Abstract
The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100–250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies.
Highlights
More than one million patients world-wide suffer from tuberous sclerosis complex (TSC) and have mutations in either of the tumor suppressor genes, TSC1 or TSC2 [1]
The majority of TSC patient develop some form of kidney cyst during their life time but the exact mechanism of kidney cystogenesis in tuberous sclerosis complex is unknown [20]
We reported that the loss of Tsc2 gene in a mouse renal principal cell affected the phenotype of intercalated cell causing an enhanced mTORC1 activity, leading to renal cystogenesis likely via an extracellular vesicle (EV)-dependent pathway [14] (Figure 5)
Summary
More than one million patients world-wide suffer from tuberous sclerosis complex (TSC) and have mutations in either of the tumor suppressor genes, TSC1 or TSC2 [1]. There is ample scientific data associating cystogenesis to the mTORC1 pathway, results from animal model and human tissue studies are difficult to reconcile with the accepted dogma about TSC-associated renal disease pathogenesis, which is based on findings in angiomyolipomata In this lesion, a somatic mutation (second hit) mechanism of TSC renal disease [7] results in an inactivating mutation and loss of heterozygosity with the loss of tuberin staining [8]. The accepted second hit mechanism is difficult to reconcile with murine Tsc cystic disease because these investigations fail to find somatic mutation in a majority of cysts, indicating that the majority of renal cysts maintain their Tsc locus integrity [9,10], and because human cysts continue to express tuberin and hamartin [11] Such a low rate of loss of heterozygosity is seen in PKD1 associated autosomal dominant polycystic kidney disease, suggesting that such cystic disease may represent a unique disease mechanism [12,13].
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