Focal Segmental Glomerulosclerosis Patients Research Articles

Identification of Genes Associated with Familial Focal Segmental Glomerulosclerosis Through Transcriptomics and In Silico Analysis, Including RPL27, TUBB6, and PFDN5.

Focal segmental glomerulosclerosis (FSGS) is a major cause of nephrotic syndrome and often leads to progressive kidney failure. Its varying clinical presentation suggests potential genetic diversity, requiring further molecular investigation. This study aims to elucidate some of the genetic and molecular mechanisms underlying FSGS. The study focuses on the use of bioinformatic analysis of gene expression data to identify genes associated with familial FSGS. A comprehensive in silico analysis was performed using the GSE99340 data set from Gene Expression Omnibus (GEO) comparing gene expression in glomerular and tubulointerstitial tissues from FSGS patients (n = 10) and Minimal Change Disease (MCD) patients (n = 8). These findings were validated using transcriptomics data obtained using RNA sequencing from FSGS (n = 3) and control samples (n = 3) from the UAE. Further validation was conducted using qRT-PCR on an independent FFPE cohort (FSGS, n = 6; MCD, n = 7) and saliva samples (FSGS, n = 3; Control, n = 7) from the UAE. Three genes (TUBB6, RPL27, and PFDN5) showed significant differential expression (p < 0.01) when comparing FSGS and MCD with healthy controls. These genes are associated with cell junction organization and synaptic pathways of the neuron, supporting the link between FSGS and the neural system. These genes can potentially be useful as diagnostic biomarkers for FSGS and to develop new treatment options.

Profiling of five urinary exosomal miRNAs for the differential diagnosis of patients with diabetic kidney disease and focal segmental glomerulosclerosis.

The objective of this study is to investigate the diagnostic utility of microRNAs (miRNAs) for distinguishing between urine samples from patients with Diabetic Kidney Disease (DKD) and those with Focal Segmental Glomerulosclerosis (FSGS). In this multicentric, cross-sectional investigation, we enrolled patients diagnosed with DKD, individuals with primary biopsy-proven FSGS, and healthy controls. The top 5 miRNAs (hsa-mir-21, hsa-mir-30a, hsa-mir-193a, hsa-mir-196a, hsa-mir-200a) were selected to quantify miRNAs in urine samples. Isolation of targeted miRNAs was performed from urinary exosomes, and the quantitative profile of the isolated miRNAs was measured by RT-qPCR. The ΔΔCt method was implemented to calculate the fold differences between disease and control samples. Thirteen DKD patients, 11 FSGS patients, and 14 healthy controls were included in this study. Hsa-mir-21 and hsa-mir-30a exhibited distinct regulation in both groups, with upregulation observed in FSGS and downregulation in DKD (hsa-mir-21 in DKD (0.668 ± 0.25, p < 0.0005) and FSGS (2.267 ± 1.138, p < 0.0077); hsa-mir-30a in DKD (0.874 ± 0.254, p = 0.079) and FSGS (1.378 ± 0.312, p < 0.0006)). Hsa-mir-193a exhibited significant dysregulation in DKD (1.017 ± 0.413, p < 0.029) but not in FSGS (4.18 ± 1.528, p = 0.058). Hsa-mir-196a and hsa-mir-200a showed upregulation in patient groups (hsa-mir-196a in DKD (1.278 ± 0.527, p = 0.074) and FSGS (2.47 ± 0.911, p < 0.0003); hsa-mir-200a in DKD (1.909 ± 0.825, p = 0.082) and FSGS (1.301 ± 0.358, p < 0.008)). Specific miRNAs, particularly miR-21, miR-30a, miR-196a, and miR-200a, might play a role in the pathogenesis of kidney diseases and could potentially serve as biomarkers to distinguish between FSGS and DKD patients.

Evaluating Predictive Factors for Lymphocele Formation Following Kidney Transplantation.

Lymphocele is a common complication post-kidney transplantation, influenced by various factors including surgical technique, graft vessel count, operator experience, body mass index, ischemia time, and immunotherapy regimens. The purpose of this study was to evaluate lymphocele risk factors, particularly focusing on the role of end-stage kidney disease. A retrospective study was conducted on renal transplant recipients from a single center (March 2020 to December 2022). Patients were categorized into those developing lymphocele and those without during the postoperative period. Data, including sociodemographic, personal history, graft-related variables, intervention, and postoperative outcomes, were collected from electronic medical records. Out of 291 renal transplant recipients, 57 (19.6%) developed postoperative lymphocele, with 15 (5.1%) being symptomatic. Patients with body mass index <24.9 kg/m2 have lower risk of developing lymphocele with an Odds Ratio of 0.538 (P=0.046). Higher lymphocele prevalence was noted in patients with chronic tubulointerstitial nephritis (46.2%; OR 3.815; P=0.024). Focal segmental glomerulosclerosis patients showed no lymphocele (0.0%; OR 0.123; P=0.048). Other factors, including autosomal dominant polycystic kidney disease, did not exhibit significant differences in lymphocele prevalence. The etiology of end-stage kidney disease can serve as a significant predictor of lymphocele development during the postoperative period following renal transplantation. Further larger prospective studies are required to comprehensively assess risk factors and explore end-stage kidney disease potential role in predicting lymphocele formation.

Incidence and Proportion of Primary Focal Segmental Glomerulosclerosis (FSGS) among a Racially and Ethnically Diverse Adult Patient Population between 2010-2021.

