Effects of steroid-resistant nephrotic syndrome serum on AA pathway in podocytes cultured in 3D in vitro glomerular model

Effects of steroid-resistant nephrotic syndrome serum on AA pathway in podocytes cultured in 3D in vitro glomerular model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of steroid-resistant nephrotic syndrome serum on AA pathway in podocytes cultured in 3D in vitro glomerular model Gaia Bianchi, Linda Bellucci, William Morello, Stefano Turolo, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4684821/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Background and hypothesis. Steroid-resistant nephrotic syndrome (SRNS) is the most severe form of nephrotic syndrome, with genetic or unidentified immunological origins and rapidly progressing to the need for kidney replacement therapy. Lipotoxicity can affect podocytes inducing kidney damage. In this study, we evaluate the effects of SRNS serum on podocyte functionality and lipid metabolism. Methods. A three-dimensional (3D) dynamic in vitro glomerulus was incubated with serum from multi-drug resistant (MDR) and genetic SRNS or healthy controls. The glomerular filtration barrier (GFB) integrity, podocyte viability, and fatty acids (FAs) composition were evaluated by serum albumin permeability estimation, cytofluorimetric analysis and gas chromatography, respectively. Expression of slit diaphragm molecules and FA-related enzymes was analyzed by immunofluorescence and PCR. Results. Serum from SRNS patients induced cell granularity, increased GFB permeability, and disrupted slit diaphragm protein structure. The podocyte damage was most severe when MDR serum was administered compared to the serum of genetic-SRNS. This was associated with a significant upregulation of the transcripts coding for nephrin, synaptopodin, and CD2AP. An alteration of fatty acid profile in MDR-treated podocytes was observed, with increased monounsaturated FAs following the decrease of saturated FAs. The exposure of cultured podocytes to MDR- and genetic-SRNS serum induced disruption of arachidonic acid (AA) synthesis pathway, with different intermediate players involved. Conclusion. This study highlights the detrimental effects of serum from SRNS patients on podocyte function and the association of AA synthesis pathway with the podocyte damage. Biological sciences/Biological techniques Biological sciences/Cell biology Health sciences/Diseases Health sciences/Nephrology steroid-resistant idiopathic nephrotic syndrome (SRNS) 3D glomerular model arachidonic acid (AA) slit diaphragm proteins Figures Figure 1 Figure 2 Figure 3 Key points We developed an in vitro glomerular millifluidic model that can capture the podocyte damage induced in SRNS patients. We showed the detrimental effects of serum from SRNS patients on podocyte function and the association of AA synthesis pathway with the podocyte damage in SRNS. The direct targeting of AA pathway could be instrumental in reducing the kidney damage progression and proteinuria in MDR patients. Introduction Idiopathic nephrotic syndrome (INS) is characterized by the presence of proteinuria, edema, and hyperlipemia 1 . In childhood-onset INS, 10–15% of patients are steroid-resistant (SRNS), as they do not achieve remission with the initial treatment with steroids. Around 50% of them, unresponsive to second-line therapies, are classified as multi-drug resistant SRNS (MDR), the most severe form of the disease. Only in 1/3 of patients, a pathogenic variant of genes coding for structural proteins of the glomerular filtration barrier (GFB) has been identified (monogenic SRNS), while in the remaining cases, a yet unidentified immunological cause is thought to be involved 2 , 3 . Both MDR and genetic SRNS are associated with poor kidney survival 4 INS is characterized by the leakage of proteins as well as lipids into the urine and a concomitant increase of blood-free fatty acids 5 . Leaked lipids mainly accumulate in the proximal tubules, inducing tubular damage and fibrosis 6 , 7 . Excessive lipid uptake in podocytes can activate an oxidative stress response, inducing cell loss, which results in glomerular damage and proteinuria 8 , 9 . Although lipid metabolism in the kidney seems to be a crucial issue, the link between podocyte lipotoxicity and INS is missing. Among fatty acids (FAs), arachidonic acid (AA) directly regulates podocyte integrity, by promoting slit diaphragm and cytoskeleton changes and cell apoptosis 10 – 12 . AA has also been described to induce TGFβ-induced fibrosis in mesangial cells by activating inflammatory and pro-fibrotic genes, which are crucial in the progression of chronic kidney disease 13 . We recently reported the presence of elevated levels of AA in the blood of INS children at different clinical INS stages, supporting its role as a sensor of a persistent inflammatory milieu 14 . In this study, we used a three-dimensional (3D) dynamic glomerular model to investigate the lipotoxic alterations in human podocytes treated with serum from different forms of SRNS. The direct effects on podocyte cell viability, cytoskeleton organization, and slit diaphragm structure, were evaluated. The FA composition of podocytes cultured in the nephrotic syndrome-induced kidney model was also assessed. Materials and Methods Patients and sample collection This study was conducted according to the principles expressed in the Declaration of Helsinki. Approve to the study was obtained by the IRCCS Ca’ Granda Institutional Review Board (ID 2633, INSiDe protocol). An informed consensus was obtained for all the participants enrolled in the study. Multi-drug resistant SRNS (MDR-SRNS, N = 5) and genetic SRNS (genetic-SRNS, N = 5) patients were recruited during the active phase of the pathology in the Paediatric Nephrology, Dialysis and Renal Transplant Unit (Fondazione Ca’ Granda IRCCS Ospedale Maggiore Policlinico, Milan). MDR-SRNS were defined as INS patients not achieving partial remission after at least 2 different lines of therapy other than steroids; genetic SRNS forms were defined as patients non-responsive to immunosuppressive treatments and harboring a pathogenetic mutation in genes coding for components of GFB. The list of mutations was reported in Supplementary Table 1. Clinical data of SRNS patients were reported in Table 1 . Furthermore, a group of healthy volunteers (N = 7) were also recruited as control group. The exclusion criteria for this group included fever, inherited, or acquired immune disorders, and the use of immunosuppressants. Serum samples were obtained by centrifuging whole blood at 3000g for 20 minutes (min). Before each experiment, sera were diluted to 2% or 10% with EndoGRO LS basal Culture Media (Merck Millipore, Burlington, Massachusetts, USA). Table 1 Clinical characteristics of the INS cohort. Multi-drug resistant (MDR) (N = 5) and genetic-SRNS (N = 5) patients were recruited in our study. Gender and ethnicity are presented as absolute numbers and relative frequency. Age of appearance and sampling are reported as mean ± standard deviation (SD). Proteinuria levels (uPr/uCr) were represented as the minimum (min)- maximum (max) range. MDR (N = 5) genetic-SRNS (N = 5) Gender Female 3.0 (60.0%) 4.0 (80.0%) Male 2.0 (40.0%) 1.0 (20.0%) Ethnicity Caucasian 5.0 (100.0%) 4.0 (80.0%) North-African 0.0 (0.0%) 1.0 (20.0%) Age of appearance Mean (SD) 16 (15.1) 5.6 (8.2) min-max 3.0–42.0 0.0–18.0 Age at sampling Mean (SD) 21.8 (18.1) 7 (8.2) min-max 4.0–52.0 0.0–19.0 uPr/uCr Mean (SD) 5.6 (3.0) 11.0 (9.3) min-max 2.0–9.0 1.4–25.9 Cell lines A podocyte cell line previously isolated 15 was used. Briefly, primary podocytes were obtained from decapsulated glomeruli isolated from normal renal tissue obtained for polar carcinoma as described by Conaldi et al. 16 , then the line was established by infection with a hybridAdeno5/SV40 virus 17 . PCR data and immunofluorescence images for podocyte-specific markers ( CD2AP , NPHS1 and SYNPO ) were obtained to ascertain their cellular identity and phenotype maintenance. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) High Glucose (Euroclone S.p.A., Pero MI, Italy) 10% fetal bovine serum (FBS), with the addition of 1% penicillin–streptomycin and 1% L-glutamine (Sigma-Aldrich). Glomerular endothelial cells (GEC) (Cell Biologics Inc., Chicago, IL, USA) of human origin were immortalized 18 and cultured in EndoGRO LS Complete Culture Media (Merck Millipore) and 10% FBS. Podocyte granularity assay Podocytes (10 5 cells/well) in monoculture were treated for 24h with 2% or 10% sera from SRNS patients and controls. The podocyte granularity was evaluated as described 18 and reported as geometric mean of side scatter (SSC-A). In vitro three-dimensional (3D) glomerular model. A 3D glomerular model was developed using a millifluidic device (IVtech Srl., Lucca, Italy) as previously described 15 . In brief, GEC and podocytes were seeded on the opposite sides of a porous membrane previously coated with type IV collagen in their complete medium (Sigma-Aldrich). After cell attachment, the bioreactor was connected to a peristaltic pump, and basal DMEM HG without FBS was allowed to flow into the podocyte part. To generate an INS kidney injury model, 2% patient or healthy donor serum was added into the endothelial compartment in the presence of basal EndoGRO LS medium, and the flow was maintained at the flow rate of 80 µl/min for 48h and in a closed circuit. TEM analysis of the 3D glomerulus was performed by direct fixation of the membranes with paraformaldehyde (PAF) 4%, HEPES 0.25 M, and glutaraldehyde 2.5% (Sigma-Aldrich). The sample was examined using a Tecnai G2 transmission electron microscope (FEI Company) operating at 100 kV. Image capture was facilitated using a Veleta digital camera (Olympus Soft Imaging System). Permeability test Albumin