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Zhe Han

$2M NIH grant for treating disease linked to APOL1

Zhe Han

Children’s researcher Zhe Han, Ph.D., has received a $2 million award from the National Institutes of Health (NIH) to study new approaches to treat kidney disease linked to inheriting Apolipoprotein L1 (APOL1) risk alleles. These risk alleles are particularly common among persons of recent African descent, and African Americans are disproportionately affected by the increased risk in kidney disease associated with these risk alleles.

Han, an associate professor in Children’s Center for Genetic Medicine Research, has established a leading research program that uses the fruit fly Drosophila as a model system to study how genetic mutations lead to disease.

“Drosophila is a very basic model, but studies in the fly have led to major breakthroughs in understanding fundamental biological processes that underlie health and disease in humans,” Han says. “Since coming to Children’s National five years ago, I have focused a significant part of my research studying particular fly cells called nephrocytes that carry out many of the important roles of human kidney glomeruli, units within the kidney where blood is cleaned. Working together with clinician colleagues here, we have demonstrated that these Drosophila cells can be used to very efficiently study different types of renal disease caused by genetic mutations.”

The APOL1 risk alleles are genetic variants, termed G1 and G2, found almost exclusively in people of African ancestry and can lead to a four-fold higher risk of end-stage kidney disease, the last of five stages of chronic kidney disease. Exactly how inheriting these risk alleles increases the risk of kidney disease remains an unanswered question and the focus of considerable research activity. Han’s laboratory has developed a Drosophila model of APOL1-linked renal disease by producing the G1 and G2 forms of APOL1 specifically in nephrocytes. This led to defects in fly renal cells that strikingly overlap with disease-associated changes in experimental model and human kidney cells expressing APOL1 risk alleles.

The new NIH award will fund large-scale screening and functional testing to identify new treatment targets and new drugs to treat kidney disease linked to APOL1. Using a genetic screening approach, Han’s lab will identify nephrocyte “modifier” genes that interact with APOL1 proteins and counter the toxic effects of risk-associated G1 and G2 variants.

The team also will identify nephrocyte genes that are turned on or off in the presence of APOL1 risk alleles, and confirm that such “downstream” APOL1-regulated genes are similarly affected in experimental model and human kidney cells. The potential of the newly identified “modifier” and “downstream” genes to serve as targets of novel therapeutic interventions will be experimentally tested in fly nephrocytes in vivo and in cultured mammalian kidney cells.

Finally, the Drosophila model will be used as a drug screening platform for in vivo evaluation of positive “hits” from a cell-based APOL1 drug screening study in order to identify compounds that are most effective with the fewest side effects.

“These types of studies can be most efficiently performed in Drosophila,” Han adds.  “They take advantage of the speed and low cost of the fly model system and the amazing array of well-established, sophisticated genetic tools available for the fly. Using this model to elucidate human disease mechanisms and to identify new effective therapies has truly become my research passion.”

fruit fly

Studying fruit flies to better understand human kidneys

fruit fly

In his latest study, Zhe Han and co-authors zeroed in on Rab genes to determine their role in fruit fly renal function.

It’s a given that fruit flies and humans are different. Beyond the obvious are a litany of less-apparent distinctions. For example, fruit flies have hemolymph instead of blood. Arranged around a single cardiac chamber, compared with humans’ four-chamber hearts, are a group of cells called nephrocytes that serve the same function as human kidneys, filtering toxins and waste from hemolymph.

But despite the dissimilarities between these two organisms, fly nephrocytes and human kidney cells are similar enough to allow the fruit fly, a common lab model that shares about 60 percent of its DNA with people, to provide insights on kidney disease in people. In a new study in fruit flies led by Zhe Han, Ph.D., principal investigator and associate professor in the Center for Cancer and Immunology Research at Children’s National Health System, researchers identified several new genes thought to be critical for renal function in humans. The findings could lend insight to the inner workings of this organ down to the molecular level and eventually help further the understanding or treatment of kidney disorders.

Han explains that recent research by his group tied 80 fruit fly genes to renal function. Many of these newly identified genes were Rab GTPases, a family of genes that make proteins whose job is to move substances around in cells through membrane-enclosed pouches called vesicles. For example, Rab proteins might put some substances on the path to destruction by moving them into lysosomes, vesicles with enzymes that break down all kinds of biomolecules. Rab proteins might help other substances be reused by steering them into recycling endosomes, vesicles that shuttle biomolecules that are still useful to where they will be used next.

In their latest study, published online Feb. 8, 2017 in Cell & Tissue Research, Han and co-authors zeroed in on these Rab genes to determine their role in fruit fly renal function. The researchers accomplished this by using genetic alterations to shut down each gene selectively in fruit fly nephrocytes. They then evaluated these transgenic flies on a number of different characteristics, including ability to effectively filter proteins from the blood, whether toxins placed in their food accumulated in their nephrocytes, how they developed and how they survived.

