The co-occurrence of sarcoidosis and anti-PLA2R-associated membranous nephropathy in a patient with underlying genetic susceptibility.

BACKGROUND: Sarcoidosis is a multisystemic inflammatory disease, characterized by the presence of non-caseating, epithelioid granulomas. Glomerular disease in patients with sarcoidosis is rare and membranous nephropathy (MN) is cited as the most common. The association between the two diseases remained unclear. This article reported a case of co-occurrence of sarcoidosis and anti-PLA2R-associated MN, to provide a possible relationship between these two entities. CASE PRESENTATION: A 61-year-old Chinese Han woman with a history of sarcoidosis was admitted to our hospital for nephrotic syndrome. Her sarcoidosis was diagnosed according to the adenopathy observed on the computed tomography scan and the biopsy of lymph nodes. The MN presented with nephrotic syndrome with a PLA2R antibody titer of 357RU/ml, and the final diagnosis was based on a renal biopsy. The patient's sarcoidosis was remitted after treatment with prednisone. One year later MN was diagnosed, and she was treated with prednisone combined with calcineurin inhibitors, based on a full dose of renin-angiotensin system (RAS) inhibitor. The patient's sarcoidosis had been in remission while the MN was recurrent, and her renal function deteriorated to end-stage renal disease 6 years later due to discontinuation of immunosuppression. A genetic test led to the identification of the HLA-DRB1*0301 and HLA-DRB1*150 genes associated with both sarcoidosis and MN, which provides a new possible explanation of the co-occurrence of these two diseases. CONCLUSION: This case suggested for the first time a potential genetic connection between idiopathic MN and sarcoidosis which needs further studies in the future.

Ectodomain shedding of PLA2R1 is mediated by the metalloproteases ADAM10 and ADAM17.

Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer and is the major autoantigen in membranous nephropathy, a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts, and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1 and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by ß- or γ-secretase, suggesting that PLA2R1 may not be a substrate for regulated intramembrane proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C activator PMA, cytokines, and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer, and membranous nephropathy.

Phospholipid metabolism-related genotypes of PLA2R1 and CERS4 contribute to nonobese MASLD.

BACKGROUND: Abnormal phospholipid metabolism is linked to metabolic dysfunction-associated steatotic liver disease (MASLD) development and progression. We aimed to clarify whether genetic variants of phospholipid metabolism modify these relationships. METHODS: This case-control study consecutively recruited 600 patients who underwent MRI-based proton density fat fraction examination (240 participants with serum metabonomics analysis, 128 biopsy-proven cases) as 3 groups: healthy control, nonobese MASLD, and obese MASLD, (n = 200 cases each). Ten variants of phospholipid metabolism-related genes [phospholipase A2 Group VII rs1805018, rs76863441, rs1421378, and rs1051931; phospholipase A2 receptor 1 (PLA2R1) rs35771982, rs3828323, and rs3749117; paraoxonase-1 rs662 and rs854560; and ceramide synthase 4 (CERS4) rs17160348)] were genotyped using SNaPshot. RESULTS: The T-allele of CERS4 rs17160348 was associated with a higher risk of both obese and nonobese MASLD (OR: 1.95, 95% CI: 1.20-3.15; OR: 1.76, 95% CI: 1.08-2.86, respectively). PLA2R1 rs35771982-allele is a risk factor for nonobese MASLD (OR: 1.66, 95% CI: 1.11-1.24), moderate-to-severe steatosis (OR: 3.24, 95% CI: 1.96-6.22), and steatohepatitis (OR: 2.61, 95% CI: 1.15-3.87), while the paraoxonase-1 rs854560 T-allele (OR: 0.50, 95% CI: 0.26-0.97) and PLA2R1 rs3749117 C-allele (OR: 1.70, 95% CI: 1.14-2.52) are closely related to obese MASLD. After adjusting for sphingomyelin level, the effect of the PLA2R1 rs35771982CC allele on MASLD was attenuated. Furthermore, similar effects on the association between the CERS4 rs17160348 C allele and MASLD were observed for phosphatidylcholine, phosphatidic acid, sphingomyelin, and phosphatidylinositol. CONCLUSIONS: The mutations in PLA2R1 rs35771982 and CERS4 rs17160348 presented detrimental impact on the risk of occurrence and disease severity in nonobese MASLD through altered phospholipid metabolism.

Combined Serologic and Genetic Risk Score and Prognostication of Phospholipase A2 receptor-Associated Membranous Nephropathy.

