TRPC6 Single Nucleotide Polymorphisms and Progression of Idiopathic Membranous Nephropathy

Background

Activating mutations in the Transient Receptor Potential channel C6 (TRPC6) cause autosomal dominant focal segmental glomerular sclerosis (FSGS). TRPC6 expression is upregulated in renal biopsies of patients with idiopathic membranous glomerulopathy (iMN) and animal models thereof. In iMN, disease progression is characterized by glomerulosclerosis. In addition, a context-dependent TRPC6 overexpression was recently suggested in complement-mediated podocyte injury in e.g. iMN. Hence, we hypothesized that genetic variants in TRPC6 might affect susceptibility to development or progression of iMN.

Methods & Results

Genomic DNA was isolated from blood samples of 101 iMN patients and 292 controls. By direct sequencing of the entire TRPC6 gene, 13 single nucleotide polymorphisms (SNPs) were identified in the iMN cohort, two of which were causing an amino acid substitution (rs3802829; Pro15Ser and rs36111323, Ala404Val). No statistically significant differences in genotypes or allele frequencies between patients and controls were observed. Clinical outcome in patients was determined (remission n = 26, renal failure n = 46, persistent proteinuria n = 29, follow-up median 80 months {range 51–166}). The 13 identified SNPs showed no association with remission or renal failure. There were no differences in genotypes or allele frequencies between patients in remission and progressors.

Conclusions

Our data suggest that TRPC6 polymorphisms do not affect susceptibility to iMN, or clinical outcome in iMN.     

Circulating TNF Receptors Are Significant Prognostic Biomarkers for Idiopathic Membranous Nephropathy

Idiopathic membranous nephropathy (iMN) is a common cause of nephrotic syndrome in adults. A biomarker to accurately indicate the severity of iMN and predict long-term prognosis is insufficient. Here, we evaluated the clinical significance of circulating tumor necrosis factor receptors (cTNFRs) as prognostic biomarkers of iMN with nephrotic syndrome. A total of 113 patients with biopsy-proven iMN and 43 healthy volunteers were enrolled in this study. Ninety patients with iMN had nephrotic range proteinuria. Levels of cTNFRs were measured by using serum samples collected at the time of initial diagnosis. Levels of cTNFRs were higher in the patients with nephrotic syndrome than in those with subnephrotic range proteinuria or in the healthy volunteers (P for trend <0.001). Estimated glomerular filtration rate and proteinuria tended to worsen as the cTNFRs levels increased. Having a cTNFR1 level within the highest tertile was a significant risk factor for renal progression after adjustment, in comparison with the other tertiles (hazard ratio [HR], 3.39; 95% confidence interval [95% CI], 1.48–7.78; P = 0.004). The cTNFR2 level within the highest tertile also significantly increased the risk of renal progression (HR, 3.29; 95% CI, 1.43–7.54; P = 0.005). Renal tubular TNFRs expression was associated with cTNFRs level. However, the cTNFRs levels were not associated with autoantibody against phospholipase A₂ receptor reactivity/levels or treatment response. This study demonstrated that cTNFRs levels at the time of initial diagnosis could predict renal progression in patients with iMN.     

Glomerular Endothelial Cell Injury and Focal Segmental Glomerulosclerosis Lesion in Idiopathic Membranous Nephropathy

Background

Focal segmental glomerulosclerosis (FSGS) lesions have often been discussed as a negative predictor in idopathic membranous nephropathy (MN). The mechanism of the development of FSGS lesion in MN is still uncertain.

Methods

From 250 cases of MN, 26 cases contained FSGS lesion. We compared the clinicopathological characteristics between MN cases with FSGS lesion [MN-FSGS(+)] and MN without FSGS lesion [MN-FSGS(−)], matched for gender, age, stage of MN.