conjugated with fluorescein isothiocyanate (BSA-FITC, Sigma-Aldrich) at a concentration of 1 mg/ml was added into the endothelial part (lower part) of the bioreactor 15 . After 6h of perfusion, the fluorescence levels in the upper part of the bioreactor (podocyte compartment) were measured. The BSA-FITC levels was measured by the detection of its levels at the beginning and after 48 hours of serum stimulation using a BSA standard curve as a normalizer and expressed as ug/mL. Fatty acid analysis by gas chromatography Cell extracts were methylated HClMe 3N, incubated for 1h at 90°C, and then refrigerated at 4°C for 10 min. Subsequently, fatty acid methyl esters (FAME) were isolated via hexane extraction and introduced into a Shimadzu Nexis GC-2030 gas chromatograph as described 19 . For the separation of FAME, a 30-meter capillary column (FAMEWAX, Restek) was employed. The Labsolution software version 5.97 SP1 (Shimadzu) was used for FA analysis, relying on the retention times of standards, including PUFA1, PUFA2, PUFA3 (Supelco, Merck), and NHI-F (AccuStandard, Restek). Single fatty acid was expressed as the relative percentage of total fatty acids (FAs). Total saturated fatty acids (SFA), total monounsaturated fatty acids (MUFA), and total polyunsaturated fatty acids (PUFA) were calculated. Immunofluorescence Membranes were sectioned, fixed in 4% PAF and then permeabilized with 0.1% Triton X100 in 1% PBS. Non-specific sites were blocked with PBS 1.5% BSA, followed by incubation with the primary antibodies, guinea pig polyclonal against nephrin (Progen, Heidelberg, Germany) and anti-human CD105-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) for 2 hours. After extensive washes, secondary Ab (1:1000, goat anti-guinea pig IgG highly cross-absorbed, Invitrogen) and Phalloidin (1:1000, Sigma-Aldrich) were added for 1h at room temperature. Cell nuclei were stained with 40,6-diamidino-2-phenylindole (1:10,000, Sigma-Aldrich). Finally, coverslips were mounted with ProLong Gold Antifade Reagent mounting medium (Thermo-Fisher Scientific). Imaging was performed using a Leica TCS SP5 Confocal System using the 409 PlanApo/1.4 NA oil dive goals. A series of x-y-z images were collected at 0.5 µm intervals throughout the sample. The fluorescence intensity was quantified using ImageJ as reported 20 . PCR analysis RNA was extracted using a TRizol and retrotranscribed using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, California, USA). Levels of mRNA were assessed by qPCR, using a mix containing 5 ng of cDNA, 100 nM of each primer (Supplementary Table 2), and 1× SYBR Green PCR Master Mix (Applied Biosystems Negative cDNA controls were cycled in parallel at each run). PCR was run in a 96-well StepOne Real-Time System (Applied Biosystems, Waltham, Massachusetts, USA). Data are shown as relative quantification (2 − ΔΔCt ). GAPDH expression was used to normalize cDNA inputs. Statistical Analysis Values were reported as mean ± SEM. Statistical significance was calculated as follows: Mann-Whitney test (for non-parametric dataset) for comparison between coupled experimental groups, while one-way ANOVA with Bonferroni's post-test (for parametric dataset) or Kruskal-Wallis (for non-parametric dataset), were performed for multiple comparisons. The data were analyzed using GraphPad Prism version 9.5.0 (GraphPad Software, San Diego, California USA). A p-value (P) < 0.05 was considered statistically significant. Results Evaluation of the loss of slit diaphragm proteins in podocytes after SRNS serum treatment. Immortalized human podocytes, cultured in two-dimensional conditions, were treated for 24h with 2% or 10% serum from MDR-SRNS or genetic-SRNS patients. A significant increase in cell granularity was also observed, after the treatment with either 2% or 10% of both MDR and genetic-SRNS patients’ sera compared to the CTRL condition ( P < 0.05 and P < 0.01 vs the corresponding CTRL) (Fig. 1 a). A three-dimensional dynamic glomerular model was then applied to study the effect of SRNS serum on the GFB (Supplementary Figure S1 ). A significant increase in the permeability to BSA-FITC was observed when the glomerulus was subjected to 2% MDR serum compared to controls (2% CTRL) ( P 0.05 ) (Fig. 1 b). Important changes in GFB permeability were accompanied by substantial modifications in relevant proteins composing the slit diaphragm structure. Immunofluorescence evaluation of the 3D glomerulus showed contraction of actin filaments, with an accumulation to the cell periphery in podocytes treated with 2% MDR and genetic-SRNS serum (Fig. 1 c, top and Supplementary Figure S1 ). Moreover, the slit diaphragm component nephrin (NPHS1) was reduced and formed cytoplasmic aggregates after both SRNS sera treatments (Fig. 1 c). TEM highlighted the loss of pedicel structure from the SRNS-treated podocytes (Fig. 1 d). qRT-PCR analysis of slit diaphragm components Nephrin ( NPHS1 ), synaptopodin ( SYNPO ), and CD2 Associated Protein ( CD2AP) , showed a statistically significant increase in their expression in podocytes treated with MDR and genetic-SRNS serum compared to cells exposed to CTRL serum (Fig. 1 e). The expression of SYNPO was also significantly reduced in MONO compared to MDR treatment ( P < 0.01 ). The administration of MDR serum was again more effective in altering the SD on podocytes than the genetic-SRNS serum treatment. Modification in the membrane localization of the vascular marker endoglin (CD105) was observed in glomerular endothelial cells (GEC) after the SRNS treatment, showing an alteration also in the endothelial compartment (Fig. 1 c, middle panel). AA synthesis pathway perturbance in podocytes induced by SRNS sera treatment. Changes in podocyte lipid components were analyzed by gas chromatography. The list of FAs changes is reported in Table 2 . A significant rise in MUFA was detected in MDR-treated podocytes compared to the CTRL-treated ones; this was accompanied by a significant reduction in SFA in this group (Fig. 2 , P < 0.05 ). Among the analyzed FAs, the AA synthesis pathway (Fig. 3 a) was the most affected in podocytes treated with SRNS serum (Table 2 ). In detail, the treatment with serum from MDR patients led to a significant increase in AA levels (Fig. 3 b). This was associated with an increase in the expression of FADS-1, converting DHLA to AA, only in MDR-treated podocytes with respect to control ( P < 0.05 ) (Fig. 3 c). Conversely, the upstream enzymes in the AA synthesis pathways were differentially affected by the diverse SRNS treatments. We identified that FADS-2 , responsible for LA to GLA conversion, was upregulated only after genetic-SRNS treatment ( P < 0.05 vs MDR and P < 0.01 vs CTRL). This was related to an increase in the LA substrate in podocytes treated with serum from genetic-SRNS ( P < 0.05 vs CTRL) (Fig. 3 b). The enzyme ELOVL-5 , converting GLA to DHLA, was induced by both MDR and genetic-SRNS administration compared to the control ( P < 0.001 and P < 0.05 , respectively) (Fig. 3 c). By immunofluorescence, we measured the changes in the density of actin filaments concomitant with AA pathway disturbance. The actin mass was mainly gathered in the podocyte periphery (Fig. 3 d) and was strongly reduced after both MDR- and genetic-SRNS administration ( P < 0.001 and P < 0.01 vs CTRL) (Fig. 3 e). Table 2 Fatty acid composition in podocytes cultured in the 3D glomerular model. Fatty acid abundance was reported as median values with the minimum to maximum range. P value was reported in the table. NS: not statistically significant; a MDR vs CTRL, b MONO vs CTRL; non-parametric Kruskal-Wallis test with Dunn’s post hoc. Fatty acids Podocytes Name Chemical name CTRL (n = 7) MDR (n = 5) MONO (n = 5) P value Palmitic acid (PA) 16:0 39.91 (24.86–49.12) 40.97 (22.30-54.79) 40.59 (25.34–49.01) NS Palmitoleic acid (POA) 16:1n7 1.64 (0.31–3.61) 3.75 (1.07–12.19) a 1.80 (0.10–7.89) P a < 0.05 Stearic acid (SA) 18:0 23.18 (14.77–50.70) 17.39 (15.05–22.23) 20.26 (15.23–34.03) NS Oleic acid (OA) 18:1n9 14.11 (3.29–22.89) 21.84 (15.06–26.80) 19.98 (8.19–30.91) NS cis-vaccenic acid (CVA) 18:1n7 2.73 (0.09–4.66) 4.23 (2.25–6.86) 4.29 (1.47–6.46) NS Linoleic acid (LA) 18:2n6 3.90 (0.66–5.75) 3.84 (2.90–5.99) 4.92 (3.49–7.08) b P b < 0.05 α-Linolenic acid (ALA) 18:3n3 0.23 (0.01–4.36) 0.20 (0.06–2.49) 0.17 (0.01–0.60) NS Mead acid (MA) 20:3n9 0.21 (0.04–3.04) 0.18 (0.02–0.62) 0.14(0.04-15.00) NS Dihomo-γ-linolenic acid (DHLA) 20:3n6 0.56 (0.42–1.29) 0.42 (0.20–0.86) 0.49 (0.10–0.60) NS Arachidonic acid (AA) 20:4n6 3.09 (1.27–4.79) 5.37 (3.39–8.37) a 3.71 (2.79–9.13) P a < 0.05 Timaodonic acid (TA) 20:5n3 0.27 (0.08–4.92) 0.34 (0.06–2.89) 0.41 (0.19–3.81) NS Behenic acid (BA) 22:0 0.74 (0.32–7.11) 0.73 (0.37–19.31) 0.57 (0.19–0.88) NS Docosapentaenoic acid (DPA) 22:5n3 0.66 (0.13–2.24) 1.38 (0.20–2.42) 0.90 (0.39–3.76) NS Lignoceric acid (LCA) 24:0 0.93 (0.30–1.52) 0.90 (0.30–1.24) 0.57 (0.42–0.88) NS Clupanodonic acid (DPAω3) 22:6n3 1.09 (0.14–2.21) 1.53 (0.11–2.69) 0.98 (0.36–3.01) NS Nervonic acid (NA) 24:1 0.38 (0.25–2.55) 0.63 (0.08–1.12) 0.58 (0.33–2.14) NS Saturated Fatty Acids (SFA) 64.72 (52.35–84.04) 55.65 (39.89–64.30) a 60.24 (35.94–77.96) P a < 0.05 Monounsaturated Fatty Acids (MUFA) 19.44 (6.24–31.10) 31.18 (19.32–38.23) a 27.94 (12.58–38.19) P a < 0.05 Polyunsaturated Fatty Acids (PUFA) 14.01 (7.03–24.74) 14.89 (9.14–21.82) 11.46 (9.38–34.69) NS Discussion SRNS is a heterogeneous disorder including immune-related and genetic etiologies 21 . The presence of unknown circulating factors has been previously related to the loss of slit diaphragm structure in children with non-genetic SRNS 22 , but their actual contribution is still undefined. Moreover, a clear understanding of the molecular mechanisms activated in damaged podocytes during SRNS is not fully recognized. We employed a