INTRODUCTION: The aim of this study was to test whether a combined risk score on the basis of genetic risk and serology can improve the prediction of kidney failure in phospholipase A2 receptor (PLA2R)-associated primary membranous nephropathy. METHODS: We performed a retrospective analysis of 519 biopsy-proven PLA2R-associated primary membranous nephropathy patients with baseline eGFR ≥25 ml/min per 1.73 m 2 . The combined risk score was calculated by combining the genetic risk score with PLA2R ELISA antibody titers. The primary end point was kidney disease progression defined as a 50% reduction in eGFR or kidney failure. Cox proportional hazard regression analysis and C-statistics were applied to compare the performance of PLA2R antibody, genetic risk score, and combined risk score, as compared with clinical factors alone, in predicting primary outcomes. RESULTS: The median age was 56 years (range, 15-82 years); the male-to-female ratio was 1:0.6, the median eGFR at biopsy was 99 ml/min per 1.73 m 2 (range: 26-167 ml/min per 1.73 m 2 ), and the median proteinuria was 5.3 g/24 hours (range: 1.5-25.8 g/24 hours). During a median follow-up of 67 (5-200) months, 66 (13%) had kidney disease progression. In Cox proportional hazard regression models, PLA2R antibody titers, genetic risk score, and combined risk score were all individually associated with kidney disease progression with and without adjustments for age, sex, proteinuria, eGFR, and tubulointerstitial lesions. The best-performing clinical model to predict kidney disease progression included age, eGFR, proteinuria, serum albumin, diabetes, and tubulointerstitial lesions (C-statistic 0.76 [0.69-0.82], adjusted R 2 0.51). Although the addition of PLA2R antibody titer improved the performance of this model (C-statistic: 0.78 [0.72-0.84], adjusted R 2 0.61), replacing PLA2R antibody with the combined risk score improved the model further (C-statistic: 0.82 [0.77-0.87], adjusted R 2 0.69, difference of C-statistics with clinical model=0.06 [0.03-0.10], P < 0.001; difference of C-statistics with clinical-serologic model=0.04 [0.01-0.06], P < 0.001). CONCLUSIONS: In patients with PLA2R-associated membranous nephropathy, the combined risk score incorporating inherited risk alleles and PLA2R antibody enhanced the prediction of kidney disease progression compared with PLA2R serology and clinical factors alone.

CENPA knockdown restrains cell progression and tumor growth in breast cancer by reducing PLA2R1 promoter methylation and modulating PLA2R1/HHEX axis.

BACKGROUND: Breast cancer is a lethal malignancy affecting females worldwide. It has been reported that upregulated centromere protein A (CENPA) expression might indicate unfortunate prognosis and can function as a prognostic biomarker in breast cancer. This study aimed to investigate the accurate roles and downstream mechanisms of CENPA in breast cancer progression. METHODS: CENPA protein levels in breast cancer tissues and cell lines were analyzed by Western blot and immunohistochemistry assays. We used gain/loss-of-function experiments to determine the potential effects of CENPA and phospholipase A2 receptor (PLA2R1) on breast cancer cell proliferation, migration, and apoptosis. Co-IP assay was employed to validate the possible interaction between CENPA and DNA methyltransferase 1 (DNMT1), as well as PLA2R1 and hematopoietically expressed homeobox (HHEX). PLA2R1 promoter methylation was determined using methylation-specific PCR assay. The biological capabilities of CENPA/PLA2R1/HHEX axis in breast cancer cells was determined by rescue experiments. In addition, CENPA-silenced MCF-7 cells were injected into mice, followed by measurement of tumor growth. RESULTS: CENPA level was prominently elevated in breast cancer tissues and cell lines. Interestingly, CENPA knockdown and PLA2R1 overexpression both restrained breast cancer cell proliferation and migration, and enhanced apoptosis. On the contrary, CENPA overexpression displayed the opposite results. Moreover, CENPA reduced PLA2R1 expression through promoting DNMT1-mediated PLA2R1 promoter methylation. PLA2R1 overexpression could effectively abrogate CENPA overexpression-mediated augment of breast cancer cell progression. Furthermore, PLA2R1 interacted with HHEX and promoted HHEX expression. PLA2R1 knockdown increased the rate of breast cancer cell proliferation and migration but restrained apoptosis, which was abrogated by HHEX overexpression. In addition, CENPA silencing suppressed tumor growth in vivo. CONCLUSION: CENPA knockdown restrained breast cancer cell proliferation and migration and attenuated tumor growth in vivo through reducing PLA2R1 promoter methylation and increasing PLA2R1 and HHEX expression. We may provide a promising prognostic biomarker and novel therapeutic target for breast cancer.

Introduction of a novel chimeric active immunization mouse model of PLA2R1-associated membranous nephropathy.

