Presentation Authors: Astgik Petrosyan, Los Angeles, CA, Paolo Cravedi, New York, NY, Valentina Villani, Los Angeles, CA, Alessandra Renieri, Siena, Italy, Roger De Filippo, Laura Perin, Stefano Da Sacco*, Los Angeles, CA
Introduction: With increasing rates of renal failure and limited options for its treatment, there is an urgent need of implementing our knowledge and resources to guide new strategies for understanding disease- and patient-specific glomerular pathophysiology and for developing more efficient drug screening tools. Recreating an ex-vivo functional glomerulus depends on our ability to generate an in vitro 3D multicellular system that allows fluid perfusion and proper interactions between podocytes and glomerular endothelial cells (hGEC) in a platform that mimics the complex architecture of the glomerular filtration barrier (GFB).
Methods: Amniotic fluid derived podocytes (hAKPC-P), human immortalized podocytes (hiPod), primary human podocytes (hpPod) and human fibroblasts or human lung endothelial cells (negative controls) were seeded on microfluidic chips with hGEC. Immunofluorescence was performed for podocyte, endothelial and GBM markers. Barrier selective-permeability was investigated. Feasibility of the system for high throughput screening was evaluated by measuring permselectivity following Puromycin Aminonucleoside (PAN) injury or culture with serum membranous nephropathy (MN) patients. Drug screening for MN and disease modeling for Alport Syndrome (AS) were also performed.
Results: We have developed an innovative, barrier-free, glomerulus-on-a-chip (GOAC) system. Podocytes formed a slit diaphragm-like structure expressing nephrin. CD31-expressing hGEC formed capillary-like structures. De-novo deposition of GBM components such as collagen IV and laminin alpha5 was verified. Albumin permselectivity was confirmed and permselectivity was impaired following PAN administration as confirmed by our assay. In the presence of MN serum the chip displayed IgG deposition on the podocytes and loss of permselectivity to albumin, to an extent proportional to urinary protein loss in vivo. ACTH successfully rescued MN-mediated damage, confirming feasibility for drug screening. Chip structure and function were impaired when we used podocytes from individuals with AS, a kidney disease due to genetically determined collagen abnormalities.
Conclusions: We have successfully developed a GOAC system that closely mimics the GFB structure and provides a powerful tool for studying renal regenerative and disease mechanisms, toxicity effects and will help the discovery of new drugs. In conclusion, this system will increase our ability to individualize treatments and drug susceptibility, thus ultimately benefiting patients affected by renal failure.
Source of Funding: Alport Syndrome FoundationTSRI Research Career Development AwardWright Foundation Pilot AwardGOFARR Fund