pathophysiological responses of proximal tubule epithelial cells...

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