Results

The glomerular filtration rate (eGFR) was significantly lower in MN-FSGS(+) cases compared to MN-FSGS(−), although nephrotic syndrome, hematuria, and systolic blood pressure levels were not significantly different between the two groups. Pathologically, glomeruli in MN-FSGS(+) cases showed narrowing and loss of glomerular capillaries with separating from GBM or disappearance of CD34+ endothelial cells, and accumulation of extracellular matrix (ECM) in capillary walls, indicating the development of glomerular capillary injury. These findings of endothelial injury were seen even in MN-FSGS(−) cases, but they were more prominent in MN-FSGS(+) than MN-FSGS(−) by computer assessed morphometric analysis. In MN-FSGS(+) cases, 44 out of 534 glomeruli (8.2%) contained FSGS lesions (n = 31, NOS lesion; n = 13, perihilar lesion). Significant thickness of GBM with ECM accumulation was evident in MN-FSGS(+) cases. Podocyte injury with effacement of foot processes was also noted, but the expression of VEGF on podocytes was not different between the two groups, which suggests that the significant thickness of capillary walls may influence the function of VEGF from podocyte resulting in the glomerular capillary injury that contribute to the development of FSGS lesion in MN.

Conclusion

Glomerular capillary injury was seen in all MN cases. Furthermore, the prominent injuries of glomerular capillaries may be associated with the deterioration of eGFR and the formation of FSGS lesions in MN.     

激素联合免疫抑制剂治疗高危因素的特发性膜性肾病46例疗效分析

目的：比较激素联合不同免疫抑制剂治疗高危因素特发性膜性肾病临床疗效,以探讨合理的治疗方案。方法：随机将2001年5月至2011年5月在我院确诊的46例高危因素的特发性膜性肾病患者分为3组,分别给予甲基泼尼松龙与环磷酰胺、环孢素A或霉酚酸酯中的一种联合使用,总疗程12个月,比较各组治疗方案的临床疗效。结果：（1）治疗前3组患者血压、血肌酐和24 h尿蛋白定量比较无显著性（P〉0.05）。（2）甲基泼尼松龙＋环磷酰胺、甲基泼尼松龙＋环孢素A和甲基泼尼松龙＋霉酚酸酯总缓解率分别为53.3%、80.0%和43.8%,甲基泼尼松龙＋环孢素A与其余两组比较总缓解率较高,且有显著性（P〈0.05）;甲基泼尼松龙＋环磷酰胺和甲基泼尼松龙＋霉酚酸酯两组之间总缓解率无明显差异（P〉0.05）。结论：激素联合免疫抑制剂治疗高危因素的特发性膜性肾病,可明显地提高患者的蛋白尿缓解率,其中甲基泼尼松龙＋环孢素A总缓解率较高,且优于其他免疫抑制剂,但其复发率较高,肾脏药物不良反应大,临床选用时需谨慎。     

Synthetic ACTH in High Risk Patients with Idiopathic Membranous Nephropathy: A Prospective,Open Label Cohort Study

New therapeutic agents are warranted in idiopathic membranous nephropathy. Synthetic ACTH may be advantageous with reported remission rates up to 85% and few side effects. We conducted a prospective open label cohort study from 2008 till 2010 (NCT00694863). We prospectively selected patients with idiopathic membranous nephropathy and high risk for progression (defined as βeta-2-microglobulin (β2m) excretion of >500 ng/min). For comparison, we selected matched historical controls treated with cyclophosphamide. The prospectively selected patients received intramuscular injections of synthetic ACTH during 9 months (maximal dose 1 mg twice a week). The primary endpoints concerned the feasibility and incidence of remissions as a primary event. Secondary endpoints included side effects of treatment and the incidence of remissions and relapses at long-term follow-up. Twenty patients (15 men) were included (age 54±14 years, serum creatinine 104 μmol/l [IQR 90–113], urine protein:creatinine ratio 8.7 g/10 mmol creatinine [IQR 4.3–11.1]). Seventeen patients (85%) completed treatment. 97% of injections were administered correctly. Cumulative remission rate was 55% (complete remission in 4 patients, partial remission 7 patients). In a group of historical controls treated with cyclophosphamide and steroids, 19 of 20 patients (95%) developed a remission (complete remission in 13 patients, partial remission in 6 patients) (p<0.01). The main limitation of our study is its small size and the use of a historical control group. We show that treatment with intramuscular injections of synthetic ACTH is feasible. Our data suggest that synthetic ACTH is less effective than cyclophosphamide in inducing a remission in high risk patients with idiopathic membranous nephropathy. The use of synthetic ACTH was also associated with many adverse events. Therefore, we advise against synthetic ACTH as standard treatment in membranous nephropathy.