Focal segmental glomerulosclerosis (FSGS) refers to a pattern of glomerular injury but also includes primary FSGS which is considered as an immune mediated glomerulopathy. We sought to determine the incidence of primary FSGS and proportion of FSGS patients with primary FSGS among a large diverse patient population in the United States. A cross-sectional study (2010-2021) was performed within an integrated health system in patients (age ≥18yrs) with biopsy-proven FSGS. Among biopsies with FSGS as the first diagnosis on pathology report, chart reviews were performed to determine primary FSGS, defined as podocyte foot process effacement ≥80% on electron microscopy. The proportion of patients with primary FSGS and annual incidence rates (per 100,000 patient-years) were calculated. Standardized incidence rates were determined by age, sex, and race and ethnicity based on US population structure of the five-year (2018-2022) American Community Survey estimates. We identified 3,838 patients with FSGS reported on biopsy. Among 1,502 with FSGS as the principal diagnosis, 637 met criteria for primary FSGS (mean [SD] age 55.5 years [17.9], 56.5% male, 35.6% Hispanic, 28.7% White, 17.9% Asian/Pacific Islander, and 16.0% Black). The mean standardized incidence rate [CI] of primary FSGS was 1.7 [0.9, 2.5] per 100,000 patient-years during the study period. The standardized annual incidence rates ranged from 1.3 - 2.4 per 100,000 patient-years. Incidence rates (per 100,000 patient-years) were highest among Black (3.2), Asian (2.7), and Pacific Islander (2.8) patients. Primary FSGS accounted for 16.6% of biopsy-proven FSGS. Primary FSGS is a likely a rare disease with incidence highest among Black, Asian, and Pacific Islander people. More precise identification of primary FSGS may facilitate work to improve understanding of this glomerulopathy and improve kidney outcomes.

Evaluation of Methodologies in Anti-nephrin Autoantibody Detection.

Recent studies discovered the prominent presence of anti-nephrin autoantibodies in minimal change disease, steroid-sensitive nephrotic syndrome and/or post-transplant recurrent focal segmental glomerulosclerosis (FSGS). However, widely different, and often unconventional autoantibody detection methods were used among these studies, making it challenging to assess the pathogenic role for the antibodies. Here we examined methods of conventional ELISA, magnetic on-beads ELISA, immunoprecipitation-immunoblotting (IP-IB), and cell- and tissue-based antibody assays with 127 plasma samples of kidney and non-kidney diseases. On the antigen side, we compared commercially available recombinant human nephrin extracelluar domain (ECD) produced from human or mouse cell lines, as well as lab-made full length, ECD, and series of ECD truncates for measuring autoantibody reactivity and specificity. Surprisingly, different assay methods and different antigen preparations led to observation of assay-specific false-positive and false-negative results. In general, a set of tests that combines magnetic beads-enhanced ELISA, followed by IP-IB, and epitope mapping showed the most robust results for anti-nephrin autoantibodies, detected in two primary FSGS patients among all cases tested. It is interesting to note that cell/tissue-based results, also supported by antigen truncation studies, clearly suggest steric hindrance of reactive epitopes, as in full length nephrin that forms compact self-associated complexes. In conclusion, anti-nephrin positivity is rare among the tested patients (2/127), including those with FSGS (2/42), and autoantibody results can be affected by the choice of detection methods.

Role of TSP-1 and its receptor ITGB3 in the renal tubulointerstitial injury of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS), a common cause of primary glomerulonephritis, has a poor prognosis and is pathologically featured by tubulointerstitial injury. Thrombospondin-1 (TSP-1) is an extracellular matrix protein that acts in combination with different receptors in the kidney. Here, we analyzed the tubular expression of TSP-1 and its receptor integrin β3 (ITGB3) in FSGS. Previously the renal interstitial chip analysis of FSGS patients with tubular interstitial injury showed that the expressions of TSP-1 and ITGB3 were up-regulated. We found that the level of TSP-1 and ITGB3 increased in the tubular cells of FSGS patients. The serum level of TSP-1 increased and was correlated to the degree of tubulointerstitial lesions in FSGS patients. THBS1/ITGB3 signaling induced renal tubular injury in HK-2 cells exposure to BSA and the ADR-induced nephropathy model. THBS1 knockout ameliorated tubular injury and renal fibrosis in ADR-treated mice. THBS1 knockdown decreased the expression of KIM-1 and caspase 3 in the HK-2 cells treated with BSA, while THBS1 overexpression could induce tubular injury. In vivo, we identified cyclo-RGDfK as an agent to block the binding of TSP-1 to ITGB3. Cyclo-RGDfK treatment could alleviate ADR-induced renal tubular injury and interstitial fibrosis in mice. Moreover, TSP-1 and ITGB3 were colocalized in tubular cells of FSGS patients and ADR-treated mice. Taken together, our data showed that TSP-1/ITGB3 signaling contributed to the development of renal tubulointerstitial injury in FSGS, potentially identifying a new therapeutic target for FSGS.

#366 Humanistic burden of immunoglobulin A nephropathy and focal segmental glomerulosclerosis on patients and care-partners (HONUS): results for Europe

