Diglycolic Acid Is the Nephrotoxic Metabolite in Diethylene Glycol Poisoning Inducing Necrosis in Human Proximal Tubule Cells In Vitro

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.

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Freshly isolated primary human proximal tubule cells as an in vitro model for the detection of renal tubular toxicity

Toxicology ◽

10.1016/j.tox.2020.152535 ◽

2020 ◽

Vol 442 ◽

pp. 152535 ◽

Cited By ~ 1

Author(s):

Piyush Bajaj ◽

Git Chung ◽

Keith Pye ◽

Tomoya Yukawa ◽

Akio Imanishi ◽

...

Keyword(s):

Proximal Tubule ◽

In Vitro Model ◽

Proximal Tubule Cells ◽

Tubular Toxicity ◽

Tubule Cells ◽

Vitro Model ◽

Renal Tubular ◽

Human Proximal Tubule

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Nephrotoxicity Testing In Vitro: The Current Situation

Alternatives to Laboratory Animals ◽

10.1177/026119299702500506 ◽

1997 ◽

Vol 25 (5) ◽

pp. 497-503

Author(s):

Jean-Paul Morin ◽

Marc E. De Broe ◽

Walter Pfaller ◽

Gabriele Schmuck

Keyword(s):

Proximal Tubule ◽

Culture Media ◽

Task Force ◽

Primary Cultures ◽

Phenotypic Expression ◽

Transepithelial Transport ◽

Specific Cell ◽

Proximal Tubule Cells ◽

Tubule Cells

An ECVAM task force on nephrotoxicity has been established to advise, in particular, on the follow-up to recommendations made in the ECVAM workshop report on nephrotoxicity testing in vitro. Since this workshop was held, in 1994, there have been several improvements in the techniques used. For example, the duration of renal slice viability, and the maintenance of functional activities in slices, have been improved by using dynamic incubation systems with higher oxygen tensions and more-appropriate cell culture media. Highly differentiated primary cultures of pig, human and rabbit proximal tubule cells have been established by using specific cell isolation procedures and/or selective culture media. To date, the most comparable phenotypic expression and transepithelial transport capacities to proximal tubules in vivo have been obtained with primary cultures of rabbit proximal tubule cells which are grown on bicompartmental supports; in this system, transepithelial substrate gradients are generated and the transepithelial transport of both organic anions and cations is highly active. This in vitro system has been selected by ECVAM for further evaluation and prevalidation. Industrial needs in the area of nephrotoxicity testing have been identified, and recommendations are made at the end of this report concerning possible future initiatives.

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