Trial Registration

ClinicalTrials.gov NCT00694863     

Prevalence of Enhanced Granular Expression of Thrombospondin Type-1 Domain-Containing 7A in the Glomeruli of Japanese Patients with Idiopathic Membranous Nephropathy

Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults. Autoantibodies against M-type phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A), which mainly belong to the IgG4 subclass, were reported as associated antibodies for the development of MN. Although PLA2R is a major target antigen for idiopathic MN, the prevalence of MN patients seropositive for PLA2R in Japan is lower than that in other countries. In this study, we conducted immunohistochemical analysis of the presence of THSD7A and PLA2R in renal specimens of MN patients to estimate the prevalence of THSD7A/PLA2R-related idiopathic MN in Japan. Enhanced granular expression of THSD7A and PLA2R was detected in 9.1% and 52.7%, respectively, of the patients with idiopathic MN. Although none of patients with secondary MN displayed enhanced granular expression of THSD7A, 5.4% of them had enhanced granular expression of PLA2R. In conclusion, the prevalence of enhanced granular expression of THSD7A in the glomeruli of Japanese patients with idiopathic MN was higher than the prevalence of MN patients seropositive for THSD7A in USA and Europe.     

Analgesic Nephropathy: Clinical Syndrome and Prognosis

Over a five-year period 86 patients presented to a renal unit with a history of prolonged analgesic abuse and no other obvious cause of renal damage. Anaemia and peptic ulceration were common, and neurological states suggestive of chronic analgesic intoxication occurred in 22 patients. Thirty-two patients died during follow-up, but the prognosis was much better in patients who ceased abuse of compound analgesics, and improvement could occur even in advanced renal failure. While 84 patients had taken mixtures containing both aspirin and phenacetin, papillary necrosis was also found in two patients who had abused only aspirin, and when phenacetin was withdrawn from several leading compound analgesics, renal function continued to deteriorate in patients ingesting those preparations.     

Autoantibodies against Phospholipase A2 Receptor in Korean Patients with Membranous Nephropathy

The data were presented in abstract form at the 45^th meeting of the American Society of Nephrology, October 30-November 04 2012, San Diego, CA, USA. Circulating autoantibodies against M-type phospholipase A₂ receptor (PLA₂R) are important pathogenic antibodies of idiopathic membranous nephropathy (MN) in adults. However, previous studies on the clinical impact of anti-PLA₂R antibodies demonstrated several limitations, including insufficient numbers of study subjects and different time points and methods for anti-PLA₂R antibody measurement. To verify the clinical significance of anti-PLA₂R antibodies in Korean patients with MN, we measured autoantibodies in serum samples obtained at the time of biopsy from a total of 100 patients with idiopathic MN who had not yet received immunosuppressive treatment. We detected anti-PLA₂R antibody in 69 patients, and we observed that autoantibody reactivity reflected the severity of disease activity. Proteinuria and hypoalbuminemia were more severe in patients with anti-PLA₂R than in those without the autoantibodies (2.95 g/g vs. 6.85 g/g, P = 0.003; 3.1 g/dL vs. 2.5 g/dL, P = 0.004, respectively). Additionally, the clinical severities worsened proportionally as the levels of anti-PLA₂R antibodies increased (P = 0.015 and P for trend <0.001 for proteinuria and hypoalbuminemia, respectively). However, neither the levels nor the presence or absence of anti-PLA₂R antibody showed a significant correlation with clinical outcomes, such as remission rate and time to remission. In conclusion, we observed that anti-PLA₂R antibodies are highly prevalent in Korean patients with idiopathic MN and that they reflect the clinical disease activity before the administration of immunosuppressive treatment. However, the levels of anti-PLA₂R antibody at the time of kidney biopsy may not predict the clinical outcomes in current clinical practice.     