Abstract Background and Aims Rare proteinuric kidney diseases are associated with clinical and economic burden, however, the humanistic burden associated with these conditions has not been directly evaluated. HONUS is a multi-national, cross-sectional survey study designed to evaluate the humanistic burden associated with two rare kidney diseases, immunoglobulin A nephropathy (IgAN) and focal segmental glomerulosclerosis (FSGS), from the patient and caregiver perspectives in the United States and Europe. The current analysis presents preliminary results from adults with IgAN or FSGS and their care-partners in Spain, Germany, France, or the United Kingdom. Method The study recruited adult patients (≥ 18 years old) with IgAN or FSGS and their adult care-partners. Participants completed an online survey including demographic and clinical characteristics, health-related quality of life (HRQoL) (i.e., Kidney Disease Quality of Life Instrument [KDQoL] including 12-Item Short Form Survey [SF-12]), anxiety and depression (i.e., Generalized Anxiety Disorder Assessment [GAD-7], Patient Health Questionnaire [PHQ-9]), and disease impact on work productivity (i.e., Work Productivity and Activity Impairment [WPAI]). Descriptive analyses were conducted. Results From Europe, 26 IgAN patients, 9 FSGS patients, and their care-partners (4 IgAN and 2 FSGS patients did not report having a care-partner) participated in the survey before November 2023. Mean age for IgAN and FSGS patients was 42.2 years and 51.4 years, with 61.5% and 44.4% being female, respectively. Over two-fifths of both IgAN (42.3%) and FSGS patients (44.4%) were in chronic kidney disease (CKD) stage 3, 4 or 5 (on dialysis), while over one-quarter had received a kidney transplant (IgAN: 26.9%; FSGS: 44.4%). Most patients had worse CKD status since diagnosis (IgAN: 61.5%; FSGS: 77.8%). Hypertension and anemia were the most common comorbidities in both groups. In terms of HRQoL, mean (SD) SF-12 physical and mental component scores (PCS, MCS) indicated IgAN patients were more impacted on the mental component (PCS: 47.3 [11.6], MCS: 43.2 [10.9]), with FSGS patients more impacted on the physical component (PCS: 40.6 [6.0], MCS: 43.8 [9.1]), reflecting generally worse HRQoL (lower scores) than previously published European general population scores (50.0 for PCS and MCS). Moderate to severe anxiety was reported by 30.8% of IgAN and 33.3% of FSGS patients, and moderate to severe depression by 30.8% of IgAN and 44.4% of FSGS patients. Among employed IgAN patients (69.2%), mean percent absenteeism (work time missed) was reported at 13.3%, presenteeism (impairment while working) at 17.7%, and overall work productivity loss at 17.7% due to IgAN-related reasons (only 3 FSGS patients [33.3%] were employed; outcomes not shown). Mean percent activity impairment was reported at 27.3% in IgAN and 43.3% in FSGS patients. Most paired care-partners were partners of patients (IgAN: 81.8%, FSGS: 71.4%), with a mean age of 46.6 years (IgAN) and 50.1 years (FSGS). Mean (SD) SF-12 PCS and MCS of IgAN care-partners were 53.3 (8.8) and 47.9 (10.8), respectively (FSGS: 47.0 [10.6], 49.4 [9.5]). Mild to moderate anxiety was reported in 31.8% (IgAN) and 42.9% (FSGS) of care-partners, with one IgAN care-partner (4.6%) reporting severe anxiety. Mild to moderate depression was reported in 27.3% (IgAN) and 71.4% (FSGS) of care-partners, with two IgAN care-partners (9.1%) reporting moderately severe or severe depression. Among employed IgAN care-partners (86.4%), mean percent absenteeism was reported at 14.2%, presenteeism at 9.3%, and overall work productivity loss at 9.9% due to IgAN-related reasons. Mean percent activity impairment was reported at 18.6% in IgAN and 17.1% in FSGS care-partners. Conclusion Patients with IgAN and FSGS in Europe experience impaired HRQoL compared to previously published European population estimates, as well as anxiety, depression, and impaired productivity. Care-partners of these patients also experience impaired HRQoL, in terms of mental components, and reduced overall productivity.

#2305 Mice with a PAX2 missense variant are susceptible to experimental focal segmental glomerulosclerosis (FSGS) and display impaired glomerular repair

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a histopathologic lesion observed in chronic kidney disease characterized by scarring of the glomerulus due to the injury and irreversible loss of highly specialized glomerular epithelial cells called podocytes. Recent studies suggest that podocytes may be regenerated by another population of glomerular epithelial cells called parietal epithelial cells (PECs), although this remains controversial. PAX2 is a transcription factor protein that regulates glomerular development and its expression persists in adult parietal epithelial cells (PECs), postulated to serve as a reservoir for podocytes in the event of injury and loss. Our team previously found that PAX2 pathogenic missense variants account for 4% of adult FSGS. In our present study, our aim is to determine whether PEC-mediated podocyte regeneration is impaired in mice with a pathogenic Pax2 missense variant (Pax2-MV). Method FSGS was induced in wildtype and Pax2-MV mice using Adriamycin. Urine samples were collected from mice every week for 28 days to measure proteinuria. At 28 days after Adriamycin injury, kidneys were collected for histopathology, transmission electron microscopy, and immunofluorescence staining for PAX2 and podocyte marker WT-1. Glomeruli from Adriamycin-injured mice were also isolated through intra-cardiac perfusion with Dynabeads and subjected to mass-spectrometry for proteomics analysis. Proteins with statistically-significant fold changes in expression levels were further analyzed by Gene Ontology to determine biological and molecular processes involved in pathogenesis. Kidney tissue from an FSGS patient with a pathogenic PAX2 missense variant was also examined by histopathology and PAX2 immunohistochemistry. Results Compared to wildtype, Pax2-MV mice were more susceptible to worsened FSGS after Adriamycin, as demonstrated by worsened glomerular scarring, increased podocyte injury and loss, and increased proteinuria, associated with increased morality in Pax2-MV mice (Fig. 1A-B). Wildtype mice showed fewer PAX2-expressing PECs after injury, accompanied by the occurrence of PAX2/WT-1 co-expressing glomerular tuft cells (Fig. 2A-B). These suggest that in Adriamycin-injured wildtype, PAX2-expressing PECs move to the glomerular tuft, where they trans-differentiate to a podocyte fate. In contrast, Pax2-MV mice showed no changes in numbers of PAX2-expressing PECs after injury, associated with fewer PAX2/WT-1 co-expressing glomerular tuft cells compared to injured wildtype (Fig. 2A-B). These suggest that in Adriamycin-injured Pax2-MV mice, PAX2-expressing PECs are unable to move to the glomerular tuft, resulting in decreased numbers of cells undergoing podocyte trans-differentiation. A subset of ectopic PAX2-expressing glomerular tuft cells after injury was increased in Pax2-MV mice (Fig. 2A), suggesting a pathologic process given the worse outcomes observed in these mice. Consistent with findings from injured Pax2-MV mice, we found that in an FSGS patient with a pathogenic PAX2 missense variant, PAX2-expressing glomerular tuft cells overlap with areas of glomerular scarring, while in a normal human kidney biopsy, PAX2 expression is only observed in PECs but not within glomerular tuft cells (Fig. 2A). Lastly, proteomics Gene Ontology analysis of isolated glomeruli demonstrated biological and molecular processes indicative of maladaptive glomerular repair in injured Pax2-MV mice, including decreased regulation of cell shape, decreased cell-cell junction assembly and decreased actin and cadherin binding compared to wildtype (Fig. 2C). Conclusion Altogether, our findings suggest that PAX2-expressing PECs migrate to the glomerular tuft to assist in podocyte regeneration in wildtype. However, this mechanism is impaired in Pax2-MV mice, leading to worsened podocyte loss and injury and further exacerbation of glomerular disease (Fig. 2C). Our findings provide a further understanding of normal and impaired regeneration mechanisms in FSGS, and our identified pathogenic mechanisms can be further harnessed for future therapeutic development.

