Nephrology

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.”

‘Trojan horse’ macrophage TNF-alpha opens door for HIV-1 to enter kidney epithelial cells, causing nephropathy

macrophage

Like a Trojan horse, the macrophage sits atop the epithelial cell with HIV hidden inside, opening a doorway into the kidney cell for high levels of HIV-1 to enter.

When nephrologist Patricio Ray, M.D., began investigating human immunodeficiency virus (HIV) as a renal fellow, children infected with the virus had a life expectancy of no more than seven years, and kids of African descent curiously were developing a type of HIV-related kidney disease.

HIV-associated nephropathy (HIVAN) is a progressive kidney disease seen in people who are both HIV-positive and of African ancestry. Kids who carry a modified protein that protects them against sleeping sickness are 80 times more likely to develop this type of kidney disease. Due to the kidney damage, they can have abnormal amounts of protein in their urine, focal segmental glomerulosclerosis, and microcystic tubular dilation, which can lead to enlarged kidneys and chronic kidney failure.

“No one understood how HIV could affect kidney cells that lack the receptors expressed in T cells and white cells,” recalls Dr. Ray, Robert Parrott Professor of Pediatrics at Children’s National Health System. Virologists said kidney epithelial cells that lacked CD4, a major receptor where HIV attaches, could not be infected with the virus. Nephrologists, meanwhile, were seeing that HIV infection was damaging these cells.

It’s taken two decades to unravel the medical mystery, aided by urine samples he coaxed kids to donate by offering them the latest music from New Kids on the Block in exchange for each urine bottle. Many of the kids died years ago, but their immortalized cells were essential in determining, through a process of elimination, which renal cell types were capable of being infected by HIV-1.

The paper represents the capstone of Dr. Ray’s career.

“This is how difficult it is to get an important contribution in science,” he says. “It’s 20 years of work involving the excellent contributions of many people, but that’s why research is called research. In the end, it’s all a learning process. But, it’s amazing how the puzzle pieces begin to fit. When the puzzle fits, it’s good.”

Dr. Ray, in collaboration with lead author Jinliang Li, Ph.D., and four additional Children’s National co-authors, published a paper November 3 in the Journal of the American Society of Nephrology that establishes a new role for transmembrane TNF-alpha, that of a facilitator that makes it easier for the HIV virus to enter certain cell types and replicate there.  Like a Trojan horse, the macrophage sits atop the epithelial cell with HIV hidden inside, opening a doorway into the kidney cell for high levels of HIV-1 to enter.

As a starting point, the research team cultured podocytes from the urine of kids with HIVAN. Through a number of steps, they isolated the unique contributions of the HIV envelope, heparan sulfate proteoglycans as attachment receptors – the glue that binds HIV to podocytes – and the essential role played by TNF-a, a 212-amino acid long type 2 transmembrane protein, in regulating at least two processes, including viral entry and fusion. They used a fluorescent marker to tag HIV-1 viruses, so it lit up bright green. Thus primed with transmembrane TNF-a, the podocytes were susceptible to HIV-1 infection when exposed to high viral loads.

Additional research is needed, such as in vitro work to help understand how HIV traffics within the cell, Dr. Ray says. Those insights could winnow the list of existing therapies that could block key steps, such as attachment to the viral envelope, which could help all people of African descent carrying the genetic mutation, including underserved kids in sub-Saharan Africa.

Another open research question is that certain cells located in the placenta and cervix express TNF-a, and may be more likely to be infected by HIV. Blocking that process could help prevent pregnant HIV-positive mothers from transmitting illness to their offspring.