Kidney Week

Abstract: TH-PO384

Influence of Gravity on Ureteric Bud Branching and Organ Morphology

Session Information

• Development, Organoids, Injury, and Regeneration
  October 24, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development, Stem Cells, and Regenerative Medicine

• 600 Development, Stem Cells, and Regenerative Medicine

Authors

• Rocha, Miguel Angel, VA Greater Los Angeles Healthcare System, Los Angeles, California, United States

Miguel Angel Rocha, MS

• Reichelt-Wurm, Simone, Universitat Regensburg Fakultat fur Medizin, Regensburg, Bayern, Germany

Simone Reichelt-Wurm, PhD

• Hamon, Morgan, University of California Los Angeles, Los Angeles, California, United States

Morgan Hamon, PhD

• Chang, Hsiao-Min, University of California Los Angeles, Los Angeles, California, United States

Hsiao-Min Chang, PhD

• Yanagawa, Norimoto, University of California Los Angeles, Los Angeles, California, United States

Norimoto Yanagawa, MD

• Banas, Miriam C., Universitat Regensburg Fakultat fur Medizin, Regensburg, Bayern, Germany

Miriam C. Banas

• Hauser, Peter V., University of California Los Angeles, Los Angeles, California, United States

Peter V. Hauser, PhD

Background

In vitro organ culture of metanephroi is a standard technique to study renal development and regeneration. While many aspects of the renal anatomy develop normally, the overall morphology of cultured kidneys appears mono-dimensional (pancake-like). We aimed to study the influence of gravity on 3D morphology and branching to grow kidneys with organotypic anatomy.

Methods

E12.5 kidneys obtained from Hoxb7 ^Venus mice were cultured in vitro under static, or dynamic conditions for up to 144h. Ureteric bud (UB) branching morphogenesis was imaged by confocal microscopy on kidneys at 72h + 96h. 3D images were used to measure Branching length, diameter, and branching angle, utilizing gradient vector-based software (TreeSurveyor). Micro Computer Tomography (mCT) images were generated from kidneys at 48h, 72h, 96h, and 120h to compare 3D morphology and volume. Microarray analysis, using GeneChip Mouse Gene 2.0 ST arrays, was performed to uncover gene expression of samples at 72h + 144h. Liquid Chromatography tandem mass spectrometry was used to compare protein expression in samples after 96h of static and dynamic culture.

Results

mCT imaging showed that dynamic culture supports organotypic morphology in contrast to static cultured kidneys, which present mono-dimensional. Cell count of kidneys showed no significant differences between the groups from 0h to 144h, demonstrating normal cell growth in both conditions. UB branching showed similar branch lengths and diameters, but the branching angle average was greater in dynamically cultured kidneys compared to static conditions. Microarray analysis displayed that dynamic culture induced significant differential regulation of genes associated with extracellular matrix (ECM) and receptor interaction, down-regulation of apoptotic processes, mechanical stimulation, bone mineralization, blood pressure regulation, cell migration, and others. Proteomic analysis identified >4000 different proteins. Dynamic culture conditions induced changes in the abundance of proteins associated with focal segmental glomerulosclerosis, Wnt signaling, ECM, pluripotency pathways, and more.

Conclusion

We think dynamic in vitro culture of metanephroi supports organotypic morphology of the developing kidney. Studies are under way to delineate the effect on specific pathways associated with mechanical stimulation.

Funding

• Veterans Affairs Support