RPTEC/TERT1 CELLS CULTURED IN MONOLAYER ARE NOT SUITABLE FOR ASSESSING APOLIPOPROTEIN A-I UPTAKE DUE TO THE LACK OF CUBILIN-AMNIONLESS (CUBAM) COMPLEX

Abstract BACKGROUND AND AIMS Renal proximal tubule epithelial cells (RPTECs) are responsible for solute and protein reabsorption. It has been described that the human RPTEC/TERT1 cell line expresses several transporters, including the cubilin–amnionless–megalin complex that is responsible for the uptake of a broad range of filtered proteins. Therefore, this cell line is considered a gold standard to perform receptor-mediated protein endocytosis assays. All lipoproteins interact with megalin to be reabsorbed except for apolipoprotein A-I (ApoA-I), which is dependent on cubilin–amnionless (CubAm) presence. Here, we aimed to confirm whether megalin and CubAm were correctly expressed and located at the cell membrane of RPTEC/TERT1 cells to later perform ApoA-I uptake assays. METHOD We first tested the specificity of the antibodies against cubilin, amnionless and megalin by immunofluorescence using histological sections of healthy human kidney tissue. Afterwards, RPTEC/TERT1 cells (purchased at ATCC, passage 1) were cultured using conventional culture plates for 7 days and also in transwells for 3 weeks to induce cell polarization. In these conditions, we studied cubilin, amnionless and megalin gene expression by RT-PCR and protein expression by immunofluorescence after fixation with 4% PFA. RESULTS As expected, in human kidney tissue, cubilin, amnionless and megalin were located at the brush border of the proximal tubular cells confirming the specificity of the tested antibodies. Although cubilin, amnionless and megalin were present at the RNA level in RPTEC/TERT1 cells cultured in conventional culture plates, immunocytochemistry analysis revealed that only megalin was located at the cell membrane in a few cells, cubilin was not expressed and amnionless was found in the nucleus (see Figure 1). When the RPTEC/TERT1 cells were cultured in transwells, even though the cells were clearly polarized, cubilin and megalin were not expressed, and amnionless was still not found at the correct location. CONCLUSION RPTEC/TERT1 cell line when cultured in monolayer expresses cubilin, amnionless and megalin at the RNA level, but only megalin could be located at the cell membrane of the cells. Although further research is needed, our results suggest that this cell model is not suitable for performing ApoA-I endocytosis assays as it lacks the CubAm complex at the cell membrane in the tested conditions.