#735 The clinicopathologic prognostic factors to estimate ESRD in patients with FSGS from national wide cohort of renal biopsy

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is one of the leading causes of glomerulonephritis in Korea. FSGS can be caused by heterogenous aetiologies with subdivided by primary, secondary, and genetical forms. The proposed clinicopathologic classification is dependent on amount of proteinuria, laboratory findings of possible secondary causes, and genetic profiles, however, that may not provide definite subtypes of FSGS. Therefore, it is important to search for prognostic markers of any form of FSGS. Method We enrolled a total of 20,404 adult patients who underwent native renal biopsy from 1979 to 2018 in 18 hospitals in Korea. Clinical, demographic, and laboratory data were collected in conjunction with pathologic findings. Among diagnosis of FSGS by pathologists of each hospital, we selected patients with definite segmental sclerosis by light microscopic examination including ≥10 glomeruli and without electron dense deposits by electron microscopic examination. We excluded patients with other forms of pathologic diagnosis except FSGS, with diabetes mellitus, or with follow-up period &amp;lt; 6 months after renal biopsy. We evaluated prognostic factors to estimate the risk of ESRD which event was found from ESRD registry of the Korean Society of Nephrology or the data from each hospital. Results Among 511 selected FSGS patients, 314 (61.4%) men were included. Mean age was 45.0 ± 17.9 years at biopsy. The laboratory finding of serum creatinine, estimated GFR, and urine protein to creatinine ratio (UPCR) was 1.38 ± 0.85 mg/dl, 71.8 ± 36.6 ml/min/1.73 m2, and 4.32 ±4.84 g/g creatinine, respectively (there were no missing data). GFR &amp;lt;60 ml/min/1.73 m2 was found in 41.5 % of patients and UPCR ≥ 3.0 g/g creatinine in 41.1 % of patients. During 117.5 ± 100.1 months of follow-up period, there were 97 (19.0%) patients with incident ESRD. Clinical prognostic factors to incident ESRD were estimated GFR, diastolic blood pressure, and year of renal biopsy. Pathologic prognostic factors were glomerular size, amount of global sclerosis and segmental sclerosis, severity of increase in glomerular matrix, interstitial fibrosis, interstitial inflammation, and tubular atrophy, and presence of artherosclerosis of intrarenal vessels. Adjusted with clinicopathologic factors related to incident ESRD, Cox's proportional hazard models revealed only pathologic findings of amount of glomerular global sclerosis and segmental sclerosis and presence of tubular atrophy were the independent prognostic factors (Model with 3.5 % of missing cases and -2 log likelihood ratio is 956.9, p &amp;lt; 0.001). GFR and UPCR showed significant relationship with incidence of ESRD, however, the significance was gone in the model adjusted with pathologic findings. AUC by ROC to estimate the event of ESRD by percentage of global sclerosis (GS) was 0.601 (range; 0.538-0.663, p = 0.002) and, by percentage of segmental sclerosis (SS), 0.679 (range; 0.616-0.743, p &amp;lt; 0.001). We grouped patients with three pathology categorical variables of a criterion of GS 30.9%, SS 10.5 %, and moderate degree in severity of tubular atrophy (TA), resulted in 8 groups, and summarized into 4 groups according to the relative risk to develop ESRD (Fig. 1). UPCR was a significant risk factor to estimate ESRD only in Group 4 with the criterion of 2 g/g creatinine by the Kaplan-Meier test (p = 0.034). GFR was not a prognostic factor in any pathology subgroup. Conclusion Important prognostic factors to estimate incident ESRD were pathologic findings of global sclerosis, segmental sclerosis, and tubular atrophy in FSGS patients. Significance of UPCR and GFR as prognostic factors was varied according to pathologic findings. We need to reconsider the criterion of UPCR to initiate immunosuppressive treatment and to break down the effect of immunosuppressive treatments for FSGS according to pathologic findings at renal biopsy.