The phospholipase A2 receptor 1 (PLA2R1) is the major target antigen in patients with membranous nephropathy (MN), an antibody-mediated autoimmune glomerular disease. Investigation of MN pathogenesis has been hampered by the lack of reliable animal models. Here, we overcome this issue by generating a transgenic mouse line expressing a chimeric PLA2R1 (chPLA2R1) consisting of three human PLA2R1 domains (cysteine-rich, fibronectin type-II and CTLD1) and seven murine PLA2R1 domains (CTLD2-8) specifically in podocytes. Mice expressing the chPLA2R1 were healthy at birth and showed no major glomerular alterations when compared to mice with a wild-type PLA2R1 status. Upon active immunization with human PLA2R1 (hPLA2R1), chPLA2R1-positive mice developed anti-hPLA2R1 antibodies, a nephrotic syndrome, and all major histological features of MN, including granular deposition of mouse IgG and complement components in immunofluorescence and subepithelial electron-dense deposits and podocyte foot process effacement in electron microscopy. In order to investigate the role of the complement system in this model, we further crossed chPLA2R1-positive mice with mice lacking the central complement component C3 (C3-/- mice). Upon immunization with hPLA2R1, chPLA2R1-positive C3-/- mice had substantially less severe albuminuria and nephrotic syndrome when compared to chPLA2R1-positive mice with a wild-type C3 status. In conclusion, we introduce a novel active immunization model of PLA2R1-associated MN and demonstrate a pathogenic role of the complement system in this model.

VEGFA promotes the occurrence of PLA2R-associated idiopathic membranous nephropathy by angiogenesis via the PI3K/AKT signalling pathway.

BACKGROUND: The M-type phospholipase A2 receptor (PLA2R)-associated idiopathic membranous nephropathy (IMN) is a common immune-related disease in adults. Vascular endothelial growth factor A (VEGFA) is the key mediator of angiogenesis, which leads to numerous kidney diseases. However, the role of VEGFA in IMN is poorly understood. METHODS: In the present study, we downloaded the microarray data GSE115857 from Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) were identified with R software. The cytoHubba plug-in were used to identify hub genes from the protein-protein interaction network. Gene set enrichment analysis (GSEA) was used to identify signalling pathway in IMN. CCK8 was performed to assess the cell viability in human vascular endothelial cells (HVECs). Then, passive Heymann nephritis (PHN) was induced in rats by a single tail vein injection of anti-Fx1A antiserum. Animals treated with VEGFA inhibitor bevacizumab (BV), with saline as a positive control. Proteinuria was evaluated by biochemical measurements. Immunohistochemistry and immunofluorescence was used to evaluate relative proteins expression. Electron microscopy was performed to observe the thickness of the glomerular basement membrane (GBM). RESULTS: We revealed 3 hub genes, including one up-regulated gene VEGFA and two down-regulated genes JUN and FOS, which are closely related to the development of PLA2R-associated IMN. Pathway enrichment analysis found that the biological process induced by VEGFA is associated with PI3K/Akt signalling. GSEA showed that the signalling pathway of DEGs in GSE115857 was focused on angiogenesis, in which VEGFA acts as a core gene. We confirmed the high expression of VEGFA, PI3K, and AKT in IMN renal biopsy samples with immunohistochemistry. In HVECs, we found that BV suppresses cell viability in a time and dose dependent manner. In vivo, we found low dose of BV attenuates proteinuria via inhibiting VEGFA/PI3K/AKT signalling. Meanwhile, low dose of BV alleviates the thickening of the GBM. CONCLUSION: VEGFA/PI3K/AKT signalling may play significant roles in the pathogenesis of IMN, which may provide new targets for the treatment of IMN.

Associations between m-type phospholipase A2 receptor,human leukocyte antigen gene polymorphisms and idiopathic membranous nephropathy.

