Sigrid Harendza

An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis

BACKGROUNDnThe recent finding that phospholipase-A(2)-receptor antibodies (PLA(2)R-AB) may play a role in the development of primary membranous glomerulonephritis (MGN) offers the opportunity to measure a marker to help diagnose, classify and eventually monitor the course of patients with MGN.nnnMETHODSnWe developed an immunofluorescence test, which allows the easy and specific analysis of the presence of PLA(2)R-AB in serum. The usefulness of this test was studied in 153 healthy blood donors, 90 patients with non-membranous glomerular injuries, 17 patients with a secondary form of MGN and 100 patients with biopsy-proven primary MGN. In addition, in five patients with biopsy-proven MGN, PLA(2)R-AB levels were monitored prospectively for up to 18 months following a single dose of rituximab (RTX) (375 mg/m(2) body surface).nnnRESULTSnPLA(2)R-AB were not found in healthy controls or patients with glomerular lesions other than biopsy-proven primary MGN. Fifty-two patients with primary MGN (52%) were positive for PLA(2)R-AB. The levels ranged from 1:10 to 1:3200. In patients who had MGN and were treated with RTX the fall in PLA(2)R-AB levels was followed by a decrease in proteinuria, whereas an increase in PLA(2)R-AB levels was associated with an increase in proteinuria.nnnCONCLUSIONSnThese studies show that the new test allows the monitoring of PLA(2)R-AB levels in patients with MGN and may help in making therapeutic decisions for these patients.

Glomerular Mesangial Cell-specific Transactivation of Matrix Metalloproteinase 2 Transcription Is Mediated by YB-1

Mesangial cell (MC) activation plays a pivotal role in the development of the end stage sclerotic lesion characteristic of most forms of chronic glomerular disease. We have previously demonstrated that MC activation is directly linked to high level expression of the matrix metalloproteinase-2 (MMP-2) enzyme (Turck, J., Pollock, A. S., Lee, L., Marti, H.-P., and Lovett, D. H. (1996) J. Biol. Chem. 25, 15074–15083), the transcription of which is regulated in a tissue-specific fashion. Recent studies (Harendza, S., Pollock, A., Mertens, P. R., and Lovett, D. H. (1995) J. Biol. Chem. 270, 18786–18796) delineated a strong cis-acting enhancer element, designated MMP-2 RE1, within the 5′-flanking region of the rat MMP-2 gene. Gel shift, DNA footprint, and transcriptional analyses mapped the enhancer element to a unique 40-base pair (bp) sequence located at −1322 to −1282 bp relative to the translational start site. Bromodeoxyuridine-substituted UV cross-linking of the 40-bp enhancer element with MC nuclear extracts yielded a single protein of 52 kDa, while Southwestern blot analysis with MMP-2 RE1 demonstrated three hybridizing nuclear proteins of 52, 62, and 86 kDa size. Screening of a human MC cDNA expression library with MMP-2 RE1 exclusively yielded clones with the identical sequence of the transcription factor YB-1. Western blot and supershift gel analysis of MC nuclear extracts with an anti-YB-1 antibody confirmed the presence of YB-1 within the shifted complex. Examination of the MMP-2 RE1 sequence revealed an incomplete Y-box sequence (CTGCTGGGCAAG), which specifically interacted with recombinant YB-1 on DMS protection footprinting analysis. YB-1 protein preferentially bound the single-stranded components of the 40-bp MMP-2 RE1 and, with increasing concentrations, formed multimeric complexes. Co-transfection of YB-1 in MC increased the enhancer activity within the context of the native MMP-2 promoter, while transfection of non-MMP-2-synthesizing glomerular epithelial cells with YB-1 led to transcriptional suppression. This study indicates that YB-1 is a major, cell type-specific transactivator of MMP-2 transcription by glomerular mesangial cells.

Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy

The M-type phospholipase A2 receptor (PLA2R) is the major target antigen in idiopathic membranous nephropathy with detectable autoantibodies in the serum of up to 70% of patients. In retrospective studies, the PLA2R-autoantibody titer in the serum was sometimes negative indicating their measurement alone may be inconclusive. In order to better differentiate between primary and secondary membranous nephropathy, we conducted a prospective study that included 88 patients with a histologic diagnosis of membranous nephropathy. Immunohistochemical analysis for PLA2R was faintly positive in kidneys from normal individuals and patients with various other glomerular injuries. In 61 of the 88 patients, PLA2R expression was strongly positive in glomeruli, and in 60 of these patients PLA2R autoantibodies were also detected in the serum. The 27 patients negative for serum PLA2R autoantibodies were faintly positive for PLA2R staining in glomeruli and in 15 of these patients a secondary cause was found. The remaining 12 patients have a yet undetected secondary cause of membranous nephropathy or have different glomerular antigens other than PLA2R. Thus, increased staining for PLA2R in glomeruli of renal biopsies tightly correlates with the presence of PLA2R autoantibodies in the serum and this may help discriminate between primary and secondary membranous nephropathy.

