Scientists from the Center for Precision Disease Modeling at the University of Maryland School of Medicine (UMSOM) say they have uncovered a mechanism that appears to explain how certain genetic mutations give rise to a rare genetic kidney disorder called nephrotic syndrome. Using a drosophila model, they found mutations in genes that code for certain proteins lead to a disruption of the recycling of the cell membrane. This disruption leads to an abnormal kidney cell structure and function, according to the study “Exocyst Genes Are Essential for Recycling Membrane Proteins and Maintaining Slit Diaphragm in Drosophila Nephrocytes” published in the Journal of the American Society of Nephrology.
“Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue,” write the investigators.
“We silenced genes encoding exocyst complex proteins specifically in Drosophila nephrocytes and studied the effects on protein reabsorption by lacuna channels and filtration by the slit diaphragm. We performed nephrocyte functional assays, carried out super-resolution confocal microscopy of slit diaphragm proteins, and used transmission electron microscopy to analyze ultrastructural changes. We also examined the colocalization of slit diaphragm proteins with exocyst protein Sec15 and with endocytosis and recycling regulators Rab5, Rab7, and Rab11.
“Silencing exocyst genes in nephrocytes led to profound changes in structure and function. Abolition of cellular accumulation of hemolymph proteins with dramatically reduced lacuna channel membrane invaginations offered a strong indication of reabsorption defects. Moreover, the slit diaphragm’s highly organized surface structure—essential for filtration—was disrupted, and key proteins were mislocalized. Ultrastructural analysis revealed that exocyst gene silencing led to the striking appearance of novel electron-dense structures that we named “exocyst rods,” which likely represent accumulated membrane proteins following defective exocytosis or recycling. The slit diaphragm proteins partially colocalized with Sec15, Rab5, and Rab11.
“Our findings suggest that the slit diaphragm of Drosophila nephrocytes requires balanced endocytosis and recycling to maintain its structural integrity and that impairment of the exocyst complex leads to disruption of the slit diaphragm and nephrocyte malfunction. This model may help identify therapeutic targets for treating kidney diseases featuring molecular defects in vesicle endocytosis, exocytosis, and recycling.”
Disruption of kidney cell function leads to nephrotic syndrome, a kidney disease that causes an abnormal amount of protein leaking into the urine due to a problem with the kidney’s filters. It occurs in about 7 in 100,000 Americans, and though rare, it is considered one of the most common kidney diseases in children. Nephrotic syndrome caused by genetic mutations often does not respond to the standard steroid treatment, so treatment depends on the identification of the genetic mutation that causes the disease, followed by targeted therapeutic development based on the disease mechanism. There are, however, no known treatments for the condition when it is caused by mutations in the genes examined in this study.
“Researchers have recently identified a mutation in one of the genes that codes for these proteins, called the exocyst complex, that has been linked to kidney disease,” says Zhe Han, PhD, associate professor at the department of medicine and director of the Center for Precision Disease Modeling at UMSOM. “However, the underlying mechanism by which the exocyst complex contributes to kidney disease had long been a mystery.”
Han’s team used Drosophila as a model to better understand the mechanism by which these gene mutations potentially give rise to kidney disease. Drosophila have specialized nephrocytes that closely resemble human podocytes, a type of kidney cell associated with nephrotic syndrome, both in structure and function. Using the power of fly genetics, the research team carried out a genetic screen with Drosophila and identified each of the eight exocyst genes that are required for nephrocytes to function properly.
Silencing the Drosophila exocyst genes in nephrocytes led to a disruption of a structure essential for filtering called the nephrocyte slit diaphragm. The researchers then used a transmission electron microscope to take a closer look at the ultrastructural changes, which revealed the appearance of unique electron-dense tubular structures which they named “exocyst rods.”
“Therefore, the formation of the exocyst rods can be used as a new biomarker for diseases caused by genetic mutations in exocyst genes,” Han said.
The finding, if replicated, also has important clinical implications for patients who are screened for genetic kidney diseases. “Our study suggests that mutations in all eight exocyst genes could lead to nephrotic syndrome and thus should be included in the sequencing panel for genetic kidney diseases,” according to Han, who added that he and his team are currently using their Drosophila model to gain additional insight into the mechanisms regulating slit diaphragm proteins and how their disruption might contribute to the podocyte pathogenesis seen in kidney disease patients.