Focal segmental glomerulosclerosis (FSGS) is the most common glomerular pathology that leads to progression and end-stage kidney disease during childhood. There is a lack of non-invasive, reliable biomarkers that predict the diagnosis, prognosis and outcome of FSGS. Furthermore, FSGS diagnosis can be delayed as most of the patients with nephrotic syndrome do not undergo kidney biopsies until they have failed steroid therapy. Increased concentrations of circulating lipids and intracellular lipid deposits is a common finding in FSGS. However how lipid anomalies are linked to progression remains unsolved. We discovered a distinct urinary lipid metabolite panel in FSGS that predicted the diagnosis and prognosis of FSGS by untargeted lipidomics analysis. Patients with FSGS displayed increased urinary concentrations of lysophosphotidycholine (LPC) and free fatty acids (FFA). We propose that implementation of this phospholipid profile as a biomarker panel is a powerful tool in prediction of diagnosis and prognosis of FSGS and to monitor response to treatment. SA-1 of this proposal will further validate the use of phospholipid and FFA panel by measuring these lipid metabolites in baseline serum and urine samples of patients collected in a national cohort of patients with FSGS and minimal change disease (MCD) (NEPTUNE) by targeted lipid analysis. The enrolled patients have completed at least 5 years of follow-up. We will assess the correlation between clinical outcomes (change in urine protein excretion and kidney function) and concentrations of the lipid metabolites in patient biosamples obtained at the entry to NEPTUNE. We will investigate sensitivity, specificity, positive predictive value, and negative predictive value of lipid biomarkers in diagnosis and prognosis of FSGS. SA- 2 of this proposal, will examine the mechanism of increased urinary LPC and FFA in an animal model of FSGS. We demonstrated increased activation of cytosolic phospholipase A2 (cPLA2) enzyme that breaks down membrane phosphotidylcholines to LPC and FFA in podocytes and tubular epithelial cells in a mouse model of FSGS. In SA-2 our goal is to investigate the role of cPLA2 in cellular injury and progression of FSGS in Fyn–/–Cd2ap+/– bigenic FSGS mouse model with cPLA2 gene knock-out. In parallel with mouse experiments, we will use BuffaloMna rats to investigate cPLA2 expression in this spontaneous FSGS model. We hypothesize that inhibition of cPLA2 will alleviate cellular injury and progression in FSGS. In this research proposal our goal is to develop a novel lipid metabolite biomarker panel that will aid in prediction of diagnosis and prognosis of FSGS. Furthermore, this novel biomarker panel will improve the way treatment response is monitored. Delineation of the mechanism of lipid mediated injury will lead to development of novel treatments in FSGS. Our overarching goal is to improve the care and outcome of FSGS patients by developing novel diagnostic and prognostic lipid bioma...