A Mechanism for Cancer-Associated Membranous Nephropathy

The cause of paraneoplastic membranous nephropathy is unclear. In the current case, new expression of THSD7A in a gallbladder carcinoma and the development of membranous nephropathy may indicate a potential mechanism for the association between cancer and this nephropathy.

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 (HRu200a=u200a3.66; 95%CI: 1.39–9.64; pu200a=u200a0.009). 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.

Angiotensin II's antiproliferative effects mediated through AT2-receptors depend on down-regulation of SM-20

Angiotensin II (ANG II) inhibits proliferation and induces differentiation through AT2 receptors. However, target genes involved in this process are not well characterized. We studied PC12 cells, a rat pheochromocytoma cell line exclusively expressing AT2 receptors. ANG II attenuated proliferation of PC12 cells without concomitant induction of apoptosis. To identify potential novel genes involved in the antimitogenic actions of ANG II, we performed differential display analysis of PC12 cells after challenge with 10−7 m ANG II for 6 hours. One identified gene selected for further study that was down-regulated by ANG II in PC12 cells was SM-20. This gene has been previously isolated from vascular smooth muscle cells treated with mitogens by differential hybridization. Recent findings show a homology of SM-20 with enzymes involved in the regulation of hypoxia inducible factor 1. ANG II suppressed mRNA expression of SM-20 in PC12 cells after only 30 minutes, as detected by Northern blotting. This effect was antagonized by an AT2 receptor blocker, but not by losartan. A rabbit polyclonal antibody was generated against a peptide sequence of SM-20 and detected a major band of the predicted size of 40 kd and a second 33-kd band that likely represents a processed form present in mitochondria. Immunohistochemistry revealed a granular staining of the cytoplasm of PC12 cells compatible with a previously described mitochondrial localization of SM-20 protein. Western blots confirmed the down-regulation of SM-20 protein in PC12 cells subsequent to incubation with ANG II. SM-20 transcripts were also reduced by ANG II acting on AT2 receptors in rat glomerular endothelial cells that express both AT1 and AT2 receptors. SM-20 antisense, but not sense, phosphothioate-modified oligonucleotides reduced base-line proliferation of PC12 cells. In contrast, inducible overexpression of SM-20 using the ecdysone system prevented the antiproliferative effects of ANG II in PC12 cells. In summary, our study identified SM-20 as an essential component of ANG IIs growth-suppressive effects mediated through AT2 receptors. This gene apparently plays an important role in the regulatory processes determining whether a cell should undergo differentiation, apoptosis, or proliferation.

An Indirect Immunofluorescence Method Facilitates Detection of Thrombospondin Type 1 Domain-Containing 7A-Specific Antibodies in Membranous Nephropathy.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target antigen identified in adult membranous nephropathy (MN) along with the major antigen phospholipase A2 receptor 1 (PLA2R1). The prevalence of THSD7A-Ab-positive patients is unknown, and it is unclear whether the clinical presentation differs between patients positive for PLA2R1-Ab or THSD7A-Ab. We screened serum samples of 1276 patients with MN from three different cohorts for the presence of THSD7A-Ab by Western blot analysis and a newly developed indirect immunofluorescence test (IFT). Compared with Western blot analysis, the IFT had a 92% sensitivity and a 100% specificity. The prevalence of THSD7A-associated MN in a prospective cohort of 345 patients with MN was 2.6%, and most were women. In this cohort, the percentage of patients with THSD7A-associated MN and malignant disease significantly exceeded that of patients with PLA2R1-associated MN and malignant disease. In all cohorts, we identified 40 patients with THSD7A-associated MN, eight of whom developed a malignancy within a median time of 3 months from diagnosis of MN. In one patient with THSD7A-associated MN and metastases of an endometrial carcinoma, immunohistochemistry showed THSD7A expression on the metastatic cells and within follicular dendritic cells of the metastasis-infiltrated lymph node. We conclude that the IFT allows sensitive and specific measurement of circulating THSD7A-Ab in patients with MN. Patients with THSD7A-associated MN differ in their clinical characteristics from patients with PLA2R1-associated MN, and more intensive screening for the presence of malignancies may be warranted in those with THSD7A-associated MN.

