Circulating Permeability Factors Research Articles

#1622 Minimal changes disease and myasthenia gravis: a logical association

Abstract Background and Aims Minimal changes disease (MCD) is responsible for 10-25% of nephrotic syndrome cases in adults. It can have a primary or secondary aetiology, being associated with diseases like infections, atopy or even haematological malignancies. There is a described association between MCD and myasthenia gravis (MG) in the literature, which we will explore in this case report. Clinical Case An autonomous 67 years old female, healthy except for overweight develops diplopia and dysphagia, being diagnosed with anti-musk MG (antibodies titters 2,2002 nmol/L). Thymoma presence was excluded by CT scan. She starts oral treatment with pyridostigmine without response. Two months later she develops generalized oedema. Analytically she had normal kidney function (Cr 0.7 mg/dL; BUN 28.5 mg/dL), hypoalbuminemia (2.09 g/dL), dyslipidaemia and nephrotic proteinuria of PCR 8g/g creat, without leucoeritrocyturia. Reno-vesical echography was normal. No alterations in immunological study except for a low IgG 396 mg/dL. Anti-PLA2R was negative. Kidney biopsy was performed, having 4 glomerulus that looked normal in optical microscopy. In our centre we do not have electronic microscopy available, so the biopsy was interpreted as MCD. She started prednisolone (PDN) 1 mg/kg/day with clinical improvement and resolution of the symptoms and proteinuria (0.062 g/g creat). In November 2022, while she was tapering the PDN, she had a myasthenic crisis, with MCD recurrency. She did 5 plasmapheresis sessions, the PDN dose was increased to 1 mg/kg/day and two rituximab administrations (1000 mg each) were done. There was an improvement in MG symptoms during 3 weeks and then she aggravated again. The proteinuria slowly decreased during 6 weeks until RPC 1.3 g/g creat and then started to aggravate when the corticosteroids were tapered. It was decided to initiate mycophenolate mofetil (MMF), there was improvement and both diseases are controlled until today, the patient is asymptomatic under 7.5 mg/day of prednisolone, with a PCR 0.2 g/g creat. Discussion The production of a circulating permeability factor by abnormal T cells, that causes the podocytes damage, is one of the possible explanations for MCD. There are reports of improvements with plasmapheresis (PF) by permeability factor remotion from circulation. MG is also caused by T cells antibodies attack, but the attack occurs against channels essential for the neuromuscular membrane function. The similarities of the mechanisms make the existence of a common branch in the t cells dysregulation seem plausible. Anti musk MG is very responsive to PF but it has a limited effect in time. Rituximab depletes B cells responsible for promoting the impaired T cells responses. It has been associated with longer remissions in cortico-sensitive MCD with relapsing disease. The exact same mechanism occurs when rituximab is used in MG. In this case the patient did not have a satisfactory response to rituximab as the patient kept dependent of high PDN doses, so we decided to initiate MMF with a complete remission of both pathologies and allowing the use of lower PDN doses. Take Home Message The association between MCD and myasthenia gravis is rare, especially in patients without thymoma. When we analyse the diseases physiopathology, we find common mechanisms that can justify the association. The identification of this relation can be advantageous making possible early rituximab use, that has been showing good results in both pathologies. In this particular clinical case, there was a recurrence with rituximab when we tapered the PDN, so the patient started MMF with good response and allowing lower PDN doses.

Current understanding of the molecular mechanisms of circulating permeability factor in focal segmental glomerulosclerosis.

The pathogenetic mechanisms underlying the onset and the post-transplant recurrence of primary focal segmental glomerulosclerosis (FSGS) are complex and remain yet to be fully elucidated. However, a growing body of evidence emphasizes the pivotal role of the immune system in both initiating and perpetuating the disease. Extensive investigations, encompassing both experimental models and patient studies, have implicated T cells, B cells, and complement as crucial actors in the pathogenesis of primary FSGS, with various molecules being proposed as potential "circulating factors" contributing to the disease and its recurrence post kidney-transplantation. In this review, we critically assessed the existing literature to identify essential pathways for a comprehensive characterization of the pathogenesis of FSGS. Recent discoveries have shed further light on the intricate interplay between these mechanisms. We present an overview of the current understanding of the engagement of distinct molecules and immune cells in FSGS pathogenesis while highlighting critical knowledge gaps that require attention. A thorough characterization of these intricate immune mechanisms holds the potential to identify noninvasive biomarkers that can accurately identify patients at high risk of post-transplant recurrence. Such knowledge can pave the way for the development of targeted and personalized therapeutic approaches in the management of FSGS.