glomerular 3D millifluidic model mimicking the GFB to elucidate the mechanisms behind glomerular damage in different forms of SRNS. Sera from multi-drug SRNS were able to induce both disruption of the slit diaphragm structure and changes in the expression and localization of slit diaphragm proteins in respect to both genetic SRNS and control group. The exposure of cultured podocytes to MDR- and genetic-SRNS serum induced disruption of arachidonic acid (AA) synthesis pathway, with different intermediate players involved. The alteration in the permeability of the GFB is the main cause of proteinuria in INS. In steroid-resistant INS children, commonly associated with focal segmental glomerulosclerosis (FSGS), the loss of GFB structure has been associated with direct podocyte damage 23 . A 3D co-culture of human glomerular endothelial cells and podocytes was established to mimic the GFB. Only glomerular endothelial cells and podocytes exposed to MDR serum, presented significantly higher permeability to albumin compared to controls. This was accompanied by an induction of granularity in podocytes expose to the serum from multi-drug resistant and to less extent to the genetic SRNS, but not in response to serum from healthy subjects. These data confirmed previous work by Li et al. 24 , showing the same activity of serum derived from recurrent SRNS using a static glomerular model. Unexpectedly, in our system, serum derived from genetic SRNS was also able to induce an effect on podocytes, without increasing albumin permeability corroborating the absence of a circulating permeability factor(s) in this group of NS. Despite that, the presence of a pro-inflammatory microenvironment in SRNS 25 could potentially explain the detrimental effect resulting in podocyte damage, notwithstanding different degrees of injury between the immune-associated and genetic forms of SRNS. Moreover, our data strongly abet the recent research, which confirms the existence of multiple etiopathogenic agents causing kidney damage progression in INS 23 . GFB loss of structure was also accompanied by morphological changes observed after 48 hours of serum incubation. In our 3D model, the treatment of podocytes resulted in nephrin redistribution from their surface. Loss of nephrin localization along the foot processes was previously correlated with progressive proteinuria resulting in the development of FSGS in adult glomeruli 26 . Interestingly, a contrasting upregulation of genes coding for NPHS1 and SYNPO , as well as for the scaffold molecule CD2AP , was observed after MDR and to less extent genetic-SRNS treatments. Noteworthy, the mRNA levels of different slit diaphragm proteins were identified as elevated in isolated glomeruli from multiple proteinuric kidney diseases 27 . In parallel, SRNS-treated glomerular endothelial cells co-cultured in the dynamic glomerular system showed a marked decrease in the endothelial marker, CD105. Our findings confirmed that podocyte impairment could be dependent on pathogenetic events happening in glomerular endothelial cells during INS 24 , 28 . Alteration in lipid metabolism is closely associated with numerous proteinuric kidney diseases 29 . Podocytes exposed to SRNS serum showed changes in FA composition. A significant increase in monounsaturated FAs and a decrease in saturated FAs was observed only in the MDR-treated group. The enhancement in monounsaturated FAs content was previously associated with CKD and inflammation 30 , 31 . The main differences in FAs in MDR-treated podocytes were observed in the polyunsaturated n-6 FA, AA. Such alterations may be produced by a synergic activity of serum components, including lipids which alteration in INS has been previously described 32 . However, we distinctly identified changes in the transcripts coding for enzymes involved in the AA synthesis pathways after 48h of podocyte treatment with MDR serum. This strongly supports that the FA changes we observed, particularly AA alterations of the podocytes, were mainly dependent on their synthesis. Furthermore, these findings are in line with studies indicating an upregulation of the AA synthesis pathway in several inflammatory diseases including INS 33 . Deregulation in AA synthesis has been shown to alter the actin cytoskeleton of podocytes, affecting foot process formation, and the integrity of the filtration barrier 11 . Consistently, our data highlighted a significant remodeling of the actin cytoskeleton probably related to the AA induction in MDR-treated podocytes and with a lesser magnitude in genetic-SRNS treatment compared to controls. In summary, our study provides important insights into the role of FA deregulation, podocyte damage, and proteinuria development in steroid-resistant INS. In our hands, even though the different etiopathology of INS forms, the podocyte damage induced by SRNS serum seems to embody the kidney response to a cumulative detrimental environment worsening the course of the INS pathology. Nevertheless, it suggests that defects in molecular pathways involved in podocytes’ AA synthesis could be related to the GFB impairment induced by the treatment with sera from MDR patients. Further studies with larger sample sizes are needed to establish the causality between AA alteration and kidney damage in MDR-SRNS, the most severe form of this disease and determine its possible therapeutic implications. Declarations Data availability statement All data generated or analyzed during this study are included in the published article [and its supplementary information files]. Ethics approval and consent to participate statement The study was approved by the IRCCS Ca’ Granda Institutional Review Board (ID 2633, INSiDe protocol). An informed consensus has been signed by the parent and/or legal guardian of all the children enrolled in the study. Acknowledgments The authors wish to thank the Microscopy facility of the Istituto Nazionale of Genetica Molecolare (INGM) for Imaging assistance. Funding This research was supported by PSR 2022, PI Starting Grant from the University of Milano (UNIMI); and Grant P-0038 from IMPACTsim S.p.A. Author contribution Conception and design: G.B., L.B., F.C.; Experiments and acquisition of experimental data: G.B., L.B., S.T., G.C., F.Ca.; Data collection: W.M., C.T., S.V., D.P.; Analysis and interpretation of data: G.B., L.B., G.C., F.C.; Draft of the manuscript: F.C.; G.B. Figures: L.B., G.B.; Funding and resources acquisition: F.C., G.M.; Revision of the manuscript: F.C., G.M. All the authors revised and approved the final version of the manuscript. Conflict of interest statement The authors declare no conflict of interest. References Seeger, H. & Fehr, T. Das nephrotische Syndrom beim Erwachsenen – Ursachen und Komplikationen. Praxis 105 , 259–267 (2016). Trautmann, A. et al. 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Assessment of increased glomerular permeability associated with recurrent focal segmental glomerulosclerosis using an in vitro model of the glomerular filtration barrier. J Nephrol 33 , 747–755 (2020). Latt, K. Z. et al. Urine Single-Cell RNA Sequencing in Focal Segmental Glomerulosclerosis Reveals Inflammatory Signatures. Kidney Int Rep 7 , 289–304 (2022). Verma, R. et al. Nephrin is necessary for podocyte recovery following injury in an adult mature glomerulus. PLoS One 13 , e0198013 (2018). Koop, K. et al. Expression of Podocyte-Associated Molecules in Acquired Human Kidney Diseases. Journal of the American Society of Nephrology 14 , 2063–2071 (2003). ‘t Hart, D. C. et al. Co-Culture of Glomerular Endothelial Cells and Podocytes in a Custom-Designed Glomerulus-on-a-Chip Model Improves the Filtration Barrier Integrity and Affects the Glomerular Cell Phenotype. Biosensors (Basel) 13 , 339 (2023). Sun, Y., Cui, S., Hou, Y. & Yi, F. The Updates of Podocyte Lipid Metabolism in Proteinuric Kidney Disease. Kidney Diseases 7 , 438–451 (2021). Szczuko, M. et al. The C18:3n6/C22:4n6 ratio is a good lipid marker of chronic kidney disease (CKD) progression. Lipids Health Dis 19 , 77 (2020). Czumaj, A. et al. Alterations of Fatty Acid Profile May Contribute to Dyslipidemia in Chronic Kidney Disease by Influencing Hepatocyte Metabolism. Int J Mol Sci 20 , 2470 (2019). Hu, L. et al. Lipidomic profiles in serum and urine in children with steroid sensitive nephrotic syndrome. Clinica Chimica Acta 555 , 117804 (2024). Tu, B. et al. Comprehensive analysis of arachidonic acid metabolism-related genes in diagnosis and synovial immune in osteoarthritis: based on bulk and single-cell RNA sequencing data. Inflammation Research 72 , 955–970 (2023). