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Rapamycin and dexamethasone during pregnancy prevents tuberous sclerosis complex associated cystic kidney disease

ABSTRACT: Chronic kidney disease is the main cause of mortality in patients with tuberous sclerosis complex disease (TSC). The mechanisms underlying TSC cystic kidney disease remain unclear with no available interventions to prevent cyst formation. Using targeted deletion of TSC1 in nephron progenitor cells, we showed that cysts in TSC1 null embryonic kidneys originate from injured proximal tubular cells with high mTOR complex 1 activity. Injection of rapamycin to pregnant mice inhibited the mTOR pathway and tubular cell proliferation in kidneys of TSC1 null offspring. Rapamycin also prevented renal cystogenesis and prolonged the life span of TSC newborns. Gene expression analysis of proximal tubule cells, identified sets of genes and pathways that were modified secondary to TSC1 deletion and rescued by rapamycin administration during nephrogenesis. Inflammation with mononuclear infiltration was observed in the cystic areas of TSC1 null kidneys. Dexamethasone administration during pregnancy decreased cyst formation not only by inhibiting the inflammatory response but also by interfering with the mTORC1 pathway. These results reveal novel mechanisms of cystogenesis in TSC disease and suggest new interventions prior to birth to ameliorate cystic disease in offspring.

ORGANISM(S): Mus musculus

PROVIDER: GSE152165 | GEO | 2020/06/10

REPOSITORIES: GEO

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Project description:Tuberous sclerosis complex (TSC) is an inherited multi-system disorder caused by mutations in the TSC1 or TSC2 gene. TSC patients are often diagnosed with a range of neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD), intellectual disability (ID), anxiety and mood disorders. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) kinase signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using neural progenitor cells (NPCs) from patient-derived induced pluripotent stem cells (iPSCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in CRISPR-modified TSC1-null NPCs, which were unaffected by mTORC1 inhibition by rapamycin,the only approved therapy for TSC1. Here, to assess TSC1-dependent gene expression programs in NPCs, we used polysome-profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level. In addition to changes in mRNA abundance, this revealed numerous TSC1-dependent alterations in translational efficiencies. To assess the relevance of these gene expression alterations we performed polysome-profiling in post-mortem brains originating from ASD donors and matched controls. Strikingly, TSC1-dependent alterations in mRNA translation observed in NPCs were largely recapitulated in human brains. Furthermore, although polysome-profiling revealed a partial reversal of TSC1-associated gene expression alterations following rapamycin treatment, most genes related to neural activity/synaptic regulation or ASD that showed TSC1-dependent translation were rapamycin-insensitive. Therefore, we also examined whether early ND rapamycin-insensitive phenotypes in TSC1-null NPCs could be rescued by a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272 (Revolution Medicines, Inc.). Unlike rapamycin, RMC-6272 strongly inhibited translation and reversed TSC1-associated proliferation and neurite outgrowth phenotypes. In summary, we reveal ample translational alterations in TSC1 patient-derived NPCs recapitulating human brain expression profiles and potential implications for treatment of TAND.

Project description:Background: Disease severity of autosomal dominant polycystic kidney disease (ADPKD) is influenced by diet. Dietary protein, a recognized cyst-accelerating factor, is catabolized into amino acids (AA) and delivered to the kidney leading to renal hypertrophy. Injury-induced hypertrophic signaling in ADPKD results in increased macrophage (MФ) activation and inflammation followed by cyst growth. We hypothesize that the cystogenesis-prompting effects of HP diet are caused by increased delivery of specific AA to the kidney, ultimately stimulating MФs to promote cyst progression. Methods: Pkd1 flox/flox mice with and without Cre (CAGG-ER) were given tamoxifen to induce global gene deletion (Pkd1KO). Pkd1KO mice were fed either a low (LP; 6%), normal (NP; 18%), or high (HP; 60%) diet for 1 week (early) or 6 weeks (chronic). Mice were then euthanized and tissues were used for histology, immunofluorescence and various biochemical assays. One week fed kidney tissue was cell sorted to isolate tubular epithelial cells for RNA sequencing. Results: Chronic dietary protein load in Pkd1KO mice increased kidney weight, number of kidney infiltrating and resident MФs, chemokines, cytokines and cystic index compared to LP diet fed mice. Accelerated cyst growth induced by chronic HP were attenuated by liposomal clodronate-mediated MФ depletion. Early HP diet fed Pkd1KO mice had larger cystic kidneys compared to NP or LP fed counterparts, but without increases in the number of kidney MФs, cytokines, or markers of tubular injury. RNA sequencing of tubular epithelial cells in HP compared to NP or LP diet group revealed increased expression of sodium-glutamine transporter Snat3, chloride channel Clcnka, and gluconeogenesis marker Pepck1, accompanied by increased excretion of urinary ammonia, a byproduct of glutamine. Conclusion: Chronic dietary protein load-induced renal hypertrophy and accelerated cyst growth in Pkd1KO mice is dependent on both infiltrating and resident MФ recruitment and subsequent inflammatory response. Early cyst expansion by HP diet, however, is relient on increased delivery of glutamine to kidney epithelial cells, driving downstream metabolic changes prior to inflammatory provocation.

Project description:Polycystic Kidney Disease is characterized by the formation of large fluid-filled cysts that eventually destroy the renal parenchyma leading to end-stage renal failure. Although remarkable progress has been made in understanding the pathologic mechanism of the disease, the precise orchestration of the early events leading to cyst formation is still unclear. Abnormal cellular proliferation was traditionally considered to be one of the primary irregularities leading to cyst initiation and growth. Consequently, many therapeutic interventions have focused on targeting this abnormal proliferation, and some have even progressed to clinical trials. However, the role of proliferation in cyst development was primarily examined at stages where cysts are already visible in the kidneys and therefore at later stages of disease development. In this study we focused on the cystic phenotype since birth in an attempt to clarify the temporal contribution of cellular proliferation in cyst development. Using a PKD2 transgenic rat model (PKD2 (1-703)) of different ages (0-60 days after birth) we performed gene expression profiling and phenotype analysis by measuring various kidney parameters. Phenotype analysis demonstrated that renal cysts appear immediately after birth in the PKD2 transgenic rat model (PKD2 (1-703)). On the other hand, abnormal proliferation occurs at later stages of the disease as identified by gene expression profiling. Interestingly, other pathways appear to be deregulated at early stages of the disease in this PKD model. Our data suggest that cystogenesis precedes deregulation of proliferation-related pathways, suggesting that proliferation abnormalities may contribute in cyst growth rather than cyst formation. In this study we focused on the cystic phenotype since birth in an attempt to clarify the temporal contribution of cellular proliferation in cyst development. Using a PKD2 transgenic rat model (PKD2 (1-703)) of different ages (0,6 and 24 days after birth) we performed gene expression profiling of kidney cells.

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Rapamycin and dexamethasone during pregnancy prevents tuberous sclerosis complex associated cystic kidney disease

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