#1389 Identification of serum lipidomic profile in nephrotic syndrome patients

Abstract Background and Aims Nephrotic syndrome describes a collection of symptoms, mainly heavy proteinuria leading to kidney function loss, that can be caused by different underlying kidney diseases. Common primary causes of nephrotic syndrome include focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD) and membranous nephropathy (MN). Nephrotic syndrome is often characterized by plasma lipid abnormalities with elevations in cholesterol, triglycerides and lipoproteins. Here we set out to generate a detailed lipidomic analysis of FSGS, MCD and MN patient's plasma to find lipid changes common to nephrotic syndrome or specific for the underlying disease. Method For this lipidomic study, we included patients with primary FSGS (2 cohorts with n = 5), genetic FSGS (n = 5), MCD (n = 5) and MN (n = 5) in whom serum had been collected at the time they presented at our center with active nephrotic syndrome. We compared the serum lipidome of nephrotic syndrome patients with age matched healthy controls (n = 5), and with age matched patients with familial hypercholesterolemia (n = 5) who, like patients with nephrotic syndrome, have significantly elevated lipid values. Serum was analyzed using the Shotgun Lipidomics platform by Lipotype GmbH (Dresden, Germany), as previously described [1]. Results The lipidomic profile of patients with nephrotic syndrome was markedly different compared to control groups (Fig. 1A). In particular, lower levels of lyso-phophatidylcholine (LPC) and higher concentrations of phosphatidylethanolamine (PE) and lyso-PE (LPE) compared to control groups were most prominent (Fig. 1B). Subspecies analysis identified LPC 16:0;0 to be highly downregulated in patients with nephrotic syndrome (Fig. 2A), whereas LPE 18:0;0 and nearly all PE subspecies were increased compared to control groups (Fig. 2B and C). The lipidomic profile was largely comparable between different underlying diseases and we could not identify lipid classes specifically different for certain diseases. Conclusion Our lipidomic data show that patients with nephrotic syndrome have a distinct lipid profile compared to healthy controls or other disease controls. We identified LPCs, PEs and LPEs to be changed in nephrotic syndrome. We could not identify lipids specific for certain diseases underlying nephrotic syndrome, but rather identified a general nephrotic syndrome lipid profile. This suggests that lipid changes are rather a consequence of disease manifestation in these patients with various underlying causes. Further research should focus on confirming this lipid profile in larger cohorts and unraveling the mechanisms leading to the observed lipid changes.

#1004 Single-nucleus RNA sequencing identifies upregulated antigen presentation, apoptosis, mTORC1 signaling and EMT in primary FSGS podocytes

Abstract Background and Aims Primary focal segmental glomerulosclerosis (FSGS) is associated with irreversible podocyte damage, but the underlying cause and pathophysiology are incompletely understood. Furthermore, diagnostic markers that allow reliable differentiation between primary and secondary FSGS subtypes are lacking. We applied single-nucleus RNA-sequencing (snRNA-seq) on podocytes of patients with primary or secondary (maladaptive) FSGS, to identify subtype-specific differentially expressed genes and pathways. Method We used droplet-based snRNA-seq (10X Genomics Chromium) on cryopreserved kidney biopsy cores from human patients with newly diagnosed primary FSGS (n = 9, all nephrotic), maladaptive FSGS (n = 9, not nephrotic), proteinuric controls (PLA2R-positive membranous nephropathy (PLA2R+ MN), n = 3), and healthy controls (pre-perfusion biopsies, n = 4). Differential gene expression (DGE) analysis was done with a pseudobulk method provided by the EdgeR R-package to reduce bias and false discoveries. The fGSEA R-package was used for subsequent gene set enrichment analysis. Results We identified a total of 120 751 high-quality nuclei, of which 2 471 were podocytes (Fig. 1A). DGE analysis identified the top 20 differentially expressed genes (DEGs) per diagnosis (Fig. 1B). Remarkably, primary FSGS podocytes showed more transcriptional similarity to PLA2R+ MN podocytes, compared to maladaptive FSGS podocytes. This underscores the heterogeneity of patients with FSGS and shows that heavy proteinuria, regardless of the underlying diagnosis, may be associated with a distinct transcriptional profile in podocytes. Upregulated genes in primary FSGS podocytes included TIMP1, BTG2, ZNF44, EHD4, ZNF250, ECM1, SEMA4G, PHLDB1, and HDAC5. Upregulation of the matrix metalloproteinase inhibitor TIMP1 may possibly contribute to the development of glomerulosclerosis. Upregulation of BTG2 has previously been found to be pathogenic in FSGS by promoting epithelial-mesenchymal transition (EMT)-induced podocyte injury [1]. Next, gene set enrichment analysis on the identified DEGs in primary FSGS podocytes showed upregulation of pathways involved in antigen presentation (driven by upregulation of HLA-A, HLA-B, HLA-C, HLA-E and B2M), mTORC1 signaling, apoptosis and EMT (Fig. 1C). In maladaptive FSGS, upregulated pathways were mainly involved in extracellular matrix interaction. This may correlate with the significantly higher degree of chronic damage on kidney biopsy in the maladaptive FSGS patients (median Mayo Clinic Chronicity Score of 6.0, IQR [4.0-7.0]) vs. primary FSGS (2.0, [1.0-4.0]) and PLA2R+ MN (1, IQR [1.0-1.5]) (Kruskal-Wallis test, P = 0.007). Conclusion Our results corroborate previous studies that implicate apoptosis, mTORC1 signaling, EMT and auto-immunity in primary FSGS. Our future work will focus on cell-cell interactions within the glomerular niche and validation studies of candidate podocyte-specific genes.

