In situ hybridization studies of stromelysin and tissue inhibitor of metalloproteinase 1 in IgA nephropathy

Summary: Accumulation of the extracellular matrix (ECM) in IgA nephropathy (IgAN) is thought to cause deterioration of glomerular function. Stromelysin and tissue inhibitor of matrix proteinase 1 (TIMP1) may play an important role in the turnover of the glomerular ECM. However, the expression of these enzymes in human renal tissues remains undefined. In the present study, non-radioactive in situ mRNA hybridization, which permitted the analysis at a cellular level, was performed to localize stromelysin and TIMP1 in renal tissue of IgAN. We also determined the percentage of cells positive for stromelysin or TIMP1 mRNA among intraglomerular cells. A total of 16 patients with IgAN were examined, including eight patients with severe histopathological changes and eight with mild changes. Three patients without glomerular disease were also studied. Stromelysin and TIMP1 mRNA were weakly expressed in the mesangium of normal kidneys and IgAN renal tissues with mild damage. However, the expression of both mRNA was significantly increased in the area of mesangial proliferation, in glomerular epithelial cells and in Bowman's capsule of advanced lesions. Several cells in the area of mesangial proliferation were double positive for stromelysin and TIMP1 mRNA, while certain cells positive for stromelysin mRNA did not express TIMP1 mRNA. In the interstitium, epithelial cells of certain tubules and some mononuclear cells were positively stained for these mRNA, especially in advanced lesions. Our results indicated that stromelysin and TIMP1 genes were expressed in glomerular resident cells, tubular epithelial cells and infiltrated mononuclear cells in IgAN, and their expression was enhanced in advanced tissue damage. the demonstration of a co-expression and discordant expression of the genes indicates that each gene expression may be regulated in a cell type-specific manner and that it could also be altered by cellular environmental factors.     

Intrarenal synthesis of IL-6 in IgA nephropathy

Summary: Recent in vitro studies have shown the synthesis of interleukin-6 (IL-6) in glomerular mesangial and epithelial cells, and suggested the involvement of IL-6 in mesangial proliferative glomerulonephritis. However, the expression site of IL-6 mRNA in renal tissue of IgA nephropathy (IgAN), the most common chronic mesangial proliferative glomerulonephritis, remains obscure. to localize IL-6 mRNA in renal biopsy specimens of IgAN, we used nonradioactive in situ hybridization (ISH) developed in our laboratory, sensitive in detecting individual cells positive for a specific mRNA. In some sections, periodic acid-Schiff staining was performed after ISH in order to identify the topographical relation between IL-6 mRNA positive cells and glomerular basement membrane and mesangial area. In situ hybridization for IL-6 mRNA and immunohistochemistry for CD3 and CD68, markers for lymphocytes and monocytes, respectively, were also performed on serial sections to examine the contribution of infiltrated mononuclear cells to cells positive for IL-6 mRNA in glomeruli. Glomerular resident cells, including glomerular mesangial and epithelial cells and cells of Bowman's capsule, as well as tubular epithelial cells and infiltrated mononuclear cells expressed IL-6 mRNA. We also compared the localization of IL-6 mRNA and protein and showed different distribution between the gene product and protein. the expression of IL-6 mRNA correlated with the degree of mesangial cell proliferation and tubulointerstitial changes. Our results indicate that IL-6 is synthesized in renal tissues of IgAN and suggest that the increased IL-6 expression may be important in the pathogenesis of IgAN.     

