A simple method for the isolation and detailed characterization of primary human proximal tubule cells for renal replacement therapy

Abstract

The main physiological functions of renal proximal tubule cells in vivo are reabsorption of essential nutrients from the glomerular filtrate and secretion of waste products and xenobiotics into urine. Currently, there are several established cell lines of human origin available as in vitro models of proximal tubule. However, these cells appeared to be limited in their biological relevance, because essential characteristics of the original tissue are lost once the cells are cultured. As a consequence of these limitations, primary human proximal tubule cells constitute a suitable and a biologically more relevant in vitro model to study this specific segment of the nephron and therefore, these cells can play an important role in renal regenerative medicine applications. Here, we describe a protocol to isolate proximal tubule cells from human nephrectomies. We explain the steps performed for an in-depth characterization of the cells, including the study of markers from others segments of the nephron, with the goal to determine the purity of the culture and the stability of proteins, enzymes, and transporters along time. The human proximal tubule cells isolated and used throughout this study showed many proximal tubule characteristics, including monolayer organization, cell polarization with the expression of tight junctions and primary cilia, expression of proximal tubule–specific proteins, such as megalin and sodium/glucose cotransporter 2, among others. The cells also expressed enzymatic activity for dipeptidyl peptidase IV, as well as for gamma glutamyl transferase 1, and expressed transporter activity for organic anion transporter 1, P-glycoprotein, multidrug resistance proteins, and breast cancer resistance protein. In conclusion, characterization of our cells confirmed presence of putative proximal tubule markers and the functional expression of multiple endogenous organic ion transporters mimicking renal reabsorption and excretion. These findings can constitute a valuable tool in the development of bioartificial kidney devices.