POS-389 PODOCYTE MATURATION IN HUMAN KIDNEY ORGANOIDS IS ACCELERATED WITH RENIN-ANGIOTENSIN SYSTEM ACTIVATION

Abstract

The global prevalence of chronic kidney disease (CKD) is approximately 10% and rising. Current renal replacement modalities (dialysis and transplant) carry significant socioeconomic burden and may struggle to meet rising demands. PSC-derived kidney organoids have emerged as a promising alternative for providing functional nephrons to CKD patients. However, this nascent technology faces numerous challenges most pressing of which is maturation. Organoid differentiation protocols rely on application of morphogens in a temporal restricted fashion mimicking ureteric bud and metanephric mesenchyme development. However, organoids are poorly vascularized and resemble first and second trimester fetal kidneys. The renin-angiotensin system (RAS) is critical for normal kidney morphogenesis guiding glomerular, vascular and ureteric formation. Indeed, prenatal exposure to RAS inhibitors is associated with kidney dysgenesis and mortality. Can RAS modulators like angiotensin 2 (ANG2) and RAS inhibitors alter kidney organoid maturation? We hypothesize that ANG2 and losartan perturb developmental programs within nephron progenitor populations, alter cell fate specification and maturation of glomerular epithelial cells (i.e. podocytes). We performed single-cell RNA sequence analyses on human iPSC derived kidney organoids treated acutely (24hrs) with vehicle, ANG2 alone, losartan alone, or ANG2 and losartan on the 28th day of the Takasato protocol. Kidney organoids expressed all major RAS genes. AGT, ACE1 and ACE2 were restricted to proximal tubule clusters while REN (renin), AGTR1 and AGTR2 were preferentially expressed in stromal cells. ACE1 and REN were also found in endothelial cells. ATP6AP2 (pro-renin receptor) was ubiquitously expressed. REN expression was downregulated by ANG2 and normalized with losartan pre-treatment suggesting a functioning RAS network. Unbiased clustering identified 3 late podocyte (LP) clusters (NPHS2hiPODXLhi), 1 early podocyte (EP) cluster (OLFM3+NPHS2loPDOXLlo), 1 parietal epithelial (PEC) cluster (CLDN1+CAV2+) and 2 nephron progenitor (NPC) clusters (SIX2+CITED1+). RNA velocity analysis revealed 2 distinct differentiation trajectories: NPC – EP – LP and NPC – EP – PEC – LP. The EP and NPC transcriptional profiles were disproportionately sensitive to ANG2 as we noted significant upregulation of podocyte differentiation genes such as BMP7, FOXC2 PODXL and NPHS2. Moreover, ANG2 depleted the immature populations (NPC and EP) and expanded mature cell cell states (PEC and LP). On the other hand, losartan downregulated the podocyte differentiation programs and increased the proportion of immature cells (EP and NPC) to mature (PEC and LP) cells. The proportion of glomerular epithelial cells relative to all cells was unchanged across treatments. Human kidney organoids developed a functioning intraorganoid RAS resembling in vivo intrarenal RAS. The lineage plasticity observed between NPCs, EPs, PECs and LPs was sensitive to acute RAS perturbation. ANG2 accelerated podocyte maturity while RAS inhibition held podocytes in an immature state. Next steps include testing different ANG2 treatment durations, validate findings using immunocytochemistry, investigate the podocyte differentiation programs closely and their relationship to RAS, and reproduce this analysis on endothelial and tubule cells.