IgA肾病外周血B淋巴细胞微核糖核酸差异表达及临床意义

目的通过检测人外周血B淋巴细胞微核糖核酸(microRNA)的差异表达,并分析其与IgA肾病临床病理特点、IgA1分子O-糖基化异常之间的关系,探索microRNA在IgA肾病发病中的可能机制。方法收集7例IgA肾病患者及4例正常人外周血5 ml,磁珠法分选出CD19~+B淋巴细胞,提取RNA,采用表达谱芯片筛选差异表达的microRNA。在另外29例IgA肾病患者和16例正常对照者中,对候选microRNAs用实时荧光定量聚合酶链反应(quantificational real-time polymerase chain reaction,QRT-PCR)进行验证,并分析它们与临床病理、IgA1分子半乳糖缺陷(galatose-defecient IgA1,Gd-IgA1)水平的关系。结果 IgA肾病患者外周血B淋巴细胞microRNA表达明显异常,其中85个明显上调,30个明显下调。对5个候选microRNA:miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p,进一步验证显示,其表达水平在IgA肾病患者与正常对照之间差异无统计学意义(P0.05)。且上述5个microRNA表达量与IgA肾病患者的临床病理指标没有明显的相关性。IgA肾病组血清IgA1、Gd-IgA1明显高于正常对照(P0.05或P0.01)。5个候选microRNA:miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p的水平与IgA肾病患者IgA1分子O-连接半乳糖缺陷之间没有明显相关性。结论 IgA肾病患者和正常人外周血CD19~+B淋巴细胞microRNA表达谱存在明显差异。初步验证显示miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p的表达水平在IgA肾病患者与正常对照之间差异无统计学意义,且与IgA肾病的临床病理特点和Gd-IgA1之间无明显相关性,提示这5个microRNA可能不是参与IgA肾病发病和进展的重要microRNA。     

IgA serology in recurrent and non-recurrent IgA nephropathy after renal transplantation

BACKGROUND: This study investigated whether abnormal circulation of macromolecularIgA and IgA with altered glycosylation or electrical chargeplays a role in the recurrence of IgA nephropathy (IgAN) aftertransplantation. STUDY DESIGN: A total of 92 renal transplant patients were enrolled; 52 IgANpatients and 40 with other non-IgAN. The IgAN group included10 patients showing IgA mesangial deposits in the grafted kidneys(recurrent group) and 10 who did not (immunohistochemicallyproven non-recurrent group). In addition another 22 IgAN transplantpatients were clinically free of recurrent disease. METHODS: The analyses included macromolecular IgA (IgAIC) detected bythe conglutinin assay (K), heavy IgA precipitated in 2.5% polyethyleneglycol (PEG), IgA-fibronectin aggregates (IgA/F Aggr), mixedIgA/IgGIC, IgA binding to mesangial matrix components (fibronectin,laminin, type IV collagen) or polycations (poly-L-lysine) andIgA with altered glycosylation (Jacalin-binding assay). RESULTS: After transplantation, IgAN patients displayed significantlyhigher mean levels for each variable measured than non-IgAN(ANOVA, P <0.05). By stepwise regression analysis, the bindingof IgA to fibronectin had the highest coefficient. By comparingdata in recurrent and clinically non-recurrent IgAN, we observedthat two groups could be distinguished by the results of thetwo assays for macromolecular IgA (conglutinin IgAIC and IgA-fibronectinaggregates) and IgA with increased affinity for type IV collagen(P <0.05). When the selected group of immunohistochemicallyproven non-recurrent IgAN was compared to the recurrent one,a statistically significant difference was found only for thebinding of IgA to type IV collagen (P<0.05). Data from thistest were significantly related with proteinuria (P<0.05)and microscopic haematuria (P <0.04). CONCLUSION: Even though the IgA serology of renal transplant IgAN patientsshows peculiar features and recurrent and non-recurrent IgANdiffer in many aspects, the prevalence of positive data in thetwo groups had no predictive value. This suggests that the recurrenceof IgAN is modulated by factors affecting the interaction betweencirculating abnormal IgA and mesangial cells and/or matrix.     

