In vitro assay for predicting renal proximal tubular cell toxicity
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一种肾近曲小管、细胞的技术，应用在预测肾近曲小管细胞毒性的体外试验领域
Inactive Publication Date: 2015-05-20
AGENCY FOR SCI TECH & RES
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A recently developed high-throughput mitochondrial nephrotoxicity assay based on rabbit cells (25) raises questions about interspecies variability
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Embodiment 1
[0071] Human primary renal proximal tubular cells (HPTC) were observed to display marked changes in cell morphology when treated with nephrotoxic substances (see figure 1 ). Also, a reduction in the number of cells due to cell death was observed. The assay uses nuclei counts and changes in cell morphology (eg, cell area and roundness) to assess toxicity to HPTC.
[0072] First, cells were seeded into multi-well plates and cultured for 3 days. It was then exposed to the test compound for 16 hours. The cells used were commercially available HPTCs (American Type Culture Collection; "ATCC") and HPTCs isolated by the inventors from fresh human kidney samples.
[0073] Cells were left untreated or treated with 0.2 mg / ml of tetracycline (ATCC cells) or gentamicin (inventors isolate).
[0074] Cells were then fixed and stained, stained with and detected for F-actin. Treatment with these nephrotoxic substances resulted in morphological changes as follows: F-actin alignment, decrea...
Embodiment 2
[0077] This example was performed using a commercially available HPTC (ATCC).
[0078] Cells were seeded into multi-well plates and cultured for 3 days. Then, the cells were left untreated, or treated with gentamicin (2.5 mg / ml), CdCl 2 (10 μg / ml), aristolochic acid (100 μg / ml), cisplatin (100 μg / ml) or DMSO (50 μg / ml).
[0079] After 24 hours, cells were imaged by phase-contrast microscopy ( figure 2 , gentamicin image and DMSO image not shown).
Embodiment 3
[0081] The following different types of renal proximal tubular cells were used: HPTC (ATCC), LLC-PK1 cells and HK-2 cells.
[0082] The cells were cultured and treated as described in Example 2, using 0-500 μg / ml of CuCl 2 , CdCl 2 、K 2 Cr 2 o 7 or tenofovir ( Figure 3-5 ) or gentamicin, tetracycline, 5-fluorouracil or As 2 o 3 ( Figure 6 and 7 )deal with. Cells were stained, imaged and counted. Different types of PTCs respond differently to compounds, and HPTC is the most sensitive ( Figure 3-5 ).
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Abstract
There is provided an in vitro assay for screening a test compound for toxicity in renal proximal tubular cells. The method comprises contacting a test compound with a test population of renal proximal tubular cells; and examining one or more cell morphology features, examining one or more cytoskeleton features, and / or determining cell numbers of the renal proximal tubular cells in the test population and comparing such cell morphology, arrangement of cytoskeletal components and / or cell count with the respective features of a control population. A change in one or more cell morphology features, a change in arrangement of one or more cytoskeleton features or a decrease in cell numbers of the test population relative to the control population is indicative that the test compound is toxic for renal proximal tubular cells.
Description
[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to US Provisional Application No. 61 / 674,024, filed July 20, 2012, the contents of which are incorporated herein by reference. technical field [0003] The present invention relates to in vitro assay methods for predicting the toxicity of compounds to renal proximal tubular cells, including predicting in vivo toxicity. Background technique [0004] The kidney is one of the main target organs of drug-induced toxicity. Nephrotoxic drugs and chemicals induce acute kidney injury (AKI) or chronic kidney disease with subsequent progression to end-stage renal disease (ESRD) (1-3). Patients with AKI and ESRD have increased morbidity and mortality and are dependent on dialysis (1,4,5). About 5% of all hospitalized patients and about 20%-30% of ICU patients develop AKI, and about 20%-25% of these cases are attributable to nephrotoxic drugs (2-4). When alternative and new age...
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