Abstract P410: Characterization of the Renin-angiotensin System in Induced Pluripotent Stem Cell-derived Human Kidney Organoids

Abstract

Intrarenal renin-angiotensin system (RAS), including proximal tubular angiotensinogen (AGT), plays crucial roles in the progression of hypertension, kidney injury, and kidney development and has been investigated in vivo using animal models. For translational relevance, we sought to further our investigations in human tissue. This study investigated RAS expression and AGT regulation by histone deacetylase 9 (HDAC9), an epigenetic repressor of AGT, in human iPSC-derived kidney organoids. After pre-treatment of human iPSC with CHIR99201, a glycogen synthase kinase inhibitor, and fibroblast growth factor 9, cells were moved to transwell membranes. Cells were harvested on day 0, 5, 12 or 18 to determine mRNA copy numbers of developmental markers and RAS genes by digital PCR. Marker genes of renal structures were induced during the culture with concomitant decrease in progenitor markers including Cited1. Immunostaining revealed that the organoids contain podocytes, proximal tubules expressing AGT and distal tubules. Angiotensin II type 1 receptor (AT1R) levels were higher than other RAS components on day 0 and the expression was downregulated on day 5. AT2R induction peaked on day 5 and reduced until day 18. Renin expression was strongly induced on day 5 and sustained until day 18. Angiotensin-converting enzyme levels were moderately augmented during the culture. AGT levels were elevated on day 12 and remained until day 18 (7.6-fold, ratio to day 0). Conversely, HDAC9 levels decreased by day 18. On day 18, AGT and HDAC9 levels were inversely correlated in a CHIR99201 concentration-dependent manner. Moreover, an HDAC9 inhibitor increased AGT expression (2.25±0.21-fold, ratio to control). These results suggest that all RAS components are expressed and independently regulated during the development of iPSC-derived human kidney organoids. The existence of all RAS components including high renin expression, their regulation, and epigenetic regulation of AGT in the organoids support previous findings in rodent models. Concerted effort, including this study, to overcome technical challenges to generate complete nephrons will provide human kidney organoids to study development, pathophysiological mechanisms, novel drugs and clinical therapies.