Story of Discovery: Engineered Kidney Tissues and Organoids: Tools for Improved Disease Modeling and Development of Therapies

Chronic kidney disease (CKD) is a major public health problem in the United States. The impact of CKD is substantial and includes increased risk of death, diminished quality of life, numerous co-associated diseases and conditions, such as cardiovascular disease, and significantly increased risk of progression to kidney failure (end-stage renal disease). As symptoms are few or non-existent, most people are unaware that they have CKD until most kidney function has been lost. Development of drugs for CKD has been hampered by non-predictive animal models, the inability to identify and prioritize molecular factors in human kidneys that could be targeted with medication, and an underlying poor understanding of human CKD. Therefore, CKD research, as well as research on other kidney-related conditions (e.g., acute kidney injury, drug toxicity, cancer), would benefit greatly from the development of improved laboratory tools to model human kidney structure and function.

Kidneys are highly complex, bean-shaped organs that cleanse metabolic waste products from the blood and maintain proper salt and mineral balance and fluid volume in the body. Each human kidney contains about 1 million individual filtration units, called nephrons, in which blood vessels intertwine with other structures to achieve these remarkable functions. Kidney formation in the developing embryo is a dynamic, orchestrated process; clusters of cells move and interact, and various cells undergo tightly regulated molecular and physical changes that ultimately drive them to mature into functional nephrons. Due to this complexity, for many years, scientists struggled to develop models that accurately recapitulate human kidney structures and function, and the lack of such human kidney models has limited the ability to develop new drugs to treat or prevent CKD. However, due to rapid technological advances made over the past few years, engineered kidney tissues and organoids—self-organizing, three-dimensional kidney assemblies often derived from adult human stem cells—have emerged as promising tools to accelerate CKD research.

KIDNEY ORGANOIDS— A LEAP FORWARD

Over the course of many years, scientists painstakingly defined a number of laboratory conditions under which human pluripotent stem cells (hPSCs)—cells that are able to become any type of cell in the body—could be coaxed to develop into individual cell types found in the nephron. However, researchers faced the challenge of assembling the different kidney cell types together into three-dimensional structures that can recapitulate human kidney functions. Between 2013 and 2015, se veral research groups reported important technological breakthroughs that defined conditions under which hPSCs could develop into higher-order kidney-like structures. For example, in 2014, sc ientists in Japan used mouse models t o carefully define a series of molecules that act sequentially to induce the stages of normal kidney development over time. The researchers then applied this knowledge from mice to a human system by adding these molecular signals step-wise to hPSCs growing in culture, coaxing the cells to grow in number, aggregate, and mature into organoids that recapitulate complex kidney structures. Other research groups also used a variety of methodologies to identify molecules and processes in normal kidney development that, when applied to cultured hPSCs, improve their ability to self-organize into organoids. Kidneys filter the blood through interactions between very thin blood vessels (capillaries) and specialized kidney cells in the nephron. The incorporation of capillaries in developing kidney structures is a process called “vascularization,” and is essential for kidney function. Therefore, vascularization of kidney organoids in culture is critical, but has been a technological hurdle for researchers. In 2018, a team of scientists, supported in part by NIDDK, made a leap forward when they showed that hPSC-derived kidney organoids, when grown in certain culture conditions and transplanted into mice, could recruit the host mouse’s blood vessels into the organoids’ budding blood vessels. They observed over time that the organoids grew in size, formed critical structures such as filtration membranes between the mouse blood vessels and kidney cells, and developed vascular connections that were fully functional. These findings demonstrated that hPSC-derived organoids exposed to a physiological environment similar to that in the body (in this case, when transplanted into a mouse) are poised to vascularize and mature.

These experiments generated a relatively small number of organoids that could, under specific conditions, mimic kidney structures and functions. But another obstacle to translating this research to wider use in the laboratory, and potentially the clinic, is scalability—the amount of hPSC-derived kidney organoids will have to be increased. To address this need, researchers, supported in part by NIDDK, modified culture conditions to optimize the yield of kidney structures that could be produced in the laboratory. In 2019, they published their finding that certain conditions favored the generation of large quantities of kidney tissue in the form of much smaller organoids, which they term “micro-organoids,” than previous studies had reported. The cellular compositions and maturity levels of the micro-organoids were similar to those of standard organoids, but these laboratory conditions generated 3 to 4 times the total amount of kidney tissue from the same number of starting cells, with a 75 percent reduction in cost.

