柴芩肾安方对IgA肾病大鼠肾小球足细胞nephrin表达的影响

目的：探讨柴芩肾安方对IgA肾病（IgAN）大鼠肾小球足细胞nephrin表达的影响。方法：通过切除单侧肾并反复静脉注射葡萄球菌肠毒素B（SEB）复制大鼠IgAN模型,分为正常组、切肾组、IgAN组、柴芩肾安方组和氯沙坦组。第12周末,检测各组大鼠24h尿蛋白量（Upr）,观察肾小球形态学和超微结构变化,并采用免疫组化和实时定量PCR法检测肾小球足细胞nephrin蛋白及mRNA的表达。结果：与正常组相比,IgAN组大鼠Upr显著升高（P〈0.01）,肾小球系膜增生,足突融合明显,足细胞nephrin蛋白及mRNA表达均明显下调（P〈0.01）。经柴芩肾安方治疗后上述指标均明显改善,与IgAN组比较差异有统计学意义（P〈0.01或P〈0.05）。结论：柴芩肾安方能减轻IgAN大鼠蛋白尿及肾小球病变,这可能与其恢复肾小球足细胞nephrin表达有关。     

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。     

肾炎康复片治疗对IgA肾病患者尿足细胞nephrin的影响

近年来,随着对肾小球足细胞研究的不断深入,人们逐渐认识到肾小球足细胞受损不仅可导致蛋白尿的发生,还是肾小球硬化的始动环节,各种损伤如血流动力学改变、足细胞基因突变（包括nephrin、a—actin4、CD2AP等）等均可直接引起足细胞脱落,进而导致肾小球基底膜裸露、肾小球硬化等一系列改变。IgA肾病是我国常见的慢性肾小球疾病之一,其预后不良,最终可导致慢性肾衰竭。我们采用肾炎康复片治疗IgA。肾病,观察其对患者尿足细胞的影响。     

罗格列酮对糖尿病肾病大鼠足细胞nephrin和podocin表达的影响

本研究采用糖尿病肾病（DN）大鼠模型，通过观察肾组织足细胞超微结构及其相关分子表达的变化，以及罗格列酮对其的影响，进一步揭示足细胞相关分子在实验性DN发病中的作用及脂质过氧化物增殖物激活受体γ（PPARγ）激动剂治疗DN的分子机制．为DN的预防和治疗提供理论依据。     

中药合剂对早期糖尿病肾病大鼠足细胞nephrin和podocin表达的影响

也页目的：观察中药合剂（ TCM mixture）对糖尿病肾病（ DKD）大鼠足细胞裂孔隔膜蛋白分子（ nephrin和podocin）表达的影响,探讨其对DKD的防治作用。方法：高脂高糖喂养8周联合小剂量STZ（30 mg/kg）建立T2DM大鼠模型,将DM模型大鼠随机分为中药合剂治疗组（B）及T2DM模型对照组（A）;另设正常对照组（NC）。各组于治疗前及干预后4、8、12周末检测大鼠血糖浓度及尿微量白蛋白;第12周末称大鼠体重（ BW）、肾重（ KW）,计算肾肥大指数（ KW/BW）,观察血清尿素氮（ BUN）、肌酐（ Scr）、总胆固醇（ TC）、三酰甘油（ TG）变化;光镜观察肾脏病理改变;应用免疫组化技术观察足细胞相关蛋白Nephrin、Podocin肾组织分布与表达;采用Western blot技术测定肾皮质Nephrin、Podocin 蛋白的表达。结果：应用中药合剂干预后与T2DM模型对照组相比,大鼠的尿微量白蛋白总量、肾肥大指数、血糖、尿素氮、血肌酐较T2DM模型组明显降低（ P〈0.05）,体重增加（P〈0.05）,病理改变减轻,nephrin和podocin蛋白表达增加（P〈0.05）。结论：中药合剂能提高nephrin和podocin的表达,促进足细胞损伤修复,减少尿微量白蛋白的排泄,减缓糖尿病肾病的进展。     

