2015 Progress Report: Organotypic Model of Human Kidney as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials

EPA Grant Number: R835738C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R835738
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials
Center Director: Faustman, Elaine
Title: Organotypic Model of Human Kidney as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
Investigators: Kelly, Edward J.
Institution: University of Washington
EPA Project Officer: Klieforth, Barbara I
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2014 through November 30,2015
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

One of the primary objectives of our project is to design, implement and test a tissue-engineered human kidney microphysiological system and to evaluate the response of exposure to engineered nanomaterials (ENMs). To this end, we evaluated the toxicological effects of two forms of silver nanoparticles (AgNPs) with an organotypic microfluidic device that utilizes the Nortis™ microphysiological (MPS) system, which accurately reflects human renal physiology with the culturing of primary human proximal tubule epithelial cells (PTEC) in a physiologically relevant 3-D configuration and an appropriately scaled lumenal flow rate. 

Progress Summary:

The AgNPs used for our ENM exposure assessment in the kidney MPS were previously assessed in human and mouse hepatocytes by Terry Kavanagh’s laboratory who is a member of the University of Washington Nanotoxicology Center (part of the NIEHS Centers for Nanotechnology Health Implications Research (NCNHIR) Consortium). Before exposing PTECs to AgNPs in an MPS, we conducted a series of dose-range finding experiments with AgNPs-exposed PTECs growing in a 96-well format and evaluated cell morphology, cell viability/health and mitochondrial function to adjust our exposure concentrations. We also used these preliminary experiments as an opportunity to optimize conditions for working with the AgNPs in our serum-free cell culture media as the AgNPs we evaluated tend to aggregate in media with low protein content and salt concentrations typically found in physiological fluids. 

For our AgNPs exposure assessments using the MPS, we exposed PTECs to either 5 or 25 µg/mL of 20 nm citrate AgNPs or 20 µg/mL of 20 nm polyvinylpyrrolidone (PVP)-coated AgNPs for 48 hours and evaluated for cell-based toxicity biomarkers with kidney injury marker (KIM-1) or heme-oxygenase (HO-1), an oxidative stress marker. Appropriate control MPS were run in parallel and sampled and analyzed for similar endpoints. Device effluents were collected for 24 hours pre-exposure and at 24 and 48-hour timepoints during the ENM exposure periods and evaluated for the shed cell-based biomarker of cellular injury kidney injury marker (KIM-1) using a Meso Scale Discovery® electrochemiluminescent assay. After the ENM exposure period, immunocytochemistry (ICC) fluorescent imaging techniques were used to evaluate cell-associated KIM-1 and heme-oxygenase (HO-1) levels in the PTEC tubules after the ENM exposure period and relative fluorescent intensities were quantified from cells in the tubule.

Dose-range finding experiments with PTECs exposed to 20 nm citrate AgNPs (0.4-25 µg/mL) in 96-well plate format for 24 and 48 hours did not reveal any obvious signs of mortality or toxicity either by gross changes in cell morphology or by a colorimetric MTS cell proliferation assay. The 20 nm citrate AgNPs tended to agglomerate in our serum-free media resulting in visible aggregates at 100X magnification. There was no indication that the cells had internalized the aggregates AgNPs. We then exposed MPS seeded with PTECs to 0, 5 and 20 µg/mL of 20 nm citrate AgNPs for 48 hours with no apparent signs of cell mortality or toxicity within the MPS.  After the exposure period, the MPS were formalin fixed and ICC fluorescent imaging techniques were used to visualize and quantify intracellular KIM-1 and HO-1. The mean fluorescence values for HO-1 in PTEC exposed to 5 and 25 µg/mL 20 nm citrate AgNPs were both 30% higher (not statistically significant) than controls suggesting that exposure to the 20 nm AgNPs may cause a mild degree of oxidative stress. There were no AgNP concentration-related changes in the MPS effluents for KIM-1 or HO-1 by ICC fluorescent imaging.

Preliminary experiments with PTECs exposed to 20 nm PVP AgNPs (1-25 µg/mL) in 96-well plate format for 24 and 48 hours also did not reveal any obvious signs of mortality or toxicity either by gross changes in cell morphology or by the colorimetric MTS cell proliferation assay. The degree of agglomeration due to the low protein/high salt concentration of our cell culture system was not as pronounced as with the 20 nm citrate AgNPs. We reduced particle agglomeration with the PVP AgNP by forming a protein corona with a human albumin/intralipid complex before adding the particles to our cell culture media. As was observed with the 20 nm citrate AgNPs, there were no signs of toxicity either visually or by ICC fluorescent imaging for KIM-1 and HO-1 after a 48-hour exposure to perfused 20 nm PVP AgNP. MPS effluent values for KIM-1 are pending analysis. In summary, PTEC exposed for up to 48 hours to 20 nm citrate or PVP AgNPs in either the MPS or 96-well plate format showed no evidence of toxicity other than a mild increase in intracellular HO-1 with 20 nm citrate AgNP exposure. Since there was no robust toxicological signal with PTEC exposure to AgNP, we did not conduct follow up with electron microscopy or RNA-seq transcriptomic analysis. 

Future Activities:

We are presently planning experiments to evaluate the toxicity of CdSe/ZnS core/shell quantum dots coated with neutral (polyethylene glycol) surface charges in an MPS format. These quantum dots are commonly used in the electronics industry, contain the toxic heavy metals Cd and Se and have already been shown to be toxic in other cell types. We have already demonstrated that we can detect PTEC toxicity with increased HO-1 and KIM-1 immunofluorescence in MPS after 48-hour exposure to 25 µM CdCl2 (Adler, Ramm et al. 2015). The results with CdCl2 are part of the control toxicants used to evaluate the MPS platform. Anticipated additional analyses include the assessment of cellular ultrastructures by electron microscopy, Adverse Outcome Pathway (AOP) determinations using RNA-seq transcriptomic analysis and immunocytochemistry (ICC) fluorescent imaging for KIM-1 and HO-1 and MPS effluent analysis for KIM-1. Other response ICC markers that will be evaluated include: SGLT2 and E-cadherin while additional effluent markers include interleukin 18 and neutrophil gelatinase-associated lipocalin (NGAL). 

References:

Adler, M., S. Ramm, M. Hafner, J. L. Muhlich, E. M. Gottwald, E. Weber, A. Jaklic, A. K. Ajay, D. Svoboda, S. Auerbach, E. J. Kelly, J. Himmelfarb and V. S. Vaidya (2015). "A Quantitative Approach to Screen for Nephrotoxic Compounds In Vitro." J Am Soc Nephrol. 

Journal Articles:

No journal articles submitted with this report: View all 64 publications for this subproject

Supplemental Keywords:

human kidney, tissue-engineered human kidney, nanomaterials, nephrotoxic compounds, human health

Relevant Websites:

The Predictive Toxicology Center (PTC) for Organotypic Cultures Exit

Progress and Final Reports:

Original Abstract
  • 2016 Progress Report
  • 2017 Progress Report
  • 2018 Progress Report
  • Final

  • Main Center Abstract and Reports:

    R835738    Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R835738C001 Airway Epithelium Organotypic Culture as a Platform forAdverseOutcomesPathway Assessment of Engineered Nanomaterials
    R835738C002 Organotypic Model of Human Kidney as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C003 Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C004 Organotypic Model of Testis as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C005 Integrating Liver, Kidney and Testis Nanomaterial Toxicity using the Adverse Outcome Pathway Approach