PROJECT SUMMARY/ABSTRACT The scope of the problem is that 50% of patients with focal segmental glomerulosclerosis (FSGS), the leading cause of primary proteinuric glomerular disease in adults, progress to chronic kidney disease. Thus, developing new treatment strategies to improve FSGS patient outcomes is of utmost importance. Yet, there is a large unmet need in our mechanistic understanding of disease progression. The traditional view in FSGS is that podocytes are central to its pathology and that they are the first cell type to be injured. A more contemporary view is an expansion of this paradigm and includes injury to neighboring glomerular cells. This grant will focus on glomerular endothelial cells (GEnCs). The rationale is that (1) in FSGS, GEnCs decrease in number, loose their glycocalyx and their fenestrae widen; (2) patients with GEnC damage have the highest rates of disease progression; (3) GEnC injury scores for all glomerular diseases are highest in FSGS; (4) GEnC genes/biomarkers are linked to the lowest remission rates and poor long-term outcomes of FSGS. Yet the understanding of how podocytes cause secondary GEnC injury is very incomplete. To begin to close the knowledge gap, we undertook an in silico approach to identify paracrine ligand-receptor signaling networks between podocytes and GEnCs. Surprisingly, we discovered that experimental and human FSGS results in a senescence associated secretory phenotype (SASP) and an activated inflammasome phenotype in non-aged podocytes, both of which are characterized by the secretion of distinct classes of signaling mediators. Our preliminary data shows that inhibiting the NLRP3 inflammasome in podocytes improves GEnC health. Our specific aims will test two hypotheses: Specific Aim #1 tests the hypothesis that SASP and inflammasome activation from injured podocytes are responsible for GEnC damage. Specific Aim #2 tests the hypothesis that interfering with the paracrine injury signals from injured podocytes is a therapeutic target to prevent GEnC dys- function in FSGS. The innovative experimental approaches we will use include: (i) In vivo loss-of-function validation using mutant mice, in which we can reduce/inhibit the SASP or inflammasome phenotypes in podocytes in the setting of experimental FSGS, and then measure the impact on GEnC health; (ii) in vivo gain- of-function validation using transgenic mice, in which we can forcibly induce either a SASP or inflammasome phenotype in podocytes to understand the impact on GEnCs; (iii) Quality-by-Design utilizing a systematic, high complexity approach based on the Design-of-Experiment theory and Multivariate Data Analysis to address the contribution of the podocyte secretome on GEnC health and function. As such, this proposal is highly significant for its short-term impact on understanding the crosstalk between podocytes and GEnCs in FSGS, and for its long-term impact in developing new therapeutic strategies to lower the risk and magnitude for se...