Their findings readily identified five Rab genes that seemed more important for these functions than the others: Rabs 1, 5, 7, 11 and 35, which all have analogous genes in humans.

Peering into the nephrocytes of flies in which these three Rabs had been silenced, the researchers made critical discoveries. Turning off Rab 7 appeared to block the path toward biomolecules in the cell entering lysosomes. Rather than biomolecules being destroyed, they instead were shuttled to the recycling route. Turning off Rab 11 had the reverse effect; recycling endosomes were drastically reduced, while lysosomes dramatically increased. Turning off Rab 5 had the most striking effect: All vesicles going in or out were blocked – like a cellular traffic jam – filling the cell with biomolecules that had no place to go, Han says.

Han, who has long tracked renal-related mutations in humans, says that no patients with kidney disease have turned up so far with Rab mutations. These genes are critical for functions throughout the body, he explains, so any embryos with these mutations are unlikely to survive. However, he adds, a host of other renal-related genes work in parallel or are controlled by different Rabs. So understanding the role of Rabs in renal function provides some insight into how these genes operate as well as what might happen when the function of these genes goes awry.

Han plans to study how Rabs 5, 7 and 11 fit into networks of renal genes as well as the role of the other Rabs that could play novel roles in the nephrocyte cell trafficking.

“These findings in fly Rabs provide the framework to study the major causes of kidney disease in human patients,” he adds.

Zhe Han, PhD

Key to genetic influence of APOL1 on chronic kidney disease

Zhe Han

Drosophila melanogaster nephrocytes share structural and functional similarities with human renal cells, making the fruit fly an ideal pre-clinical model for studying how the APOL1 gene contributes to renal disease in humans.

Using the Drosophila melanogaster pre-clinical model, a Children’s National Health System research team identified a key mechanism by which the APOL1 gene contributes to chronic kidney disease in people of African descent. The model exploits the structural and functional similarities between the fruit fly’s nephrocytes and renal cells in humans to give scientists an unprecedented ability to study gene-to-cell interactions, identify other proteins that interact with APOL1 in renal disease, and target novel therapies, according to a paper published November 18 in the Journal of the American Society of Nephrology.

“This is one of the hottest research topics in the kidney field. We are the first group to generate this result in fruit flies,” says Zhe Han, Ph.D., a senior Drosophila specialist and associate professor in the Center for Cancer & Immunology Research at Children’s National. Han, senior author of the paper, presented the study results this month during Kidney Week 2016, the American Society of Nephrology’s annual gathering in Chicago that was expected to draw more than 13,000 kidney professionals from around the world.

The advantages of Drosophila for biomedical research include its rapid generation time and an unparalleled wealth of sophisticated genetic tools to probe deeply into fundamental biological processes underlying human diseases. People of African descent frequently inherit a mutant version of the APOL1 gene that affords protection from African sleeping sickness, but is associated with a 17- to 30-fold greater chance of developing certain types of kidney disease. That risk is even higher for individuals infected with the human immunodeficiency virus (HIV). Drosophila renal cells, called nephrocytes, accurately mimic pathological features of human kidney cells during APOL1-associated renal disease.

“Nephrocytes share striking structural and functional similarities with mammalian podocytes and renal proximal tubule cells, and therefore provide us a simple model system for kidney diseases,” says Han, who has studied the fruit fly for 20 years and established the fly nephrocyte as a glomerular kidney disease model in 2013 with two research papers in the Journal of the American Society of Nephrology.

In this most recent study, Han’s team cloned a mutated APOL1 gene from podocyte cells cultured from a patient with HIV-associated nephropathy. They created transgenic flies making human APOL1 in nephrocytes and observed that initially the transgene caused increased cellular functional activity. As flies aged, however, APOL1 led to reduced cellular function, increased cell size, abnormal vesicle acidification, and accelerated cell death.

“The main functions of nephrocytes are to filter proteins and remove toxins from the fly’s blood, to reabsorb protein components, and to sequester harmful toxins. It was surprising to see that these cells first became more active and temporarily functioned at higher levels,” says Han. “The cells got bigger and stronger but, ultimately, could not sustain that enhancement. After swelling to almost twice their normal size, the cells died. Hypertrophy is the way that the human heart responds to stress overload. We think kidney cells may use the same coping mechanism.”

The Children’s research team is a multidisciplinary group with members from the Center for Cancer & Immunology Research, the Center for Genetic Medicine Research, and the Division of Nephrology. The team also characterized fly phenotypes associated with APOL1 expression that will facilitate the design and execution of powerful Drosophila genetic screening approaches to identify proteins that interact with APOL1 and contribute to disease mechanisms. Such proteins represent potential therapeutic targets. Currently, transplantation is the only option for patients with kidney disease linked to APOL1.

“This is only the beginning,” Han says. “Now, we have an ideal pre-clinical model. We plan to start testing off-the-shelf therapeutic compounds, for example different kinase inhibitors, to determine whether they block any of the steps leading to renal cell disease.”