.14%vs3.52±2.55%after HS stimulation,8.11±3.98%vs0.77±0.41%, in the TH2population. The mean±SD values for HS stimulation group in IgAN group vs CT group were0.87±0.25vs4.57±1.92.
4. The values of the Th1:Th2ratio varied widely among the patients in IgAN group, and we selected those patients who had undergone proteinuria. The Th1:Th2ratio significantly negatively correlated with proteinuria.
Conclusion:The value of Thl/Th2in CT group was significantly higher than that in IgAN group without or with stimulation. We hypothesized that a polarization toward TH2response at the stimulated lymphocyte level may lead to immune abnormalities in IgAN. THO cells are differentially mobilized during contact sensitization and by adjuvants such as LPS that induce T helper typel (Th1) responses, or HS that induces T helper type2(Th2) responses.
Chapter Ⅲ. Effects of IL-4, LPS and HS on STAT6, HIPK2, C1GALT1and COSMC expression, IgAl secretion and O-Glycosylation
Objective:To detect the Effects of IL-4, LPS and HS on IL-4/STAT6, HIPK2, C1GALT1and COSMC expression, IgAl secretion and O-Glycosylation.
Methods:Tonsillar lymphocyte were cultured with or without10ng/mL of recombinant human IL-4or HS (1*108/ml) or LPS (10ug/ml) for72hours. The expression of STAT6, HIPK2, C1GALT1and COSMC were examined by RT-PCR and Western blot. IgAl content in the supernatant was measured in duplicate using ELISA. The levels of IgA1O-glycosylation were determined by Vicia villosa (VV) lectin-binding assay
Results:
1. The levels of mRNA and protein encoding IL-4, STAT6and HIPK2in cells coincubated with IL-4, LPS, HS were significantly higher than that in control without stimulation after72h in IgAN group. Whereas the levels of mRNA and protein encoding C1GALT1and COSMC was significantly lower compared to that without stimulation in IgAN group. The C1GALT1and COSMC mRNA and protein expression from cells cultured with HS was significantly lower than cells cultured with IL-4or LPS in IgAN group (P<0.05). Moreover, the C1GALT1and COSMC expression of TMCs in IgAN patients after stimulation was significantly lower than that in CT patients.
2. The levels of mRNA and protein encoding IL-4, STAT6and HIPK2in cells coincubated with IL-4, LPS, HS were significantly higher than that in control in CT group. While no significantly difference could be seen in C1GALT1and COSMC expression of CT group among different stimulations compared to control group.
3. The IgAl production of TMCs stimulated with IL-4, LPS or HS was significantly higher compared to that without stimulation in both groups. Mean IgAl levels of supernatants were remarkable higher after IL-4or HS stimulation compared with cells cultured with LPS. As expected, VV lectin binding to IgAl derived from cells stimulated by HS, IL-4and LPS was significantly higher than that from unstimulated cells in IgAN group. No significant increase in lectin binding was observed for IgA stimulated with IL-4, LPS or HS relative to the unstimulated cultures in CT group.
Conclusion:IL-4/STAT6signaling pathway in tonsil of IgA nephropathy can be activated by IL-4, LPS and HS, which can together promoting the production of HIPK2, and downregulation the expression of C1GALT1and COSMC. All the above lead to the production of Under-O-glycosylation IgAl
Chapter IV. The effect of silencing HIPK2gene expression in IgAl secretion and O-Glycosylation
Objective:HIPK2was highly activated in tonsil of IgAN group, and it has a close relationship with IgAl secretion and O-Glycosylation. In this chapter, we silenced the gene expression of HIPK2to observe the influence in IgAl secretion and O-Glycosylation by HIPK2-siRNA.
Methods:To assess the percentage of apoptotic and viable cell fractions after transfection of HIPK2siRNA, a human Annexin V-FITC/PI staining kit was used. The expression of C1GALT1C1and COSMC in tonsil mononuclear cells (TMCs) were tested by RT-PCR and Western-blot. IgAl content and the levels of O-glycosylation in the supernatant were measured in duplicate using ELISA.
Results:
1. We detected the apoptosis of mononuclear cells after24hours since transfection of HIPK2siRNA. The early stage of apoptosis is (13.801±4.371)%, while the late stage of apoptosis is (10.204±3.441)%. The apoptosis of mononuclear cells increased after HIPK2-siRNA transfection.
2. Our results showed that the expression of C1GALT1and COSMC protein and mRNA was increased significantly in IgAN-HIPK2siRNA-treated group.While in CT group the expression of C1GALT1and COSMC protein and mRNA remains stable after transfection of HIPK2siRNA.
3. IgAl protein detected by ELISA decreased after HIPK2-siRNA transfection, while in CT group, IgAl level maintain stable. In IgAN-HIPK2siRNA-treated group, VV lectin binding to IgAl was significantly lower than that from normal controls. While in CT group the OD level of VV lectin binding ELISA remains stable.
Conclusion:HIPK2-siRNA negatively regulates IL-4/STAT6pathway-induced IgA secretion. Importantly, HIPK2-siRNA attenuates the aberrant glycosylation of IgAl secretion. Thus, these results suggest that HIPK2is indispensable for IgA production and may play a key role in IgA glycosylation.
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