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.     

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。     

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、     

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.     

The Calcineurin Inhibitor Tacrolimus Reduces Proteinuria in Membranous Nephropathy Accompanied by a Decrease in Angiopoietin-Like-4

Tacrolimus is an anticalcineurinic agent with potent immunosuppressive activity that has recently been shown to have the added benefit of reducing proteinuria in membranous nephropathy (MN) patients. However, its potential mechanisms remain unknown. To reveal the mechanism, rat cohorts were administered tacrolimus or vehicle from days 7 to 28 after the induction of passive Heymann nephritis (PHN). PHN induction resulted in heavy proteinuria and increased expression of desmin, a marker of injured podocytes. We also showed that the glomerular expression of angiopoietin-like-4 (Angptl4) was markedly upregulated in PHN rats and human MN followed by an increase in urine Angptl4 excretion. In addition, increased Angptl4 expression may be related to podocyte injury and proteinuria. Furthermore, upregulated Angptl4 expression primarily colocalized with podocytes rather than endothelial or mesangial cells, indicating that podocytes may be the source of Angptl4, which then gradually migrated to the glomerular basement membrane over time. However, tacrolimus treatment markedly reduced glomerular and urinary Angptl4, accompanied by a reduction in the established proteinuria and the promotion of podocyte repair. Additionally, glomerular immune deposits and circulating IgG levels induced by PHN clearly decreased following tacrolimus treatment. In conclusion, this is the first demonstration that the calcineurin inhibitor tacrolimus can reduce Angptl4 in podocytes accompanied by a decrease in established proteinuria and promotion of podocyte repair in MN.     

The Occurrence of Warfarin-Related Nephropathy and Effects on Renal and Patient Outcomes in Korean Patients

Background

Warfarin-related nephropathy (WRN) is a recently described disease entity, in which excessive warfarinization (international normalized ratio (INR) >3.0) causes acute kidney injury. Previous reports regarding WRN included few Asian patients who might have differed from the western WRN patients in terms of genetic and environmental factors.

Methods

During the period of March 2003 to December 2011, the data about a total of 1297 patients who had serum creatinine (sCr) level measured within 1 week after INR >3.0 and within 6 months before INR >3.0 was analyzed through the retrospective review of electronic medical records of a single tertiary hospital in Korea.

Result

WRN developed in 19.3% of patients having excessive warfarinization. The incidence was higher in the chronic kidney disease (CKD) group than the non-CKD group. The risk of WRN increased as the basal serum albumin level decreased and was strongly associated with highest quartile serum AST level at post INR elevation and the presence of congestive heart failure. But the presence of atrial fibrillation was protective against the development of WRN. Neither the presence of CKD nor basal estimated glomerular filtration rate (eGFR) was an independent risk factor for WRN. Despite no difference in the basal sCr level, the sCr level was higher in patients with WRN than those without WRN after follow-up. The mortality rates were also higher in patients with WRN.

Conclusions

WRN developed in 19.3% of patients having excessive warfarinization. A lower basal serum albumin, highest quartile serum AST level at post INR elevation, and congestive heart failure were associated with the occurrence of WRN. The development of WRN adversely affected renal and patient outcomes.     

PLA2R Antibody Levels and Clinical Outcome in Patients with Membranous Nephropathy and Non-Nephrotic Range Proteinuria under Treatment with Inhibitors of the Renin-Angiotensin System

Patients with primary membranous nephropathy (MN) who experience spontaneous remission of proteinuria generally have an excellent outcome without need of immunosuppressive therapy. It is, however, unclear whether non-nephrotic proteinuria at the time of diagnosis is also associated with good prognosis since a reasonable number of these patients develop nephrotic syndrome despite blockade of the renin-angiotensin system. No clinical or laboratory parameters are available, which allow the assessment of risk for development of nephrotic proteinuria. Phospholipase A₂ Receptor antibodies (PLA₂R-Ab) play a prominent role in the pathogenesis of primary MN and are associated with persistence of nephrotic proteinuria. In this study we analysed whether PLA₂R-Ab levels might predict development of nephrotic syndrome and the clinical outcome in 33 patients with biopsy-proven primary MN and non-nephrotic proteinuria under treatment with blockers of the renin-angiotensin system. PLA₂R-Ab levels, proteinuria and serum creatinine were measured every three months. Nephrotic-range proteinuria developed in 18 (55%) patients. At study start (1.2±1.5 months after renal biopsy and time of diagnosis), 16 (48%) patients were positive for PLA₂R-Ab. A multivariate analysis showed that PLA₂R-Ab levels were associated with an increased risk for development of nephrotic proteinuria (HR = 3.66; 95%CI: 1.39–9.64; p = 0.009). Immunosuppressive therapy was initiated more frequently in PLA₂R-Ab positive patients (13 of 16 patients, 81%) compared to PLA₂R-Ab negative patients (2 of 17 patients, 12%). PLA₂R-Ab levels are associated with higher risk for development of nephrotic-range proteinuria in this cohort of non-nephrotic patients at the time of diagnosis and should be closely monitored in the clinical management.