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Kidney Week

Abstract: PO0390

An Improved Protocol for the Isolation and Monoculture of Primary Murine Renal Peritubular Endothelial Cells to Enhance Long-Term Phenotypic Retention

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Thompson, Austin D., The University of Arizona College of Pharmacy, Tucson, Arizona, United States
  • Janda, Jaroslav, The University of Arizona College of Pharmacy, Tucson, Arizona, United States
  • Schnellmann, Rick G., The University of Arizona College of Pharmacy, Tucson, Arizona, United States

Group or Team Name

  • Rick G. Schnellmann's Lab
Background

Acute kidney injury (AKI) is often followed by a persistent reduction in mitochondrial function, microvasculature (MV) dysfunction/rarefication, and tubular injury/necrosis. While the functional restoration of the renal MV is crucial, the mechanisms by which MV angiogenesis improves renal recovery remain understudied. Unfortunately, primary cultures of renal microvascular endothelial cells (RMEC) exhibit variability in purity and outgrowth, and undergo phenotypic losses in monocultures. Thus, we focused on refining the isolation and phenotypic retention of monocultured mouse renal peritubular endothelial cells (MRPEC).

Methods

MRPEC were initially isolated using the method of Zhao et al (2014). MRPEC were then subjected to a second round of CD146+ magnetic bead purification. Twice purified MRPEC (TP-MRPEC) were seeded in cloning cylinders within 35mm culture dishes precoated with fibronectin or gelatin, and incubated overnight at 37°C with 5% CO2 in ScienceCell™ EC media. Cloning cylinders were removed the next day, and media was changed every 24h. After 48h, cells were placed onto a circular rotor in a 37°C incubator. After 10 days, purity and phenotype were assessed by flow cytometry analysis using a CD146-PE+ antibody (Biolegend™) and confirmed by immunofluorescent (IF) staining of endothelial markers VE-Cadherin and CD31.

Results

Brightfield micrographs revealed that TP-MRPEC seeded in cloning cylinders increased seeding density, promoted faster outgrowth, and preserved MRPEC morphology. MRPEC seeded onto fibronectin exhibited faster outgrowth; however, MRPEC seeded onto gelatin reduced morphological variability. MRPEC placed onto a circular rotor set to ~45rpm enhanced endothelial cell polarization and paracrine signaling. Flow cytometry analysis revealed that standard MRPEC had an average PE+ purity of ~76% compared to the IgG isotype and unstained controls. Conversely, TP-MRPEC average purity was ~93% (N=5, T(x)=829.09). Immunofluorescence staining confirmed high levels of CD-31 and VE-Cadherin 10 days after isolation (EVOS-M500).

Conclusion

In conclusion, we have developed a modified MRPEC isolation and cell monoculture protocol that enhances the uniformity, purity, and long-term phenotypic retention of primary MRPEC monocultures.

Funding

  • Veterans Affairs Support