#4013 LIPIDOMIC ANALYSIS OF PFSGS PATIENTS' SERUM TO IDENTIFY POTENTIAL CANDIDATES FOR THE CIRCULATING PERMEABILITY FACTOR(S) IN THE LIPID DOMAIN

Abstract Background and Aims Patients with primary focal segmental glomerulosclerosis (pFSGS) present with nephrotic syndrome. Whereas the exact pathogenesis is largely unknown, there is strong evidence that one or more circulating permeability factors in the circulation induce podocyte injury. Podocyte injury eventually leads to foot process effacement, massive proteinuria and kidney function loss. Historically, research into the cause of pFSGS has focused primarily on identifying the circulating factor in the protein domain, but reports of successful treatment with lipid apheresis suggest that it could also be a lipid compound. Here, we set out to investigate the lipidome of pFSGS patients’ serum to identify potential candidates for the circulating permeability factor(s) in the lipid domain. Method For this lipidomics study, we included 5 patients with pFSGS in whom serum had been collected at the time they presented at our center with active nephrotic syndrome. We compared the serum lipidome of the pFSGS patients with age matched healthy controls (n = 5), and with age matched patients with familial hypercholesterolemia (n = 5) who, like pFSGS patients, have significantly elevated lipid values. Serum was analyzed using the Shotgun Lipidomics platform by Lipotype GmbH (Dresden, Germany), as previously described [1]. The amount of lipids were normalized to the total amount of lipids present within the sample. Results The lipidomic profile of patients with pFSGS was markedly different from the profile of healthy controls and patients with hypercholesterolemia. We found differences in various lipid classes in pFSGS patients, including cholesterol esters (CE), lyso-phophatidylcholine (LPC), phophatidylethanolamine (PE), diacylglycerol (DAG) and triacylglycerol (TAG) that were significantly changes compared to both healthy controls and FH patients. Diacylglycerols (DAG) were identified as the most significantly increased lipid species in pFSGS. By analyzing the subspecies we discovered that DAGs with carbon chains greater than 38 carbon atoms in both chains combined (long carbon chain DAGs, lccDAGs) were exclusively present in patients with pFSGS. Conclusion Our lipidomics data show that patients with pFSGS have a substantially different lipid profile than healthy controls and patients with familial hypercholesterolemia. We identified lccDAGs as unique lipids in patients with pFSGS, that were not present in controls. This is an interesting finding since the DAG-analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) is known to be able to induce podocyte injury in vitro [2]. In further research, we will focus on confirming whether lccDAGs are unique to pFSGS patients, whether it is suitable as a biomarker, and whether it plays a causal role in the disease process.

#3269 EPIDEMIOLOGY OF PRIMARY FSGS INCLUDING CLUSTER ANALYSIS OVER A 20-YEAR PERIOD

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is one of the leading causes of nephrotic syndrome (NS) in adults. This epidemiological study describes a renal centre's 20 year ‘real world' experience of patients with primary FSGS including cluster analysis. Method This retrospective analysis was conducted on patients diagnosed with primary FSGS at a tertiary renal centre with a catchment population of 1.55 million, over 2 decades. Data collection included baseline demographics, laboratory results, immunosuppression (IS) and outcomes: Complete & partial remission, relapse, progression to renal replacement therapy (RRT) and mortality. A two-step cluster analysis was used to reveal groupings to aid prognostication and treatment decisions. Pre-specified cluster variables were: Serum albumin (sAlb), urine protein: creatinine ratio (uPCR) & estimated glomerular filtration rate (eGFR). Results 87 patients were identified with primary FSGS after exclusion of secondary causes. Mean age was 49.3 years, 60.9% male, 86.2% white with median eGFR 46 ml/min, uPCR 573 mg/mmol, sAlb 33g/l (Table 1). Rates of partial and complete remission were 23% & 49.4% respectively. Progression to RRT occurred in 27.4%, death in 27.4%. Comparing those receiving IS vs not: sAlb was lower 23 g/L vs 40 g/L (p<0.001), uPCR was higher 795 mg/mmol vs 318 mg/mmol (p <0.001). The IS group was more likely to achieve complete remission (62% vs 40%, p = 0.041), but relapsed more 48.6% vs 22% (p<0.027). There were no statistically significant differences between mortality or RRT. The 2-step cluster analysis separated the cohort into 3 clusters, see Fig. 1: Cluster 1 (n = 26) with ‘nephrotic range proteinuria’ mean sAlb 30 g/L, uPCR 778 mg/mmol and eGFR 72 ml/min; Cluster 2 (n = 43) with ‘non-nephrotic range proteinuria’, mean sAlb 39 g/L, uPCR 284 mg/mmol and eGFR 46 ml/min; Cluster 3 (n = 18) ‘NS’ sAlb 20 g/L, uPCR 1117 mg/mmol and eGFR 25 ml/min. IS use was comparable in the NS (cluster 3) and nephrotic range proteinuria (cluster 1) cohorts, but lower in cluster 2 (non-nephrotic range proteinuria) 77.8% & 69.2% vs 11.6% p <0.001. Rates of complete remission were greatest in clusters 1 & 3 vs cluster 2: 57.7% &66.7% vs 37.2% though this did not achieve statistical significance (p 0.067). Conclusion Although there have been several recent US & Asian reviews of primary FSGS, this study provides a review of the epidemiology of FSGS over a 20-year period in a predominantly white cohort in the UK. In general, the patients who received IS had lower sAlb and achieved remission more frequently. Although the aetiology of primary FSGS is unknown, it is hypothesised that it is due to an undiscovered circulating permeability factor. After the exclusion of secondary causes, KDIGO suggests only making the diagnosis in the presence of NS. Despite this the average sAlb in most epidemiological studies of primary FSGS have been above 30g/l. This is likely because of the inclusion of undiagnosed genetic or adaptive FSGS, but it may also be true that NS phenotype alone is too insensitive to diagnose primary ‘antibody driven’ FSGS. Our cluster analysis highlighted 3 potential FSGS phenotypes: A nephrotic cluster that clearly require IS; a cohort with preserved sAlb and non-nephrotic range proteinuria who will benefit from supportive care; lastly a cluster with heavy proteinuria but a sAlb > 30g/l. This group may still have antibody driven disease and thus benefit from immunosuppression and potentially genetic testing. The study received grant support from CSL Vifor