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterialdef.pdf Cite Share Download PDF Status: Published Journal Publication published 14 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 07 Aug, 2024 Reviews received at journal 06 Aug, 2024 Reviewers agreed at journal 16 Jul, 2024 Reviewers agreed at journal 16 Jul, 2024 Reviewers invited by journal 14 Jul, 2024 Editor assigned by journal 08 Jul, 2024 Editor invited by journal 08 Jul, 2024 Submission checks completed at journal 08 Jul, 2024 First submitted to journal 04 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4684821","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":332263639,"identity":"da62a6c6-1935-4dbc-9f7d-eaaa82ecc5fc","order_by":0,"name":"Gaia Bianchi","email":"","orcid":"","institution":"University of Milano","correspondingAuthor":false,"prefix":"","firstName":"Gaia","middleName":"","lastName":"Bianchi","suffix":""},{"id":332263640,"identity":"8af28e3f-da20-4b06-9b0e-c781dfe67e6c","order_by":1,"name":"Linda Bellucci","email":"","orcid":"","institution":"Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico","correspondingAuthor":false,"prefix":"","firstName":"Linda","middleName":"","lastName":"Bellucci","suffix":""},{"id":332263641,"identity":"7db5a9df-b8bc-4b9f-929f-320dd222772c","order_by":2,"name":"William Morello","email":"","orcid":"","institution":"Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Morello","suffix":""},{"id":332263642,"identity":"40301ecf-fa70-4f86-9878-55f2c6ce58f2","order_by":3,"name":"Stefano Turolo","email":"","orcid":"","institution":"Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Turolo","suffix":""},{"id":332263643,"identity":"ab4deb6b-3a37-4f30-919a-8eb1d66d87f8","order_by":4,"name":"Giulia Cricrì","email":"","orcid":"","institution":"Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Cricrì","suffix":""},{"id":332263644,"identity":"242e024b-a2d2-4aff-ac72-d4c6b851e233","order_by":5,"name":"Federico Caicci","email":"","orcid":"","institution":"University of Padova","correspondingAuthor":false,"prefix":"","firstName":"Federico","middleName":"","lastName":"Caicci","suffix":""},{"id":332263645,"identity":"cb30b2c5-cf6f-4b02-8984-b0fee1b4d277","order_by":6,"name":"Chiara Tamburello","email":"","orcid":"","institution":"Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico","correspondingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Tamburello","suffix":""},{"id":332263646,"identity":"6ae8162e-f744-405a-9b8e-ce07fc5d2b6d","order_by":7,"name":"Stefania Villa","email":"","orcid":"","institution":"Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico","correspondingAuthor":false,"prefix":"","firstName":"Stefania","middleName":"","lastName":"Villa","suffix":""},{"id":332263647,"identity":"f163236b-fb03-4114-813b-e60ad1018724","order_by":8,"name":"Daniele Prati","email":"","orcid":"","institution":"Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico","correspondingAuthor":false,"prefix":"","firstName":"Daniele","middleName":"","lastName":"Prati","suffix":""},{"id":332263648,"identity":"5d2931bf-d50f-4cd3-bc2b-6d635527f6ef","order_by":9,"name":"Giovanni Montini","email":"","orcid":"","institution":"University of Milano","correspondingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"","lastName":"Montini","suffix":""},{"id":332263649,"identity":"81d9a013-88d0-4d97-9916-04150dc2c80c","order_by":10,"name":"Federica Collino","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBAC9gYGAwYQAgLGAw+ADH4g6wBQhAeXFp4DCC0MBxKADMkGqBZceiBaGGBagITBAZgULi3szRsfVxTYyTOwHz5wIKHgjpzxjdyDh24UMMjY49LCc6zY8IxBsmEDT1oC0GHPjM1u5CUczsHjMHuJHDPJBoMDjA0SPAZALYcTt93IMcCrhUf+jflPoBZ7uJbNMwhpkeAxYwRqSYRr2SBBSAtPWjHQYcnJbRC/HDaWOPMGpEWCBxiYOELs8MaPDX/sbPvZDx988OHPYTn+9hzjzzl/bOyBsYwfsKHxJQioHwWjYBSMglGADwAAOzBW8Af4jD4AAAAASUVORK5CYII=","orcid":"","institution":"University of Milano","correspondingAuthor":true,"prefix":"","firstName":"Federica","middleName":"","lastName":"Collino","suffix":""}],"badges":[],"createdAt":"2024-07-04 08:07:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4684821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4684821/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-95216-2","type":"published","date":"2025-04-14T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62140890,"identity":"701bd195-db0c-4fea-b935-ad42452b7611","added_by":"auto","created_at":"2024-08-09 17:08:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6574417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of serum of SRNS children on glomerular filtration barrier using a 3D dynamic glomerular model.\u003c/strong\u003e (a) Podocytes were exposed for 24 hours (24h) to 2% or 10% of serum from MDR-SRNS (MDR, N=5) and genetic-SRNS (MONO, N=5); or healthy subjects (CTRL, N=7). Each sample was tested in at least three different experiments. Granularity was expressed as geometric mean of SSC-A measured in flow cytometry. *\u003cem\u003eP \u0026lt;0.05,\u003c/em\u003e **\u003cem\u003eP \u0026lt;0.01 vs \u003c/em\u003eCTRL; non-parametric Kruskal-Wallis test with Dunn’s post hoc. (b) Filtration assay measuring the passage of BSA-FITC across the GFB after treatment with CTRL, MDR or MONO serum. Values were expressed as BSA concentration (μg/ml); **\u003cem\u003eP \u0026lt;0.01\u003c/em\u003e \u003cem\u003evs\u003c/em\u003e CTRL, non-parametric Kruskal-Wallis test with Dunn’s post hoc. (c) Representative immunofluorescence staining of podocytes (PODO, top) and glomerular endothelial cells (GEC, bottom) layered on the 3D glomerulus. Labeling of actin (red), in combination with the slit diaphragm protein nephrin (green) in PODO and CD105 (yellow) in GEC was evaluated. Nuclei were stained with DAPI (blue). (d) Transmission electron microscopy (TEM) analysis of podocytes subjected to CTRL, MDR, and MONO sera treatment; scale bars= 1μm. (e) qRT-PCR analysis of the expression of slit diaphragm molecules, \u003cem\u003eNPHS1\u003c/em\u003e, \u003cem\u003eSYNPO\u003c/em\u003e, and \u003cem\u003eCD2AP \u003c/em\u003ein serum-treated podocytes. Data are shown as mean ± SEM (N=3 for each condition). *\u003cem\u003eP \u0026lt;0.05,\u003c/em\u003e **\u003cem\u003eP \u0026lt;0.01\u003c/em\u003e, ***\u003cem\u003eP \u0026lt;0.001 \u003c/em\u003e****\u003cem\u003eP \u0026lt;0.0001 vs\u003c/em\u003e the corresponding treatment. One-way ANOVA with Bonferroni's post-test.\u003c/p\u003e","description":"","filename":"Fig1SC.png","url":"https://assets-eu.researchsquare.com/files/rs-4684821/v1/5b9f689b4c7c8a62c793a1c7.png"},{"id":62139284,"identity":"7a7e25ad-439c-4cec-8aba-d969daf37fb5","added_by":"auto","created_at":"2024-08-09 16:52:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of steroid-resistant nephrotic syndrome serum on fatty acid composition and podocyte metabolism.\u003c/strong\u003e Gas chromatography analysis of podocytes was performed to evaluate SFA, MUFA, and PUFA as median % of total lipids after CTRL (N=7), MDR (N=5), and MONO (N=5) serum treatments, \u003cem\u003e*P\u0026lt; 0.05\u003c/em\u003e \u003cem\u003evs\u003c/em\u003eCTRL. Non-parametric Kruskal-Wallis test with Dunn’s post hoc. CTRL, treatment with healthy donor serum; MDR, treatment with multi-drug resistant nephrotic syndrome serum; MONO, treatment with monogenic steroid-resistant nephrotic syndrome serum; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.\u003c/p\u003e","description":"","filename":"Fig2SC.png","url":"https://assets-eu.researchsquare.com/files/rs-4684821/v1/0f5118e157721794774e8fab.png"},{"id":62139287,"identity":"fc6cbd76-fb1e-4739-8f7b-d6322127c684","added_by":"auto","created_at":"2024-08-09 16:52:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1466197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of steroid-resistant nephrotic syndrome serum on AA synthesis pathway. \u003c/strong\u003e(a) Schematic representation of the AA synthesis pathway. (b) Gas chromatography analysis of LA, DHLA, and AA in podocytes treated with CTRL (N=7), MDR (N=5), and MONO (N=5) sera. Data are expressed as the relative percentage of total fatty acids analyzed (% of tot); non-parametric Kruskal-Wallis test with Dunn’s post hoc. \u003cem\u003e*P\u0026lt; 0.05 vs\u003c/em\u003e the corresponding treatment. (c) qRT-PCR analysis of the enzymes \u003cem\u003eFADS-2\u003c/em\u003e, \u003cem\u003eELOVL-5,\u003c/em\u003e and \u003cem\u003eFADS-1\u003c/em\u003e, involved in the AA synthesis pathway (d) Representative immunofluorescence images of actin cytoskeleton changes (Phallodin-AF568) in podocytes after 48 hours of exposure to 2% CTRL, MDR, or MONO serum. (e) Actin fluorescence intensity measured as corrected total cell fluorescence (CTCF) as described in the Method’s section. Data are shown as mean ± SEM, One-way ANOVA with Bonferroni's post hoc. \u003cem\u003e**P\u0026lt; 0.01; ***P\u0026lt; 0.001 vs\u003c/em\u003e the corresponding treatment. CTRL, treatment with healthy donor serum; MDR, treatment with multi-drug resistant nephrotic syndrome serum; MONO, treatment with monogenic steroid-resistant nephrotic syndrome serum; LA, linoleic acid; DHLA, dihomo-γ-linolenic acid; AA, arachidonic acid; \u003cem\u003eFADS-2\u003c/em\u003e, Fatty acid desaturase 2; \u003cem\u003eELOVL-5\u003c/em\u003e, elongation of very long chain fatty acids protein 5; \u003cem\u003eFADS-1\u003c/em\u003e, Fatty acid desaturase 1.\u003c/p\u003e","description":"","filename":"Fig3SC.png","url":"https://assets-eu.researchsquare.com/files/rs-4684821/v1/946c0ff81205e6d242a9c128.png"},{"id":81051222,"identity":"be985269-8d0b-4e3d-b459-847d8082a4fb","added_by":"auto","created_at":"2025-04-21 16:10:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11984259,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4684821/v1/e05ed282-f110-4b81-bce7-e35158cab29c.pdf"},{"id":62139285,"identity":"ae459b1a-b783-46a0-a865-e18aade54cc0","added_by":"auto","created_at":"2024-08-09 16:52:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1384586,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialdef.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4684821/v1/f56b0fd28ea9b1c50bd2e152.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of steroid-resistant nephrotic syndrome serum on AA pathway in podocytes cultured in 3D in vitro glomerular model","fulltext":[{"header":"Key points","content":"\u003col\u003e\n \u003cli\u003eWe developed an \u003cem\u003ein vitro\u003c/em\u003e glomerular millifluidic model that can capture the podocyte damage induced in SRNS patients.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWe showed the detrimental effects of serum from SRNS patients on podocyte function and the association of AA synthesis pathway with the podocyte damage in SRNS.\u003c/li\u003e\n \u003cli\u003eThe direct targeting of AA pathway could be instrumental in reducing the kidney damage progression and proteinuria in MDR patients.