Primary membranous nephropathy, also known as idiopathic membranous nephropathy, is an autoimmune disease. As an autoimmune disease, genetic factors are essential in the pathogenesis of IMN. People pay more and more attention to genetics and bioinformatics. With the continuous improvement and development of high-throughput gene sequencing and genotyping technology, it has been confirmed that many genes and their single nucleotide polymorphisms are strongly correlated with IMN disease susceptibility. However, there are few studies on HLA-DQA1 and PLA2R gene polymorphisms and IMN susceptibility in China. The purpose of this study was to investigate whether PLA2R rs2715928 and rs16844715 are related to IMN, the correlation between five SNP loci of PLA2R and HLA-DQA1 and IMN, and the effect of gene-gene interaction among different genotypes of each locus on disease. In this study, 86 patients with IMN confirmed by renal biopsy in the first hospital of Harbin Medical University and 90 healthy controls were selected. All subjects were excluded from secondary membranous nephropathy, pregnant or breastfeeding women, severe primary disease of vital organs, severe infection, major surgery, and severe trauma. Seven selected SNP loci were genotyped using the IMLDR multiple SNP typing kit. Chi-square test and logistic regression were used to analyze the correlation between each SNP and IMN. The general clinical data and laboratory indicators of each subject were recorded, and the relationship between different genotypes and clinical manifestations was analyzed. Among the 7 SNP loci included in the study, except HLA-DQA1 rs2187668, the other 6 loci all met Hardy-Weiberg equilibrium test (P > 0.05). The allele distribution of PLA2R rs2715928 and rs16844715 was significantly different between the IMN group and the healthy control group, and it was closely related to IMN (P < 0.05). There was no statistical difference in the distribution of alleles of rs2715918 between the IMN group and the control group (P* > 0.05), and there was also statistical difference in the distribution of alleles of rs35771982, rs3749117, and rs4664308 between the IMN group and the healthy control group (P < 0.05).The C allele of rs16844715 (OR = 2.03, 95%CI: 1.29-3.19, P* = 0.0140) and the A allele of rs2715928 (OR = 3.18, 95%CI: 1.94-5.24, P* = 3.54E-5), G allele of rs35771982 (OR = 4.07, 95%CI: 2.34-7.08, P* = 4.96E-6), T allele of rs3749117 (OR = 4.07, 95%CI: 2.34-7.08, P* = 4.96E-6), the A allele of rs4664308 (OR = 2.63, 95%CI: 1.54-4.49, P* = 0.0028) was the risk gene of IMN.Through the establishment of different genetic models, we found that,in the additive model, the three SNPs of PLA2R rs2715928 (OR = 5.40, 95%CI: 1.77-16.50, P* = 0.0217) and rs35771982 (OR = 15.15, 95%CI: 2.92-78.48, P* = 0.0084), rs3749117 (OR = 15.15, 95%CI: 2.92-78.48, P* = 0.0084) had a strong correlation with IMN. In the stealth model,homozygous gene risk type of the five SNPs,PLA2R rs16844715 (OR = 2.52, 95%CI: 1.38-4.61, P* = 0.0189) and rs2715928 (OR = 4.30, 95%CI: 2.31-8.03, P* = 3.14E-5), rs35771982 (OR = 4.85, 95%CI: 5.53-9.31, P* = 1.42E-5), rs3749117 (OR = 4.85, 95%CI: 5.53-9.31, P* = 1.42E-5) and rs4664308 (OR = 3.16, 95%CI: 1.67-5.97, P* = 0.0028) had a strong correlation with IMN. The distribution of GT haplotypes and CC haplotypes of rs35771982 and rs3749117 and CA haplotypes and TG haplotypes of rs16844715 and rs4664308 were significantly different between IMN group and control group (P < 0.05). When GMDR software was used to establish a model to analyze the interaction between various SNP sites, it was found that the combination of GG genotype at rs35771982 and AA genotype at rs2715928 was the highest risk of disease. The risk genotypes of rs16844715, rs2715928 and rs4664308 had no effect on the clinical manifestations of IMN (P > 0.05). PLA2R rs2715928 and rs16844715 are associated with susceptibility to IMN. The C allele of rs16844715, the A allele of rs2715928, the G allele of rs35771982, the T allele of rs3749117, and the A allele of rs4664308 are the dangerous genes of IMN. The combination of GG genotype at rs35771982 and AA genotype at rs2715928 poses the greatest risk of disease. Haplotype may affect susceptibility to IMN. The risk genotype had no effect on the clinical manifestations of IMN.

Development and validation of a discrimination model between primary PLA2R-negative membranous nephropathy and minimal change disease confirmed by renal biopsy.

Membranous nephropathy (MN) and minimal change disease (MCD) are two common causes leading to nephrotic syndrome (NS). They have similar clinical features but different treatment strategies and prognoses. M-type phospholipase A2 receptor (PLA2R) is considered as a specific marker of membranous nephropathy. However, its sensitivity is only about 70%. Therefore, there is a lack of effective and noninvasive tools to distinguish PLA2R-negative MN and MCD patients without renal biopsy. A total 949 patients who were pathologically diagnosed as idiopathic MN or MCD were enrolled in this study, including 805 idiopathic MN and 144 MCD. Based on the basic information and laboratory examination of 200 PLA2R-negative MN and 144 MCD, we used a univariate and multivariate logistic regression to select the relevant variables and develop a discrimination model. A novel model including age, albumin, urea, high density lipoprotein, C3 levels and red blood cell count was established for PLA2R-negative MN and MCD. The discrimination model has great differential capability (with an AUC of 0.904 in training group and an AUC of 0.886 in test group) and calibration capability. When testing in all 949 patients, our model also showed good discrimination ability for all idiopathic MN and MCD.