雷公藤内酯醇对IgA肾病大鼠蛋白尿和nephrin、podocin蛋白及mRNA表达的影响

目的:观察雷公藤内酯醇(TP)对IgA肾病(IgAN)大鼠尿蛋白和足细胞nephrin、podocin蛋白及mRNA表达的影响。方法:采用牛血清白蛋白+四氯化碳+脂多糖的方法建立IgAN大鼠模型。将60只大鼠随机分为正常组、模型组、洛丁新组、TP剂量组、TP中剂量组、TP高剂量组,每组10只。于0,10,14周收集血尿标本,并处死大鼠留取肾组织标本。检测24 h尿蛋白定量,血生化指标,分别采用实时定量PCR及ELISA法检测肾组织nephrin、podocin mRNA和蛋白表达。结果:(1)实验前各组大鼠尿蛋白差异无统计学意义(P>0.05),第10周末各造模组蛋白尿均明显高于正常组(P<0.01),第14周末TP各剂量组及洛丁新组蛋白尿均较模型组明显减少(P<0.01),TP高剂量组Alt值高于其余各组(P<0.05);(2)与正常组相比,模型组nephrin、podocin蛋白、mRNA表达明显降低(P<0.01);TP各剂量组及洛丁新组nephrin、podocin蛋白、mRNA表达均较模型组明显增高(P<0.05);TP中、高剂量组较洛丁新组nephrin、podocin蛋白浓度显著增高(P<0.05);TP各剂量组与洛丁新组及TP各剂量组间nephrin、podocin mRNA表达均差异无统计学意义(P>0.05)。结论:TP能够明显减轻IgAN大鼠的蛋白尿,且能明显提高肾组织nephrin、podocin mRNA及蛋白的表达。提示TP能调节IgAN足细胞相关分子nephrin、podocin基因及蛋白的表达,减少足细胞损害,减少尿蛋白。     

Rac1抑制剂上调糖尿病小鼠肾小球nephrin的表达

目的：探讨Rac1抑制剂（NSC33766）对STZ诱导的糖尿病小鼠肾小球内nephrin水平的影响。方法：采用腹腔单剂注射链脲佐菌素（streptozotocin,STZ,150mg/kg）的方法建立小鼠糖尿病模型,检测NSC33766对正常和糖尿病小鼠尿白蛋白排泄率、血清肌酐水平的影响,采用PAS染色观察肾脏组织学的改变,透射电镜观察超微结构的改变;采用免疫组化观察NSC33766对肾组织内足细胞骨架蛋白nephrin表达的影响。结果：NSC33766降低糖尿病小鼠尿白蛋白排泄率;减少肾组织内系膜区细胞外基质的积聚和基底膜的厚度,改善肾小球滤过膜足突融合,上调nephrin表达,未发现NSC33766具有降低血糖的作用。结论：Rac1抑制剂NSC33766可能通过上调肾小球内nephrin水平,改善足细胞骨架的方式,对糖尿病肾脏损伤具有保护作用。     

