ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: PO0507

Functional Maturation of Kidney Organoid Tubules: Mechanosensitive Ca2+ Signaling

Session Information

Category: Bioengineering

  • 300 Bioengineering

Authors

  • Carrisoza-Gaytan, Rolando, Icahn School of Medicine at Mount Sinai Department of Pediatrics, New York, New York, United States
  • Soong, Joanne, Icahn School of Medicine at Mount Sinai Department of Pediatrics, New York, New York, United States
  • Hanss, Basil G., Icahn School of Medicine at Mount Sinai Department of Medicine, New York, New York, United States
  • Kroll, Katharina T., Harvard University John A Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts, United States
  • Hiratsuka, Ken, Harvard Medical School Department of Medicine, Boston, Massachusetts, United States
  • Gupta, Navin R., Harvard Medical School Department of Medicine, Boston, Massachusetts, United States
  • Morizane, Ryuji, Harvard Medical School Department of Medicine, Boston, Massachusetts, United States
  • Lewis, Jennifer A., Harvard University Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, United States
  • Satlin, Lisa M., Icahn School of Medicine at Mount Sinai Department of Pediatrics, New York, New York, United States
Background

When grown under static conditions, kidney organoids derived from human pluripotent stem cells exhibit glomerular- and tubular-like structures. However, static organoids possess an “immature” gene expression profile and their vascular development is limited (Homan et al., 2019; Takasato et al., 2015). As the technology for culturing organoids advances, aiming to promote terminal differentiation, the need to better characterize their physiological function has become a priority. To begin to functionally phenotype static organoids, we focused on characterizing mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and signaling pathways in tubules isolated from maturing static organoids.

Methods

Tubular structures, microdissected from organoids between 21-57 d of culture, were microperfused in vitro or affixed to the base of a specimen chamber, and loaded with Fura-2 AM (20 µM) to measure [Ca2+]i. Digital ratio imaging was performed in individually identified cells by epifluorescence microscopy using commercial software.

Results

The average baseline [Ca2+]i in microperfused tubules was 189±13 nM (n=6). A rapid increase in [Ca2+]i was observed when tubules were subject to luminal filling, sufficient to cause circumferential stretch and turbulent flow, reaching values of 404±186 and 719±78 nM in organoids at 40 and 57 d of culture, respectively (n=3, p≤0.002 vs. baseline). Luminal flow-induced increases in [Ca2+]i were not detected in tubules isolated from organoids <40 d in culture (n=3). Mechanosensitive Piezo1 channels contribute to the flow-induced [Ca2+]i response in the fully differentiated distal tubule (Carrisoza-Gaytan et al., EB 2019). Nonperfused organoid tubules exposed to basolateral Piezo1 activator Yoda 1 (20 µM) exhibited increases in [Ca2+]i from 110±36 to 272±114 nM (24-31 d in culture; n=4, p≤0.00001) and from 130±36 to 504±197 nM (43-67 d in culture; n=4, p≤0.002).

Conclusion

These preliminary results are consistent with a maturational increase in flow/stretch-sensitive Ca2+ channels, including Piezo1, and/or associated signaling pathways, in tubules of static organoids in culture.

Funding

  • NIDDK Support