Abstract: FR-PO0166
Superphysiologic Insulin Concentrations in Media Undermine Differentiation of Cultured Primary Renal Tubule Cells
Session Information
- Diabetic Kidney Disease: Metabolic, Mitochondrial, and Inflammatory Mechanisms
October 23, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Cardiovascular-Kidney-Metabolic Health
- 601 Cardiovascular-Kidney-Metabolic Health: Basic
Authors
- Evans, Rachel C., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Roy, Shuvo, University of California San Francisco, San Francisco, California, United States
- Fissell, William Henry, University of Alberta Faculty of Medicine & Dentistry, Edmonton, Alberta, Canada
Background
Tubule cells in culture rapidly lose the transcriptional, functional, and ultrastructural features of their in vivo counterparts. Attenuation of phenotype undermines the application of cell culture to many medical applications. Cell culture protocols often include superphysiologic concentrations of insulin, even though renal tubule cells do not require insulin for glucose transport into the cytoplasm. We examined the in vitro effect of varying insulin concentrations on apicobasal function and localization of proteins involved in brush border assembly.
Methods
Primary human renal proximal tubule cells (hRPTC, Lonza) were cultured on permeable supports in 50:50 DMEM/F12 with hydrocortisone, T3, selenium, transferrin, antibiotics, insulin-like growth factor 1, and insulin (0, 0.5, 5, and 1720 pmol/ml). Apicobasal transport was measured gravimetrically. PIP2, PIP3, Ezrin and EPS8 distributions in the cell were imaged with indirect immunofluorescence.
Results
Cells grew to confluence in all cases. Ezrin and EPS8 demonstrated the most distinct apical localization at physiologic insulin concentrations, compared to 0 and 1720pmol/ml. There was subtly less PIP2 evident by immunofluorescence, although PIP3 concentrations seemed similar between conditions. Apicobasal fluid transport was similar between conditions.
Conclusion
Insulin signaling activates PIP3K, converting (4,5)PIP2 to (3,4,5)PIP3, a crucial step in Akt/Protein Kinase B signaling. PIP2 is an essential phospholipid anchoring ezrin and the actin cytoskeleton to the apical membrane. We hypothesized that superphysiologic concentrations of insulin might alter PIP2/PIP3 balance and undermine brush border assembly. We showed that indeed in conventional high-insulin media, ezrin and EPS8 seem less organized and PIP2 seems less abundant in the apical cytoskeleton. However, physiologic insulin concentrations did not increase apicobasal transport compared to high insulin.
Funding
- Private Foundation Support