Expression of renal cell protein markers is dependent on initial mechanical culture conditions

Abstract

The rotating wall vessel is optimized for suspension culture, with laminar flow and adequate nutrient delivery, but minimal shear. However, higher shears may occur in vivo. During rotating wall vessel cultivation of human renal cells, size and density of glass-coated microcarrier beads were changed to modulate initial shear. Renal-specific proteins were assayed after 2 days. Flow cytometry antibody binding analysis of vitamin D receptor demonstrated peak expression at intermediate shears, with 30% reduction outside this range. Activity of cathepsin C showed the inverse pattern, lowest at midshear, with twofold increases at either extreme. Dipeptidyl-peptidase IV had no shear dependence, suggesting that the other results are specific, not universal, changes in membrane trafficking or protein synthesis. On addition of dextran, which changes medium density and viscosity but not shear, vitamin D receptor assay showed no differences from controls. Neither cell cycle, apoptosis/necrosis indexes, nor lactate dehydrogenase release varied between experiments, confirming that the changes are primary, not secondary to cell cycling or membrane damage. This study provides direct evidence that mechanical culture conditions modulate protein expression in suspension culture.

Gene expression and survival changes in Saccharomyces cerevisiae during suspension culture

“…For example, the flow of biological fluid such as blood or renal tubular fluid exerts the same type of surface shear on venous and arterial endothelium, and renal tubular cells, as the cells experience in the RWV (Malek and Izumo, 1995;Nerem et al, 1998;Resnick and Gimbrone, 1995). A number of biological differences have been identified in prokaryotic and eukaryotic cells grown under different shear levels, with different shear levels optimal for different cells types or features in the same cells (Cowger et al, 2002;Kaysen et al, 1999;Petersen et al, 1988). The S. cerevisiae deletion series is a powerful tool that can be used to identify specific steps in the metabolic response to shear and help piece together the complicated relationship between transcriptional activation and strain survival (Winzeler, 1999).…”

“…Strains were grown to stationary phase, diluted, and grown in RWVs placed at 0 (static), 0.2 (rotator), or 1 dyne/cm 2 (shaker) (Cowger et al, 2002). Yeast produce a large amount of CO 2 when grown for extended time periods at a high cell density (>3e06 cells/mL), which can overwhelm the gas exchange capacity of the RWV and lead to a toxic buildup within the vessel (Hammond and Hammond, 2001).…”

The Bonn Criteria: Minimal Experimental Parameter Reporting for Clinostat and Random Positioning Machine Experiments with Cells and Tissues

“…In our own lab, we examined whether the expression of renal cell protein markers is dependent on initial mechanical culture conditions (Cowger et al 2002). During rotating wall vessel cultivation of human renal cells, size and density of glass-coated microcarrier beads were changed to modulate initial shear, and renal-specific proteins were assayed after 2 days.…”

Metabolic inhibition-induced transient Ca2+increase depends on mitochondria in a human proximal renal cell line

“…SV-40 immortalized HCRE cells (primary cells obtained from Clonetics prior to immortalization) were used in this study. HCRE cells were previously characterized and used as a model of renal proximal epithelial cells (3,9,25). They show proximal tubular features including proximal tubular enzyme markers (␥-glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase), form a polarized monolayer with apical microvilli, and express the proximal tubular receptors cubulin and megalin (25).…”

“…Human cortical renal epithelial cells (HCRE) (3,9,25), which possess the characteristic brush border markers of the proximal tubule and have been used in the past as a model for this segment, were selected. Inhibitors of ATP production were utilized because they have been used extensively to study ischemic injury in polarized epithelial cells (2,11,28,46).…”