3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening

Lorna J Hale,Sean Wilson,Rachel Lennon,Pei X Er,Shahnaz Khan,Salman Hosawi,Andrew Lonsdale,Alicia Oshlack,Melissa H Little,Sara E Howden,Belinda Phipson,Irene Ghobrial,Kynan T Lawlor,Catherine Quinlan

doi:10.1038/s41467-018-07594-z

Abstract

The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.

Highlights

The human kidney regulates fluid homoeostasis, electrolyte balance, and waste product removal by filtering the blood via glomeruli, the specialised filtration unit within each nephron
A number of kidney diseases leading to proteinuria and/or haematuria, including congenital nephrotic syndrome (CNS) and Alport syndrome, result from defects in the glomerular basement membrane (GBM), or functional and structural alterations to the podocyte that lead to foot process effacement and loss of slit diaphragms[8]
Within human induced pluripotent stem cell (iPSC)-derived kidney organoids, we observed the formation of podocyte clusters that were resistant to enzymatic dissociation

Summary

Introduction

The human kidney regulates fluid homoeostasis, electrolyte balance, and waste product removal by filtering the blood via glomeruli, the specialised filtration unit within each nephron. Podocytes are postmitotic cells with a highly specialised morphology[2] They possess elaborate interdigitating cellular processes which are anchored to the glomerular basement membrane (GBM) via a network of integrins and dystroglycans. The genetic basis of many podocytopathies has been elucidated[9] These include mutations in genes encoding components of the podocyte actin cytoskeleton, slit diaphragm, and GBM. The generation of a temperature-sensitive SV40 conditionally immortalised podocyte cell line, which allows proliferation at 33 °C and terminal differentiation at 37 °C in vitro[11], began to address this challenge Studies using this two-dimensional podocyte model, primary human podocyte cultures and murine podocyte cell lines have substantially advanced our understanding of podocyte biology. Preliminary analysis of the glomeruli within these kidney organoids showed tightly interdigitated podocytes while transcriptional profiling of whole organoids revealed the induction of podocyte gene expression, including NPHS1, NPHS2 and PODXL17, implicating this approach as a promising source of human podocytes

Methods

Results

Conclusion