3D vascularized proximal tubule model improves cell phenotype
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE198822 | GEO | 2022/03/20
REPOSITORIES: GEO
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Project description:Angiotensin II (AngII), the effector of the renin angiotensin system causes kidney disease progression by signalling through AT-1 receptor, but there are no measures of AngII activity in the kidney. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular epithelial cells (PTEC) in order to identify potential markers of AngII activity in the kidney. PTECs were labeled with SILAC heavy arginine(+6) and lysine(+8) OR light arginine and lysine for 6 doubling times. Heavy-labeled PTECs were stimulated with AngII and light-labeled PTECs were stimulated with the control medium. AngII-treated and control-treated PTEC lysates were mixed in 1:1 total protein ratio and their proteomes were compared. We generated 5 biological replicates (1 supernatant, 1 replicate with reverse labeling). LTQ-Orbitrap mass spectrometer was used for LC-MSMS analysis. Of 4618 quantified proteins, 83 were differentially regulated by AngII in 4 biological replicates of PTEC lysates. We subsequently confirmed and verified 18 differentially regulated proteins by using SRM, RT-PCR and ELISA. We also went on to validate the main functional, enriched networks by using systems biology approach and an in vivo mouse model.
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Project description:Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular-vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium-endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.
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Project description:Overexpression of a caspase-resistant form of Bcl-2 (D34A) in human umbilical vein endothelial cells (EC) implanted into immunodeficient mice promotes the maturation of human EC-lined microvessels invested by vascular smooth muscle cells (VSMC) of mouse origin. In contrast, EC implants not overexpressing Bcl-2 form only simple, uncoated EC tubes. Here we compare the phenotypes of vessels formed in vivo and the transcriptomes in vitro of EC expressing different forms of Bcl-2. Wild type Bcl-2, like the caspase resistant D34A Bcl-2 mutant, is anti-apoptotic in vitro and promotes VSMC recruitment in vivo, whereas a G145E mutant that has diminished anti-apoptotic activity in vitro does not promote vessel maturation in vivo. The D34A and wild type forms of Bcl-2, but not the G145E mutant form of Bcl-2 significantly regulate RNA transcripts previously associated with EC-VSMC interactions and VSMC biology, including matrix Gla protein, insulin like growth factor binding protein (IGFBP)-2, matrix metaloproteinase-14 (MMP14), ADAM17 and Stanniocalcin-1. These effects of Bcl-2 on the transcriptome are detected in EC cultured as angiogenic 3-D tubes but are attenuated in EC cultured as 2-D monolayers. Bcl-2-regulated transcription in EC may contribute to vascular maturation, and support design of tissue engineering strategies using EC. Experiment Overall Design: This study consists of the analysis of human umbilical vein endothelial cells (HUVEC) cultured under several conditions, including retroviral transduction, as well as on different culture substrates and in different geometries. For a full description please see our paper in the journal Endothelium, 2008. For
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