IgA肾病患者血清IgA1与系膜细胞共培养上清诱导足细胞凋亡

目的 探讨IgA肾病(IgAN)患者血清IgA1与系膜细胞共培养上清对足细胞凋亡的影响。 方法 用Jacalin 亲和层析柱和Sephacryl S-200 分子筛纯化蛋白。单体IgA1（mIgA1）热聚合为聚合体IgA1（aIgA1）。实验分为患者上清组、健康上清组和对照组，系膜细胞分别与IgAN患者的aIgA1、健康对照的aIgA1和5%胎牛血清共培养，收集上清，与同步化的足细胞作用。流式细胞仪检测细胞凋亡情况。实时定量PCR 检测凋亡相关基因Bcl-2、Bax、Fas和Fas-L表达情况。 结果 患者上清可诱导足细胞凋亡，其凋亡率显著高于健康上清组和对照组[（28.5±5.9）%比（22.5±5.8）%、（20.5±4.5）%， 均P < 0.05]。患者上清可诱导足细胞Fas mRNA 升高，为对照组的1.89倍（P < 0.05）， 而Bcl-2 mRNA下调为对照组的72%（P < 0.05）。患者上清组的AngⅡ和TGF-β1水平均高于健康上清组[（13.2±3.4） ng/L比（8.2±2.3） ng/L，P < 0.05；（15.4±3.4） ng/L比（10.8±3.2） ng/L，P < 0.05]。 结论 IgAN患者血清IgA1与系膜细胞共培养上清可诱导足细胞凋亡，可能参与IgAN的进展。     

Engagement of transferrin receptor by polymeric IgA1: evidence for a positive feedback loop involving increased receptor expression and mesangial cell proliferation in IgA nephropathy

IgA nephropathy (IgAN), the most common primary glomerulonephritis in the world, is characterized by IgA immune complex-mediated mesangial cell proliferation. The transferrin receptor (TfR) was identified previously as an IgA1 receptor, and it was found that, in biopsies of patients with IgAN, TfR is overexpressed and co-localizes with IgA1 mesangial deposits. Here, it is shown that purified polymeric IgA1 (pIgA1) is a major inducer of TfR expression (three- to four-fold increase) in quiescent human mesangial cells (HMC). IgA-induced but not cytokine-induced HMC proliferation is dependent on TfR engagement as it is inhibited by both TfR1 and TfR2 ectodomains as well as by the anti-TfR mAb A24. It is dependent on the continued presence of IgA1 rather than on soluble factors released during IgA1-mediated activation. In addition, pIgA1-induced IL-6 and TGF-beta production from HMC was specifically inhibited by mAb A24, confirming that pIgA1 triggers a TfR-dependent HMC activation. Finally, upregulation of TfR expression induced by sera from patients with IgAN but not from healthy individuals was dependent on IgA. It is proposed that deposited pIgA1 or IgA1 immune complexes could initiate a process of auto-amplification involving hyperexpression of TfR, allowing increased IgA1 mesangial deposition. Altogether, these data unveil a functional cooperation between pIgA1 and TfR for IgA1 deposition and HMC proliferation and activation, features that are commonly implicated in the chronicity of mesangial injuries observed in IgAN and that could explain the recurrence of IgA1 deposits in the mesangium after renal transplantation.     

骨形成蛋白7和转化生长因子β1在不同病理类型IgA肾病的变化

目的 观察转化生长因子β1（TGF-β1）和骨形成蛋白7（BMP-7）在不同病理类型IgA肾病的变化,并探讨其意义。方法 89例IgA肾病患者分成3组：A组为47例轻度系膜增生性IgA肾病;B组为29例中重度系膜增生性IgA肾病;C组为13例增生硬化或硬化性IgA肾病。检测患者的血压、尿蛋白量（24 h）、Scr和Ccr。免疫组化和ELISA方法测定患者肾组织冰冻切片及其血、尿中TGF-β1和BMP-7水平。计算患者病理切片硬化肾小球数、新月体数和间质纤维化面积百分比。结果 随着IgA肾病患者肾小球病变的加重,肾小管萎缩和肾间质纤维化增多,其血压、尿蛋白量（24 h）、Scr逐渐增加,除B、C两组间尿蛋白量（24 h）无显著差异外,其余各组间差异均有统计学意义（P＜0．05）。与A组比较,B组肾组织及血、尿TGF-β1明显增多,C组显著降低（P＜0．01）。肾组织冰冻切片及血、尿BMP-7随着肾脏病变的加重,水平逐渐下降（P＜0．01）;而且与Ccr呈正相关;与血压、Scr、尿蛋白量（24 h）、硬化肾小球数、新月体数、肾间质纤维化面积呈负相关。结论 TGF-β1在IgA肾病系膜增生严重时明显增加,肾脏广泛纤维化时明显降低,可能参与了IgA肾病肾间质纤维化的发生。BMP-7随肾脏病变的加重而明显降低,可能导致其抗肾纤维化作用减弱。     