PLA2R1 promotes DNA damage and inhibits spontaneous tumor formation during aging.

Although aging is a major risk factor for most types of cancers, it is barely studied in this context. The transmembrane protein PLA2R1 (phospholipase A2 receptor) promotes cellular senescence, which can inhibit oncogene-induced tumor initiation. Functions and mechanisms of action of PLA2R1 during aging are largely unknown. In this study, we observed that old Pla2r1 knockout mice were more prone to spontaneously develop a wide spectrum of tumors compared to control littermates. Consistently, these knockout mice displayed increased Parp1, a master regulator of DNA damage repair, and decreased DNA damage, correlating with large human dataset analysis. Forced PLA2R1 expression in normal human cells decreased PARP1 expression, induced DNA damage and subsequent senescence, while the constitutive expression of PARP1 rescued cells from these PLA2R1-induced effects. Mechanistically, PARP1 expression is repressed by a ROS (reactive oxygen species)-Rb-dependent mechanism upon PLA2R1 expression. In conclusion, our results suggest that PLA2R1 suppresses aging-induced tumors by repressing PARP1, via a ROS-Rb signaling axis, and inducing DNA damage and its tumor suppressive responses.

PLA2R antibody, PLA2R rs4664308 polymorphism and PLA2R mRNA levels in Tunisian patients with primary membranous nephritis.

BACKGROUND: Primary membranous nephritis (PMN) is an autoimmune disease induced by the deposit of antibodies (Ab) to the phospholipase receptor A2 receptor (PLA2R) on podocytes. In this context, we aimed to assess the relationships between anti-PLA2R Ab, PLA2R rs4664308 SNP, PLA2R mRNA levels and PMN susceptibility and outcome. METHODS: Sixty-eight PMN patients, 30 systemic lupus erythematosus (SLE) patients with secondary MN and 30 healthy control subjects served for anti-PLA2R Ab measurement by ELISA and PLA2R rs4664308 SNP genotyping by a commercial real-time PCR. Twenty patients with tubulo-interstitial nephritis (TIN) were used as controls for renal PLA2R mRNA quantification in PMN patients from kidney biopsies. PLA2R mRNA quantification was carried-out by real-time PCR after RNA extraction. RESULTS: Forty-three (63.2%) PMN patients received initial therapy consisting of alternating monthly cycles of corticosteroids and cyclophosphamide. Twelve (17.6%) patients had resistant PMN to initial therapy and were consecutively treated by cyclosporine or tacrolimus. Anti-PLA2R Ab were positive in 54 (79.4%) PMN patients, while all SLE patients and controls were negative, p<0.0001. Moreover, anti-PLA2R Ab levels were significantly higher in PMN patients (134.85 [41.25-256.97] RU/ml) than in SLE patients (3.35 [2.3-4.35] RU/ml) and controls (2 [2-2.3]), p<0.0001. Consequently, a ROC curve showed for 100% specificity a sensitivity of 94.1% at a threshold of 2.6 RU/ml. Besides, Anti-PLA2R antibodies levels were significantly associated to non-remission; p = 0.002. The rs4664308*A wild-type allele was significantly more frequent in PMN patients (0.809) than in controls (0.633) and SLE patients (0.65); p = 0.008, OR [95% CI] = 2.44 [1.24-4.82] and p = 0.016, OR [95% CI] = 2.27 [1.15-4.5], respectively. Renal PLA2R mRNA levels were significantly higher in PMN patients (218.29 [66.05-486.07]) than in TIN patients (22.09 [13.62-43.34]), p<0.0001. Moreover, PLA2R mRNA levels were significantly higher in non-remission patients (fold-factor vs. partial remission = 2.46 and fold-factor vs. complete remission = 12.25); p = 1.56 10E-8. In addition, PLA2R mRNA and anti-PLA2R Ab levels were significantly correlated, Spearman Rho = 0.958, p<0.0001. CONCLUSION: Anti-PLA2R Ab and renal PLA2R mRNA could be useful markers for PMN outcome predicting. The PLA2R rs6446308 SNP is associated with PMN susceptibility in Tunisians.

Clinical Significance of Expression Changes and Promoter Methylation of PLA2R1 in Tissues of Breast Cancer Patients.