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eIdiopathic nephrotic syndrome (INS) is characterized by the presence of proteinuria, edema, and hyperlipemia\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In childhood-onset INS, 10\u0026ndash;15% of patients are steroid-resistant (SRNS), as they do not achieve remission with the initial treatment with steroids. Around 50% of them, unresponsive to second-line therapies, are classified as multi-drug resistant SRNS (MDR), the most severe form of the disease. Only in 1/3 of patients, a pathogenic variant of genes coding for structural proteins of the glomerular filtration barrier (GFB) has been identified (monogenic SRNS), while in the remaining cases, a yet unidentified immunological cause is thought to be involved\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Both MDR and genetic SRNS are associated with poor kidney survival\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eINS is characterized by the leakage of proteins as well as lipids into the urine and a concomitant increase of blood-free fatty acids\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Leaked lipids mainly accumulate in the proximal tubules, inducing tubular damage and fibrosis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Excessive lipid uptake in podocytes can activate an oxidative stress response, inducing cell loss, which results in glomerular damage and proteinuria\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Although lipid metabolism in the kidney seems to be a crucial issue, the link between podocyte lipotoxicity and INS is missing. Among fatty acids (FAs), arachidonic acid (AA) directly regulates podocyte integrity, by promoting slit diaphragm and cytoskeleton changes and cell apoptosis\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. AA has also been described to induce TGFβ-induced fibrosis in mesangial cells by activating inflammatory and pro-fibrotic genes, which are crucial in the progression of chronic kidney disease\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We recently reported the presence of elevated levels of AA in the blood of INS children at different clinical INS stages, supporting its role as a sensor of a persistent inflammatory milieu\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we used a three-dimensional (3D) dynamic glomerular model to investigate the lipotoxic alterations in human podocytes treated with serum from different forms of SRNS. The direct effects on podocyte cell viability, cytoskeleton organization, and slit diaphragm structure, were evaluated. The FA composition of podocytes cultured in the nephrotic syndrome-induced kidney model was also assessed.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and sample collection\u003c/h2\u003e \u003cp\u003e This study was conducted according to the principles expressed in the Declaration of Helsinki. Approve to the study was obtained by the IRCCS Ca\u0026rsquo; Granda Institutional Review Board (ID 2633, INSiDe protocol). An informed consensus was obtained for all the participants enrolled in the study. Multi-drug resistant SRNS (MDR-SRNS, N\u0026thinsp;=\u0026thinsp;5) and genetic SRNS (genetic-SRNS, N\u0026thinsp;=\u0026thinsp;5) patients were recruited during the active phase of the pathology in the Paediatric Nephrology, Dialysis and Renal Transplant Unit (Fondazione Ca\u0026rsquo; Granda IRCCS Ospedale Maggiore Policlinico, Milan). MDR-SRNS were defined as INS patients not achieving partial remission after at least 2 different lines of therapy other than steroids; genetic SRNS forms were defined as patients non-responsive to immunosuppressive treatments and harboring a pathogenetic mutation in genes coding for components of GFB. The list of mutations was reported in Supplementary Table\u0026nbsp;1. Clinical data of SRNS patients were reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Furthermore, a group of healthy volunteers (N\u0026thinsp;=\u0026thinsp;7) were also recruited as control group. The exclusion criteria for this group included fever, inherited, or acquired immune disorders, and the use of immunosuppressants. Serum samples were obtained by centrifuging whole blood at 3000g for 20 minutes (min). Before each experiment, sera were diluted to 2% or 10% with EndoGRO\u003csup\u003eLS\u003c/sup\u003e basal Culture Media (Merck Millipore, Burlington, Massachusetts, USA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eClinical characteristics of the INS cohort.\u003c/b\u003e Multi-drug resistant (MDR) (N\u0026thinsp;=\u0026thinsp;5) and genetic-SRNS (N\u0026thinsp;=\u0026thinsp;5) patients were recruited in our study. Gender and ethnicity are presented as absolute numbers and relative frequency. Age of appearance and sampling are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Proteinuria levels (uPr/uCr) were represented as the minimum (min)- maximum (max) range.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMDR\u003c/p\u003e \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003egenetic-SRNS\u003c/p\u003e \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0 (60.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0 (80.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.0 (40.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEthnicity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCaucasian\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.0 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0 (80.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNorth-African\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0 (0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge of appearance\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean (SD)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16 (15.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.6 (8.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003emin-max\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0\u0026ndash;42.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u0026ndash;18.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge at sampling\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean (SD)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21.8 (18.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7 (8.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003emin-max\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.0\u0026ndash;52.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u0026ndash;19.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003euPr/uCr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean (SD)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.6 (3.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.0 (9.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003emin-max\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.0\u0026ndash;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.4\u0026ndash;25.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eA podocyte cell line previously isolated\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e was used. Briefly, primary podocytes were obtained from decapsulated glomeruli isolated from normal renal tissue obtained for polar carcinoma as described by Conaldi et al.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, then the line was established by infection with a hybridAdeno5/SV40 virus\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. PCR data and immunofluorescence images for podocyte-specific markers (\u003cem\u003eCD2AP\u003c/em\u003e, \u003cem\u003eNPHS1\u003c/em\u003e and \u003cem\u003eSYNPO\u003c/em\u003e) were obtained to ascertain their cellular identity and phenotype maintenance. Cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) High Glucose (Euroclone S.p.A., Pero MI, Italy) 10% fetal bovine serum (FBS), with the addition of 1% penicillin\u0026ndash;streptomycin and 1% L-glutamine (Sigma-Aldrich). Glomerular endothelial cells (GEC) (Cell Biologics Inc., Chicago, IL, USA) of human origin were immortalized\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and cultured in EndoGRO\u003csup\u003eLS\u003c/sup\u003e Complete Culture Media (Merck Millipore) and 10% FBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePodocyte granularity assay\u003c/h2\u003e \u003cp\u003ePodocytes (10\u003csup\u003e5\u003c/sup\u003e cells/well) in monoculture were treated for 24h with 2% or 10% sera from SRNS patients and controls. The podocyte granularity was evaluated as described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and reported as geometric mean of side scatter (SSC-A).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro three-dimensional (3D) glomerular model.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA 3D glomerular model was developed using a millifluidic device (IVtech Srl., Lucca, Italy) as previously described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In brief, GEC and podocytes were seeded on the opposite sides of a porous membrane previously coated with type IV collagen in their complete medium (Sigma-Aldrich). After cell attachment, the bioreactor was connected to a peristaltic pump, and basal DMEM HG without FBS was allowed to flow into the podocyte part. To generate an INS kidney injury model, 2% patient or healthy donor serum was added into the endothelial compartment in the presence of basal EndoGRO\u003csup\u003eLS\u003c/sup\u003e medium, and the flow was maintained at the flow rate of 80 \u0026micro;l/min for 48h and in a closed circuit. TEM analysis of the 3D glomerulus was performed by direct fixation of the membranes with paraformaldehyde (PAF) 4%, HEPES 0.25 M, and glutaraldehyde 2.5% (Sigma-Aldrich). The sample was examined using a Tecnai G2 transmission electron microscope (FEI Company) operating at 100 kV. Image capture was facilitated using a Veleta digital camera (Olympus Soft Imaging System).