IgA肾病患者血清aIgA1对足细胞增殖及nephrin分子表达的影响

目的：观察IgA肾病（IgA nephropathy,IgAN）患者与正常人的血清热聚合IgA1（aggregated IgA1,aIgA1）对足细胞增殖及nephrin分子表达的影响。方法：利用亲和层析联合分子筛层析法分离获得原发性IgAN患者与健康人血清单体IgA1（monomeric IgA1,mIgA1）,将mIgA1热聚合为aIgA1,分别刺激小鼠MPC5足细胞株,利用MTT法检测aIgA1对足细胞增殖的影响,用RT-PCR法检测aIgA1干预后足细胞nephrin分子基因表达水平的改变。结果：不同浓度aIgA1刺激组间足细胞MTT吸光度的差异有统计学意义（P〈0.05）。RT-PCR实验发现正常人及患者的aIgA1刺激足细胞后,均可以时间依赖性及剂量依赖性的方式下调nephrin mRNA的表达,且患者的aIgA1下调nephrin表达的作用更明显（P〈0.01）。结论：IgAN患者与正常人的aIgA1均可以影响足细胞增殖,并下调足细胞nephrin mRNA的表达。患者aIgA1对足细胞nephrin mRNA表达的下调作用较正常人明显,在疾病过程中IgA1可能是加重足细胞损伤的原因之一。     

Effect of aggregated immunoglobulin A1 from immunoglobulin A nephropathy patients on nephrin expression in podocytes

Aim: Abnormal immunoglobulin (Ig)A1 is considered to play a pivotal role in IgA nephropathy. We used mouse podocytes as the experimental model to investigate the effect of aggregated IgA1 (aIgA1) isolated from IgA nephropathy (IgAN) patients on nephrin expression in podocytes through direct and indirect pathways. Methods: Jacalin affinity chromatography and Sephacryl S‐200 molecular sieve chromatography were used to isolate IgA1 from blood of IgAN patients which was therefore became aIgA1. Podocytes were incubated with aIgA1 or special mesangial medium. Nephrin expression in podocytes was measured by real‐time polymerase chain reaction and western blot analysis. Results: Aggregated IgA1 from IgAN patients and healthy controls reduced nephrin expression in podocytes at mRNA and protein levels when compared with podocytes incubated with control medium (RPMI‐1640 with 0.5% foetal bovine serum) (P < 0.05). While medium from mesangial cells incubated with aIgA1 from IgAN inhibited nephrin expression in podocytes at mRNA and protein levels when compared with podocytes incubated with medium from mesangial cells with aIgA1 from healthy controls (P < 0.05). Conclusion: Our findings implicate that aIgA1 from IgAN patients could inhibit nephrin expression through direct and indirect pathways, although these mechanisms remain to be clarified.     

Mechanisms of tubulointerstitial injury in IgA nephropathy

BACKGROUND: IgA nephropathy (IgAN) runs a highly variable clinical course, with frequent involvement of tubulointerstitial damage. A subgroup of IgAN with severe tubulointerstitial damage is often associated with the most rapid progression to end-stage renal failure. In IgAN, mesangial sclerosis and tubulointerstitial damage were found to be correlated with the increase in pore size of the glomerular barrier. METHODS: The direct toxicity of proximal tubular epithelial cells (PTEC) by IgA in IgAN is still unresolved. Activation of PTEC by mediators released from infiltrating cells or resident kidney cells that induce tubular inflammation is the common final pathway in most chronic renal diseases. We hypothesize that mediators released from human mesangial cells (HMC) triggered by IgA deposition may lead to PTEC activation. RESULTS: We found that IgA binding to PTEC was less than one tenth that of HMC. The binding was nonspecific and exhibited no increased cell proliferation or enhanced synthesis of cytokines or adhesion molecules. However, when PTEC were cultured with IgA-HMC spent medium prepared from IgAN patients, there was enhanced proliferation of PTEC and increased synthesis of cytokines and adhesion molecules. CONCLUSION: These findings implicate a glomerulotubular cross-talk with mediators released from the mesangium, contributing to the pathogenesis of tubulointerstitial damage in IgAN. There are preliminary data to suggest that the expression of angiotensin II subtype-1 receptor and angiotensin II subtype-2 receptor in PTEC differs from that of HMC. These novel findings may provide clinicians new therapeutic approach for selective blockade of the tubulointerstitial injury in IgAN.     