Expression of connective tissue growth factor mRNA in human IgA nephropathy

Background. Connective tissue growth factor (CTGF) is a cysteine-rich member of a new family of growth regulators. Its upregulation is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis. Methods. We evaluated the expression and localization of CTGF mRNA in renal tissues of 20 patients with IgA nephropathy (IgA-N) and 5 normal human kidneys (NHK), using high-resolution in situ hybridization with digoxigenin-labeled oligonucleotide. The expression level of CTGF mRNA was quantitated by counting all nuclei, as well as nuclei surrounded by CTGF mRNA-positive cytoplasm in at least ten randomly selected cross-sections of nonsclerotic glomeruli, and expressing the results as percentage of positive cells. Results. In both IgA-N and NHK, CTGF mRNA was mainly expressed in glomerular intrinsic cells, including mainly glomerular mesangial and epithelial cells, and some endothelial cells and cells of Bowman's capsule. CTGF mRNA-positive cells were abundant in tubulointerstitial fibrotic areas, especially in IgA-N with severe tissue damage. CTGF mRNA expression was also increased in vascular cells in IgA-N. The percentage of cells positive for CTGF mRNA was significantly higher in IgA-N than in NHK. Furthermore, the percentage of cells positive for CTGF mRNA was significantly greater in IgA-N with moderate mesangial proliferative lesions than in IgA-N with mild mesangial proliferative lesions and/or sclerotic lesions. Conclusions. Our study indicates that CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in IgA-N. Received: July 17, 2000 / Accepted: October 21, 2000     

IgA肾病520例临床病理分析

目的研究IgA肾病(IgAN)的临床和病理特点及其相互关系。方法对1992年11月～2003年6月温州医学院附属第一医院肾内科病理室肾活检诊断的原发性IgAN520例进行临床与病理分型关系的分析。结果520例IgAN临床表现以无症状性尿检异常最常见,占346例(66.5%),其次是慢性肾炎和肾病综合征,分别占77例(14.8%)和66例(12.7%)。病理类型以局灶节段硬化性肾小球肾炎最常见,占186例(35.8%),其次是系膜增生性肾小球肾炎、轻微病变肾小球肾炎和局灶节段增生性肾小球肾炎,分别为116例(22.3%)、104例(20%)和63例(12.1%)。结论IgAN的临床病理表现多样化并具有一定特点。临床表现最常见为无症状性尿检异常,在病理上最常见的是局灶性肾小球病变类型。     

Contribution of cellular infiltration to the progression of IgA nephropathy: A longitudinal, immunocytochemical study on repeated renal biopsy specimens

Summary: A retrospective immunocytochemical study was performed on repeated renal biopsy specimens from 47 patients with IgA nephropathy, 23 of whom received steroid therapy after the initial biopsy. Immune cells in renal tissues were detected by the immunoperoxidase method using monoclonal antibodies against common leukocyte antigens, T cells, B cells and monocytes/macrophages.
Overall, glomerular infiltration of total leukocytes and monocytes/macrophages was significantly correlated with proteinuria. Interstitial infiltration of total leukocytes, T cells and monocytes/macrophages was significantly correlated with histological injury and renal dysfunction. In the steroid-treated group (group S), urinary protein and glomerular infiltration of total leukocytes and macrophages were significantly reduced at the follow-up biopsy, while these parameters remained unchanged in the nonsteroid-treated group (group N). In group S, interstitial infiltration of almost all of the various cell types, histological renal damage and renal function remained unchanged at the follow-up biopsy, while group N showed a significant increase in the number of interstitial total leukocytes, T cells and macrophages and showed a significant progression of histological renal injury.
These findings suggest that glomerular immune cells, especially monocytes/macrophages, are involved in inducing proteinuria and interstitial immune cells are involved in renal deterioration. Furthermore, steroid therapy appears to reduce urinary protein and prevent the progression of tissue injury through suppression of renal infiltration of these inflammatory cells.     

Multiplicity of histopathologic renal lesions in IgA nephropathy: Retrospective analysis of histologic grade and stage in serial biopsy specimens

Background A slit system for scoring histologic grade and stage has been proposed for the evaluation of renal tissue injury in IgA nephropathy. This grade-stage system (G-S system) represents the histologic analysis of the disease state. Methods The first and the subsequent renal biopsy specimens were analyzed using the G-S system in periodic acid methenamine silver stained sections. The sequential change of histologic grade and stage seen in the serial sections was examined to show the natural histologic events occurring during the prolonged course of this disease. Results The histologic multiplicity of IgA nephropathy was simplified by subclassifying the findings into 4 groups: low active, active, active sclerosing, and sclerosing. Since the histologic grade represents activity or acuteness of the disease, and stage correlates with sclerosis, the low active group can be expected to be the major population, and the other 3 groups will include cases with more advanced IgA nephropathy. Conclusion The G-S system was useful for measuring the disease state of IgA nephropathy. Such a slit system is recommended to aid in the selection of therapy and the follow-up of patients.     