Clinicopathologic Features and Prognosis of Type 2 Diabetes Mellitus and Diabetic Nephropathy in Different Age Groups: More Attention to Younger Patients

Objective: Our study sought to investigate the clinicopathologic features and renal prognosis of patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) in different age groups.Methods: A total of 315 patients with T2DM and biopsy-proven DN were enrolled and divided into three groups by age: the Youth group (≤44 years old), the Middle-aged group (45 to 59 years old), and the Elderly group (≥60 years old).Results: The Youth group, Middle-aged group, and Elderly group accounted for 19.05% (60/315), 59.37% (187/315), and 21.59% (68/315) of the patients in our study, respectively. The patients in the Youth group had a higher estimated glomerular filtration rate (calculated using the Chronic Kidney Disease–Epidemiology collaboration formula) (P<.001), a higher incidence of diabetic retinopathy (P = .044), and a higher incidence of being in the lower-risk chronic kidney disease heat map category (P = .046) but lower duration of diabetes (P = .016). Histologically, patients in the Youth group had the highest incidence of glomerular classification in class I (P = .006) and arteriolar hyalinosis score of 0 (P = .005). The renal survival among the three groups was comparable (P>.05).Conclusion: This study indicated that there were different clinicopathologic features among Chinese DN patients in different age groups. Although the Youth group had a relatively lower rapid kidney disease progression rate, there were no significant differences in renal survival rate among the three groups, which calls more attention to early supervision and prevention for younger DN patients.Abbreviations: CKD = chronic kidney disease; DN = diabetic nephropathy; DR = diabetic retinopathy; eGFR = estimated glomerular filtration rate; ESRD = end-stage renal disease; G&Y&O = green and yellow and orange; IFTA = interstitial fibrosis and tubular atrophy; T2DM = type 2 diabetes mellitus     

MMF联合泼尼松治疗成人特发性膜性肾病的疗效及安全性研究

目的:探究吗替麦考酚酯(MMF)联合泼尼松(Pre)治疗成人特发性膜性肾病(IMN)的疗效及安全性。方法:选取2015.06-2017.06我院收治的102例行IMN患者列为研究对象,将患者按随机数字表法以1:1比例分为对照组与观察组,每组各51例,对照组患者使用Pre进行治疗,观察组患者使用MMF+Pre进行治疗,治疗12个月。比较两组治疗12个月后疗效,治疗前、治疗后6个月及12个月后肾功能相关指标[24 h尿蛋白定量(24 h UP)、血清胱抑素C(CysC)、血清尿素氮(BUN)、血清肌酐(Scr)、血清白蛋白(Alb)]、脂代谢指标[总胆固醇(TC)、甘油三酯(TG)],并记录用药间期出现的药物不良反应。结果:治疗12个月后,研究组患者总有效率为90.20%,显著高对照组74.51%(P0.05);治疗6个月、12个月后,两组患者24 h UP、Cys C、TC、TG水平较治疗前均依次显著下降而Alb水平显著上升(P0.05),且观察组上述指标与同期对照组对比差异显著(P0.05);而两组BUN和Scr水平较治疗前差异不显著(P0.05),两组对比无统计学意义(P0.05)。治疗期间,观察组患者药物不良反应率为11.76%,显著低于对照组27.45%(P0.05)。结论:应用MMF联合泼尼松治疗成人IMN疗效更佳,可显著改善患者肾功能,并改善患者脂代谢,药物方案安全性较高,具有较高应用价值。     

影响早期糖尿病肾病预后危险因素探讨分析

目的：探讨研究影响早期糖尿病肾病（DN）预后的主要危险因素,为延缓早期糖尿病肾病向糖尿病终末期肾病进展提供依据。方法：回顾性分析52例旱期DN的临床、实验室及治疗等,临床资料,了解年龄、控制血糖、血压、血脂、尿蛋白对旱期糖尿病肾病预后的影响。结果：50岁以下早期糖尿病肾病患者5年进展为终末期肾病高达25％,明显低于50岁以上的患者（75％）。血糖、血压、血红蛋白、血浆白蛋白、血脂、尿蛋白等指标控制良好的早期糖尿病肾病的预后明显好于控制不佳者（P〈0．05）。结论：控制血糖、血压、血脂、尿蛋白和不吸烟或戒烟对改善早期糖尿病肾病的预后、提高患者生活质量、延长患者的肾存活期和生存期有着十分重要的意义、     

