Single Cell Gene Expression Studies Reveal Kidney Clues

Bridget M. Kuehn Jihwan Park, et al. Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease. Science2018. DOI: 10.1126/science.aar2131

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A new genetic technique called single cell genetic sequencing is helping to reveal new insights about the cells that make up the kidney—insights that are essential to understanding what goes wrong in kidney disease and how it might be reversed.

Many types of cells make up the kidney, each with a distinct role in kidney health, making it a challenge for scientists to study. Traditionally, scientists have tried to distinguish these various cell types by their location in the kidney and appearance under a microscope, according to Katalin Susztak, MD, PhD, a professor of medicine at the Perelman School of Medicine at the University of Pennsylvania. But rapid advances in genetic techniques are giving scientists new tools for studying these cells.

Whole genome sequencing studies have allowed scientists to document an individual’s entire genetic blueprint. Now the technology has advanced to allow scientists to look at which genes are turned on or off in individual cells.

“Every cell in your body has a full complement of DNA coding for approximately 21,000 proteins,” said Mark Knepper, MD, PhD, a senior investigator at the National Heart, Lung and Blood Institute. “However, each cell expresses only around 6000 to 8000 of these genes.”

Single cell RNA sequencing allows scientists to determine which 6000 to 8000 genes are turned on in each cell, which “provides a road map” for studying specific cell types, Knepper said.

“The single cell RNA sequencing approach and other next generation sequencing methods will provide essential information at a basic science level that will ultimately result in a better understanding of many renal diseases,” Knepper said.

Emerging insights

A groundbreaking study by Susztak and colleagues using single cell sequencing identified several new types of cells in the kidney, and showed that some cells in the kidney can transition back and forth between two cell types to help the kidney adapt to changing conditions.

In their study, published in Science, Susztak and her colleagues took one kidney from seven different male mice and used droplet-based single cell RNA sequencing to analyze the gene expression in the more than 43,745 cells in each kidney. They used a special machine to connect each cell with a bead that is able to capture all the genes that are active in that cell. Then, they sorted the cells based on these genes. This method is far cheaper than previous RNA sequencing methods, costing just 10 to 20 cents per sample, Susztak said, compared with older techniques that could cost $300 for a single sample.

“Because the cost is much lower, we can actually sequence a large number of cells,” she explained.

Another advantage of this approach is that it provides an “unbiased” way of grouping cells, she said. The approach classifies cells only on the genetic information being used in each cell instead of more superficial physical cell characteristics that might be shared by multiple types of cells. This allowed the researchers to discover that 1 of the 3 types of cells previously identified in the collecting duct of the kidney are actually just cells in transition from one type to another. These results reinforce findings from a previous study by Knepper and colleagues that had suggested a transitional cell type in the collecting duct.

“Both of us found hybrid cells that express markers of both principal cells and intercalated cells,” Knepper said. “This finding adds to additional evidence from ‘fate mapping’ studies that principal cells may convert into intercalated cells. This is currently a hot area of research.”

Scientists know that the collecting duct and its cells help balance salt, water, and acid–base levels in the body, according to Knepper. To keep up with changing demands, it appears the cells may be able to transition from being principal cells that transport water, sodium, and potassium to intercalated cells that regulate acid–base balance by transporting hydrogen ions.

“We think in healthy adult kidneys this type of interconversion happens on a regular basis to kind of balance the water and acid, but this interconversion also happens more profoundly in [kidney disease] where the kidneys might need to focus on water balance,” said Rojesh Shrestha, BS, a research specialist in Susztak’s laboratory.

Both Susztak and Knepper’s studies also found that most principal cells in the collecting duct expressed the Notch2 gene, and that the gene for its receptor is expressed mostly by intercalated cells. This may help “explain how principal cells and intercalated cells are able to ‘talk’ with one another,” said Knepper. It may also have clinical implications, noted Susztak, who explained that it might be possible to use treatments that manipulate these messages to intervene in diseases where the acid–base balance has gone awry.

The insights were just part of a huge amount of data generated in the study. Susztak and colleagues also showed that the genes for specific kidney diseases were expressed by just one type of cell. For example, genes linked with high and low blood pressure were traced to one type of cell. This insight may help scientists trying to pinpoint the cause of certain diseases. Susztak suggested this likely means that there is a very clear division of labor among kidney cells, and if one type of cell is not working properly, for example because of a gene mutation, the others do not pick up the slack.

The next step for Susztak’s research will be to start cataloguing single gene expression in kidneys affected by disease to understand how gene expression changes. In the meantime, she hopes the data from her current study will help fuel other researchers’ work.

“We generated the periodic table for the kidney, so now we know where all the elements in the kidney are,” she said. “All the researchers who study kidney physiology or kidney homeostasis will be able to put the elements together and understand disease development, and how the kidney works under [healthy] conditions.”

Clinical implications

Already some groups have begun to apply single cell sequencing techniques to samples taken from patients with kidney disease. For example, a network of researchers from the Accelerating Medicines Partnership studying rheumatoid arthritis and systemic lupus erythematosus recently used single cell gene sequencing to analyze 16 kidney and 12 skin tissue samples taken from patients with lupus nephritis during routine care. The National Institutes of Health, nonprofit groups, and industry are jointly funding the network with the aim of accelerating the development of new treatments.

The study provided a proof of concept that single cell RNA sequencing might reveal useful information from clinical samples. From just several millimeters of kidney tissue the investigators were able to glean important information that added on to earlier work with standard light and electron microscopy studies of biopsies. In addition to providing information that might help classify patients, the study suggested that the sequencing data might also hint at a patient’s prognosis 6 months later.

“We’re getting a remarkable amount of information which has direct clinical relevance,” said Chaim Putterman, MD, chief of the division of rheumatology at Albert Einstein College of Medicine and Montefiore Medical Center, who was the co-principal investigator, along with Jill P. Buyon, MD, director of the division of rheumatology at New York University School of Medicine, and Thomas Tuschl, PhD, head of the Laboratory for RNA Molecular Biology at Rockefeller University in New York.

Putterman said that if other larger studies confirm the potential prognostic value of single cell RNA sequencing, it might encourage physicians to intensify initial treatments for patients exhibiting molecular predictors of a more aggressive disease.

The researchers also did single cell sequencing on skin cells collected from patients with lupus to see if it might provide useful information about the progression of the disease. Putterman explained that kidney biopsies are critical to assessing patients with lupus nephritis, but its invasive nature together with the potential risks associated with the procedure limit how many times it can be repeated. Skin cells could be more easily and safely collected over time. The study showed that some of the same lupus-linked genetic changes occurring in kidney cells may also be seen in skin cells.

“If we can use the skin to reflect what’s happening in the kidney, that would be a major advance forward,” Putterman said.

While single cell sequencing is an enormously promising technique and will likely lead to many new insights in nephrology research, Knepper was cautious in his assessment of the clinical potential of the technique. He noted there are still many practical issues that would have to be resolved before such technology could be used in the clinic. For example, it’s difficult to apply to glomerular cells, although some groups are working to solve this problem, he said. It’s also not clear whether the process itself might alter gene expression in the cells or if the genes expressed by cells in isolation are the same ones expressed by cells in the kidney that may be interacting with neighboring cell types.

“That kind of thing limits the potential direct clinical use,” Knepper said. “This is predominantly a research method. With present technology, I don’t see this being applied directly to a patient admitted to the hospital. But the information the technique provides is marvelous and unique.”