Specific binding of circulating IgA antibodies in patients with IgA nephropathy

Detection of circulating IgA antibodies which are specific in patients with IgA nephropathy is described. Freeze and thawed extracts of pharyngeal cells obtained from patients with IgA nephropathy, other glomerular diseases, and healthy adults were cultured with fibroblasts such as Vero or Hel cells at 37 degrees C for 2 weeks. Serum samples were obtained from these patients and healthy adults. The cultured fibroblasts were fixed on slide glasses, and then incubated with the serum samples from the same or other patients with IgA nephropathy. The cells were stained with FITC-labeled heavy-chain specific anti-human IgA antiserum and then examined with a fluorescent microscope. It was demonstrated that the IgA antibodies in sera obtained from patients with IgA nephropathy or HSP nephritis were bound with the nuclear regions of such fibroblasts. It was suggested that IgA antibodies in sera could be bound with some antigenic substances which were transferred from pharyngeal cells of patients with IgA nephropathy to fibroblasts in vitro.     

IgA肾病患者肾脏组织中差异细胞因子的探索研究

目的筛选IgA肾病(IgAN)患者肾脏组织中的差异蛋白质-细胞因子,进一步探讨IgAN的发病机制。 方法入选年龄18～60岁,解放军总医院肾脏病科住院肾活检确诊为IgAN患者10例;泌尿外科行肾癌肾脏切除的患者10例,取癌旁6 cm以外正常肾脏组织作为对照。肾脏组织利用L-493细胞因子抗体芯片进行检测,筛选差异蛋白质,并对差异蛋白质进行基因本体论(GO)分析。以IL-33(白介素-33)利用酶联免疫吸附试验(ELISA)进行验证。采用IBM SPSS Statistics 19.0软件对结果进行统计分析,利用T检验对差异蛋白质进行筛选,P<0.05,并且差异蛋白质差异倍数>1.4认为是差异蛋白质。 结果L-493细胞因子抗体芯片检测,在20例患者中(IgAN组10例,对照组10例),发现了25种差异蛋白质,GO分析结果表明：这些蛋白质主要与应激、细胞粘附、蛋白质代谢、信号转导相关。ELISA验证结果显示：IgAN患者肾组织中IL-33表达水平显著高于对照组。 结论IgAN患者肾脏组织中差异表达蛋白质直接或间接调控着炎症、细胞增殖和凋亡及纤维化过程。IL-33可能与IgAN发病机制有关。     