影响早期糖尿病肾病预后危险因素探讨分析

目的:探讨研究影响早期糖尿病肾病(DN)预后的主要危险因素,为延缓早期糖尿病肾病向糖尿病终末期肾病进展提供依据。方法:回顾性分析52例早期DN的临床、实验室及治疗等临床资料,了解年龄、控制血糖、血压、血脂、尿蛋白对早期糖尿病肾病预后的影响。结果:50岁以下早期糖尿病肾病患者5年进展为终末期肾病高达25%,明显低于50岁以上的患者(75%)。血糖、血压、血红蛋白、血浆白蛋白、血脂、尿蛋白等指标控制良好的早期糖尿病肾病的预后明显好于控制不佳者(P<0.05)。结论:控制血糖、血压、血脂、尿蛋白和不吸烟或戒烟对改善早期糖尿病肾病的预后、提高患者生活质量、延长患者的肾存活期和生存期有着十分重要的意义。     

特发性膜性肾病患者外周血中性粒细胞-淋巴细胞比值的临床与病理价值分析

摘要 目的：探讨外周血中性粒细胞与淋巴细胞比值（NLR）在特发性膜性肾病（IMN）中的临床及病理价值。方法：收集2017年1月至2019年12月确诊为IMN患者221例作为IMN组,将2019年7月至2019年9月体检且尿常规和肾功能指标正常的87例健康体检者作为正常对照组,计算每个研究对象的NLR值,比较两组间NLR值的差异。记录IMN患者的血生化指标及患者的肾脏病理分期及纤维化程度,并且根据MDRD公式计算肾小球滤过率(eGFR),分析NLR与IMN患者的血生化指标及病理特征的相关性。ROC曲线分析外周血NLR评估IMN患者肾间质纤维化的敏感性和特异性。结果：IMN组外周血NLR值高于对照组,差异有统计学意义（P<0.05）。外周血NLR值与IMN患者年龄和肾间质纤维化有关联（P均<0.05）,但与IMN患者性别及肾脏病理分期无关联（P均>0.05）。IMN外周血NLR值与IMN患者hs-CRP、SCr、BUN呈正相关（P<0.05）,与eGFR呈负相关（P<0.05）,与ESR、UA、TP、Alb、24小时蛋白尿定量均无相关性（P均>0.05）。不同程度肾间质纤维化的IMN患者外周血NLR值不同,差异有统计学意义（P<0.05）,且肾间质纤维化程度在1、2、3级时,纤维化程度越重,NLR值越大;3个级别间两两比较,差异均有统计学意义（P均<0.05）。外周血NLR值预测IMN患者肾间质纤维化的ROC曲线下面积为0.715[95%CI(0.626,0.803)],其截断值为1.858时,灵敏度为68.6%,特异度为66.7%。结论：外周血NLR可作为IMN肾脏功能水平的一个有效评价指标,且与IMN患者肾间质纤维化有关,可作为判断肾间质纤维化的参考指标。     

Identification and Characterization of a New Autoimmune Protein in Membranous Nephropathy by Immunoscreening of a Renal cDNA Library

Membranous Nephropathy (MN) represents a large amount of Nephrotic Syndromes in the adult population and its definitive diagnosis is currently carried out through biopsy. An autoimmune condition has been demonstrated in idiopathic MN (iMN) in which some kidney structures are targeted by patient autoantibodies. Some candidate antigens have been described and other likely involved target proteins responsible for the disease are not known yet. In this work our aim is to identify these proteins by screening a lambda-phage library with patients’ sera. We enrolled four groups of patients: two MN groups of 12 full iMN patients; one control group of 15 patients suffering from other renal diseases; one control group of 15 healthy individuals. A commercial cDNA phagemide library was screened using the above described sera, in order to detect positive signals due to antigen-antibody bond. We detected one phagemide clone expressing a protein which was shown to be targeted by the antibodies of the iMN sera only. Control sera were negative. The sequence analysis of cDNA matched the Synaptonemal Complex protein 65 (SC65) coding sequence. Further proteomic analyses were carried out to validate our results. We provide evidence of an involvement of SC65 protein as an autoimmune target in iMN. Considering the invasiveness and the resulting risk coming from renal biopsy, our ongoing aim is to set a procedure able to diagnose affected patients through a little- or non-invasive method such as blood sampling rather than biopsy.     

An Anti-Phospholipase A2 Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

The phospholipase A₂ receptor (PLA₂R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA₂R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA₂R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA₂R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA₂R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA₂R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA₂R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA₂R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA₂R.