Primary Focal Segmental Glomerulosclerosis Plasmas Increase Lipid Droplet Formation and Perilipin-2 Expression in Human Podocytes.

Many patients with primary focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation. Several circulating permeability factors (CPFs) responsible for recurrence have been suggested, but were never validated. We aimed to find proteins involved in the mechanism of action of CPF(s) and/or potential biomarkers for the presence of CPF(s). Cultured human podocytes were exposed to plasma from patients with FSGS with presumed CPF(s) or healthy and disease controls. Podocyte proteomes were analyzed by LC-MS. Results were validated using flow cytometry, RT-PCR, and immunofluorescence. Podocyte granularity was examined using flow cytometry, electron microscopy imaging, and BODIPY staining. Perilipin-2 protein expression was increased in podocytes exposed to presumed CPF-containing plasmas, and correlated with the capacity of plasma to induce podocyte granularity, identified as lipid droplet accumulation. Elevated podocyte perilipin-2 was confirmed at protein and mRNA level and was also detected in glomeruli of FSGS patients whose active disease plasmas induced podocyte perilipin-2 and lipid droplets. Our study demonstrates that presumably, CPF-containing plasmas from FSGS patients induce podocyte lipid droplet accumulation and perilipin-2 expression, identifying perilipin-2 as a potential biomarker. Future research should address the mechanism underlying CPF-induced alterations in podocyte lipid metabolism, which ultimately may result in novel leads for treatment.

Novel mouse strains to study circulating permeability factor(s) in primary focal segmental glomerulosclerosis.

Recurrence of proteinuria after kidney transplantation in primary focal segmental glomerulosclerosis (FSGS) is unpredictable. Several putative circulating permeability factors (CPFs) have been suggested, but none have been validated. A clinically relevant experimental model is required that demonstrates the presence of CPF(s) in patient material, to study CPF(s) and possibly predict recurrence in patients. We aimed to develop a FSGS-prone Thy-1.1 transgenic mouse model with accelerated proteinuria after injection of samples from patients with FSGS. The Thy-1.1 transgene was backcrossed into 5 mouse strains. The age of onset and severity of spontaneous proteinuria varied between the different genetic backgrounds. 129X1/SvThy-1.1 and 129S2/SvPasThy-1.1 mice displayed proteinuria at 4 weeks, whereas Balb/cThy-1.1 and C57BL/6JThy-1.1 mice developed proteinuria from 6 weeks, and were used further. We determined the maximum protein dose that could be injected without causing protein overload in each background. Balb/cThy-1.1 and C57BL/6JThy-1.1 males and females were injected with presumably CPF-containing plasmapheresis effluent from 6 FSGS patients, which induced albuminuria particularly in Balb/cThy-1.1 males. Unfortunately, no response could be detected when using sera instead of plasmapheresis effluent, serum being more clinically relevant in the context of predicting FSGS recurrence. Considering the differences between responses elicited by serum and plasmapheresis effluent, simultaneously collected serum, plasma, and plasmapheresis effluent were tested. Whereas we could detect responses using a validated in vitro model, none of these presumably CPF-containing samples induced proteinuria in Balb/cThy-1.1 males. Thus, we have extensively tested the Thy-1.1 mouse model on different genetic backgrounds with proteinuria after injection of FSGS patient material as clinically relevant readout. The Balb/cThy-1.1 male mouse strain demonstrated the most promising results, but to detect CPF activity in FSGS serum e.g. prior to kidney transplantation, this strain clearly lacks sensitivity and is therefore not yet clinically applicable. It could, however, still be used as research tool to study CPFs in patient samples that did induce proteinuria.