M-type Phospholipase A2 Receptor Autoantibodies and Renal Function in Patients with Primary Membranous Nephropathy

BACKGROUND AND OBJECTIVESnLoss of renal function in patients with primary membranous nephropathy cannot be reliably predicted by laboratory or clinical markers at the time of diagnosis. M-type phospholipase A2 receptor autoantibodies have been shown to be associated with changes in proteinuria. Their eventual effect on renal function, however, is unclear.nnnDESIGN, SETTING, PARTICIPANTS, & MEASUREMENTSnIn this prospective, open, multicenter study, the potential role of M-type phospholipase A2 receptor autoantibodies levels on the increase of serum creatinine in 118 consecutive patients with membranous nephropathy and positivity for serum M-type phospholipase A2 receptor autoantibodies was analyzed. Patients were included in the study between April of 2010 and December of 2012 and observed until December of 2013. The clinical end point was defined as an increase of serum creatinine by ≥ 25% and serum creatinine reaching ≥ 1.3 mg/dl.nnnRESULTSnPatients were divided into tertiles according to their M-type phospholipase A2 receptor autoantibody levels at the time of inclusion in the study: tertile 1 levels=20-86 units/ml (low), tertile 2 levels=87-201 units/ml (medium), and tertile 3 levels ≥ 202 units/ml (high). The median follow-up time of all patients in the study was 27 months (interquartile range=18-33 months). The clinical end point was reached in 69% of patients with high M-type phospholipase A2 receptor autoantibodies levels (tertile 3) but only 25% of patients with low M-type phospholipase A2 receptor autoantibodies levels. The average time to reach the study end point was 17.7 months in patients with high M-type phospholipase A2 receptor autoantibodies levels and 30.9 months in patients with low M-type phospholipase A2 receptor autoantibodies levels. A multivariate Cox regression analysis showed that high M-type phospholipase A2 receptor autoantibodies levels-in addition to men and older age-are an independent predictor for progressive loss of renal function.nnnCONCLUSIONSnHigh M-type phospholipase A2 receptor autoantibodies levels were associated with more rapid loss of renal function in this cohort of patients with primary membranous nephropathy and therefore, could be helpful for treatment decisions.

Spontaneous remission of proteinuria is a frequent event in phospholipase A2 receptor antibody negative patients with membranous nephropathy

BACKGROUNDnPhospholipase A2 receptor antibodies (PLA2R-Ab) and thrombospondin type-1 domain-containing 7A antibodies (THSD7A-Ab) are present in 70-80% of patients with membranous nephropathy (MN). Little, however, is known about the pathogenesis of MN and the clinical outcome in PLA2R-Ab- and THSD7A-Ab-negative patients.nnnMETHODSnIn this prospective multicentre observational study, the clinical outcome of 37 patients with biopsy-proven MN who were negative for PLA2R-Ab and THSD7A-Ab in the serum was analysed.nnnRESULTSnA total of 198 patients were screened for inclusion in the study. Of these, 157 patients were positive for PLA2R-Ab and 4 patients for THSD7A-Ab. The remaining 37 patients were negative for both antibodies were and included in this study. Six patients died during the follow-up, five because of malignant diseases and one of an infection. One patient went into end-stage renal disease, and two patients were lost to follow-up. The remaining 28 patients were followed for at least 24 months (35.6 ± 8.9 months). Seventeen patients received immunosuppressive (IS) therapy, and 11 received supportive care only. At the end of the follow-up, 14 of the 17 patients treated with immunosuppressants and 10 of 11 patients on supportive therapy had a remission of proteinuria. The time to reach remission of proteinuria and serum creatinine levels at the end of the follow-up were not different between both groups. A univariate Cox regression analysis indicated that the use of immunosuppression did not alter the chance to reach a remission of proteinuria.nnnCONCLUSIONSnA high number of PLA2R-Ab- and THSD7A-Ab-negative patients with MN have a good prognosis and might not need IS therapy.

The CTLA-4 +49GG genotype is associated with susceptibility for nephrotic kidney diseases

BACKGROUNDnThe pathogenesis of primary nephrotic kidney diseases is not completely understood. As T-cell involvement is suspected, cytotoxic T-lymphocyte antigen 4 (CTLA-4) expressed on activated T cells could play a role in the immune response. Single-nucleotide polymorphisms (SNPs) in the CTLA-4 gene are associated with several autoimmune-related diseases.nnnMETHODSnOur goal was to study the occurrence of the SNPs -318C/T, +49A/G and CT60 on the CTLA-4 gene in healthy blood donors (N = 156) compared with nephrotic patients with biopsy-proven minimal-change disease (MCD, N = 160), focal segmental glomerulosclerosis (FSGS, N = 159) and membranous nephropathy (MN, N = 185). Odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated to estimate the strength of the association.nnnRESULTSnThe +49GG genotype was significantly (P < 0.001) associated with the risk for MCD, FSGS and MN (AA versus GG: OR = 3.403, 95% CI = 1.748-6.622, OR = 3.846, 95% CI = 1.945-7.604 and OR = 2.381, 95% CI = 1.257-4.511, respectively). No further significant associations, neither with the heterozygous genotype of +49A/G nor for the -318C/T or CT60 SNP, were detected.nnnCONCLUSIONSnThe +49GG genotype of the +49A/G SNP in the CTLA-4 gene is associated with the risk for MCD, FSGS and MN, suggesting a possible role for CTLA-4 in a proposed common final pathway in the pathogenesis of primary nephrotic kidney diseases.