一种成人肾病综合征患者尿沉渣足细胞分子nephrin及Podocalyxin检测方法

目的:探讨采用ELISA方法以尿AQP(Aquaporin)-2校正检测肾病综合征尿nephrin podocalyxin排泌,以帮助评估成人肾病综合征的诊断和病情。方法:入组71例经肾穿刺明确病理诊断的成人肾病综合征(NS)患者,包括20例增殖性肾小球疾病的患者,49例非增殖的患者和2例淀粉样变性患者。22例FSGS患者(其中13例确定FSGS亚型诊断)。选取10例健康志愿者作为研究对照组。用ELISA法测定尿nephrin,podocalyxin和AQP-2。以尿AQP2作为内参照校正尿nephrin(neph/AQP)和podocalyxin(PCX/AQP)。结果:尿neph/AQP和PCX/AQP正相关(R=0.616,P<0.001),正常人尿neph/AQP和PCX/AQP显著低于NS患者(P<0.05),增殖和非增殖性肾病综合征患者的尿neph/AQP和PCX/AQP差异无统计学意义(P>0.05),FSGS患者尿neph/AQP和PCX/AQP中显著高于其他NS患者(P<0.05),在IgMN患者尿neph/AQP和PCX/AQP增加,肾淀粉样变性患者的尿neph/AQP和PCX/AQP较少。对13例明确FSGS病理亚型诊断的患者尿neph/AQP和PCX/AQP进行评估,发现各组间差异无统计学意义(P>0.05),但以细胞型FSGS尿neph/AQP排泌最高,NOS型其次,顶端型再次,以脐部型最低。尿PCX/AQP则以细胞型FSGS排泌最高,NOS型其次,脐部型再次,顶端型最低。对于肾病综合征患者FSGS的诊断,ROC曲线分析表明,当尿neph/AQP取临界值为0.165时其灵敏度为0.864,而特异性为0.603,尿PCX/AQP取临界值3.056时其灵敏度为0.773,特异性为0.672。结论:采用ELISA方法的以AQP-2作为内参检测成人肾病综合征尿足nephrin,podocalyxin较为简便。IgMN患者尿neph/AQP和PCX/AQP增高,肾淀粉样变性患者的尿neph/AQP和PCX/AQP较低。FSGS患者尿neph/AQP和PCX/AQP高于其他肾病综合征患者,在FSGS亚型中以细胞型及NOS型稍高于其他亚型FSGS患者,该方法还有助于在临床肾病综合征患者中帮助诊断FSGS。     

尿足细胞阳性IgA肾病患者不同蛋白尿水平临床病理分析

目的：探讨尿足细胞阳性的IgA肾病患者不同蛋白尿水平的临床病理特点.方法：选取原发性IgA肾病尿足细胞阳性患者41例,收集患者临床病理资料,根据尿蛋白水平分成3组：组1（≤0.5 g/24 h）（n=9）,组2（〉0.5 g/24 h,〈1.0 g/24 h）（n=14）,组3（≥1.0 g/24 h）（n=18）,比较3组临床病理特点.结果：（1）临床特点：组1患者收缩压、eGFR、UA均较组3差异有统计学意义（P〈0.05）,且这些指标在三组间随蛋白尿水平呈现递进表现.组2和组3仅在收缩压、UA方面差异有统计学意义（P〈0.05）;（2）病理特点：组1在节段性肾小球病变比例及间质炎细胞浸润方面较组3轻,差异有统计学意义（P〈0.05）;组1在节段性肾小球病变比例及节段损伤积分方面较组2轻,差异有统计学意义（P〈0.05）;组2和组3两组病理改变差异无统计学意义（P〉0.05）.结论：尿足细胞阳性的IgA肾病患者中,尿蛋白水平仍然是肾脏病轻重的一个指标,与临床病理关系密切.     

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。     

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.     

Targeted downregulation of extracellular nephrin in human IgA nephropathy

BACKGROUND: The identification of nephrin and CD2AP, podocyte proteins which modulate the properties of glomerular barrier selectivity, has made it possible to unravel the mechanisms undergoing foot process effacement and proteinuria. Here we explored the role of nephrin and CD2AP together with the integrity of the slit diaphragm in the pathogenesis of proteinuria in patients with acquired glomerular diseases. METHODS: Nephrin mRNA and protein expression were systematically evaluated in 28 renal biopsy samples from adult patients with primary glomerular disease and proteinuria by in situ hybridization and immunohistochemistry using antibodies directed against extra- and intracellular nephrin and compared with biopsy samples from normal controls. CD2AP protein expression by immunohistochemistry was also assessed. Morphometrical analysis of the filtration slit was performed by transmission electron microscopy. RESULTS: Nephrin mRNA and expression of the extracellular nephrin were markedly reduced in IgA nephropathy. No changes were found in patients with minimal change nephrosis and focal segmental glomerulosclerosis. The staining of intracellular nephrin and CD2AP did not change among patients. A comparable frequency of the filtration slits was observed in all patient groups, except in minimal change disease patients, due to extensive foot process effacement. The percentage of slit diaphragms with a filamentous image was markedly reduced in IgA nephropathy, but was comparable to controls in minimal change disease. Slit pore width showed a tendency to decrease both in patients with minimal change disease and IgA nephropathy. CONCLUSIONS: These results indicate that the ultrastructure of the filtration slit diaphragm is altered in patients with IgA nephropathy as a consequence of targeted downregulation of extracellular nephrin. Further studies are needed to evaluate the pathophysiological meaning of nephrin abnormality in IgA nephropathy and how these changes can be modulated by antiproteinuric therapy.     

