Project description:Glucocorticoid resistance complicates the treatment of ~20% of children with nephrotic syndrome, yet the molecular basis for resistance remains unclear. We generated the transcriptome profile by RNA sequencing of peripheral blood leukocytes from children obtained both at initial nephrotic syndrome presentation and after ~7 weeks of glucocorticoid therapy to identify genes or a gene panel able to differentiate steroid sensitive from steroid resistant nephrotic syndrome. RNA -seq analysis was followed by in-silico algorithm-based approaches and subsequent biochemical analyses on relevant candidate gene with important roles in podocyte and glomerular pathophysiology, using both patient samples and experimental models of nephrotic syndrome and podocyte injury.
Project description:This study aimed to evaluate gene expression patterns in urinary sediment samples of children with steroid-resistant nephrotic syndrome (SRNS).
Project description:Paired citrate plasma samples were collected from 27 steroid-sensitive and 14 steroid-resistant nephrotic syndrome participants prior to beginning treatment and after an average of 7 weeks (range 3-19 wks) of treatment with a corticosteroid.
Project description: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.
2022-10-14 | GSE215231 | GEO
Project description:A gut microbiota study about steroid children steroid-dependent primary nephrotic syndrome
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