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePermeability test\u003c/h2\u003e \u003cp\u003eAlbumin conjugated with fluorescein isothiocyanate (BSA-FITC, Sigma-Aldrich) at a concentration of 1 mg/ml was added into the endothelial part (lower part) of the bioreactor\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. After 6h of perfusion, the fluorescence levels in the upper part of the bioreactor (podocyte compartment) were measured. The BSA-FITC levels was measured by the detection of its levels at the beginning and after 48 hours of serum stimulation using a BSA standard curve as a normalizer and expressed as ug/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFatty acid analysis by gas chromatography\u003c/h2\u003e \u003cp\u003eCell extracts were methylated HClMe 3N, incubated for 1h at 90\u0026deg;C, and then refrigerated at 4\u0026deg;C for 10 min. Subsequently, fatty acid methyl esters (FAME) were isolated via hexane extraction and introduced into a Shimadzu Nexis GC-2030 gas chromatograph as described\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. For the separation of FAME, a 30-meter capillary column (FAMEWAX, Restek) was employed. The Labsolution software version 5.97 SP1 (Shimadzu) was used for FA analysis, relying on the retention times of standards, including PUFA1, PUFA2, PUFA3 (Supelco, Merck), and NHI-F (AccuStandard, Restek). Single fatty acid was expressed as the relative percentage of total fatty acids (FAs). Total saturated fatty acids (SFA), total monounsaturated fatty acids (MUFA), and total polyunsaturated fatty acids (PUFA) were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eMembranes were sectioned, fixed in 4% PAF and then permeabilized with 0.1% Triton X100 in 1% PBS. Non-specific sites were blocked with PBS 1.5% BSA, followed by incubation with the primary antibodies, guinea pig polyclonal against nephrin (Progen, Heidelberg, Germany) and anti-human CD105-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) for 2 hours. After extensive washes, secondary Ab (1:1000, goat anti-guinea pig IgG highly cross-absorbed, Invitrogen) and Phalloidin (1:1000, Sigma-Aldrich) were added for 1h at room temperature. Cell nuclei were stained with 40,6-diamidino-2-phenylindole (1:10,000, Sigma-Aldrich). Finally, coverslips were mounted with ProLong Gold Antifade Reagent mounting medium (Thermo-Fisher Scientific). Imaging was performed using a Leica TCS SP5 Confocal System using the 409 PlanApo/1.4 NA oil dive goals. A series of x-y-z images were collected at 0.5 \u0026micro;m intervals throughout the sample. The fluorescence intensity was quantified using ImageJ as reported \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePCR analysis\u003c/h2\u003e \u003cp\u003eRNA was extracted using a TRizol and retrotranscribed using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, California, USA). Levels of mRNA were assessed by qPCR, using a mix containing 5 ng of cDNA, 100 nM of each primer (Supplementary Table\u0026nbsp;2), and 1\u0026times; SYBR Green PCR Master Mix (Applied Biosystems Negative cDNA controls were cycled in parallel at each run). PCR was run in a 96-well StepOne Real-Time System (Applied Biosystems, Waltham, Massachusetts, USA). Data are shown as relative quantification (2\u0026thinsp;\u0026minus;\u0026thinsp;\u003csup\u003eΔΔCt\u003c/sup\u003e). GAPDH expression was used to normalize cDNA inputs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eValues were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was calculated as follows: Mann-Whitney test (for non-parametric dataset) for comparison between coupled experimental groups, while one-way ANOVA with Bonferroni's post-test (for parametric dataset) or Kruskal-Wallis (for non-parametric dataset), were performed for multiple comparisons. The data were analyzed using GraphPad Prism version 9.5.0 (GraphPad Software, San Diego, California USA). A p-value (P)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEvaluation of the loss of slit diaphragm proteins in podocytes after SRNS serum treatment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eImmortalized human podocytes, cultured in two-dimensional conditions, were treated for 24h with 2% or 10% serum from MDR-SRNS or genetic-SRNS patients. A significant increase in cell granularity was also observed, after the treatment with either 2% or 10% of both MDR and genetic-SRNS patients\u0026rsquo; sera compared to the CTRL condition (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs\u003c/em\u003e the corresponding CTRL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA three-dimensional dynamic glomerular model was then applied to study the effect of SRNS serum on the GFB (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A significant increase in the permeability to BSA-FITC was observed when the glomerulus was subjected to 2% MDR serum compared to controls (2% CTRL) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In contrast, in genetic SRNS-treated podocytes, the increase in filtration didn\u0026rsquo;t reach a statistical significance (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eImportant changes in GFB permeability were accompanied by substantial modifications in relevant proteins composing the slit diaphragm structure. Immunofluorescence evaluation of the 3D glomerulus showed contraction of actin filaments, with an accumulation to the cell periphery in podocytes treated with 2% MDR and genetic-SRNS serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, top and Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Moreover, the slit diaphragm component nephrin (NPHS1) was reduced and formed cytoplasmic aggregates after both SRNS sera treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). TEM highlighted the loss of pedicel structure from the SRNS-treated podocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). qRT-PCR analysis of slit diaphragm components Nephrin (\u003cem\u003eNPHS1\u003c/em\u003e), synaptopodin (\u003cem\u003eSYNPO\u003c/em\u003e), and CD2 Associated Protein (\u003cem\u003eCD2AP)\u003c/em\u003e, showed a statistically significant increase in their expression in podocytes treated with MDR and genetic-SRNS serum compared to cells exposed to CTRL serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The expression of \u003cem\u003eSYNPO\u003c/em\u003e was also significantly reduced in MONO compared to MDR treatment (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). The administration of MDR serum was again more effective in altering the SD on podocytes than the genetic-SRNS serum treatment. Modification in the membrane localization of the vascular marker endoglin (CD105) was observed in glomerular endothelial cells (GEC) after the SRNS treatment, showing an alteration also in the endothelial compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, middle panel).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAA synthesis pathway perturbance in podocytes induced by SRNS sera treatment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eChanges in podocyte lipid components were analyzed by gas chromatography. The list of FAs changes is reported in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A significant rise in MUFA was detected in MDR-treated podocytes compared to the CTRL-treated ones; this was accompanied by a significant reduction in SFA in this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, P\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Among the analyzed FAs, the AA synthesis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) was the most affected in podocytes treated with SRNS serum (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In detail, the treatment with serum from MDR patients led to a significant increase in AA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This was associated with an increase in the expression of FADS-1, converting DHLA to AA, only in MDR-treated podocytes with respect to control (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Conversely, the upstream enzymes in the AA synthesis pathways were differentially affected by the diverse SRNS treatments. We identified that \u003cem\u003eFADS-2\u003c/em\u003e, responsible for LA to GLA conversion, was upregulated only after genetic-SRNS treatment (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs\u003c/em\u003e MDR and \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs\u003c/em\u003e CTRL). This was related to an increase in the LA substrate in podocytes treated with serum from genetic-SRNS (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs\u003c/em\u003e CTRL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The enzyme \u003cem\u003eELOVL-5\u003c/em\u003e, converting GLA to DHLA, was induced by both MDR and genetic-SRNS administration compared to the control (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). By immunofluorescence, we measured the changes in the density of actin filaments concomitant with AA pathway disturbance. The actin mass was mainly gathered in the podocyte periphery (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) and was strongly reduced after both MDR- and genetic-SRNS administration (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs\u003c/em\u003e CTRL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eFatty acid composition in podocytes cultured in the 3D glomerular model.\u003c/b\u003e Fatty acid abundance was reported as median values with the minimum to maximum range. P value was reported in the table. NS: not statistically significant; \u003cb\u003ea\u003c/b\u003e MDR \u003cem\u003evs\u003c/em\u003e CTRL, \u003cb\u003eb\u003c/b\u003e MONO \u003cem\u003evs\u003c/em\u003e CTRL; non-parametric Kruskal-Wallis test with Dunn\u0026rsquo;s post hoc.