Abstract: PO0673
Human Proximal Tubular Cells in a 3D In Vitro Culture as a Model for Exploring Diabetic Lesions
Session Information
- Diabetic Kidney Disease: Basic - I
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 601 Diabetic Kidney Disease: Basic
Authors
- Palau, Vanesa, Consorci Parc de Salut MAR de Barcelona, Barcelona, Catalunya, Spain
- Barrios, Clara, Consorci Parc de Salut MAR de Barcelona, Barcelona, Catalunya, Spain
- Márquez, Eva, Consorci Parc de Salut MAR de Barcelona, Barcelona, Catalunya, Spain
- Rodriguez, Eva, Consorci Parc de Salut MAR de Barcelona, Barcelona, Catalunya, Spain
- Crespo, Marta, Consorci Parc de Salut MAR de Barcelona, Barcelona, Catalunya, Spain
- Pascual, Julio, Consorci Parc de Salut MAR de Barcelona, Barcelona, Catalunya, Spain
- Riera, Marta, Consorci Parc de Salut MAR de Barcelona, Barcelona, Catalunya, Spain
Background
We have previously demonstrated, the beneficial effect of ADAM17 deletion on human proximal tubular kidney cells (HKC-8) in 3D in vitro spheroids incubated with high glucose resembling the human kidney diabetic environment. Galectin-3 (Gal-3) is a potent pro-fibrotic protein and modulates the activity of fibroblasts and macrophages in chronically inflamed organs through activation of the TGF-β/Smad3 pathway and it is postulated as regulator of cardiac oxidative stress which can facilitate the development of fibrosis. Also, Dynamin related protein 1 (DRP-1) is a key regulator of the mitochondrial fission and ATP production under stress condition. As ADAM17 has been associated with TGF-β modulation during renal fibrosis, we wanted to evaluate the effect of ADAM17 deletion on Gal-3, Fibronectin and DRP-1 in HKC-8 spheroids incubated under high glucose conditions
Methods
ADAM17 deletion of renal tubular cells was performed using the CRISPR/Cas9 technology. HKC-8 cells grew inside an RGD-functionalized dextran hydrogel to obtain 3D spheroids. 13 days post-seeding, the spheroids were incubated with 35mM of D-glucose (HG), 5mM of D-glucose (LG) or 35mM of mannitol as osmotic control for 72h. Immunofluorescence for Gal-3, pDRP-1 and Fibronectin was performed
Results
HG increased the expression of fibronectin and pDRP-1 and tends to increase Gal-3 in wild-type (WT) spheroids. Interestingly, ADAM17 deletion decreased fibronectin expression in spheroids incubated with HG as compared to WT spheroids. Moreover, ADAM17 deletion abrogates the effect of HG on Gal-3 expression (see table and images, scale bar 50μm). The osmotic control, mannitol, did not affect the expression of the analysed proteins
Conclusion
ADAM17 blockade protects against fibrosis by decreasing fibronectin and Gal-3 and modulated the mitochondrial dynamic in human kidney tubular spheroids under high glucose conditions