The effect of serum from children with nephrotic syndrome on transcriptome of human immortalized podocytes cultured in vitro

Nephrotic syndrome (NS) is an unfavorable disease with heterogeneous causes and variable prognosis. One of the clinically crucial prognostic feature is the response to initial glucocorticoid treatment. Whereas in some children with steroid-sensitive nephrotic syndrome (SSNS) the treatment may induce long-term remission of the disease, steroid-resistant cases (SRNS) are at risk of renal failure requiring renal replacement therapy. The aim of this project was to explore whether sera from children with the two main clinically defined types of NS induce changes in transcriptome of in-vitro cultures of immortalized human podocytes. After 3 days of culture, total RNA was isolated and then library from polyadenylated RNA was prepared and RNA sequencing was performed. Log2 (fold-changes) were calculated to describe differential gene expression. Within the Reactome online tool, Camera methodology was implemented to identify functionally linked gene groups (pathways) that could distinguish the two patient groups. Significant transcriptome differences were found between samples from children with SSNS and SRNS. This may help to reveal the molecular mechanisms of the disease and open up for an effective individualized treatment. Overall design: SSNS serum samples (n = 9) were obtained at disease manifestation, before the administration of glucocorticoids. SRNS serum samples (n = 4) were obained from patients, who had long-term remission and did not take any immunosuppressive drugs for at least 6 months before blood sampling. Fully differentiated immortalized human podocytes were incubated with 10 % serum samples at 37 °C in 5 % CO2 for 72 hours, in triplicates. The cells were lysed and the RNA was isolated using spin-column method. The RNA libraries were prepared from polyadenylated RNA and sequenced by NextSeq instrument. Sequencing data were processed by STAR program and DESeq2. Pathway analysis was performed by Reactome online software.