Phospholipase A2 receptor 1 (PLA2R1) expression and its role in the initiation and progression of breast cancer are an unresolved issue. PLA2R1 was found to endorse several tumor suppressive responses, including cellular senescence and apoptosis. Previous in vitro studies demonstrated that DNA hypermethylation was highly associated with the epigenetic silencing of PLA2R1 in breast cancer cell lines. Our objective was to study the level of PLA2R1 mRNA expression and the methylation of its promoter in different histological grades and molecular subtypes of breast cancer. We performed bioinformatics analyses on available human breast cancer expression datasets to assess the PLA2R1 mRNA expression. We used qRT-PCR to evaluate the PLA2R1 mRNA expression and its promoter's methylation in breast cancer tissue in comparison to breast fibroadenomas. Our results describe, for the first time, the expression of PLA2R1 and the methylation of its promoter in human breast cancer tissues. A significant downregulation of PLA2R1, together with hypermethylation of the promoter was detected in breast cancers of different histological grades and molecular subtypes when compared to benign breast tissues. PLA2R1 promoter hypermethylation was associated with aggressive subtypes of breast cancer. In conclusion, PLA2R1 promoter hypermethylation is a potentially useful diagnostic and prognostic biomarker and could serve as a possible therapeutic target in breast cancer.

Methylation of the Phospholipase A2 Receptor 1 Promoter Region in Childhood B Cell Acute Lymphoblastic Leukaemia.

Acute lymphoblastic leukaemia (ALL) is the most common form of paediatric cancer and epigenetic aberrations are determinants of leukaemogenesis. The aim of this study was to investigate the methylation degree of a distinct phospholipase A2 receptor 1 (PLA2R1) promoter region in paediatric ALL patients and to evaluate its relevance as new biomarker for monitoring treatment response and burden of residual disease. The impact of PLA2R1 re-expression on proliferative parameters was assessed in vitro in Jurkat cells with PLA2R1 naturally silenced by DNA methylation. Genomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of 44 paediatric ALL patients. PLA2R1 methylation was analysed using digital PCR and compared to 20 healthy controls. Transfected Jurkat cells were investigated using cell growth curve analysis and flow cytometry. PLA2R1 was found hypermethylated in BM and PB from pre-B and common ALL patients, and in patients with the disease relapse. PLA2R1 methylation decreased along with leukaemic blast cell reduction during ALL induction treatment. In vitro analysis revealed an anti-proliferative phenotype associated with PLA2R1 re-expression, suggesting a tumour-suppressive function of PLA2R1. Collected data indicates that PLA2R1 promoter methylation quantitation can be used as biomarker for ALL induction treatment control, risk stratification, and early detection of ALL relapse.

Generation of a conditional transgenic mouse model expressing human Phospholipase A2 Receptor 1.

The Phospholipase A2 Receptor 1 (PLA2R1) was first identified for its ability to bind some secreted PLA2s (sPLA2s). It belongs to the C-type lectin superfamily and it binds different types of proteins. It is likely a multifunctional protein that plays a role i) in inflammation and inflammatory diseases, ii) in cellular senescence, a mechanism participating in aging and age-related diseases including cancer, and iii) in membranous nephropathy (MN), a rare autoimmune kidney disease where PLA2R1 is the major autoantigen. To help study the role of PLA2R1 in these pathophysiological conditions, we have generated a versatile NeoR-hPLA2R1 conditional transgenic mice which will allow the specific expression of human PLA2R1 (hPLA2R1) in relevant organs and cells following Cre recombinase-driven excision of the NeoR-stop cassette flanked by LoxP sites. Proof-of-concept breeding of NeoR-hPLA2R1 mice with the ubiquitous adenoviral EIIa promoter-driven Cre mouse line resulted in the expected excision of the NeoR-stop cassette and the expression of hPLA2R1 in all tested tissues. These Tg-hPLA2R1 animals breed normally, with no reproduction or apparent growth defect. These models, especially the NeoR-hPLA2R1 conditional transgenic mouse line, will facilitate the future investigation of PLA2R1 functions in relevant pathophysiological contexts, including inflammatory diseases, age-related diseases and MN.

Membranous nephropathy associated with thrombospondin type-1 domain-containing 7A (THSD7A) in an adult woman with eosinophilia.

A 30-year-old woman on steroid therapy for eosinophilia presented with nephrotic syndrome during steroid tapering. She was diagnosed with membranous nephropathy (MN) stage II-III (positive for IgG1 and IgG4) by renal biopsy. There was no evidence of secondary MN. Her urinary protein level was controlled to 0.5 g/day or less, and her eosinophil count in white blood cell differential was stabilized at less than 10% without increasing the steroid dosage. The renal specimen did not show any enhanced granular expression of PLA2R along the glomerular basement membrane, and PLA2R was not detected in the patient's serum. On retrospective analysis, enhanced granular staining for thrombospondin type-1 domain-containing 7A (THSD7A) in the glomeruli was detected in the biopsy, and anti-THSD7A IgG was detected in the serum using a commercial indirect immunofluorescence test (IFT). Based on these, the case was considered as THSD7A-associated MN with comorbid eosinophilia. The causal relationship between THSD7A-related MN and eosinophilia was unclear. However, a few cases of THSD7A-associated MN with eosinophilia have been reported, and further clarification on the relationship between THSD7A-related MN and eosinophilia is warranted.