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eFatty acids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003ePodocytes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChemical name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTRL (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMDR (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMONO (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePalmitic acid (PA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e16:0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.91 (24.86\u0026ndash;49.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.97 (22.30-54.79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.59 (25.34\u0026ndash;49.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePalmitoleic acid (POA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e16:1n7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.64 (0.31\u0026ndash;3.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.75 (1.07\u0026ndash;12.19) \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.80 (0.10\u0026ndash;7.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003cem\u003ea\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStearic acid (SA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e18:0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23.18 (14.77\u0026ndash;50.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.39 (15.05\u0026ndash;22.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.26 (15.23\u0026ndash;34.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOleic acid (OA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e18:1n9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.11 (3.29\u0026ndash;22.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.84 (15.06\u0026ndash;26.80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.98 (8.19\u0026ndash;30.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ecis-vaccenic acid (CVA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e18:1n7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.73 (0.09\u0026ndash;4.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.23 (2.25\u0026ndash;6.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.29 (1.47\u0026ndash;6.46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLinoleic acid (LA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e18:2n6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.90 (0.66\u0026ndash;5.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.84 (2.90\u0026ndash;5.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.92 (3.49\u0026ndash;7.08) \u003cb\u003eb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003cem\u003eb\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eα-Linolenic acid (ALA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e18:3n3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.23 (0.01\u0026ndash;4.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.20 (0.06\u0026ndash;2.49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17 (0.01\u0026ndash;0.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMead acid (MA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20:3n9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.21 (0.04\u0026ndash;3.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.18 (0.02\u0026ndash;0.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.14(0.04-15.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDihomo-γ-linolenic acid (DHLA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20:3n6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.56 (0.42\u0026ndash;1.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42 (0.20\u0026ndash;0.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.49 (0.10\u0026ndash;0.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eArachidonic acid (AA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20:4n6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.09 (1.27\u0026ndash;4.79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.37 (3.39\u0026ndash;8.37) \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.71 (2.79\u0026ndash;9.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003cem\u003ea\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTimaodonic acid (TA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20:5n3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.27 (0.08\u0026ndash;4.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.34 (0.06\u0026ndash;2.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.41 (0.19\u0026ndash;3.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBehenic acid (BA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e22:0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.74 (0.32\u0026ndash;7.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73 (0.37\u0026ndash;19.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.57 (0.19\u0026ndash;0.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDocosapentaenoic acid\u0026nbsp;(DPA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e22:5n3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.66 (0.13\u0026ndash;2.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.38 (0.20\u0026ndash;2.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.90 (0.39\u0026ndash;3.76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLignoceric acid (LCA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e24:0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.93 (0.30\u0026ndash;1.52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.90 (0.30\u0026ndash;1.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.57 (0.42\u0026ndash;0.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClupanodonic acid\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(DPAω3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e22:6n3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.09 (0.14\u0026ndash;2.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.53 (0.11\u0026ndash;2.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.98 (0.36\u0026ndash;3.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNervonic acid (NA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e24:1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.38 (0.25\u0026ndash;2.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.63 (0.08\u0026ndash;1.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.58 (0.33\u0026ndash;2.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSaturated Fatty Acids (SFA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64.72 (52.35\u0026ndash;84.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.65 (39.89\u0026ndash;64.30) \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.24 (35.94\u0026ndash;77.96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003cem\u003ea\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMonounsaturated Fatty Acids (MUFA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.44 (6.24\u0026ndash;31.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.18 (19.32\u0026ndash;38.23) \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.94 (12.58\u0026ndash;38.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003cem\u003ea\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePolyunsaturated Fatty Acids (PUFA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.01 (7.03\u0026ndash;24.74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.89 (9.14\u0026ndash;21.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.46 (9.38\u0026ndash;34.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSRNS is a heterogeneous disorder including immune-related and genetic etiologies\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The presence of unknown circulating factors has been previously related to the loss of slit diaphragm structure in children with non-genetic SRNS\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, but their actual contribution is still undefined. Moreover, a clear understanding of the molecular mechanisms activated in damaged podocytes during SRNS is not fully recognized. We employed a glomerular 3D millifluidic model mimicking the GFB to elucidate the mechanisms behind glomerular damage in different forms of SRNS. Sera from multi-drug SRNS were able to induce both disruption of the slit diaphragm structure and changes in the expression and localization of slit diaphragm proteins in respect to both genetic SRNS and control group. The exposure of cultured podocytes to MDR- and genetic-SRNS serum induced disruption of arachidonic acid (AA) synthesis pathway, with different intermediate players involved.\u003c/p\u003e \u003cp\u003eThe alteration in the permeability of the GFB is the main cause of proteinuria in INS. In steroid-resistant INS children, commonly associated with focal segmental glomerulosclerosis (FSGS), the loss of GFB structure has been associated with direct podocyte damage\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A 3D co-culture of human glomerular endothelial cells and podocytes was established to mimic the GFB. Only glomerular endothelial cells and podocytes exposed to MDR serum, presented significantly higher permeability to albumin compared to controls. This was accompanied by an induction of granularity in podocytes expose to the serum from multi-drug resistant and to less extent to the genetic SRNS, but not in response to serum from healthy subjects. These data confirmed previous work by Li et al.