The renin-angiotensin system is present and functional in ipsc-derived kidney organoids

A.S. Shankar, S.S. Korevaar, I.M. van den Berg-Garrelds, J. Gribnau, C.C. Baan, A.H.J. Danser, E.J. Hoorn, M.J. Hoogduijn

Thursday 14 march 2019

11:20 - 11:30h at Tropentheater

Categories: Best abstracts, Parallelsessie

Parallel session: Parallelsessie XII – Best abstracts III

Background: The intrarenal renin-angiotensin system (RAS) arises early during kidney development and is important for proper nephrogenesis. The lack of a suitable human model to study the intrarenal RAS hampers deeper investigation into the relevance of this system. Recently protocols for the in vitrogeneration of kidney organoids from induced pluripotent stem cells (iPSC) have been developed.

Methods: Therefore, in this study we investigated the presence and functionality of the RAS in iPSC-derived kidney organoids. Four human iPSC lines were grown on Geltrex and treated with CHIR99201 (a Wnt agonist) and fibroblast growth factor 9, after which the cells were pelleted and transferred to a transwell membrane. The resulting organoids showed a 10- to 40-fold increase in mRNA for kidney-specific markers after 25 days of culture, including a marker for renin-producing stromal cells, FOXD1. Essential renal structures were observed using immunohistochemistry. Moreover, there was a 10-fold increase in the expression of the organic anion transporters OAT1 and OAT3, suggesting tubular function.

Results: Interestingly, the mRNA level of angiotensinogen (AGT) increased more than 100-fold as early as day 7 of the culture in comparison to iPSC and remained stable until day 25. Also, angiotensin receptor type 1 and type 2 mRNA expression increased and remained highly expressed throughout the culture, while high levels of ACE were also maintained in kidney organoids. Finally, a 10- to 100-fold increase in the mRNA expression of renin was observed at day 25. The use of an indirect enzyme-kinetic assay revealed the functionality of renin in the kidney organoids at day 25, as measured by the conversion of exogenously administered AGT to angiotensin I. Moreover, analysis of the medium harvested from kidney organoid cultures at day 25 exhibited varying amounts of renin activity, ranging from 12 to 200 ng angiotensin I/ml per hour. The addition of the cyclic AMP-elevating agents forskolin and dibutyryl cyclic AMP to the culture for 24 hours increased the mRNA expression of renin drastically (up to 1000-fold), indicating that the production of renin in the kidney organoids may be a regulated and inducible process.

Conclusions: In summary, we demonstrate the presence and functionality of components of the RAS in human iPSC-derived kidney organoids. This provides the opportunity to study the intrarenal RAS and its regulation in an in vitrohuman model.