Mesangial expression of angiotensin II receptor in IgA nephropathy and its regulation by polymeric IgA1

BACKGROUND: Enhanced gene expression for the renin-angiotensin system (RAS) is detected in glomerular mesangial cells in IgA nephropathy (IgAN). Preliminary studies showed a reduced glomerular gene expression of angiotensin II subtype 1 receptor (AT1R), suggesting a regulatory response to high intrarenal angiotensin II (Ang II) concentration in IgAN. METHODS: We examined the effect of polymeric IgA1 (pIgA1) from patients with IgAN on the expression of Ang II receptors in cultured human mesangial cells (HMC). RESULTS: Polymeric IgA1 from patients with IgAN down-regulated the expression of AT1R in HMC in a dose-dependent manner. When similar experiments were conducted with addition of an angiotensin-converting enzyme inhibitor (captopril) or an AT1R antagonist (losartan), there was a significant increase in the expression of AT1R. Blockade of Ang II with captopril or losartan alone resulted in a stepwise increase of AT1R in cultured HMC. Down-regulation of Ang II subtype 2 receptor (AT2R) was not observed in HMC cultured with pIgA1 from patients with IgAN. The acute suppressive effect of pIgA1 from IgAN on the expression of AT1R was confirmed in HMC incubated with IgA isolated from 15 IgAN patients, 15 healthy subjects, and other glomerulonephritides control subjects. Reduced glomerular expression of AT1R (but not AT2R) was also demonstrated in renal biopsies from patients with IgAN. CONCLUSION: Our findings demonstrate an altered AT1R expression in HMC in response to raised intrarenal Ang II in IgAN. Our in vitro studies also support that an imbalance of AT1R and AT2R activity in HMC following exposure to pIgA plays a significant pathogenetic role in the inflammatory injury in IgAN.     

Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy

Transferrin receptor (TfR) has been identified as a candidate IgA1 receptor expressed on human mesangial cells (HMC). TfR binds IgA1 but not IgA2, co-localizes with mesangial IgA1 deposits, and is overexpressed in patients with IgA nephropathy (IgAN). Here, structural requirements of IgA1 for its interaction with mesangial TfR were analyzed. Polymeric but not monomeric IgA1 interacted with TfR on cultured HMC and mediates internalization. IgA1 binding was significantly inhibited (>50%) by soluble forms of both TfR1 and TfR2, confirming that TfR serves as mesangial IgA1 receptor. Hypogalactosylated serum IgA1 from patients with IgAN bound TfR more efficiently than IgA1 from healthy individuals. Serum IgA immune complexes from patients with IgAN containing aberrantly glycosylated IgA1 bound more avidly to TfR than those from normal individuals. This binding was significantly inhibited by soluble TfR, highlighting the role of TfR in mesangial IgA1 deposition. For addressing the potential role of glycosylation sites in IgA1-TfR interaction, a variety of recombinant dimeric IgA1 molecules were used in binding studies on TfR with Daudi cells that express only TfR as IgA receptor. Deletion of either N- or O-linked glycosylation sites abrogated IgA1 binding to TfR, suggesting that sugars are essential for IgA1 binding. However, sialidase and beta-galactosidase treatment of IgA1 significantly enhanced IgA1/TfR interaction. These results indicate that aberrant glycosylation of IgA1 as well as immune complex formation constitute essential factors favoring mesangial TfR-IgA1 interaction as initial steps in IgAN pathogenesis.     