Expression of human nephrin mRNA in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is associated with functional changes in the filtration barrier, and microalbuminuria is a strong predictor of the development of overt DN. Nephrin is a novel podocyte-specific protein which localizes at the slit diaphragm. This study examines the expression of nephrin mRNA in the kidneys of type 2 diabetics with DN. METHODS: Renal tissues were obtained from 13 type 2 diabetics with DN. We also examined samples from five patients with minimal change nephrotic syndrome (MCNS) and five normal kidneys (normals) as control. The severity of DN was classified into two grades based on histopathological findings. DN grade 1 (DN1 = seven patients) presented mild mesangial expansion, and DN grade 2 (DN2 = six patients) moderate mesangial expansion. Nephrin mRNA was quantitated and localized by in situ hybridization. RESULTS: Cells positive for nephrin mRNA were detected exclusively in glomerular epithelial cells. The percentage of cells positive for nephrin mRNA in DN2 was significantly lower than in MCNS and normal kidneys. Furthermore, there was an inverse correlation between the percentage of cells positive for nephrin mRNA and extent of proteinuria. CONCLUSION: The low expression of nephrin mRNA may be closely linked to development and/or progression of proteinuria in human diabetic nephropathy.     

IgA肾病肾组织中基质金属蛋白酶2和单核细胞趋化因子1的表达

目的探讨基质金属蛋白酶2（MMP-2）和单核细胞趋化因子-1（MCP-1）在IgA肾病肾组织的表达强度及其在该病早期肾损害发病机制中的作用。方法将30例IgA肾病患者经肾脏活体组织检查的。肾组织和1（）例切除肾肿瘤患者正常肾组织,进行MMP-2、MCP-1、Ⅳ型胶原免疫组织化学检测并进行半定量分析,按其组织学改变Lee分级标准和肾小管间质病变程度进行分组和分级,同时检测肾功能、24h尿蛋白定量等临床指标,并与组织学、免疫组织化学检测结果进行相关分析。结果在IgA肾病轻度肾损害阶段,MMP2和MCP-1表达水平显著高于正常对照（P〈0．05）,随着病变级别加重,MMP-2和MCP-1在肾小管间质的表达呈逐渐减少趋势（P〈0．05）,而Ⅳ型胶原在肾小管间质表达随肾损害加重呈逐渐增加趋势（P〈0．01）。Spearman相关分析显示,MMP-2和MCP-1表达水平与肾小管间质炎症细胞浸润程度呈正相关（P〈0．05）,与Ⅳ型胶原表达水平呈负相关（P〈0．05）。结论MMP-2和MCP-1可能参与IgA肾病早期单个核细胞浸润等早期肾损害发病机制。     

Detection of enriched Thomsen-Friedenrich antigen on IgA1 from IgA nephropathy patients

BACKGROUND: Human serum IgA1 has a mucin-like structure on its hinge portion which is composed of a mucin-type sugar chain and amino acid sequence rich in proline, serine and threonine. There are incompletely glycosylated O-linked oligosaccharide(s) on the IgA1 hinge region in some nephropathy patients. METHODS: We made a detailed analysis of the incompleteness of the sugar chain by digesting IgA1 with various glycosidases. To verify the incompleteness of the sugar chains, the galactosamine/glucosamine ratio (O/N ratio) was introduced as a specific value for each IgA1 preparation. RESULTS: When IgA1 from serum was treated with alpha-N-acetylgalactosaminidase and/or neuraminidase or endo-beta-N-acetylglucosaminidase H (Endo-H), the O/N ratio did not change. However, endo-alpha-N-acetylgalactosaminidase (Endo-A) reduced the O/N ratio of IgA1 from the IgA nephropathy patient whereas before treatment, the O/N ratio had been similar in the normal control and IgA nephropathy. CONCLUSIONS: This result means there is a small amount of the unsubstituted and the sialylated N-acetylgalactosamine residues (Tn and sialyl Tn antigen, respectively), and abundant asialo Galbeta1,3GalNAc (TF antigen) in the IgA1 molecule. In view of the incompleteness of the IgA1 sugar chain, the decrease in the sialic acid content of the mucin-type sugar chain on IgA1 from an IgA nephropathy patient became obvious in this experiment.     