Circulating Permeability Factors in Focal Segmental Glomerulosclerosis: In Vitro Detection

The recurrence of proteinuria after kidney transplantation in patients with focal segmental glomerulosclerosis (FSGS) is considered proof of the presence of circulating permeability factors (CPFs). The aim of this study is to demonstrate the presence of plasma CPFs using series of invitro assays. Podocytes and endothelial cells (glomerular microvascular endothelial cells [GMVECs]) were incubated with plasma from FSGS patients with presumed CPFs in relapse and remission and from steroid-resistant nephrotic syndrome (SRNS), steroid-sensitive nephrotic syndrome (SSNS), membranous nephropathy (MN), and healthy controls (hCtrls). Cell viability, podocyte actin cytoskeleton architecture, and reactive oxygen species (ROS) formation with or without ROS scavenger were investigated by Cell Counting Kit-8 assay, immunofluorescence staining, and CM-H2DCFDA probing, respectively. Presumed CPF-containing plasma causes a series of events in podocytes but not in GMVECs. These events include actin cytoskeleton rearrangement and excessive formation of ROS, which results in podocyte loss. These effects were solely observed in response to CPF plasma collected during relapse, but not in response to plasma of hCtrls, or patients with SRNS, SSNS, and MN. The copresence of dimethylthiourea, a scavenger of ROS, abolished the aforementioned effects of CPF plasma. We provide a panel of invitro bioassays to measure podocyte injury and predict the presence of CPFs in plasma of patients with nephrotic syndrome (NS), providing a new framework for monitoring CPF activity that may contribute to future NS diagnostics or used for disease monitoring purposes. Moreover, our findings suggest that the inhibition of ROS formation or facilitating rapid ROS scavenging may exert beneficial effects in patients with CPFs.

Biomarkers in Primary Focal Segmental Glomerulosclerosis in Optimal Diagnostic-Therapeutic Strategy.

Focal segmental glomerulosclerosis (FSGS) involves podocyte injury. In patients with nephrotic syndrome, progression to end-stage renal disease often occurs over the course of 5 to 10 years. The diagnosis is based on a renal biopsy. It is presumed that primary FSGS is caused by an unknown plasma factor that might be responsible for the recurrence of FSGS after kidney transplantation. The nature of circulating permeability factors is not explained and particular biological molecules responsible for inducing FSGS are still unknown. Several substances have been proposed as potential circulating factors such as soluble urokinase-type plasminogen activator receptor (suPAR) and cardiolipin-like-cytokine 1 (CLC-1). Many studies have also attempted to establish which molecules are related to podocyte injury in the pathogenesis of FSGS such as plasminogen activator inhibitor type-1 (PAI-1), angiotensin II type 1 receptors (AT1R), dystroglycan(DG), microRNAs, metalloproteinases (MMPs), forkheadbox P3 (FOXP3), and poly-ADP-ribose polymerase-1 (PARP1). Some biomarkers have also been studied in the context of kidney tissue damage progression: transforming growth factor-beta (TGF-β), human neutrophil gelatinase-associated lipocalin (NGAL), malondialdehyde (MDA), and others. This paper describes molecules that could potentially be considered as circulating factors causing primary FSGS.

Focal segmental glomerulosclerosis: Risk for recurrence and interventions to optimize outcomes following recurrence.

