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

doi:10.21203/rs.3.rs-4684821/v1

Download PDF

Article

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

https://doi.org/10.21203/rs.3.rs-4684821/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

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

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.

Idiopathic nephrotic syndrome (INS) is characterized by the presence of proteinuria, edema, and hyperlipemia¹. 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 survival⁴

INS is characterized by the leakage of proteins as well as lipids into the urine and a concomitant increase of blood-free fatty acids⁵. 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 disease¹³. 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 milieu¹⁴.

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.

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 isolated¹⁵ 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 virus¹⁷. 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 immortalized¹⁸ and cultured in EndoGRO^LS Complete Culture Media (Merck Millipore) and 10% FBS.

Podocyte granularity assay

Podocytes (10⁵ cells/well) in monoculture were treated for 24h with 2% or 10% sera from SRNS patients and controls. The podocyte granularity was evaluated as described¹⁸ 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 described¹⁵. 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 bioreactor¹⁵. 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 described¹⁹. 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.

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. 1a).

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 < 0.01) (Fig. 1b). In contrast, in genetic SRNS-treated podocytes, the increase in filtration didn’t reach a statistical significance (P > 0.05) (Fig. 1b).

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. 1c, top and Supplementary Figure S1). Moreover, the slit diaphragm component nephrin (NPHS1) was reduced and formed cytoplasmic aggregates after both SRNS sera treatments (Fig. 1c). TEM highlighted the loss of pedicel structure from the SRNS-treated podocytes (Fig. 1d). 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. 1e). 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. 1c, 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. 3a) 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. 3b). 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. 3c). 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. 3b). 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. 3c). 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. 3d) and was strongly reduced after both MDR- and genetic-SRNS administration (P < 0.001 and P < 0.01 vs CTRL) (Fig. 3e).

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)	Pa < 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	Pb < 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)	Pa < 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)	Pa < 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)	Pa < 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

SRNS is a heterogeneous disorder including immune-related and genetic etiologies²¹. The presence of unknown circulating factors has been previously related to the loss of slit diaphragm structure in children with non-genetic SRNS²², 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 damage²³. 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 SRNS²⁵ 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 INS²³.

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 glomeruli²⁶. 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 diseases²⁷. 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 diseases²⁹. 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 described³². 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 INS³³. Deregulation in AA synthesis has been shown to alter the actin cytoskeleton of podocytes, affecting foot process formation, and the integrity of the filtration barrier¹¹. 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.

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.

Seeger, H. & Fehr, T. Das nephrotische Syndrom beim Erwachsenen – Ursachen und Komplikationen. Praxis 105, 259–267 (2016).
Trautmann, A. et al. Spectrum of Steroid-Resistant and Congenital Nephrotic Syndrome in Children. Clinical Journal of the American Society of Nephrology 10, 592–600 (2015).
Ye, Q. et al. The immune cell landscape of peripheral blood mononuclear cells from PNS patients. Sci Rep 11, 13083 (2021).
Tullus, K., Webb, H. & Bagga, A. Management of steroid-resistant nephrotic syndrome in children and adolescents. Lancet Child Adolesc Health 2, 880–890 (2018).
Claudio, P. & Gabriella, M. Nephrotic syndrome: pathophysiology and consequences. J Nephrol 36, 2179–2190 (2023).
Gai, Z. et al. Lipid Accumulation and Chronic Kidney Disease. Nutrients 11, 722 (2019).
Yonezawa, S., Kawasaki, Y., Natori, Y. & Sugiyama, A. Improvement of LXR-mediated lipid metabolism in nephrotic model kidney accompanied by suppression of inflammation and fibrosis. Biochem Biophys Res Commun 666, 122–127 (2023).
Shi, Y. et al. Downregulation of PTEN promotes podocyte endocytosis of lipids aggravating obesity-related glomerulopathy. American Journal of Physiology-Renal Physiology 318, F589–F599 (2020).
Yang, X. et al. CD36 Promotes Podocyte Apoptosis by Activating the Pyrin Domain-Containing-3 (NLRP3) Inflammasome in Primary Nephrotic Syndrome. Medical Science Monitor 24, 6832–6839 (2018).
PAN, Q. et al. Role of basophils in the pathogenesis of minimal change nephrotic syndrome: A literature review. Exp Ther Med 8, 1027–1031 (2014).
Yang, L. et al. Excessive arachidonic acid induced actin bunching remodeling and podocyte injury via a PKA-c-Abl dependent pathway. Exp Cell Res 388, 111808 (2020).
Martínez-García, C. et al. Renal Lipotoxicity-Associated Inflammation and Insulin Resistance Affects Actin Cytoskeleton Organization in Podocytes. PLoS One 10, e0142291 (2015).
Priante, G., Musacchio, E., Valvason, C. & Baggio, B. EPA and DHA suppress AngII- and arachidonic acid-induced expression of profibrotic genes in human mesangial cells. J Nephrol 22, 137–43 (2009).
Turolo, S. et al. Role of Arachidonic Acid and Its Metabolites in the Biological and Clinical Manifestations of Idiopathic Nephrotic Syndrome. Int J Mol Sci 22, 5452 (2021).
Bellucci, L., Montini, G., Collino, F. & Bussolati, B. Mesenchymal Stromal Cell-Derived Extracellular Vesicles Pass through the Filtration Barrier and Protect Podocytes in a 3D Glomerular Model under Continuous Perfusion. Tissue Eng Regen Med 18, 549–560 (2021).
Conaldi, P. G. et al. Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells. J Virol 71, 9180–9187 (1997).
Collino, F. et al. Preeclamptic sera induce nephrin shedding from podocytes through endothelin-1 release by endothelial glomerular cells. American Journal of Physiology-Renal Physiology 294, F1185–F1194 (2008).
den Braanker, D. J. W. et al. Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis. Nephrology Dialysis Transplantation 36, 247–256 (2021).
Turolo, S. et al. Persistent Abnormalities of Fatty Acids Profile in Children With Idiopathic Nephrotic Syndrome in Stable Remission. Front Pediatr 8, (2021).
Chan, P.-M., Tan, Y.-S., Chua, K.-H., Sabaratnam, V. & Kuppusamy, U. R. Attenuation of Inflammatory Mediators (TNF-α and Nitric Oxide) and Up-Regulation of IL-10 by Wild and Domesticated Basidiocarps of Amauroderma rugosum (Blume & T. Nees) Torrend in LPS-Stimulated RAW264.7 Cells. PLoS One 10, e0139593 (2015).
Lee, J. M., Kronbichler, A., Shin, J. Il & Oh, J. Current understandings in treating children with steroid-resistant nephrotic syndrome. Pediatric Nephrology 36, 747–761 (2021).
Saleem, M. A. Molecular stratification of idiopathic nephrotic syndrome. Nat Rev Nephrol 15, 750–765 (2019).
Veissi, S. T. et al. Circulating Permeability Factors in Focal Segmental Glomerulosclerosis: In Vitro Detection. Kidney Int Rep 7, 2691–2703 (2022).
Li, M. et al. 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).

No competing interests reported.

Supplementarymaterialdef.pdf

Download PDF

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

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

Status:

Version 1

Abstract

Background and hypothesis.

Methods.

Results.

Conclusion.

Figures

Key points

Introduction

Materials and Methods

Patients and sample collection

Cell lines

Podocyte granularity assay

Permeability test

Fatty acid analysis by gas chromatography

Immunofluorescence

PCR analysis

Statistical Analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1