KIM-1 expression predicts renal outcomes in IgA nephropathy

Background

Kidney injury molecule-1 (KIM-1) is a sensitive biomarker for proximal tubular injury. Recently, a few studies have shown that urinary KIM-1 has clinical implications in IgA nephropathy (IgAN). We performed this study to determine whether tissue KIM-1 has clinical implications for predicting long-term outcome and whether urinary KIM-1 is correlated with tissue KIM-1 and kidney injury in IgAN patients.

Methods

We assessed the prognostic prediction capability of tissue KIM-1 expression in 69 adult patients with IgAN retrospectively. Renal biopsies from all patients were scored by a pathologist who was blinded to the clinical data for the pathologic variables. The primary outcome was the composite of a 50 % reduction in eGFR or end-stage renal disease. Tissue KIM-1 expression was assessed semiquantitatively by counting the stained tubules per 100× power field; 0 tubule indicates grade 0; 1–5 tubules, grade 1; 6–10 tubules, grade 2; and more than 10 tubules, grade 3. Comparing urinary KIM-1 and tissue KIM-1 expression, 50 consecutive IgAN patients were prospectively enrolled to measure urinary KIM-1 levels and examine their biopsy specimens by KIM-1 immunohistochemistry.

Results

Univariate analysis showed that tissue KIM-1 expression was associated with the renal outcome in IgAN. Multivariate regression analysis, as the relationship of tissue KIM-1 with prognosis, was consistent. Proteinuria at biopsy and tissue KIM-1 grade 3 were shown to have a prognostic value. The concentration of urinary KIM-1/Cr in patients with IgAN was significantly higher than that in the normal controls.

Conclusion

Tissue KIM-1 expression is an independent predictor of adverse renal outcomes in IgA nephropathy patients.     

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。     

The association of WISP-1 expression in IgA nephropathy patients with renal interstitial fibrosis

Objective To investigate the relationship between the expression of Wnt induced secreted protein-1 (WISP-1) and the fibrosis of renal biopsy tissue in IgA nephropathy (IgAN) patients. Methods Fifty-three patients firstly diagnosed as IgA nephropathy by renal biopsy were included and classified according to Oxford and Lee's classification. Sixteen patients with MCD entered the fibrosis negative control group, and fourteen healthy adults entered the normal control group. The expression of WISP-1 in renal tissues and serum of all subjects were detected by immunohistochemistry and ELISA respectively. Results Immunohistochemistry results showed that WISP-1 was not expressed in MCD patients and normal human kidney tissues, which was abundantly deposited in renal tissue of patients with focal proliferative IgAN with renal interstitial fibrosis. The serum level of WISP-1 in IgAN patients was significantly higher than that in normal subjects (P=0.015) and MCD patients (P=0.030). In the subgroup analysis of IgAN renal fibrosis, the serum concentration of WISP-1 of fibrosis grade between 0-10% (F1 group) and fibrosis＞25% (F3 group) were significantly higher than that in the normal group and the MCD group (all P＜0.05). There was no significant difference between F2 group (10%＜fibrosis≤25%) and normal group or MCD group (P＞0.05). Conclusions The expression of WISP-1 in serum and renal tissue of renal interstitial fibrosis IgAN patients is higher than that of normal and MCD patients without renal fibrosis, and the IgAN patients' serum level of WISP-1 is significantly increased in fibrosis lower score group. The expressions of WISP-1 in serum and renal tissue are related to the occurrence of IgAN renal interstitial fibrosis, in which WISP-1 may play an important role as an early precursor factor in the pathogenesis of IgAN renal interstitial fibrosis.