Pathophysiological Responses of Proximal Tubule Epithelial Cells to Human Serum
Modeling the Effects of Proteinuria on the Proximal Tubule
Ranita Patel, MD
Pediatric Nephrology Fellow
Seattle Children’s Hospital
University of Washington Department of Pharmacy
Background
Jefferson, J., Shankland, S., & Pichler, R. (2008). Proteinuria in diabetic kidney disease: A mechanistic viewpoint. Kidney International,74(1), 22-36. doi:10.1038/ki.2008.128
Background
Modified from Abbate, Zoja, and Remuzzi. How does proteinuria cause progressive renal damage? JASN. 2006 17(11):2974-2984
Project rationale
Proteinuria is a clinical biomarker of kidney damage •May be a contributing cause, not just the result of, renal injury
Proximal tubule cells acquire a pro-inflammatory, pro-fibrotic phenotype during sustained proteinuria
•May contribute to loss of renal function in progressive kidney disease
Our goals are to: •Develop a model of proteinuria to understand the molecular changes that occur in proximal tubule epithelial cells (PTECs) •Identify potential avenues for pharmacological intervention •Evaluate the impact of microgravity on PTEC response to proteinuria
Hypothesis
Hypothesis: Changes in renal ultrafiltrate composition during proteinuria drive PTECs towards a pro-inflammatory and pro-fibrotic phenotype
2D Methods: Static PTEC Cultures
3 donors
Cells were seeded into 24 well plates at ~40,000 cells/well.
Following day cells were treated with 0, 0.5, 1, or 2% albumin containing serum
At 8 hours, 48 hours, and 7 days, supernatant collected every 24 hours - quantified [KIM-1] Immunocytochemistry - staining for proliferative marker Ki67 RNA extraction - qPCR and RNAseq (MCP-1, Endothelin-1, HO-1, Megalin, Cubilin, KIM-1, GusB)
The Triple Channel Microphysiological System
Dual Channel Microphysiological System
Forming a channel/tubule
3D Methods: Fluidic PTEC Cultures
4 donors, 5 experimental sets
Cells seeded then cultured in devices under flow for 7 days
Treated with varying % human serum At 8, hours, 48 hours, 7 days:
supernatant collected every 24 hours - quantified [KIM-1] Immunocytochemistry - staining for proliferative marker Ki67 RNA extraction - qPCR and RNAseq (MCP-1, Endothelin-1, HO-1, Megalin, Cubilin, KIM-1, GusB)
Human serum promotes shedding of KIM-1 without increasing KIM-1 gene expression or cell proliferation in static PTEC cultures
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