MODELING BIOLOGICAL FUNCTIONS BY MOUNTING KIDNEY TISSUES TO PLATFORMS

While the laboratory protocols for growing organoids in culture have made impressive strides in producing tissue structures that resemble the kidney, kidney development and maturation has not been fully replicated under simple culture conditions. As an example, organoids in the previously described study required grafting into mice to achieve vascularization. To more closely replicate the conditions of normal kidney development, researchers have turned to “microfluidic” platforms, which are devices on which kidney cells or organoids can be mounted and exposed to tiny, controllable volumes of liquid. These modifiable platforms, or “chips,” can therefore be used to test various culture conditions with extraordinary precision to identify factors that promote maturation of kidney cells and organoids.

In recent studies in 2018 and 2019, two teams of NIDDK-funded scientists sought to use chips to improve functional models of tubules, which are specific portions of the nephron where various molecules (e.g., water, proteins, salts, sugars) are exchanged between the nephron and surrounding capillaries to achieve proper balance in the body. One research team developed a chip-mounted renal vascular-tubular unit (hRVTU), consisting of a permeable membrane with vascular (capillary) components on one side and tubule cells on the other. Over time, molecules produced by the cells “remodeled” the membrane between the two compartments to closely resemble the normal interface between tubules and capillaries in human kidneys. Moreover, proteins and sugars that normally pass between tubules and capillaries could flow between these two compartments, but others could not; this selectivity revealed the great extent to which the hRVTU could replicate kidney function.

The other group of scientists used “3-D bioprinting” technology to create chips on which three- dimensional tubules and capillaries were “printed” directly adjacent to one another and are embedded in an engineered matrix that resembles the environmental conditions surrounding these structures within nephrons in the body. By measuring the contents within the two compartments, they observed the exchange of molecules between the two structures, demonstrating that the chip modeled normal tubule function. The scientists then exposed the chips to hyperglycemic (excess sugar in the fluid) conditions to model the effects of diabetes on nephron tubules and capillaries. Hyperglycemia led to cellular damage and dysfunction in the capillary and tubular cells, effects that were prevented by treating the chips with a drug that reduces the transport of sugar between the compartments. Thus, the chips proved useful for accurately modeling tubule-capillary dynamics in both normal conditions and immediately following high sugar exposure; future studies are needed to determine whether these chips could be useful for modeling the effects of long-term, chronic high sugar exposure characteristic of diabetes.

While these studies focused on creating chips modeling particular parts of the nephron, other researchers have sought to mount and develop entire organoids on microfluidic platforms. In a recent study published in 2019, NIDDK-supported scientists sought to overcome the barrier of organoid vascularization by reasoning that because developing kidneys normally are exposed to fluid flow, perhaps adding the stress of fluid flow to chips could mimic the natural environment. When the researchers grew chip-mounted kidney organoids in the presence of a high rate of fluid flow, they developed an array of blood vessels with varying diameters; by contrast, organoids exposed to low fluid rate or none at all had far fewer blood vessels. Under high flow conditions, the developing blood vessels successfully transported fluids and even assembled as networks connecting neighboring organoids, demonstrating that they were physiologically mature.

ONGOING AND FUTURE RESEARCH EFFORTS

Tools developed through these advances are already proving their utility for accelerating CKD research. For example, one study used hPSC-derived kidney organoids to help overcome a long-standing technological roadblock. Previously, researchers had tried to treat damaged kidney cells with viral gene therapy, but research protocols to deliver genes to appropriate human cells were unsuccessful. NIDDK-supported scientists recently identified a specific subtype of virus that could successfully deliver gene therapy and prevent damage to kidney cells in mice. However, it is well known that many discoveries in mice cannot be translated to humans due to inter-species differences between kidneys. Therefore, the scientists tested the virus in hPSC-derived organoids, determining that the gene delivery vehicle could successfully work in these human cell-derived structures and providing a foundation for potential future therapeutic studies. This study exemplifies how using organoids that faithfully mimic human kidney structures and physiology can help predict drug or other treatment effectiveness and toxicity at a relatively low cost.

NIH and the International Space Station U.S. National Laboratory are currently collaborating on another fascinating use for kidney chips, as well as chips modeling other organs and diseases. Researchers will use the tissue chips in space to study aging and certain disease states that appear to be accelerated in microgravity and then later to test the potential effects of drugs on those tissues. The projects aim to provide insights that will speed the development of treatments for kidney stones, arthritis, and other conditions that affect us here on Earth.

Research to develop engineered kidney tissues and organoids have taken extraordinary leaps over the past few years. As technology continues to improve, these laboratory tools will undoubtedly play central roles in understanding human kidney development, modeling disease, accelerating drug discovery, and catalyzing innovation in renal replacement therapy.