FSGS is a common indication for kidney transplant with a high-risk of posttransplant recurrence. In this review, we summarize current knowledge about FSGS recurrence after kidney transplantation, including epidemiology, pretransplant planning, posttransplant management, and investigational treatments. FSGS recurs in 14%-60% of first transplants, likely associated with a circulating permeability factor. Pretransplant counseling regarding recurrence is critical, and patients with FSGS should undergo pretransplant genetic screening. Rapid progression to ESKD, initial steroid responsiveness, younger age at diagnosis, race/ethnicity, and mesangial hypercellularity or minimal change histology on native biopsy may be associated with recurrence. Living donation is not contraindicated but does not result in improved graft survival relative to deceased donation. Pretransplant nephrectomy may be performed for a variety of reasons, but does not decrease recurrence. Pretransplant therapy with rituximab and/or PE is understudied but not clearly effective at preventing recurrence. Patients with FSGS typically present early with rapid-onset severe proteinuria. Diagnosis can be confirmed by biopsy showing foot process effacement; typical FSGS lesions are not seen on light microscopy in the early stages. There is no established effective treatment for recurrent FSGS, but renin-angiotensin-aldosterone system inhibition and extracorporeal therapies, including PE and IA, are most commonly used. Adjunct or alternative therapies may include rituximab, lipopheresis, and cyclosporine.

Serum levels of plasminogen activator urokinase receptor and cardiotrophin-like cytokine factor 1 in patients with nephrotic syndrome.

41 patients with chronic glomerulonephritis (CGN) were included in our study. 17 patients had FSGS, 7 patients had MCD, 5 patients had membranoproliferative glomerulonephritis (MPGN), 6 patients had IgA nephropathy, and 6 patients had membranous nephropathy (MN). The laboratory data were compared with the clinical and histological features of nephritis. Serum levels of plasminogen activator urokinase receptor (uPAR) and cardiotrophin-like cytokine factor 1 (CLCF-1)were measured by ELISA. The serum levels of uPAR were higher in FSGS patients before treatment than in patients with other morphological forms (MCD, IgA nephropathy, MN, and MPGN). The levels of uPAR in serum did not correlate with daily proteinuria, serum creatinine/eGFR, arterial hypertension, the number of sclerosed glomeruli, or tubulointerstitial fibrosis. No correlations were found between the levels of CLCF-1 in serum and creatinine levels/glomerular filtration rate, the percentage of sclerosed glomeruli, or the severity of tubulointerstitial fibrosis. There were no significant differences between the histological variants of nephritis. However, we found correlations between CLCF-1 levels and proteinuria and lipid levels. The data indicate an increase in the serum uPAR levels of FSGS before treatment. CLCF-1 levels in serum do not depend on histological forms of CGN, kidney function, or immunosuppressive treatment, but they correlate with proteinuria and serum lipids in patients with NS.

Elevated urinary CD80 excretion in children with steroid-responsive nephrotic syndrome

Background: Idiopathic nephrotic syndrome (INS) is one of the most common glomerular diseases in children with different pathological types and different responses to corticosteroids. A definitive diagnosis is essential for planning the treatment and determining the prognosis of these patients and currently, kidney biopsy is the only method for definitive diagnosis. However, this is an invasive procedure. In addition, in some cases, the biopsy is contraindicated or tissue obtained on biopsy is insufficient and may not represent the underlying disease. According to the recent hypothesis about the role of circulating permeability factors in the pathogenesis of INS, urine protein analysis as a noninvasive method to determine the specific biomarkers of the disease is of great interest to nephrologists and can be useful. Methods: In this case − control study, we analyzed urinary CD80 (uCD80) levels in 51 patients with INS using a special CD80 enzyme-linked immunosorbent assay kit and were compared between different groups of patients. Results: The highest urine CD80/creatinine ratio was found in patients with active minimal change disease and steroid-responsive nephrotic syndrome in the relapse stage of the disease. Conclusion: A significant level of uCD80 is correlated with better renal function and a more favorable response to steroids in patients with INS. Therefore, it can be concluded that a high level of uCD80 is correlated with a good prognosis in these patients.

Recurrent Focal Segmental Glomerulosclerosis After Kidney Transplantation in African Americans: Review of the Current Evidence.

In many countries of sub-Saharan Africa, the most common causes of end-stage kidney disease are hypertension, chronic glomerulonephritis, and diabetes mellitus. So far, literature on recurrent focal segmental glomerulosclerosis in sub-Saharan African populations is limited. With the intention of providing guidance for best practices in sub-Saharan Africa, we reviewed available evidence for African Americans, a population with a similar genetic background. We chose this population as a pseudo-population to show how similar genetic backgrounds can predict disease occurrence in similar populations residing in different continents. Our extended PubMed and Scopus literature search used these key words: "focal segmental glomerulosclerosis in African Americans" (search 1), "recurrent focal segmental glomerulosclerosis after kidney transplantation" (search 2), "risk factors for recurrent focal segmental glomerulosclerosis" (search 3); and "APOL1 gene and kidney transplantation" (search 4). Search 1 yielded 4 articles, search 2 yielded 44 articles, search 3 yielded 6 articles, and search 4 yielded 8 articles. African Americans were shown to be disproportionately predisposed to endstage kidney disease, traceable to focal segmental glomerulosclerosis (the most common cause of glomerulonephritis leading to end-stage kidney disease). Apolipoprotein L1 presence in 22% of African Americans explained the odds ratio of 17 in developing focal segmental glomerulosclerosis and 8 times lifetime risk of end-stage kidney disease. Focal segmental glomerulosclerosis recurred in 30% of kidney transplant recipients; risk factors included young age, rapid progression to end-stage kidney disease, and White race recipient. Circulating permeability factors played a central role in primary and recurrent focal segmental glomerulosclerosis. For recurrent cases, transplant biopsy has remained the gold standard for diagnosis, with treatment involving a multi-modal approach, often resulting in partial or complete remission of proteinuria; allograft loss can occur if treatment is not successful. More randomized clinical trials are needed to chart the way forward for prolonged allograft function.

