Abstract: TH-PO785
Isolation of Functional Podocytes from Urine of Alport Patients and Establishment of a Kidney Glomerulus Chip
Session Information
- Cellular Crosstalk in Glomerular Diseases - I
October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix
Authors
- Bussolati, Benedetta, University of Torino, Torino, Italy
- Iampietro, Corinne, Universita'' degli studi di Torino, Turin, Italy
- Bellucci, Linda, Universita'' degli studi di Torino, Turin, Italy
- Arcolino, Fanny Oliveira, KU Leuven , Leuven, Belgium
- Levtchenko, Elena N., University Hospitals Leuven, Herent, Belgium
Background
Alport syndrome (AS) is a genetic disorder characterized by mutations in genes of collagen IV α3α4α5 network. In AS patients, podocyte loss starts at birth and results in progressive reduction in podocyte number per glomerulus with time, correlating with renal damage. However, data on the phenotypic characteristics of human AS podocytes are currently limited. Our goal was to set up a novel human in vitro model representing the functional and molecular alterations typical of human AS glomerulus.
Methods
We generated conditionally immortalized podocytes from urine of three different patients (aged 16.5 ± 5.5 years) with Alport syndrome (AS podocytes). Patient 1 showed mutation in Col4α3 gene, while Patient 2 and Patient 3 were mutated in Col4α5 gene in heterozygosity and in hemizygosity, respectively. AS podocytes were analyzed for specific podocyte markers by RT-PCR and Western Blot and release of collagen IV chains by ELISA. Podocyte motility was tested in a wound-healing assay. We generated a kidney glomerulus chip using primary human glomerular endothelial cells and control or AS podocytes using a millifluidic device for continuous perfusion of co-culture. Passage of FITC-labeled albumin and inulin was measured by fluorimeter.
Results
Podocytes were immortalized with temperature-sensitive SV40T and telomerase reverse transcriptase. Cell showed growth at 33°C (permissive condition) and differentiation at 37°C, with expression of typical podocyte markers. Levels of nephrin, but not of podocin or synaptopodin, were significantly reduced in respect to control podocytes. Significant reduction of specific collagen IV chains were detected, confirm specific functional alterations. AS podocytes had significantly reduced motility in respect to control podocytes. Moreover, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip millifluidic device, AS podocytes presented increased permeability.
Conclusion
In conclusion, we generated Alport podocytes and identified specific molecular and functional alterations. Moreover, we developed a human Alport glomerulus-on-a-chip that replicates kidney disease phenotype in vitro. This will be pivotal to test pharmacological approaches for glomerular alterations in Alport disease.
Funding
- Government Support - Non-U.S.