Kidney Week

Abstract: PO0507

Functional Maturation of Kidney Organoid Tubules: Mechanosensitive Ca2+ Signaling

Session Information

• Bioengineering: Organoids and Organs-on-a-Chip
  November 04, 2021 | Location: On-Demand, Virtual Only
  Abstract Time: 10:00 AM - 12:00 PM

Category: Bioengineering

• 300 Bioengineering

Authors

• Carrisoza-Gaytan, Rolando, Icahn School of Medicine at Mount Sinai Department of Pediatrics, New York, New York, United States

Rolando Carrisoza-Gaytan, PhD

• Soong, Joanne, Icahn School of Medicine at Mount Sinai Department of Pediatrics, New York, New York, United States

Joanne Soong,

• Hanss, Basil G., Icahn School of Medicine at Mount Sinai Department of Medicine, New York, New York, United States

Basil G. Hanss,

• Kroll, Katharina T., Harvard University John A Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts, United States

Katharina T. Kroll,

• Hiratsuka, Ken, Harvard Medical School Department of Medicine, Boston, Massachusetts, United States

Ken Hiratsuka,

• Gupta, Navin R., Harvard Medical School Department of Medicine, Boston, Massachusetts, United States

Navin R. Gupta,

• Morizane, Ryuji, Harvard Medical School Department of Medicine, Boston, Massachusetts, United States

Ryuji Morizane,

• Lewis, Jennifer A., Harvard University Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, United States

Jennifer A. Lewis,

• Satlin, Lisa M., Icahn School of Medicine at Mount Sinai Department of Pediatrics, New York, New York, United States

Lisa M. Satlin,

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