血脂与IgA肾病患者肾脏预后的相关性研究

目的了解原发性IgA肾病(IgAN)血脂异常患者的临床、病理特征,探讨血脂对IgAN肾脏预后的影响。 方法回顾性分析2000年1月1日至2018年12月31日在我院肾活检确诊的原发性IgAN患者的资料,随访截止2020年1月1日,随访的终点事件是终末期肾病(ESRD)或估算的肾小球滤过率(eGFR)下降≥50%,未达终点事件者随访最少1年。按肾活检时的基线血脂水平并根据血脂异常诊断标准,将IgAN患者分为血脂异常组(450例)及血脂正常组(331例),血脂异常组包括高胆固醇组(高TC组)、高甘油三酯组(高TG组)、高低密度脂蛋白组(高LDL组)及低高密度脂蛋白组(低HDL组)4个单一指标亚组。参照IgAN牛津分型进行病理评分,Logistic回归和Cox回归模型分析影响IgAN患者预后的风险因素,采用Kaplan-Meier生存曲线比较血脂异常组和血脂正常组IgAN患者生存率的差异。 结果血脂异常组年龄、身体质量指数(BMI)、血压、血肌酐、血尿酸、尿蛋白定量高于血脂正常组,而血白蛋白、eGFR低于血脂正常组(P<0.05)。根据牛津分型评分,与其它组比较,低HDL组IgAN患者的肾小管间质病变程度更重(P<0.05)。Logistic回归分析提示,年龄大(OR 1.044,95%CI：1.023～1.066,P<0.001)、高平均动脉压(OR 1.025,95%CI：1.008～1.043,P＝0.004)、低血红蛋白(OR 0.963,95%CI：0.950～0.976,P<0.001)、高TG(OR 1.008,95%CI：1.005～1.010,P<0.001)、低HDL(OR 0.546,95%CI：0.311～0.959,P＝0.035)、高24 h尿蛋白定量(OR 1.185,95%CI：1.039～1.352,P＝0.011)和高牛津分型T评分(OR 9.115,95%CI：5.297～15.685,P<0.001)是IgAN基线肾功能下降的风险因素。多因素Cox回归模型分析结果显示,低血红蛋白(OR 0.965,95%CI：0.949～0.980,P<0.001)、低基线eGFR(OR 0.984,95%CI：0.973～0.996,P＝0.008)、高24 h尿蛋白定量(OR 1.151,95%CI：1.043～1.271,P＝0.005)、高牛津分型T评分(OR 1.680,95%CI：1.033～2.732,P＝0.036)和高TG(OR 1.177,95%CI：1.038～1.334,P＝0.011)是IgAN肾脏不良预后的风险因素。Kaplan-Meier生存曲线分析显示,随访血脂异常组IgAN患者的肾脏中位生存时间显著短于血脂正常组(χ²＝8.316,P＝0.004)。 结论HDL与肾小管间质病变相关,高TG是IgAN肾脏预后不良的风险因素,临床上应加强对IgAN患者的血脂监测。     

IgA肾病患者IgA1糖基化异常及其致病机制

IgA肾病(IgAN)是导致终末期肾病最常见的原发性肾小球疾病。其病理特点为IgA1在肾小球系膜区沉积,IgA1分子的异常糖基化是导致IgAN发病的关键因素。多种与IgAN相关的基因位点已经被发现。这些基因编码的细胞因子参与了IgA1糖基化异常的发病机制。此外糖基化酶缺乏、分子伴侣甲基化异常都可能导致IgA1异常糖基化。异常糖基化的IgA1可通过自我聚集或形成免疫复合物沉积于系膜区,进而刺激系膜细胞增殖、分泌系膜基质、细胞因子、趋化因子、生长因子等,导致肾小球损伤。对IgA1异常糖基化的深入研究有助于了解IgA肾病的发病机制并提供新的诊断与治疗措施。     

IgA肾病患者肾组织中增殖细胞核抗原表达

用免疫组化方法研究了２０例原发性ＩｇＡ肾病患者肾组织中增殖细胞核抗原（ＰＣＮＡ）表达。结果显示ＩｇＡ肾病患者肾组织中ＰＣＮＡ表达增加，肾小球内ＰＣＮＡ阳性细胞数、肾小管和间质中ＰＣＮＡ阳性细胞百分数均与肾组织学损害程度呈正相关；临床－病理研究显示肾小管中ＰＣＮＡ阳性细胞百分数分别与２４小时尿蛋白量、血清肌酐浓度（Ｓｃｒ）呈正相关；肾间质中ＰＣＮＡ阳性细胞百分数亦与２４小时尿蛋白量呈正相关。肾脏细胞增殖程度作为一项判断肾小球肾炎组织学损害程度和预后的指标值得进一步研究。     

Demonstration of secretory IgA in kidneys of patients with IgA nephropathy.