MO247NOVEL DIAGNOSTIC AND THERAPEUTIC TECHNIQUES REVEAL CHANGED METABOLOMIC PROFILE IN RECURRENT FOCAL SEGMENTAL GLOMERULOSCLEROSIS*

Abstract Background and Aims Idiopathic forms of Focal Segmental Glomerulosclerosis (FSGS) are caused by circulating permeability factors, which can lead early recurrence of FSGS and kidney failure after kidney transplantation. In the past three decades, many research endeavors were undertaken to identify these unknown factors. Even though some potential candidates have been recently discussed in the literature, “the” actual factor remains elusive. Therefore, there is an increased demand in FSGS research for the use of novel technologies that can allow us to study FSGS from a yet unexplored angle. Method We used novel treatment options, a personalized in vitro and in vivo assay as well as Raman spectroscopy and mass spectrometry for recurrent FSGS. Results Here, we report the successful treatment of recurrent FSGS in a patient after living related kidney transplantation by removal of circulating factors with CytoSorb apheresis (Fig. 1). Interestingly, the classical published circulating factors were all in normal range in this patient but early disease recurrence in the transplant kidney and immediate response to CytoSorb apheresis were still suggestive for pathogenic circulating factors. To proof the functional effects of the patient’s serum on podocytes and the glomerular filtration barrier we used a podocyte cell culture model and a proteinuria model in zebrafish to detect pathogenic effects on the podocyte’s actin cytoskeleton inducing a functional phenotype (Fig. 2, Fig. 3). We then performed Raman spectroscopy in the <50 kD serum fraction (Fig. 4), on cultured podocytes treated with the FSGS serum (Fig. 5), and in kidney biopsies of the same patient at the time of transplantation and at the time of disease recurrence (Fig. 6). The analysis revealed metabolomic changes in podocytes induced by the FSGS serum as well as in focal glomerular and parietal epithelial cell regions in the FSGS biopsy. Several disturbed Raman spectra were identified in the fractionated serum and metabolome analysis by mass spectrometry detected lipid profiles in the FSGS serum, which corresponded to disturbances in the Raman spectra. Conclusion Our novel innovative analysis reveals changed lipid metabolome profiles associated with idiopathic FSGS that might reflect a new subtype of the disease.

MO210SERUM LEVELS OF PLASMINOGEN ACTIVATOR UROKINASE RECEPTOR AND CARDIOTROPHIN-LIKE CYTOKINE FACTOR 1IN PATIENTS WITH NEPHROTIC SYNDROME

Abstract Background and Aims The pathogenesis of primary focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) remains unknown to date. Some circulating permeability factors are discussed. This work assessed molecule candidates for permeability in serum samples of patients with nephrotic syndrome (NS). Method Forty-one patients with chronic glomerulonephritis (CGN) were included in our study. Seventeen patients had FSGS, 7 patients had MCD, 5 patients had membranoproliferative glomerulonephritis (MPGN), 6 patients had IgA nephropathy, and 6 patients had membranous nephropathy (MN). The laboratory data were compared with the clinical and histological features of nephritis. Serum levels of uPAR and CLCF-1 were measured by ELISA. Results The serum levels of plasminogen activator urokinase receptor (uPAR) were higher in FSGS patients before treatment than in patients with other morphological forms (MCD, IgA nephropathy, MN and MPGN). The levels of uPAR in serum did not correlate with daily proteinuria, serum creatinine/eGFR, arterial hypertension, the number of sclerosed glomeruli or tubulointerstitial fibrosis. No correlations were found between the levels of cardiotrophin-like cytokine factor 1 (CLCF-1) in serum and creatinine levels/glomerular filtration rate, the percentage of sclerosed glomeruli or the severity of tubulointerstitial fibrosis. There were no significant differences between the histological variants of nephritis. However, we found correlations between CLCF-1 levels and proteinuria (Rs = 0. 397, p = 0.015) and triglycerides levels (Rs = 0. 475, p = 0.003). Conclusion The data indicate an increase in the serum uPAR levels of FSGS before treatment. CLCF-1 levels in serum do not depend on histological forms of CGN, kidney function or immunosuppressive treatment, but they correlate with proteinuria and serum lipids in patients with NS.