#1442 Retrospective evaluation of etiology, clinical and laboratory findings in patients diagnosed with secondary and genetic FSGS

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a glomerulonephritis characterized by segmental sclerosis in at least one of the glomeruli and may develop due to primary, secondary, genetic etiology. We compared clinical, histopathological, and laboratory findings, treatment options, and response to treatment in patients with secondary and genetic FSGS. Method This study analyzed 172 patients with secondary and genetic FSGS at our Glomerular Diseases Clinic (1987-2022). Criteria included at least 16 weeks of follow-up, absence of other primary glomerular diseases, and age over 18. Retrospective data were collected on demographics, diagnosis, renal biopsies, genetic tests, imaging, treatments, treatment responses, end-stage renal disease (ESRD) development, and transplantation. The focus was on predicting genetic FSGS and ESRD. Results 103 of our patients were female (59.8%), and the mean age at diagnosis was 38.46 ± 13.75 years. Of these patients, 29 were diagnosed with genetic FSGS and 141 with non-genetic secondary FSGS. Forty-one patients with non-genetic secondary FSGS were obese, and 21 had a single kidney. The patients had a median follow-up of 78 (38-134) months. 28 patients developed ESRD during follow-up, 16 of whom underwent renal transplantation. Familial renal disease (p &amp;lt; 0.001), familial ESRD (p &amp;lt; 0.001), and hematuria at diagnosis (p = 0.005) were more common in patients with genetic FSGS. Comparison of genetic FSGS and non-genetic secondary FSGS patients shown in Figure 1. The number of glomeruli with global sclerosis (p = 0.006) and C3 accumulation (p = 0.048) on renal biopsy was more frequent in patients with ESRD. Comparison of ESRD and non-ESRD patients are shown in Figure 2. Conclusion Differentiating genetic and non-genetic secondary FSGS is vital in clinical practice. For those with a family history of renal disease, hematuria, or unresponsive to immunosuppressive therapy, consider genetic FSGS and request genetic testing. Patients with secondary FSGS, diagnosed with high creatinine, a substantial presence of sclerosing glomeruli on biopsy, and C3 accumulation, should be closely monitored for ESRD.

#1166 ACTION3 pivotal Phase 3 &amp; open-label extension study assessing DMX-200 in adult &amp; paediatric patients with focal segmental glomerulosclerosis (FSGS)

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a major cause of glomerular disease that affects both adults and children. FSGS can rapidly progress to end-stage kidney disease. Lack of safe and effective treatment represents a significant unmet need for patients with FSGS. Development of treatments has been hampered by complexities including the rare nature of the disease, varying drivers of pathogenesis, and the evolving diagnostic and clinical trial designs, necessitating innovative research approaches. DMX-200 (repagermanium) is a C-C motif chemokine receptor 2 (CCR2) pathway inhibitor. Activation of the angiotensin II type 1 receptor (AT1R) and CCR2 have been independently implicated in the pathogenesis and progression of FSGS. These receptors form a functional complex so inhibition of both receptors may provide an efficient approach to FSGS treatment. In vivo data shows co-administration of DMX-200 and the angiotensin receptor blocker (ARB) irbesartan demonstrated a synergistic reduction in proteinuria, macrophage infiltration, and podocyte loss compared with vehicle or either antagonist alone. A subsequent Phase 2a cross-over study (NCT03649152) in N = 8 patients with biopsy-proven primary FSGS showed that the combination of DMX-200 and irbesartan (compared to placebo) was well tolerated with most patients experiencing a reduction in proteinuria. Based on these data, the global Phase 3 study (ACTION3) was initiated to confirm the proteinuria lowering effects of DMX-200 and assess effects on eGFR decline. ACTION3 is a randomised, placebo-controlled, double-blind, long-duration study of FSGS patients and on completion will provide a resource for the wider nephrology community and advocacy groups on the natural history of FSGS patients and the effects of a CCR2-inhibitor on the background of an ARB. Here we present the baseline data from the first 90 patients enrolled in ACTION3 at 12th January 2024. Method The ACTION3 (NCT05183646) study is a pivotal Phase 3, multicentre study of the efficacy and safety of DMX-200, 120 mg twice daily compared with placebo in adult patients with biopsy-proven FSGS receiving maximal tolerated ARB, with persistent proteinuria &amp;gt;1.5g/ day and eGFR &amp;gt;25 ml/min/1.73 m2. The study plans to enroll approximately 286 patients with primary FSGS, genetic FSGS and FSGS of undetermined cause with the primary objective of evaluating the efficacy of DMX-200 in percent change in 24-hour urine PCR and eGFR slope. The initial 104-week randomized period will be extended in a 2-year open label extension study. A futility analysis by an Independent Data Monitoring Committee following the first 72 patients completing 35 weeks of treatment confirm that continuation of the study is non-futile. Results The study is actively recruiting at 71 sites in 11 countries listed in Table 1. Demographic data of randomised patients is listed in Table 2. Patients are predominantly male (65%), with an average age of 46.6 yrs. and mean eGFR 56.3 ± 27.6 (ml/min/1.73 m2). The study is expanding recruitment into additional countries in the Asia Pacific, Europe, and Latin American regions. Further, the study protocol has sought and received regulatory approval to enroll adolescent patients aged 12-18 in Argentina, Mexico, UK and the US. Conclusion DMX-200 (repagermanium) has shown encouraging signs of safety and efficacy in combination with an ARB for the treatment of FSGS. The contributing countries and baseline characteristics of the patients enrolled in ACTION3 are similar to the populations studied in other recent FSGS trials. No clinically significant safety concerns have been observed in patients enrolled to date in the ACTION3 Phase 3 study.

Identification of Plasma hsa_circ_0001230 and hsa_circ_0023879 as Potential Novel Biomarkers for Focal Segmental Glomerulosclerosis and circRNA-miRNA-mRNA Network Analysis