Current insights into functions of phospholipase A2 receptor in normal and cancer cells: More questions than answers.

Lipid signaling network was proposed as a potential target for cancer prevention and treatment. Several recent studies revealed that phospholipid metabolising enzyme, phospholipase A2 (PLA2), is a critical regulator of cancer accelerating pathologies and apoptosis in several types of cancers. In addition to functioning as an enzyme, PLA2 can activate a phospholipase A2 receptor (PLA2R1) in plasma membrane. While the list of PLA2 targets extends to glucose homeostasis, intracellular energy balance, adipocyte development, and hepatic lipogenesis, the PLA2R1 downstream effectors are few and scarcely investigated. Among the most addressed PLA2R1 effects are regulation of pro-inflammatory signaling, autoimmunity, apoptosis, and senescence. Localized in glomeruli podocytes, the receptor can be identified by circulating anti-PLA2R1 autoantibodies leading to development of membranous nephropathy, a strong autoimmune inflammatory cascade. PLA2R1 was shown to induce activation of Janus-kinase 2 (JAK2) and estrogen-related receptor α (ERRα)-controlled mitochondrial proteins, as well as increasing the accumulation of reactive oxygen species, thus leading to apoptosis and senescence. These findings indicate the potential role of PLA2R1 as tumor suppressor. Epigenetic investigations addressed the role of DNA methylation, histone modifications, and specific microRNAs in the regulation of PLA2R1 expression. However, involvement of PLA2R1 in suppression of malignant growth and metastasis remains controversial. In this review, we summarize the recent findings that highlight the role of PLA2R1 in the regulation of carcinogenesis-related intracellular signaling.

Anti-PLA2R1 Antibodies Containing Sera Induce In Vitro Cytotoxicity Mediated by Complement Activation.

The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy (MN). However, the pathogenic role of anti-PLA2R1 autoantibodies is unclear. Our aim was to evaluate the in vitro cytotoxicity of anti-PLA2R1 antibodies mediated by complement. Forty-eight patients with PLA2R1-related MN from the prospective cohort SOURIS were included. Anti-PLA2R1 titer, epitope profile, and anti-PLA2R1 IgG subclasses were characterized by ELISA. Cell cytotoxicity was evaluated by immunofluorescence in HEK293 cells overexpressing PLA2R1 incubated with patient or healthy donor sera in the presence or absence of rabbit complement or complement inhibitors. Mean cytotoxicity of anti-PLA2R1 sera for HEK293 cells overexpressing PLA2R1 was 2 ± 2%, which increased to 24 ± 6% after addition of rabbit complement (p < 0.001) (n = 48). GVB-EDTA, which inhibits all complement activation pathways, completely blocked cell cytotoxicity, whereas Mg-EGTA, which only inhibits the classical and lectin pathways, highly decreased suggesting a limited role of the alternative pathway. A higher diversity of IgG subclasses beyond IgG4 and high titer of total IgG anti-PLA2R1 were associated with increased cytotoxicity (p = 0.01 and p = 0.03 respectively). In a cohort of 37 patients treated with rituximab, high level of complement-mediated cytotoxicity was associated with less and delayed remission at month 6 after rituximab therapy (5/12 vs. 20/25 (p = 0.03) in 8.5 months ± 4.4 vs. 4.8 ± 4.0 (p = 0.02)). Kaplan-Meier analysis demonstrated that high level of cytotoxicity (≥40%) (p = 0.005), epitope spreading (defined by immunization beyond the immunodominant CysR domain) (p = 0.002), and high titer of anti-PLA2R1 total IgG (p = 0.01) were factors of poor renal prognosis. Anti-PLA2R1 antibodies containing sera can induce in vitro cytotoxicity mediated by complement activation, and the level of cytotoxicity increases with the diversity and the titer of anti-PLA2R1 IgG subclasses. These patients with high level of complement-mediated cytotoxicity could benefit from adjuvant therapy using complement inhibitor associated with rituximab to induce earlier remission and less podocyte injury.

MiR-130a-5p prevents angiotensin II-induced podocyte apoptosis by modulating M-type phospholipase A2 receptor.