Therapeutic trials in adult FSGS: lessons learned and the road forward.

Focal segmental glomerulosclerosis (FSGS) is not a specific disease entity but a lesion that primarily targets the podocyte. In a broad sense, the causes of the lesion can be divided into those triggered by a presumed circulating permeability factor, those that occur secondary to a process that might originate outside the kidneys, those caused by a genetic mutation in a podocyte or glomerular basement membrane protein, and those that arise through an as yet unidentifiable process, seemingly unrelated to a circulating permeability factor. A careful attempt to correctly stratify patients with FSGS based on their clinical presentation and pathological findings on kidney biopsy is essential for sound treatment decisions in individual patients. However, it is also essential for the rational design of therapeutic trials in FSGS. Greater recognition of the pathophysiology underlying podocyte stress and damage in FSGS will increase the likelihood that the cause of an FSGS lesion is properly identified and enable stratification of patients in future interventional trials. Such efforts will facilitate the identification of effective therapeutic agents.

POS-452 SERUM LEVELS OF PLASMINOGEN ACTIVATOR UROKINASE RECEPTOR AND CARDIOTROPHIN-LIKE CYTOKINE FACTOR 1 IN PATIENTS WITH NEPHROTIC SYNDROME

The pathogenesis of primary focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) remains unknown to date. Some circulating permeability factors are discussed. This work assessed molecule candidates for permeability in serum samples of patients with nephrotic syndrome (NS). Forty-one patients with chronic glomerulonephritis (CGN) were included in our study. Seventeen patients had FSGS, 7 patients had MCD, 5 patients had membranoproliferative glomerulonephritis (MPGN), 6 patients had IgA nephropathy, and 6 patients had membranous nephropathy (MN). The laboratory data were compared with the clinical and histological features of nephritis. Serum levels of uPAR and CLCF-1 were measured by ELISA. Levels of uPAR in the serum of patients with CGN The levels of uPAR in serum did not correlate with daily proteinuria (Rs=-0.114, p=0.51), serum creatinine/eGFR (Rs=0.032, p=0.847 and Rs=-0.054, p=0.747, respectively), arterial hypertension (Rs=0.015, p=0.926), glomerulosclerosis percentage (Rs=0.364, p=0.07) or tubulointerstitial fibrosis (Rs=-0.189, p=0.517). There was no correlation with serum C-reactive protein levels (Rs = 0.226, p=0.173) Regarding the morphological forms of glomerulonephritis, the levels of uPAR were not significantly higher in FSGS patients than in MCD, IgA nephropathy, membranous nephropathy or membranoproliferative glomerulonephritis patients (Figure 1). However, the uPAR levels were significantly higher in FSGS patients before immunosuppressive treatment. In FSGS patients who already received immunosuppressive treatment, the uPAR levels were lower and comparable to those of the other groups (Figure 2). CLCF-1 in the serum of patients with CGN No correlations were found between the levels of CLCF-1 in serum and creatinine levels/glomerular filtration rate (Rs=0.285, p=0.079/Rs=-0.268, p=0.099), arterial hypertension (Rs=0.212, p=0.195), percentage of sclerosed glomeruli (Rs=0.28, p=0.127) or severity of tubulointerstitial fibrosis (Rs=0.238, p=0.413). There were no significant differences between the histological variants of nephritis (Figure 1). The levels of CLCF-1 did not depend on the therapy (Figure 2).However, we found correlations between CLCF-1 levels and proteinuria (Rs = 0. 397; p = 0.015) and lipid levels (Rs = 0. 475; p = 0.003). Additionally, correlations between CLCF-1 levels and triglyceride levels (Figure 3), very low-density lipoprotein (Rs = 0.467, p=0.045), and high-density lipoprotein levels (Rs = - 0.599, p=0.031) were found. View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our data indicate a significant increase in the serum suPAR levels in untreated FSGS; immunosuppressive treatment can decrease the suPAR levels. CLCF-1 levels in serum did not depend on histological forms of nephritis or treatment, but it correlated with proteinuria and lipid levels in patients with nephrotic syndrome.

Immunopathogenesis of idiopathic nephrotic syndrome in children: two sides of the coin.