Future directions in the treatment of IgA nephropathy

Summary: In IgA nephropathy (IgAN), there is a defect of clearance of immune complexes. Some of these patients are genetically predisposed to the development of the nephritis. Poorly solubilized polymeric IgA immune complexes are then deposited in the mesangium. Recent data indirectly suggest IgA-immune complexes from patients with IgAN are different from those of healthy subjects and they can exert pathophysiologic effect on target cells. Mesangial reactivity to the immune complexes triggers off release of cytokines, with decreased prostaglandin E2 synthesis and increase in thromboxane A2 production promoting mesangial cell proliferation. Angiotensin II (ATII)-induced mesangial cell contraction with efferent arteriolar vasodilatation initiates glomerular injury and may eventually lead to glomerulosclerosis due to increased synthesis of transforming growth factor-β (TGF-β) and platelet-derived growth factor PDGF). This paper highlights the possible therapeutic strategies in the future based on the recently reported pathogenetic findings in IgAN. These strategies include: (i) decreasing the synthesis of IgA-immune complexes; (ii) limiting the mesangial uptake of IgA-immune complexes; (iii) antagonizing the effect of PDGF and TGF-β to reduce mesangial proliferation and glomerulosclerosis; and (iv) reducing the noxious glomerular injury due to infiltrating neutrophils. The effective treatment of IgAN requires a further clarification of the pathogenesis of the nephropathy. Future therapeutic attempts to slow down the rate of renal deterioration in IgAN rest on the better understanding of the mechanisms mediating inflammatory injury in the kidney.     

Activation of tubular epithelial cells by mesangial-derived TNF-alpha: glomerulotubular communication in IgA nephropathy

BACKGROUND: IgA nephropathy (IgAN), characterized by mesangial IgA deposition, runs a variable clinical course with tubulointerstitial damage and renal failure in no less than 30% of patients. Histologically, IgA is rarely detected in renal tubules. The direct toxicity by IgA on renal tubules remains uncertain. We hypothesize that mediators released from human mesangial cells (HMC) triggered by IgA deposition may lead to activation of proximal tubular epithelial cells (PTEC). METHODS: The binding of IgA to PTEC or HMC was assessed by flow cytometry. IgA-HMC medium was prepared by collecting the spent medium in which growth arrested HMC were incubated with IgA isolated from patients with IgAN, healthy control subjects, or other nephritic control patients. PTEC was cultured with the IgA-HMC medium in the presence or absence of neutralizing antibodies to TNF-alpha, IL-1beta, TGF-beta, or PDGF. Gene expression and protein synthesis of TNF-alpha, MIF, or ICAM-1 by PTEC were determined by RT-PCR and ELISA, respectively. RESULTS: The binding of IgA isolated from patients with IgAN to PTEC was increased when compared to binding of IgA from healthy control subjects (P < 0.005). However, the binding to PTEC was less than one tenth that of HMC in IgAN. The binding to PTEC was not mediated through known IgA receptors, as shown by competitive binding assays and gene expression of the receptors. Despite the in vitro binding, PTEC cultured with isolated IgA exhibited no increased cell proliferation or enhanced synthesis of TNF-alpha, MIF, or sICAM-1. However, when PTEC were cultured with IgA-HMC medium prepared from IgAN patients, there was enhanced proliferation of PTEC (P < 0.001) and increased synthesis of TNF-alpha, MIF, and sICAM-1 when compared with PTEC cultured with IgA-HMC medium from control subjects (P < 0.001). The synthesis of MIF and sICAM-1 by PTEC cultured with IgA-HMC medium was reduced by neutralizing antibodies to TNF-alpha (P < 0.001) but not by neutralizing antibodies to IL-1beta, TGF-beta, or PDGF. CONCLUSION: Our finding implicates that TNF-alpha released from the mesangium after IgA deposition activates renal tubular cells. The glomerulotubular communication could play an important role in the pathogenesis of tubulointerstitial damage in IgAN.     

Polymeric IgA1 from patients with IgA nephropathy upregulates transforming growth factor-beta synthesis and signal transduction in human mesangial cells via the renin-angiotensin system

The effects of polymeric IgA1 (pIgA1) and monomeric IgA1 (mIgA1) from patients with IgA nephropathy (IgAN) on the renin-angiotensin system (RAS) and TGF-beta synthesis were examined in cultured human mesangial cells (HMC). Both pIgA1 and mIgA1 induced renin gene expression in HMC, in a dose-dependent manner. Similar findings were observed for TGF-beta gene and protein expression. The values measured in HMC incubated with pIgA1 were significantly higher than those in HMC incubated with equivalent amounts of mIgA1. When similar experiments were performed with the addition of either captopril or losartan, there was a significant increase in the renin gene expression by HMC, whereas the synthesis of TGF-beta was markedly reduced. The TGF-beta signal transduction pathways in HMC were studied by measuring the receptor-regulated Smad proteins (Smad 2 and 3) and common-partner Smad proteins (Smad 4). pIgA1 from patients with IgAN upregulated Smad activity in HMC, and the activity observed in HMC that had been preincubated with pIgA1 was readily suppressed with optimal concentrations of captopril or losartan. The effects of pIgA1 on the RAS were further examined in HMC incubated with IgA isolated from 30 patients with IgAN, 30 healthy subjects, and disease control subjects with other diseases. pIgA1 induction of angiotensin II or TGF-beta synthesis in HMC was significantly greater with preparations from patients with IgAN, compared with healthy or disease control subjects. The findings support a pathogenetic role of pIgA1 in IgAN through upregulation of the RAS and TGF-beta, leading to chronic renal failure with renal fibrosis.     

IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells

BACKGROUND: Sera of patients with IgA nephropathy (IgAN) contain circulating immune complexes (CIC) composed of galactose-deficient IgA1 complexed with antiglycan antibodies. The role of these CIC in the pathogenesis of IgAN is not known. METHODS: We studied how proliferation of cultured mesangial cells (MC) is affected by CIC prepared from sera of IgAN patients and healthy control subjects using size-exclusion chromatography. CIC-containing fractions were added to serum-starved MC in culture, and cell proliferation was measured using (3)H-thymidine incorporation. The results were confirmed by staining MC using an antibody against proliferating cell nuclear antigen. RESULTS: The incubation of starved MC with serum fractions with M(r) 800 to 900 kD, rich with galactose-deficient IgA1, stimulated proliferation, while fractions with smaller complexes were inhibitory. Furthermore, CIC-containing larger molecular mass fractions isolated from serum of an IgAN patient collected during an episode of macroscopic hematuria stimulated MC proliferation more than CIC obtained during a subsequent quiescent phase. To examine the role of IgA, we removed IgA1 from serum before fractionation. The resultant IgA1-depleted fractions were devoid of stimulatory IgA-CIC. Sera of IgAN patients were also fractionated after addition of desialylated galactose-deficient polymeric IgA1 to form additional immune complexes. Supplementation with a small quantity of this IgA1 increased cellular proliferation in assays using serum fractions of M(r)>/=800 to 900 kD; uncomplexed IgA1 did not affect MC proliferation significantly. In contrast, supplementation with a larger quantity of this IgA1 inhibited cellular proliferation in assays using serum fractions of M(r) 700 to 800 kD. CONCLUSION: Overall, these findings suggest that CIC containing aberrantly glycosylated IgA1 affect proliferation of MC in vitro and, thus, likely play a role in the pathogenesis of IgAN.     

雷公藤内酯醇对IgA肾病大鼠的治疗作用及对肾系膜区CD71表达的影响

目的：观察雷公藤内酯醇（triptolide,TP）对IgA肾病（IgAN）大鼠肾系膜区CD71表达的影响及其对IgAN的治疗作用。方法：将雄性SD大鼠随机分为正常组、IgAN模型组（模型组）、IgAN＋TP干预组（TP组）、IgAN＋泼尼松干预组（Pred组）,每组8只。采用牛血清白蛋白（BSA）＋葡萄球菌肠毒素（SEB）＋四氯化碳（CCl4）的方法建立IgAN大鼠模型,TP组给予TP100μg·kg^-1·d^-1,Pred组给予Pred5.0mg·kg^-1·d^-1灌胃。分别于0、7、11周测24h尿蛋白定量及尿红细胞计数。12周处死大鼠取血测血肌酐（Scr）、尿素氮（BUN）;肾组织行光镜、荧光、电镜观察病理学改变。并对系膜区IgA沉积及CD71表达水平作半定量分析。RT-PCR法检测CD71mRNA在肾组织中的表达水平。结果：造模7周后模型组、TP组、Pred组大鼠尿蛋白及红细胞均较正常组显著增多,第11周时TP组及Pred组尿蛋白及红细胞较模型组显著减少,TP组尿红细胞下降尤为明显。模型组大鼠光镜下肾系膜基质增生,系膜细胞增多,部分毛细血管攀闭塞;荧光下IgA呈团块状在系膜区沉积;电镜下足突融合,系膜增生,有块状电子致密物沉积,TP组及Pred组病理较模型组显著改善,正常组未见病理改变。CD71的蛋白及mRNA水平在模型组大鼠肾组织高表达,TP组及Pred组表达显著减弱,正常组微弱表达。CD71在肾系膜区的表达与IgA的沉积呈正相关。结论：IgAN大鼠肾系膜区CD71高表达,与IgA的沉积呈正相关,TP能显著减少CD71在肾系膜区的表达,减少IgA沉积,改善临床及病理损伤指标。可见TP通过下调系膜区中CD71的表达,进而减少IgA的沉积是TP治疗IgAN的机制之一。     