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, showing the same activity of serum derived from recurrent SRNS using a static glomerular model. Unexpectedly, in our system, serum derived from genetic SRNS was also able to induce an effect on podocytes, without increasing albumin permeability corroborating the absence of a circulating permeability factor(s) in this group of NS. Despite that, the presence of a pro-inflammatory microenvironment in SRNS\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e could potentially explain the detrimental effect resulting in podocyte damage, notwithstanding different degrees of injury between the immune-associated and genetic forms of SRNS. Moreover, our data strongly abet the recent research, which confirms the existence of multiple etiopathogenic agents causing kidney damage progression in INS\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGFB loss of structure was also accompanied by morphological changes observed after 48 hours of serum incubation. In our 3D model, the treatment of podocytes resulted in nephrin redistribution from their surface. Loss of nephrin localization along the foot processes was previously correlated with progressive proteinuria resulting in the development of FSGS in adult glomeruli\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Interestingly, a contrasting upregulation of genes coding for \u003cem\u003eNPHS1\u003c/em\u003e and \u003cem\u003eSYNPO\u003c/em\u003e, as well as for the scaffold molecule \u003cem\u003eCD2AP\u003c/em\u003e, was observed after MDR and to less extent genetic-SRNS treatments. Noteworthy, the mRNA levels of different slit diaphragm proteins were identified as elevated in isolated glomeruli from multiple proteinuric kidney diseases\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In parallel, SRNS-treated glomerular endothelial cells co-cultured in the dynamic glomerular system showed a marked decrease in the endothelial marker, CD105. Our findings confirmed that podocyte impairment could be dependent on pathogenetic events happening in glomerular endothelial cells during INS\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlteration in lipid metabolism is closely associated with numerous proteinuric kidney diseases\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Podocytes exposed to SRNS serum showed changes in FA composition. A significant increase in monounsaturated FAs and a decrease in saturated FAs was observed only in the MDR-treated group. The enhancement in monounsaturated FAs content was previously associated with CKD and inflammation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The main differences in FAs in MDR-treated podocytes were observed in the polyunsaturated \u003cem\u003en-6\u003c/em\u003e FA, AA. Such alterations may be produced by a synergic activity of serum components, including lipids which alteration in INS has been previously described\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, we distinctly identified changes in the transcripts coding for enzymes involved in the AA synthesis pathways after 48h of podocyte treatment with MDR serum. This strongly supports that the FA changes we observed, particularly AA alterations of the podocytes, were mainly dependent on their synthesis. Furthermore, these findings are in line with studies indicating an upregulation of the AA synthesis pathway in several inflammatory diseases including INS\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Deregulation in AA synthesis has been shown to alter the actin cytoskeleton of podocytes, affecting foot process formation, and the integrity of the filtration barrier\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Consistently, our data highlighted a significant remodeling of the actin cytoskeleton probably related to the AA induction in MDR-treated podocytes and with a lesser magnitude in genetic-SRNS treatment compared to controls.\u003c/p\u003e \u003cp\u003eIn summary, our study provides important insights into the role of FA deregulation, podocyte damage, and proteinuria development in steroid-resistant INS. In our hands, even though the different etiopathology of INS forms, the podocyte damage induced by SRNS serum seems to embody the kidney response to a cumulative detrimental environment worsening the course of the INS pathology. Nevertheless, it suggests that defects in molecular pathways involved in podocytes\u0026rsquo; AA synthesis could be related to the GFB impairment induced by the treatment with sera from MDR patients. Further studies with larger sample sizes are needed to establish the causality between AA alteration and kidney damage in MDR-SRNS, the most severe form of this disease and determine its possible therapeutic implications.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in the published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the IRCCS Ca\u0026rsquo; Granda Institutional Review Board (ID 2633, INSiDe protocol). An informed consensus has been signed by the parent and/or legal guardian of all the children enrolled in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank the Microscopy facility of the Istituto Nazionale of Genetica Molecolare (INGM) for Imaging assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by PSR 2022, PI Starting Grant from the University of Milano (UNIMI); and Grant P-0038 from IMPACTsim S.p.A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: G.B., L.B., F.C.; Experiments and acquisition of experimental data: G.B., L.B., S.T., G.C., F.Ca.; Data collection: W.M., C.T., S.V., D.P.; Analysis and interpretation of data: G.B., L.B., G.C., F.C.; Draft of the manuscript: F.C.; G.B. Figures: L.B., G.B.; Funding and resources acquisition: F.C., G.M.; Revision of the manuscript: F.C., G.M. All the authors revised and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSeeger, H. \u0026amp; Fehr, T. Das nephrotische Syndrom beim Erwachsenen \u0026ndash; Ursachen und Komplikationen. \u003cem\u003ePraxis\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 259\u0026ndash;267 (2016).\u003c/li\u003e\n\u003cli\u003eTrautmann, A. \u003cem\u003eet al.\u003c/em\u003e Spectrum of Steroid-Resistant and Congenital Nephrotic Syndrome in Children. \u003cem\u003eClinical Journal of the American Society of Nephrology\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 592\u0026ndash;600 (2015).\u003c/li\u003e\n\u003cli\u003eYe, Q. \u003cem\u003eet al.\u003c/em\u003e The immune cell landscape of peripheral blood mononuclear cells from PNS patients. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 13083 (2021).\u003c/li\u003e\n\u003cli\u003eTullus, K., Webb, H. \u0026amp; Bagga, A. 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Lipotoxicity can affect podocytes inducing kidney damage. In this study, we evaluate the effects of SRNS serum on podocyte functionality and lipid metabolism.\u003c/p\u003e\u003ch2\u003eMethods.\u003c/h2\u003e \u003cp\u003eA three-dimensional (3D) dynamic \u003cem\u003ein vitro\u003c/em\u003e glomerulus was incubated with serum from multi-drug resistant (MDR) and genetic SRNS or healthy controls. The glomerular filtration barrier (GFB) integrity, podocyte viability, and fatty acids (FAs) composition were evaluated by serum albumin permeability estimation, cytofluorimetric analysis and gas chromatography, respectively. Expression of slit diaphragm molecules and FA-related enzymes was analyzed by immunofluorescence and PCR.\u003c/p\u003e\u003ch2\u003eResults.\u003c/h2\u003e \u003cp\u003eSerum from SRNS patients induced cell granularity, increased GFB permeability, and disrupted slit diaphragm protein structure. The podocyte damage was most severe when MDR serum was administered compared to the serum of genetic-SRNS. This was associated with a significant upregulation of the transcripts coding for nephrin, synaptopodin, and CD2AP. An alteration of fatty acid profile in MDR-treated podocytes was observed, with increased monounsaturated FAs following the decrease of saturated FAs. The exposure of cultured podocytes to MDR- and genetic-SRNS serum induced disruption of arachidonic acid (AA) synthesis pathway, with different intermediate players involved.\u003c/p\u003e\u003ch2\u003eConclusion.\u003c/h2\u003e \u003cp\u003eThis study highlights the detrimental effects of serum from SRNS patients on podocyte function and the association of AA synthesis pathway with the podocyte damage.\u003c/p\u003e","manuscriptTitle":"Effects of steroid-resistant nephrotic syndrome serum on AA pathway in podocytes cultured in 3D in vitro glomerular model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 16:52:53","doi":"10.21203/rs.3.rs-4684821/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-07T04:07:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-06T19:00:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248383159461551333778036823390883475460","date":"2024-07-16T15:13:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266698634999842837096954684122506089220","date":"2024-07-16T14:54:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-14T13:31:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-08T12:11:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-08T10:08:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-08T10:06:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-04T08:05:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"da63e9f6-3ec0-4784-888d-9d71a4895cec","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35183225,"name":"Biological sciences/Biological techniques"},{"id":35183226,"name":"Biological sciences/Cell biology"},{"id":35183227,"name":"Health sciences/Diseases"},{"id":35183228,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2025-04-21T16:06:17+00:00","versionOfRecord":{"articleIdentity":"rs-4684821","link":"https://doi.org/10.1038/s41598-025-95216-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-14 15:57:45","publishedOnDateReadable":"April 14th, 2025"},"versionCreatedAt":"2024-08-09 16:52:53","video":"","vorDoi":"10.1038/s41598-025-95216-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-95216-2","workflowStages":[]},"version":"v1","identity":"rs-4684821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4684821","identity":"rs-4684821","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}