Serum-independent modulation of hemicyst formation by dissolved oxygen in postconfluent epithelial monolayers

Abstract

Hemicyst formation is considered a manifestation of either transepithelial solute and fluid movement or secretory activity in culture. This study shows that hemicyst formation in postconfluent monolayers of rhesus monkey kidney (LLC-MK2) cells is modulated by the dissolved oxygen concentration (PO2) of the culture medium. Either daily replacement of serum-free medium or displacement of the gas phase with 18% vol/vol O2 (initial medium PO2 = 125 to 135 mm Hg) enhances formation of hemicysts. Use of 30% O2 (medium PO2 approximately equal to 175 mm Hg) does not further increase the incidence, but neither 10% O2 (medium PO2 = 90 to 95 mm Hg) nor 1% O2 (medium PO2 = 35 to 50 mm Hg), the approximate range of dissolved oxygen values in blood, supports hemicyst formation unless cultures are gently rocked to disrupt diffusion gradients. Phase photomicrography of living cultures shows that the surface of a turgid hemicyst is furrowed, and cinephotomicrography reveals that the walls vibrate subtly. When hypoxic conditions (0 to 1% O2) are introduced this vibration ceases within 2 to 3 h, whereas collapse and disappearance of turgid hemicysts requires 18 to 20 h, seems virtually synchronous, and is reversible. Hemicysts form in a broad osmotic range, and increased electrolyte concentration increases the incidence. Hemicysts persist in locally dense areas when cell-free strips are etched in the postconfluent monolayer; no DNA synthesis is detected under these conditions, but two-dimensional cell spreading into the denuded area is seen along the edge of the wound. We conclude that the dissolved oxygen supply in the cellular microenvironment modulates functional expression by differentiated kidney epithelial cells in culture and that increased electrolyte concentration also enhances expression of this phenotypic marker.