Introduction: Focal segmental glomerulosclerosis (FSGS) is a common glomerulopathy with an unclear mechanism. The demand for FSGS clinical diagnostic biomarkers has not yet been met. Circular RNA (circRNA) is a novel non-coding RNA with multiple functions, but its diagnostic value for FSGS remains unexplored. This study aimed to identify circRNAs that could aid in early clinical diagnosis and to investigate their mechanisms in podocyte injury. Methods: The signature of plasma circRNAs for FSGS was identified by circRNA microarray. The existence of circRNAs was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR), RNase R assay, and DNA sequencing. Plasma levels of circRNAs were evaluated by qRT-PCR. The diagnostic value was appraised by the receiver operating characteristic curve. The circRNA-miRNA-mRNA network was built with Cytoscape 7.3.2. Statistically significant differences were calculated by the Mann-Whitney U test. Results: A total of 493 circRNAs (165 upregulated, 328 downregulated) were differentially expressed in the plasma of FSGS patients (n = 3) and normal controls (n = 3). Eight candidate circRNAs were demonstrated to be circular and stable transcripts. Among them, hsa_circ_0001230 and hsa_circ_0023879 were significantly upregulated in FSGS patients (n = 29) compared to normal controls (n = 51). The areas under the curve value of hsa_circ_0001230 and hsa_circ_0023879 were 0.668 and 0.753, respectively, while that of the two-circRNA panel was 0.763. The RNA pull-down analysis revealed that hsa_circ_0001230 and hsa_circ_0023879 could sponge hsa-miR-106a. Additionally, hsa_circ_0001230 and hsa_circ_0023879 positively regulated hsa-miR-106a target genes phosphatase and tensin homolog (PTEN) and Bcl-2-like protein 11 (BCL2L11) in podocytes. Conclusion: hsa_circ_0001230 and hsa_circ_0023879 are novel blood biomarkers for FSGS. They may regulate podocyte apoptosis by competitively binding to hsa-miR-106a.

A new index for the outcome of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a common pathological form of nephrotic syndrome. This study analyzed the value of pathological lesions and clinical prognosis of different segmental glomerulosclerosis ratios in FSGS. Two hundred and six FSGS patients were collected from Dec 2013 to Apr 2016. The patients were divided into two groups according to the proportion of glomerular segmental sclerosis: F1 (SSR ≤ 15%, n = 133) and F2 (SSR > 15%, n = 73). The clinical and pathological data were recorded and analyzed, and statistical differences were observed between the serum uric acid level and the percentage of chronic renal failure. The pathological results showed significant differences in interstitial fibrosis and tubular atrophy (IFTA), degree of mesangial hyperplasia, vascular lesions, synaptopodin intensity, and foot process effacement between the two groups. Multivariate logistic regression analysis showed significant differences in creatinine (OR: 1.008) and F2 group (OR: 1.19). In all patients, the prognoses of urine protein and serum creatinine levels were statistically different. Multivariate Cox regression analysis revealed that F2 (hazard ratio: 2.306, 95% CI 1.022–5.207) was associated with a risk of ESRD (end stage renal disease). The proportion of segmental glomerulosclerosis provides a guiding value in the pathological diagnosis and clinical prognosis of FSGS.

Adult primary glomerular diseases due to podocytopathies: a single center experience on patient characteristics, treatment and outcomes

Purpose: This study aims to evaluate the demographic, clinical, and pathologic characteristics and response to immunosuppressive therapy, particularly corticosteroids, in adult patients with primary focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD), which are classified as podocytopathies. &#x0D; Materials and Methods: Between January 1998 and December 2014, this study included 44 patients (27 with primary FSGS and 17 with MCD) aged older than 18 years with a histopathologic diagnosis, symptoms of nephrotic syndrome, and a minimum follow-up of six months. Patients were divided into two groups according to the treatment they received and three groups according to their response to treatment. Patients diagnosed with primary FSGS and MCD were evaluated based on clinical, demographic, and laboratory findings, as well as response to treatment, and a comparison was conducted between the two groups.&#x0D; Results: 59.1% of the patients were male with a mean age of 44.8±17.7 years. At the time of diagnosis, there were no statistically significant differences in clinical and demographic characteristics between MCD and primary FSGS patients. However, in patients with MCD, the mean creatinine clearance (118.0±46.7 ml/min) was higher and the rate of microscopic hematuria (11.8%) was lower at the time of diagnosis. There was an increased need for alternative immunosuppressive treatments besides corticosteroids in patients with primary FSGS to achieve partial or complete remission. At both the third and sixth-month follow-ups, MCD patients achieved a higher rate of complete remission (proteinuria

Urinary C4d and progression of kidney disease in IgA vasculitis.

Immunoglobulin A (IgA) vasculitis nephritis (IgAVN) is the most common secondary IgA nephropathy (IgAN). Urinary C4d have been identified associated with the development and progression in primary IgAN; however, its role in kidney disease progression of IgAVN is still unclear. This study enrolled 139 patients with IgAVN, 18 healthy subjects, 23 focal segmental glomerulosclerosis patients and 38 IgAN patients. Urinary C4d levels at kidney biopsy were measured using enzyme-linked immunosorbent assay. The association between urinary C4d/creatinine and kidney disease progression event, defined as 40% estimated glomerular filtration rate decline or end-stage kidney disease, was assessed using Cox proportional hazards models and restricted cubic splines. The levels of urinary C4d/creatinine (Cr) in IgAVN and IgAN patients were higher than in healthy controls. Higher levels of urinary C4d/Cr were associated with higher proteinuria and severe Oxford C lesions, and glomerular C4d deposition. After a median follow-up of 52.79 months, 18 (12.95%) participants reached composite kidney disease progression event. The risk of kidney disease progression event was higher with higher levels of Ln(urinary C4d/Cr). After adjustment for clinical data, higher levels of urinary C4d/Cr were associated with kidney disease progression in IgAVN [per Ln-transformed urinary C4d/Cr, hazard ratio 1.573, 95% confidence interval (CI) 1.101-2.245; P=.013]. Compared with the lower C4d/Cr group, the hazard ratio was 5.539 (95% CI 1.135-27.035; P=.034) for the higher levels group. Higher levels of urinary C4d/Cr were associated with kidney disease progression event in patients with IgAVN.

Five-Year study on renal outcomes in biopsy-proven focal segmental glomerulosclerosis patients in Shiraz, Iran.

