Novel regulatory mechanisms of the glomerular endothelium

University Of Southern California, Los Angeles CA

Investigators

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Abstract

Glomerular endothelial cells (GEnC) play pivotal roles in the maintenance and function of the glomerular filtration barrier (GFB), and endothelial injury is a primary event in the development of chronic kidney disease (CKD). Several systemic and local secreted factors derived from podocytes and mesangial cells including insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF) have been established to help maintain the specialized GEnCs and the GFB. In the previous grant cycle our group discovered a key, but formerly overlooked, regulatory role of macula densa (MD) cells in glomerular endothelial function in health and disease. Unexpectedly, the roles include the secretion of novel MD-specific angiogenic, tissue growth, patterning, and extracellular matrix (ECM) remodeling factors and their paracrine mechanistic actions to control resident progenitor cells and regulate endogenous kidney tissue remodeling and regeneration. This renewal proposal aims to extend the novel, paradigm-shifting concept of glomerular endothelial regulation by the MD. We focus on the #1 top enriched MD-specific factor - pregnancy-associated plasma protein A2 (Pappa2), as the most proximal and principal GEnC regulating cellular and molecular pathway. Specifically, we hypothesize that MD- derived secreted Pappa2 and cell communication network (CCN1) are key determinants in the maintenance of a healthy glomerular endothelium and GFB by acting as central physiological regulators of IGF1-VEGF signaling efficiency in GEnCs. Preliminary work identified MD and GEnC-specific high expression and functional activity as well as angiogenic effects of the complementary enzyme/ligand/receptor pairs of MD Pappa2/CCN1 and GEnC Igf1r/Vegfr2/Itg/ECM signaling axis in mouse, rat, and human kidney. Single-cell genetic cell fate tracking with serial intravital multiphoton microscopy (MPM) confirmed clonal GEnC remodeling by endothelial precursor cells localized in the glomerular arterioles closest to the MD. Pappa2 knockout or Vegfr2 blockade resulted in endothelial injury, GFB dysfunction, glomerulosclerosis (GS), albumin leakage, all improved with CCN1 treatment. This project will use a comprehensive experimental approach that includes GEnC and MD cell cultures in vitro, new transgenic Cdh5-Confetti BalbC mouse and Pappa2 knockout rat models with both males/females in consideration of expected sex-specificity. Genetic single-cell fate tracking, disease models, serial MPM, and AAV9-mediated MD-specific gene delivery in vivo will investigate and therapeutically translate the key endothelial regulatory functions of MD-derived Pappa2. The specific aims are to (i) Examine the functional importance of maintaining the MD Pappa2-CCN1 balance for normal function of the glomerular endothelium in physiological conditions, (ii) Determine the disease modifying role of the MD Pappa2-CCN1 balance in endothelial injury, (iii) Test the therapeutic potential of MD-targeting Pappa2/CCN1 gene therapy in vivo for CKD.