Idiopathic nephrotic syndrome is a common form of glomerular nephropathy in children, with an incidence rate of 1.15-16.9/100,000 depending on different nationalities and ethnicities. The etiological factors and mechanisms of childhood idiopathic nephrotic syndrome have not yet been fully elucidated. This review summarizes the progress of the immunopathogenesis of idiopathic nephrotic syndrome in children. We review the literature on the immunopathogenesis of idiopathic nephrotic syndrome in children. Databases including Medline, Scopus, and Web of Science were searched for studies published in any language with the terms "children", "idiopathic nephrotic syndrome", "immunopathogenesis", "T cells", "circulating permeability factors", and "B cells". Dysfunction in T lymphocytes and pathogenic circulatory factors were indicated to play key roles in the pathogenesis of idiopathic nephrotic syndrome. Recently, some studies have shown that cellular immune dysfunction may also be involved in the pathogenesis of idiopathic nephrotic syndrome. Both T- and B-cell dysfunction may play significant roles in the pathogenesis of idiopathic nephrotic syndrome, like two sides of one coin, but the role of B cell seems more important than T cells.

Novel diagnostic and therapeutic techniques reveal changed metabolic profiles in recurrent focal segmental glomerulosclerosis

Idiopathic forms of Focal Segmental Glomerulosclerosis (FSGS) are caused by circulating permeability factors, which can lead to early recurrence of FSGS and kidney failure after kidney transplantation. In the past three decades, many research endeavors were undertaken to identify these unknown factors. Even though some potential candidates have been recently discussed in the literature, “the” actual factor remains elusive. Therefore, there is an increased demand in FSGS research for the use of novel technologies that allow us to study FSGS from a yet unexplored angle. Here, we report the successful treatment of recurrent FSGS in a patient after living-related kidney transplantation by removal of circulating factors with CytoSorb apheresis. Interestingly, the classical published circulating factors were all in normal range in this patient but early disease recurrence in the transplant kidney and immediate response to CytoSorb apheresis were still suggestive for pathogenic circulating factors. To proof the functional effects of the patient’s serum on podocytes and the glomerular filtration barrier we used a podocyte cell culture model and a proteinuria model in zebrafish to detect pathogenic effects on the podocytes actin cytoskeleton inducing a functional phenotype and podocyte effacement. We then performed Raman spectroscopy in the < 50 kDa serum fraction, on cultured podocytes treated with the FSGS serum and in kidney biopsies of the same patient at the time of transplantation and at the time of disease recurrence. The analysis revealed changes in podocyte metabolome induced by the FSGS serum as well as in focal glomerular and parietal epithelial cell regions in the FSGS biopsy. Several altered Raman spectra were identified in the fractionated serum and metabolome analysis by mass spectrometry detected lipid profiles in the FSGS serum, which were supported by disturbances in the Raman spectra. Our novel innovative analysis reveals changed lipid metabolome profiles associated with idiopathic FSGS that might reflect a new subtype of the disease.

Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis.

Many patients with idiopathic focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation (TX). Although several circulating permeability factors (CPFs) responsible for recurrence have been suggested, there is no consensus. To facilitate CPF identification and predict recurrence after TX, there is a need for robust methods that demonstrate the presence of CPFs. Cultured human podocytes (hPods) and human and mouse glomerular endothelial cells (ciGEnC, mGEnC) were exposed to plasmas of FSGS patients with presumed CPFs, and of (disease) controls. A visual scoring assay and flow cytometry analysis of side scatter were used to measured changes in cellular granularity after exposure to plasma. Nine out of 13 active disease plasmas of 10 FSGS patients with presumed CPFs induced granularity in hPod in a dose- and time-dependent manner. Corresponding remission plasmas induced no or less granularity in hPod. Similar results were obtained with ciGEnC and mGEnC, although induced granularity was less compared with hPod. Notably, foetal calf serum, healthy plasma and a remission plasma partially blocked FSGS plasma-induced hPod granularity. We developed a novel assay in which active disease, presumably CPF-containing, FSGS plasmas induced granularity in cultured hPod. Our results may indicate the presence of CPF inhibitor(s) in healthy and remission plasma. We suggest the presence of a delicate balance between CPF and a CPF inhibitory factor, which is disturbed in patients with active disease. Our novel assays can be applied in future research to identify CPF and CPF inhibitors, and possibly to predict recurrence after TX.

Sera From Patients With Minimal Change Disease Increase Endothelial Permeability to Sodium

Edema is the main symptom of nephrotic syndrome associated to minimal change disease (MCD). Besides the increase in glomerular permeability, possibly induced by a circulating permeability factor,1 an increase in systemic vascular permeability could participate in the constitution of edema.