Effect of tet methylcytosine dioxygenase 2 on the regulation of transforming growth factor-β1 expression in mesangial cells induced by high glucose

Objective To investigate the role of tet methylcytosine dioxygenase 2 (TET2) in the regulation of transforming growth factor-β1 (TGF-β1) expression in human glomerular mesangial cells induced by high glucose. Methods Cultured human glomerular mesangial cells were divided into normal control group (5.5 mmol/L glucose) and high glucose group (30.0 mmol/L glucose) which was cultured for 12 h to 72 h. The gene expression of TET2 in mesangial cells were inhibited by small molecule chemical called SC1, and which were divided into high glucose group (30.0 mmol/L glucose+DMEM), DMSO group (30.0 mmol/L glucose+0.1%DMSO) and SC1 group (30.0 mmol/L glucose+3 μmol/L SC1). The mRNA and protein expression of TGF-β1, TET1 to 3 and α-smooth muscle actin (α-SMA) was detected by quantitative real-time PCR and Western blotting. Methylation of CpG islands in the regulation region of TGF-β1 was detected by bisulfite sequencing PCR (BSP). The activity of mesangial cell proliferation was assessed by colorimetry of thiazolyl blue (MTT). Results Compared with normal control group, the mRNA and protein expression of TET2 in mesangial cells induced by high glucose was increased significantly in a time-dependent manner (all P＜0.05), but the expression of TET1 and TET3 was not affected. Meanwhile methylation rate of 4 CG sites from 24 h to 72 h were decreased in the first exon of TGF-β1 (P＜0.01), but not in the promoter. Compared with high glucose group, when the expression of TET2 was inhibited by SC1, the methylation rate of TGF-β1 was recovered evidently (P＜0.05), the mRNA and protein expression of TGF-β1 and α-SMA was suppressed, and the proliferation of mesangial cells was decreased (all P＜0.05). Conclusions Demethylation of the CpG island mediated by TET2 may play an important role in the expression of TGF-β1 and mesangial cell phenotype transformation induced by high glucose.     

p38丝裂原活化蛋白激酶介导抵抗素调节高糖刺激下人肾小球系膜细胞增殖的作用

目的 观察巨噬细胞因子抵抗素过度表达对高糖刺激作用下人肾小球系膜细胞p38丝裂原活化蛋白激酶（MAPK）信号通路的影响，探讨抵抗素调控肾小球系膜细胞增殖及细胞外基质积聚的作用机制。 方法 通过转染携带野生型抵抗素基因的腺病毒载体 （Ad-resistin）构建过度表达抵抗素的人巨噬细胞模型，并与高糖刺激后的人肾小球系膜细胞共培养。3H-氚标胸腺嘧啶掺入法检测肾小球系膜细胞增殖。免疫细胞化学法检测系膜细胞激活蛋白1（AP-1）的表达。免疫荧光检测细胞外基质层粘连蛋白（LN）的表达。Western印迹检测系膜细胞内p38MAPK、转化生长因子(TGF)β1的表达并测定Smad2的磷酸化水平。 结果 Ad-resistin转染后，人巨噬细胞抵抗素 mRNA水平及蛋白表达均显著升高（P < 0.01）。与对照组比较，与过度表达抵抗素的人巨噬细胞共培养后，人肾小球系膜细胞p38MAPK、TGF-β1的蛋白表达显著增强；细胞内Smad2的磷酸化水平显著升高（P < 0.05）；肾小球系膜细胞出现明显的增殖（P < 0.01）；细胞外基质的合成增多（P < 0.05）。 结论 巨噬细胞因子抵抗素的过度表达可能通过p38MAPK信号通路，促进高糖刺激作用下肾小球系膜细胞的增殖及细胞外基质的异常积聚。     

