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

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. 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. 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. 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.