Podocyte apoptosis is considered as the important element that promotes the development and progress of membranous nephropathy (MN). Unfortunately, the underlying mechanism of podocytes apoptosis in MN remains elusive. We compared the renal expressions of miR-130a-5p and M-type phospholipase A2 receptor (PLA2R) between MN patients (n = 30) and 30 controls by qRT-PCR and western blot, respectively. The podocyte damage model in vitro was established by angiotensin II (Ang II, 100 nmol/L) exposure for 24 h. Interaction between miR-130a-5p and PLA2R was determined using dual-luciferase reporter gene assay. MN mice were induced by intravenous injection of cBSA. In this study, miR-130a-5p expression was significantly decreased both in the renal biopsy specimens from MN patients and podocyte cell line AB8/13 following stimulation of Ang II. Overexpressed miR-130a-5p in AB8/13 cells significantly attenuated the Ang II induced-apoptosis in vitro. In contrast, down-regulated miR-130a-5p induced podocyte apoptosis. PLA2R was identified as the target of miR-130a-5p in AB8/13 cells. And up-regulated or down-regulated PLA2R could obviously attenuate the effect of miR-130a-5p overexpression or knockdown on the apoptosis of AB8/13 cells. Furthermore, it was also observed that overexpressed miR-130a-5p by miR-130a-5p agomir could obviously alleviate renal injury in MN mice. In conclusion, decreased miR-130a-5p was contributed to the pathological mechanism of MN through increasing PLA2R expression, which induced podocyte apoptosis.

Identification and Quantification of Heterogeneously-methylated DNA Fragments Using Epiallele-sensitive Droplet Digital Polymerase Chain Reaction (EAST-ddPCR).

BACKGROUND/AIM: DNA methylation plays an important role in the initiation and propagation of carcinogenesis; however, the role of heterogeneously methylated epialleles is currently not well studied, also due to the lack of sensitive, unbiased and high throughput methods. Here, a newly developed droplet digital PCR (ddPCR)-based method was evaluated regarding its ability to quantify such heterogeneously methylated epialleles with sufficient analytical sensitivity and specificity. MATERIALS AND METHODS: Genomic DNA from blood leukocytes and bone marrow aspirate of an 8-year old male with B-cell acute lymphoblastic leukemia (B-ALL) and from normal and malignant prostate cell lines were analysed using ddPCR. RESULTS: By using these DNA samples, the specificity of an applied set of fluorescence-labeled probes was demonstrated as a proof of concept. CONCLUSION: All individual heterogeneously-methylated epialleles were quantifiable by a set of fluorescence-labeled probes with complementary sequences to epialleles in a closed-tube and high-throughput manner. The new method named epiallele-sensitive droplet digital PCR (EAST-ddPCR) may give new insights in the generation and regulation of epialleles and may help in finding new biomarkers for the diagnosis of benign und malignant diseases.

[Clinical features and expression of PLA(2)R in renal tissue with idiopathic membranous nephropathy in children].

Objective: To explore the clinical features and expression of PLA(2)R in renal tissue of children with idiopathic membranous nephropathy. Methods: Retrospective study was performed in patients with membranous nephropathy diagnosed through renal biopsy and the follow-up time was at least half a year in Shanghai Children's Hospital from January 2010 to February 2017. We compared their clinicopathological and pathological findings of IMN. Indirect immunofluorescence assay was used to detect glomerular PLA(2)R expression. We analyzed the differences of clinical features between the PLA(2)R negative and positive groups. T test, rank-sum test and Fisher exact test were used. Results: Eleven cases had hematuria and proteinuria, 9 cases presented with nephrotic syndrome, and 2 cases showed isolated proteinuria. Of the 22 cases of children with IMN, 16 patients had complete remission (complete remission rate was 72.8%), and 22 patients had partial remission. The renal function of all cases was normal and in all cases the estimated glomerular filtration rate was > 90 ml/(min·1.73m(2)). Of 22 cases with IMN, 7 cases were PLA(2)R-positive in renal tissue and 15 cases were PLA(2)R-negative. The age of positive group (10 years old) was older than the negative group (6 years old)(Z=-2.483, P<0.05) and the time of positive group (6 months) for urine protein to return to negative was longer than the negative group (2.5 months) through treatment. These differences were significantly different (Z=-2.072, P<0.05). Conclusions: Hematuria and proteinuria can be found in most children with idiopathic primary membranous nephropathy. Prednisone combined with immunosuppressant was effective. The positive rate of PLA(2)R in renal tissue of children with IMN was about 32%. The age of PLA(2)R positive group was older than the negative group. And the time of urine protein turning to negative in positive group was longer than that in the negative group.