BACKGROUND: Recently we reported a possible role for secretory IgA (SIgA) in IgA nephropathy (IgAN), as suggested by increased serum levels in patients with active disease and accumulation of SIgA in a glomerular eluate. Therefore, we attempted to find support for these findings by analysis of the presence of SIgA in biopsies of IgAN patients. METHODS: Renal biopsies of 26 patients with biopsy-proven IgAN were analysed for the presence of SIgA and complement proteins. RESULTS: In 15% mesangial deposition of SIgA was demonstrated, using a specific staining for secretory component (SC) and colocalization with IgA. The presence of SIgA in these biopsies showed a strong correlation with deposition of mannose-binding lectin (MBL) and C4d. Moreover, we observed a strong colocalization between SIgA and MBL or C4d. This local complement activation has previously been linked to more severe renal disease. CONCLUSIONS: Therefore, these data provide additional evidence for a pathogenic role for SIgA in IgA nephropathy.     

Ⅰ型纤溶酶原激活物抑制物在IgA肾病肾小管间质损伤中的作用研究

目的 探讨Ⅰ型纤溶酶原激活物抑制物（ＰＡＩ－１）在ＩｇＡ肾病肾小管间质损害中的作用。方法 采用原位杂交和免疫组织化学技术,分别在基因和蛋白质水平检测３８例ＩｇＡ肾病患者肾小管间质中的ＰＡＩ－１表达,同时观察α－平滑肌肌动蛋白和增殖细胞核抗原（ＰＣＮＡ）的表达变化。     

Characteristics of patients with coexisting IgA nephropathy and membranous nephropathy

Background: Coexistence of IgA nephropathy (IgAN) and membranous nephropathy (MN) in the same patient is rare. Few studies have reported the clinical and pathological features of patients with combined IgAN and MN (IgAN–MN).

Methods: The clinico-pathological features, levels of galactose-deficient IgA1 (Gd-IgA1) and autoantibodies against M-type transmembrane phospholipase A₂ receptor (anti-PLA₂R) in sera were compared among IgAN–MN, IgAN, and MN patients.

Results: Twenty-six patients with biopsy-proven IgAN–MN were enrolled. The mean age at biopsy was 43.6?±?15.9?years, and 65.4% were male. Proteinuria and estimated glomerular filtration rate (eGFR) levels in patients with IgAN–MN were similar to that of MN patients. Compared with the IgAN patients, IgAN–MN patients showed a higher median proteinuria level (4.3 vs. 1.2?g/day, p?2, p?p?=?.801). Percentage of IgAN–MN patients with detectable serum levels of anti-PLA₂R was lower than that of MN patients (38.5% vs. 68.6%, p?=?.011).

Conclusions: IgAN–MN patients display similar clinical features to MN patients and milder pathological lesions than IgAN patients. IgAN–MN patients have similar levels of Gd-IgA1 to those of IgAN patients, and a lower proportion of anti-PLA₂R than MN patients.     

Losartan reduces proteinuria and preserves renal function in hypertensive patients with IgA nephropathy

Background. The objectives of this study were to evaluate the effects of the angiotensin II receptor antagonist losartan on blood pressure (BP), proteinuria, and renal function in hypertensive patients with IgA nephropathy. Method. The study subjects comprised 18 patients with biopsy-proven IgA nephropathy with mild hypertension. Patients were classified into three groups (good/relatively good, relatively poor, poor) according to renal histologic findings and treated once a day with losartan 50 mg for 12 months. Changes in BP, proteinuria, renal function, and biochemical parameters were prospectively evaluated before and after the treatment. Results. BP began to fall after 1 month and proteinuria decreased significantly after 9 months of therapy. Glomerular filtration rate (GFR), renal plasma flow (RPF), and filtration fraction (FF) did not change throughout the observation period. There was no significant difference between the three different histologic groups in relation to the effects of losartan on BP, proteinuria, and renal function. We found that in patients with a proteinuria reduction rate of more than 50%, RPF increased and FF decreased significantly. Although it has been reported that losartan has a uric acid lowering effect, this study could not confirm such an effect. Conclusions. Losartan lowers blood pressure and decreases proteinuria in hypertensive patients with IgA nephropathy. These effects appear to be independent of renal histologic findings. This study suggests that the antiproteinuric effect of losartan is primarily mediated by changes in glomerular hemodynamics. Received: April 13, 2001/Accepted: July 15, 2002 Correspondence to:H. Ohashi