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National Toxicology Program

National Toxicology Program

UPDATE NewsletterUPDATE NewsletterSeptember 2015

Kidney toxicity screening tool developed by grantee and NTP team

By Kelly Lenox
Reprinted from Environmental Factor, September 2015

A team of researchers, including NIEHS grantees and National Toxicology Program (NTP) scientists, developed the first method to test for kidney toxicity using high throughput screening (HTS). Because drugs and environmental chemicals can cause injury to kidneys, the new test could help reduce a significant health burden for patients and decrease the financial risk for pharmaceutical and chemical companies.

The team, led by Vishal Vaidya, Ph.D., of Harvard Medical School, and including molecular toxicologist Scott Auerbach, Ph.D., from NTP, published their approach Aug. 10 in the Journal of the American Society of Nephrology. “This high-throughput assay allows, for the first time, rapid and robust screening of kidney toxic compounds, to support chemical risk assessment and facilitate elimination of drug candidates early in the process before they reach humans,” Vaidya said.

High throughput screens were lacking

HTS, which has been advanced by the Tox21 collaboration, is rapidly becoming the standard tool for predictive toxicology, which aims for early detection of adverse health effects from chemical compounds, including medicines.

“The lack of adequate models to accurately predict human toxicity contributes to an underestimation of the kidney toxic potential of new therapeutic candidates,” the scientists wrote, “which also explains why nephrotoxic effects in patients are often only detected during late phase clinical trials, or in some cases, after regulatory approval.”

Using the right cells

The authors reported three important advances. First, they confirmed that the cells they used were suitable for the job. A particular type of kidney cell, called human proximal tubular epithelial cells (HPTECs), is the predominant target of most substances that are toxic to the kidney.

The researchers characterized the structure and function of HPTECs. The cells were shown to possess characteristics of differentiated epithelial cells, which made them desirable for use in in vitro systems.

Finding a biomarker

Next, the team identified a biomarker more sensitive than the currently used assays — cell viability and cell death — to indicate toxicity before changes occurred that might damage the cells.

“A few years ago at SOT [the Society of Toxicology annual meeting], Vishal stopped by the NIEHS booth, where we were demonstrating DrugMatrix,” Auerbach said. DrugMatrix is a molecular toxicology database that contains toxicogenomic profiles for hundreds of compounds. “DrugMatrix enabled us to provide a list of prototype kidney toxicants for him to test,” he said.

After exposing the HPTEC cells to nine kidney toxins and analyzing the expression of 1,000 genes, the researchers found that expression of one gene, HO-1, was significantly increased in the presence of the toxins.

The team validated the HO-1 biomarker in two ways. Edward Kelly, Ph.D., and Jonathan Himmelfarb, M.D., at the University of Washington, found that, following a kidney toxicant challenge, HO-1 was induced in a kidney-on-a-chip system that uses living tissue to accurately model organ function. Auerbach, collaborating with Dan Svoboda, Ph.D., of Sciome, LLC, examined two rat toxicogenomic databases and identified a significant association between HO-1 expression and kidney injury.

Additional analyses helped the team refine the assay. “Sensitivity and specificity can be improved even further by combining the readout for HO-1 concentration and the total cell number, measured in the same well,” reported the authors.

Developing and validating the test

Finally, the team developed a new assay to measure HO-1 in a rapid and cost-efficient manner. They used a test called a homogeneous time resolved fluorescence (HTRF) assay (see image below). “The HO-1 levels obtained in response to most of the [39 tested] compounds … correlated well with immunofluorescence,” the authors wrote.

The authors noted some limitations to the method. For example, a chemical might undergo modification in the liver and produce a metabolite toxic to the kidney. This could be missed by the new tool.

However, the authors remarked that it is an important step forward, providing a test that is robust — it detects kidney toxicants from multiple classes of chemicals; it is sensitive, because it picks up toxicants at a high rate; and it is specific, which means it correctly identifies compounds that are not kidney toxicants.

Citation: Adler M, Ramm S, Hafner M, Muhlich JL, Gottwald EM, Weber E, Jaklic A, Ajay AK, Svoboda D, Auerbach S, Kelly EJ, Himmelfarb J, Vaidya VS. 2015. A Quantitative Approach to Screen for Nephrotoxic Compounds In Vitro. J Am Soc Nephrol; doi:10.1681/ASN.2015010060 [Online 10 Aug 2015].

Scheme of the HTRF assay performed in a 384-well plate

Scheme of the HTRF assay performed in a 384-well plate. When the acceptor labeled antibody and the donor labeled antibody bind to HO-1, the two dyes are brought into close proximity with each other. Excitation of the donor with a light source triggers a fluorescence resonance energy transfer toward the acceptor. The emission fluorescence can be detected after incubation for four hours. This signal is proportional to the amount of human HO-1 present in the cell lysate. (Photo courtesy of Vishal Vaidya)

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