The Renin-Angiotensin System is present and functional in human ipsc-derived kidney organoids

A.S. Shankar, H. Tejeda Mora, T.P.P. van den Bosch, S.S. Korevaar, I.M. van den Berg -Garrelds, J. Gribnau, M.C. Clahsen-van Groningen, C.C. Baan, A.H.J. Danser, E.J. Hoorn, M. Hoogduijn

Wednesday 4 march 2020

12:40 - 12:50h at Theaterzaal

Parallel session: Plenaire sessie II - Best abstracts

Background: Renin production in the adult kidney dictates salt, blood pressure and fluid volume homeostasis. The lack of suitable human models to study the RAS and the regulation of renin hampers deeper investigation into the relevance of this system. Recently protocols for the in vitro generation of kidney organoids from human induced pluripotent stem cells (iPSC) have been developed.

Methods: We wanted to examine whether the hormonal function of the kidney can be emulated in this in vitro model.

Results: To confirm that we could successfully generate kidney organoids, single cell sequencing analysis (SC-seq) of a kidney organoid after 25 days of culture was performed. Differentiated kidney-specific cell populations consisting of podocytes, proximal tubular cells, distal tubular cells, stromal cells and a small cluster of endothelial cells were revealed. Subsequently, these presence of these cell types was confirmed in situ using immunohistochemistry. Second, components of the RAS were expressed in kidney organoids. Angiotensinogen mRNA increased 100-fold from day 1 to day 7 and was shown to be primarily present in proximal tubuli, while 75-fold increase in angiotensin receptor type 1 and 10-fold increase type 2 mRNA during differentiation could be attributed to expression in the stromal cell cluster. During kidney organoid differentiation renin-producing cells appear in this stromal cluster, which contains cells characteristic of pericytes and mesenchymal stem cells. The addition of the cyclic AMP-elevating agent forskolin to the culture for 24 hours further increased the mRNA expression of renin up to 1000-fold (p<0.05). Moreover, the use of an enzyme-kinetic assay revealed a 20-fold (p<0.05) increased renin activity in the forskolin-treated kidney organoids. Analysis of the medium harvested from forskolin-treated kidney organoid cultures exhibited a similar significant increase of renin activity, confirming secretion of functional renin by the kidney organoids. Immunostaining confirmed the presence of renin-producing cells which could be observed in the stromal compartment in close association with glomerular and tubular cells. Lastly, kidney organoids were subcutaneously implanted in immune deficient mice(IL2Ry^-/- RAG2^-/-DKO) and harvested after 1 month follow-up. Implantation led to vascularisation and further maturation, as demonstrated by the presence of glomerular and tubular structures using immunohistochemistry. When stimulation with forskolin was performed on explanted kidney organoids, there was a significant 10-fold increase of functional renin secreted into the supernatant.

Conclusions: Therefore, kidney organoids have the ability to maintain kidney-specific hormonal production after implantation. This may have beneficial implications for future use in transplantation and kidney regeneration. Furthermore, the kidney organoid model can provide novel insights into the regulation of renin in an in vitro setting.