On May 20, 2020, President Tsai Ing-wen declared in her inaugural address that reaching global competitiveness in the animal and medical sciences is one of the country’s six core strategic industries. Additionally, Minister Wu Tsung-Tsong of the Ministry of Science and Technology (MOST) also highlighted the importance of deepening the biotech medical industry and precision medicine in his proposed six core areas for achieving 2030 goals in science and technology. In line with these goals, the National Laboratory Animal Center (NLAC) of National Applied Research Laboratories (NARLabs) established the “Platform for Next-generation Nephrotoxicity Testing in Mice” (also known as the “Shennong Mouse Nephrotoxicity Screening Platform”) to supply much-needed lab mice to domestic developments in nephrotoxicity testing and kidney treatment products, while also reducing the possibility of nephrotoxicity-inducing substances from affecting the citizenry.
Currently, global markets lack truly effective testing methods for the renal toxicity of drugs, health foods or food additives. NLAC’s Shennong Mouse Nephrotoxicity Screening Platform binds the kidney-specific enzyme myo-inositol oxygenase (MIOX) with foreign luminescence enzymes, and when the kidney is damaged, the organ’s cells will release this luminescent enzyme into the blood or urine. Through measuring the activity of luminescence enzyme in the blood or urine of the lab mice, the degree of kidney damage can then be assessed. This method is both more economical and effective than the analysis method of rat urine kidney damage protein jointly developed by the "Predictive Safety Testing Consortium", which had been established in 2005 by major international pharmaceutical companies and the public drug management agencies of the US, EU and Japan.
Improper medication routines cause excessive exposure to nephrotoxic substances
In recent years, food and drug safety issues have attracted widespread public attention. According to statistics from the National Health Insurance Agency, in 2019, the number of people with renal failure and dialysis in Taiwan was as high as 92,000, and more than 422,000 people sought medical care for chronic kidney disease, which cost about TWD $53.3 billion (around USD $1.86 billion) in health insurance. The causes of kidney disease are complicated, with population ageing and poor control of diabetes, hypertension, and hyperlipidemia being the main causes for kidney dialysis. Statistics from the American Kidney Foundation also show that about 3-5% of new cases of chronic kidney disease every year in the United States are caused by the abuse of painkillers.
Traditionally, food additives, functional health foods or drugs must undergo safety assessment and testing before they are approved for use and entering the market. These mainly include acute toxicity and long-term feeding to observe any chronic effects, and only after passing these procedures can they begin efficacy and clinical trials. Although regulations have strict guidelines and supervision mechanisms, every year around the world, 2~3% of new drugs approved for marketing are still removed from the shelves due to toxicity problems that were not detected during testing, causing health issues to consumers and huge losses to pharmaceutical companies.
The world lacks effective nephrotoxicity testing methods
This absence is because original safety assessment methods are still incomplete and need to be improved. Regarding nephrotoxicity tests, there is currently no sufficiently specific biological indicator in the kidney to rely on, so tests measure the blood metabolic waste products urea nitrogen (BUN), creatinine concentration, urine protein and other renal metabolite indexes produced by kidneys to estimate whether the organ’s function is normal. However, as long as the kidneys still have at least 50% renal function, they can avoid the urea nitrogen and creatinine concentrations levels in the blood from increasing. Therefore, this method is not sensitive enough to detect of mild and moderate renal impairment. The index of renal metabolites such as urine protein is easily influenced by other physiological factors such as a high-protein diet, dehydration, infections, gastrointestinal bleeding and other issues, reducing its specificity and not necessarily reflect results caused by the kidney itself.
To this end, major international pharmaceutical companies and the public drug regulatory agencies of the US, EU and Japan had jointly founded the "Predictive Safety Testing Consortium" (PSTC) in 2005 to accelerate the establishment of new safety assessment standards, and to break barriers experienced by pharmaceutical companies by jointly developing a test platform using mice to measure the concentration of seven kidney injury-related proteins in urine to enhance the sensitivity of kidney injury detection. To avoid repeating previous safety hazards, in the early safety assessment stage of drug development, even if a considerable additional cost is required, this new type of nephrotoxicity analysis method may be added to reduce any risks of development. However, it still cannot fully ensure that the drug is not nephrotoxic.
Mice at NLAC’s "Shennong Rat Kidney Toxicity Screening Platform" do the herb-tasting for consumers
In order to strengthen the safety assessment tools in nephrotoxicity testing, professor at National Cheng Kung University (NCKU)’s College of Medicine and director of the affiliated hospital’s Pediatric Nephrology Department, Dr. Yuan-You Chiou, who is also a co-employed researcher at NLAC, combined his long-term clinical experience with NLAC’s unique large-segment genetic modification technology to create the Shennong Mouse Nephrotoxicity Screening Platform.
Dr. Chiou selected the kidney-specific enzyme myo-inositol oxygenase (MIOX) as the detection index, which can be released from the cells into the blood serum and urine within 24 hours of kidney injury, thereby facilitating early detection. MIOX is also only expressed in the proximal epithelial cells of the kidney, which is specific to this organ alone. However, the detection of enzymes is not easy and cannot be directly applied to the detection of nephrotoxicity. Therefore, using the unique design of NLAC’s large-segment genetic modification technology, the foreign luminescence enzyme gene’s expression can be regulated by the mouse’s MIOX gene, simultaneously indicating the location of MIOX gene expressions and amplifying the luminescence expression for cell labeling. When the kidney is damaged, the luminescence enzyme is driven by the MIOX gene and released out of the kidney cells along with the endogenous MIOX protein into the blood or urine. A luminescence enzyme analysis or image analysis is then used to quantify the enzyme within the blood or urine, and finally assess the degree of kidney damage.
The Shennong Mouse Nephrotoxicity Screening Platform is equipped with early predictability, high specificity, and high sensitivity, which can provide earlier, more sensitive, and stable evidence of kidney damage than the analysis method of rat urine kidney damage protein used by the "Predictive Safety Testing Consortium". The platform uses a more economical and effective method to safely test and taste products before consumers do, and also assists pharmaceutical companies and health food manufacturers in preliminary checks, reducing development risks, and reducing the possibility of nephrotoxic substances sneaking their way clinical trials or average citizens’ lives.