Abstract
Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.
Highlights
The kidney, a key organ of the urinary system, plays a pivotal role in many physiological processes such as the maintenance of homeostasis, the excretion of nitrogen catabolism waste and the secretion of endocrine factors
Cells were isolated by FACS using antibodies to CD10 and CD13, two markers previously described in PT cells [2,8]
Only about 15% of CD10/CD13 cells that were initially double negative remained negative for both markers from the second cell passage onward (Figure 8D). This de novo expression of proximal tubule markers suggests the dedifferentiation of primary distal tubular epithelial cells in culture (Figure 8D). Cell models such as renal cell lines and primary cultures are currently used for studies of renal physiology and nephrotoxicicity
Summary
The kidney, a key organ of the urinary system, plays a pivotal role in many physiological processes such as the maintenance of homeostasis, the excretion of nitrogen catabolism waste and the secretion of endocrine factors. In renal pathology and injury, all these processes are altered and accompanied by several symptoms: hypertension due to the alteration of the renin/angiotensin system and/or an imbalance of calcium and phosphorus metabolism induced by the deficiency of calcitriol [1] Studying these pathophysiological mechanisms requires the use of in vitro models such as renal cell cultures. Among the different cell types, proximal tubular epithelial cells (PT cells) play a major role in the reabsorption of substances such as glucose and amino acids and the control of acid-base balance by the excretion of almost all the bicarbonate and the synthesis of ammonia [3]. PT cells are sensitive to ischemic injury, and represent a primary target for xenobiotics, such as nephrotoxins (and their metabolites), whose effects can extend up to the kidney failure [5,6]
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