Focal segmental glomerulosclerosis (FSGS) is a prevalent glomerular disease that often leads to nephrotic syndrome. It is characterized by consolidating a portion of the glomerular capillary tuft connected to Bowman's capsule. This retrospective cohort study aimed to determine the demographic characteristics, risk factors, and prognostic indicators associated with FSGS in Shiraz, Iran. The study included 53 primary FSGS patients aged over 18 years who were referred to clinics affiliated with Shiraz University of Medical Sciences. Data were collected through a comprehensive data-gathering sheet encompassing demographic information, medical history, laboratory test results, and histopathological findings. Statistical analysis was performed using SPSS 18, considering a significance level of p<0.05. A five-year follow-up was conducted on the 53 patients, with the mean age of 41.0±13.3 years. The most common FSGS variants observed were "not otherwise specified" (NOS, 13.2%) and tip variant (7.5%). Older patients exhibited higher disease activity, whereas remission rates were higher among younger individuals (P=0.012). Patients achieving remission had lower creatinine and Pro/Cr ratios and higher glomerular filtration rates (p<0.05). Treatment involving a combination of corticosteroids and mycophenolate mofetil showed a significant correlation with remission (P=0.036). Older patients with higher creatinine levels, higher Pro/Cr ratios, and lower glomerular filtration rates at disease onset may require more aggressive treatment. Combination therapy with mycophenolate mofetil and corticosteroids yields better outcomes, leading to increased remission rates. These findings provide valuable insights for managing FSGS patients.

Clinicopathological Outcomes in Focal Segmental Glomerulosclerosis: A Retrospective Cohort Study

Introduction: Focal Segmental Glomerulosclerosis (FSGS) is a nephrotic syndrome with a variety of clinicopathological presentations and varied responses to treatment. Hence, this study attempts to classify FSGS based on clinical presentation and pathological findings on kidney biopsy, which is essential for appropriate treatment and avoidance of inappropriate use of immunosuppressants. Aim: To analyse clinicopathological findings and responses to immunosuppressants in FSGS. Materials and Methods: A retrospective cohort study was conducted at Department of Nephrology, Vydehi Institute of Medical Sciences and Research Centre Bengaluru, Karnataka, India, to analyse clinicopathological parameters such as urine analysis, 24-hour urine protein, serum creatinine, serum albumin, lipid profile, renal biopsy details, and response to treatment in 97 patients. The study was planned, analysed, and executed between January 2023 and February 2023. All variables were expressed as mean±standard deviation or percentage. Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) software version 16.0. Results: Among a total of 97 patients, 64% were males. Sudden onset oedema was observed in 90% of the cases, while nephrotic proteinuria was seen in 71%. The Not Otherwise Specified (NOS) variant was noted in 60% of the cases. Complete remission was observed in 61%, suggesting a possible primary FSGS. Persistent nephrotic proteinuria with a poor response to therapy was noted in 32%, indicating a possible secondary/genetic FSGS, despite adequate immunosuppressive therapy. Therefore, differentiating between primary and secondary forms of FSGS has therapeutic and prognostic implications. Accurate diagnosis of each form of FSGS is vital to avoid unnecessary immunosuppressive-based therapy and establish appropriate treatment. Conclusion: Resistance to steroid therapy was observed in one-third of FSGS patients. It is likely that unrecognised genetic FSGS or secondary FSGS were included among the study group of primary FSGS, leading to misinterpretation of treatment responses in primary FSGS. Hence, a clinicopathological approach for correctly differentiating between primary FSGS, secondary (maladaptive, viral, or toxic) FSGS, and genetic FSGS helps in making correct treatment decisions.

The most common founder pathogenic variant c.868G > A (p.Val290Met) in the NPHS2 gene in a representative adult Czech cohort with focal segmental glomerulosclerosis is associated with a milder disease and its underdiagnosis in childhood.

Genetic focal segmental glomerulosclerosis (FSGS) is caused by pathogenic variants in a broad spectrum of genes that have a variable representation based on subjects' ethnicity and/or age. The most frequently mutated autosomal recessive gene in FSGS is NPHS2. In this study, we analyzed the spectrum of NPHS2 variants and their associated phenotype in Czech adult FSGS patients. A representative cohort of 234 adult patients with FSGS, derived from 225 families originating from all regions of Czechia, was analyzed by massively parallel sequencing. In this study, we focused on the comprehensive analysis of the NPHS2 gene. The histological classification of FSGS followed the Columbia classification. We detected seven (3%) cases bearing homozygous or compound heterozygous pathogenic NPHS2 variants. A single pathogenic variant c.868G > A (p.Val290Met) was found in the majority of NPHS2-positive cases (86%; 6 out of 7) in histologically confirmed instances of FSGS. Its allele frequency among unrelated NPHS2-associated FSGS patients was 50% (6/12), and Haplotype analysis predicted its origin to be a result of a founder effect. There is an identical V290M-related haplotype on all V290M alleles spanning a 0,7 Mb region flanking NPHS2 in Central European FSGS populations. The phenotype of the p.Val290Met NPHS2-associated FSGS demonstrated a later onset and a much milder course of the disease compared to other NPHS2 pathogenic variants associated with FSGS. The mean age of the FSGS diagnosis based on kidney biopsy evaluation was 31.2 ± 7.46 years. In 50% of all cases, the initial disease manifestation of proteinuria occurred only in adulthood, with 83% of these cases not presenting with edemas. One-third (33%) of the studied subjects progressed to ESRD (2 out of 6) at the mean age of 35.0 ± 2.82 years. We identified the most prevalent pathogenic variant, p.Val290Met, in the NPHS2 gene among Czech adult FSGS patients, which has arisen due to a founder effect in Central Europe. The documented milder course of the disease associated with this variant leads to the underdiagnosis in childhood. We established the histopathological features of the NPHS2-associated adult FSGS cases based on the Columbia classification. This might improve patient stratification and optimize their treatment.