黏膜方对IgA肾病大鼠异常糖基化IgA1及肾间质纤维化的干预及机制研究

目的:研究血清和肾组织中的异常糖基化IgA1水平与IgA肾病的间质纤维化程度的相关性;阐明黏膜方治疗IgA肾病的作用机制。方法:采用口服牛血清白蛋白、四氯化碳皮下注射和尾静脉注射脂多糖免疫复合法建立IgA肾病大鼠模型,随机分为正常组、模型组、中药组(黏膜方)及西药组(福辛普利钠)、中西医结合组(黏膜方+福辛普利),观察治疗后各组血清和肾组织异常糖基化的IgA1水平、血清IgA-纤维连接蛋白(IgA-FN)、肾组织转化生长因子-β1(TGF-β1)和α-平滑肌肌动蛋白(α-SMA)及其mRNA的表达量。结果:血清IgA-FN,中药组及中西医结合组低于模型组(P<0.001);血清异常糖基化IgA1量,中药组及西药组均低于模型组(P<0.05);肾组织中的异常糖基化IgA1水平,模型组高于正常组(P<0.05),各治疗组低于模型组(P<0.05);肾组织中TGF-β1mRNA及蛋白表达量模型组均显著高于正常组(P<0.01),各治疗组TGF-β1mRNA及蛋白表达均低于模型组(P<0.05);模型组α-SMAmRNA高于正常组(P<0.05),各治疗组低于模型组(P<0.001),模型组α-SMA蛋白表达阳性面积高于正常组(P<0.001),中西医结合组和西药组阳性面积低于模型组(P<0.05);肾组织α-SMAmRNA及蛋白表达与血清及肾组织中异常糖基化IgA1呈正相关(P<0.005);肾组织TGF-β1mRNA与血清及肾组织异常糖基化IgA1呈正相关(P<0.005)。结论:血清中异常糖基化IgA1与肾间质纤维化程度密切相关,黏膜方能减轻实验性IgAN大鼠血清及肾脏异常糖基化的IgA1水平,改善肾间质纤维化。     

Polymeric IgA increases the synthesis of macrophage migration inhibitory factor by human mesangial cells in IgA nephropathy.

BACKGROUND: It has been suggested that polymeric IgA (pIgA) or IgA immune complexes play a significant pathogenic role in IgA nephropathy (IgAN). Macrophage migration inhibitory factor (MIF) shares many activities with other pro-inflammatory cytokines. In human glomerulonephritis, including IgAN, glomerular expression of MIF is found to correlate with progressive renal injury. We hypothesized that deposition of pIgA within the kidney may lead to enhanced synthesis of MIF by mesangial cells. METHODS: In this study we examined the effect of pIgA and monomeric IgA (mIgA) from randomly selected patients with IgAN in clinical quiescence on the gene expression and protein synthesis of MIF in cultured human mesangial cells (HMC). RESULTS: Both pIgA and mIgA from IgAN patients or matched healthy controls increased MIF gene expression and protein synthesis in a dose-dependent fashion. The magnitude of MIF protein induction by pIgA (100 microg/ml) was similar to that of tumour necrosis factor-alpha (TNF-alpha) at 10 pg/ml. In all subjects, the induction of MIF was higher for pIgA when compared with mIgA (P < 0.01). Furthermore, the up-regulation of MIF synthesis by either pIgA or mIgA was significantly higher in IgAN patients than in healthy controls (P < 0.05). Similarly, pIgA and mIgA were able to induce TNF-alpha gene expression and protein synthesis in mesangial cells. Incubation of mesangial cells with neutralizing antibody to TNF-alpha reduced the MIF synthesis induced by pIgA. CONCLUSION: We demonstrate that pIgA is capable of inducing MIF and TNF-alpha production in HMC, which may play a major pathogenic role in IgAN. Induction of MIF can be partially blocked by neutralizing antibody to TNF-alpha, suggesting the possibility that up-regulation of MIF synthesis in HMC is mediated via an amplifying proinflammatory loop involving TNF-alpha.     

三种实验性IgA肾病模型的比较

目的探讨建立一种理想的IgA肾病（IgAN）动物模型方法。方法分别采用葡聚糖G200、大肠杆菌外膜蛋白和金葡菌的细胞膜20肽抗原决定簇诱导小鼠IgA肾病模型。用分子生物学和病理学方法对3组IgAN模型小鼠进行鉴定和比较。结果（1）葡聚糖组尿蛋白增高，伴有血尿；免疫荧光显示部分肾小球大量IgA沉积；光镜下肾小球系膜细胞增多，肝和脾可见弥漫性的粉染物质沉积；电镜下肾小球系膜区少量低电子密度的致密沉积物，肝和脾可见淀粉丝样物质沉积。（2）大肠杆菌外膜蛋白组尿蛋白增高，伴有血尿；免疫荧光显示肾小球有少量IgA沉积；光镜下肾小球系膜细胞轻度增多，间质炎细胞浸润明显；电镜下肾小球系膜区无电子致密沉积物。（3）金葡菌细胞膜20肽抗原决定簇组尿蛋白增高，伴有血尿；免疫荧光显示多数肾小球均可见大量IgA沉积；光镜下肾小球系膜细胞增多，伴系膜基质轻度增生；电镜下肾小球系膜区和基底膜的内皮细胞下可见高电子密度的致密沉积物。结论金葡菌细胞膜20肽抗原决定簇组诱导的IgAN模型从临床表现和病理学变化与人IgAN极其相似，是3种IgAN模型中最理想的IgAN模型。     

IgA肾病患者腭扁桃体单个核细胞IgA类别转换基因表达变化

目的 研究脂多糖(LPS)或溶血性链球菌(HS)刺激IgA肾病和非肾脏疾病慢性扁桃体炎患者腭扁桃体单个核细胞Iα-Cα胚系转录本、激活诱导的胞嘧啶脱氨酶(AID)mRNA和蛋白的表达,以探讨IgA肾病腭扁桃体单个核细胞IgA及IgA1产生异常的分子机制.方法 入组2009年1月到2010年2月在我院住院的IgA肾病患者27例,非肾脏疾病慢性扁桃体炎患者27例作为对照.通过单个核细胞分离液和密度梯度离心法分离出腭扁桃体单个核细胞.IgA肾病组及非肾脏疾病慢性扁桃体炎组腭扁桃体单个核细胞分别分为3组:LPS刺激组,HS刺激组和未刺激组.ELISA法检测培养上清中IgA和IgA1的浓度.实时PCR检测Iα-Cα胚系转录本和AID mRNA的表达;Western印迹检测AID蛋白的表达.结果 IgA肾病组腭扁桃体单个核细胞IgA和IgA1的分泌,特别是IgA1/IgA较慢性扁桃体炎组显著增加(P＜0.05),Iα-Cα和AID mRNA和AID蛋白的表达较慢性扁桃体炎组显著增加(均P＜0.05).IgA肾病组腭扁桃体单个核细胞IgA和IgA1的水平在刺激后明显增加(P＜0.05);Iα-Cα和AID mRNA的表达明显上调(均P＜0.05);AID蛋白表达明显增加(LPS刺激组P＜0.05,HS刺激组P＜0.01).结论 LPS和HS均能够诱导IgA肾病患者腭扁桃体单个核细胞IgA和IgA1的分泌、AID和Iα-Cα的表达增加,提示IgA肾病患者腭扁桃体IgA和IgA1的分泌增加可能与IgA类别转换相关基因AID和Iα-Cα高表达有关.     

Differential effects of circulating IgA isolated from patients with IgA nephropathy on superoxide and fibronectin production of mesangial cells.

BACKGROUND: IgA nephropathy (IgAN) is characterized by predominant deposition of IgA in the glomerular mesangium. Serum IgA is often elevated in patients with IgAN, and it has been postulated that it is responsible for the mesangial lesions. However, the direct effect of circulating IgA on mesangial cells is not clear. METHODS: We investigated the effects of sera and IgA which were isolated from patients with IgAN on thymidine uptake, superoxide and fibronectin production and fibronectin mRNA expression of cultured rat mesangial cells, and we compared the findings to the effects of IgA isolated from patients with non-IgA mesangial proliferative glomerulonephritis (MsPGN) and normal controls. IgA was isolated with affinity chromatography using cyanogen bromide activated Sepharose 4B coupled to sheep antihuman IgA antiserum. RESULTS: Our results demonstrated that both sera and IgA from patients with IgAN dose-dependently increased mitogenesis of mesangial cells as measured by (3)H-labeled thymidine uptake. The thymidine uptake by sera and IgA isolated from patients with IgAN was significantly higher than that of sera and IgA isolated from patients with MsPGN and normal controls. Sera and IgA from patients with IgAN significantly enhanced superoxide and fibronectin production and fibronectin mRNA expression of mesangial cells. The superoxide and fibronectin production was also significantly higher as compared with patients with MsPGN and normal controls. CONCLUSIONS: Our results indicate that circulating IgA isolated from patients with IgAN is different from that of patients with MsPGN and normal controls and may potentially induce oxidative injury and production of extracellular matrix of glomerular mesangial cells in IgAN.