Unravelling the Role of PAX2 Mutation in Human Focal Segmental Glomerulosclerosis.

Lorena Longaretti,Piera Trionfini,Susanna Tomasoni,Elena Romano,Ariela Benigni,Christodoulos Xinaris,Roberta Giampietro,Valerio Brizi,Caterina Mele,Giuseppe Remuzzi,Matteo Breno

doi:10.3390/biomedicines9121808

Abstract

No effective treatments are available for familial steroid-resistant Focal Segmental Glomerulosclerosis (FSGS), characterized by proteinuria due to ultrastructural abnormalities in glomerular podocytes. Here, we studied a private PAX2 mutation identified in a patient who developed FSGS in adulthood. By generating adult podocytes using patient-specific induced pluripotent stem cells (iPSC), we developed an in vitro model to dissect the role of this mutation in the onset of FSGS. Despite the PAX2 mutation, patient iPSC properly differentiated into podocytes that exhibited a normal structure and function when compared to control podocytes. However, when exposed to an environmental trigger, patient podocytes were less viable and more susceptible to cell injury. Fixing the mutation improved their phenotype and functionality. Using a branching morphogenesis assay, we documented developmental defects in patient-derived ureteric bud-like tubules that were totally rescued by fixing the mutation. These data strongly support the hypothesis that the PAX2 mutation has a dual effect, first in renal organogenesis, which could account for a suboptimal nephron number at birth, and second in adult podocytes, which are more susceptible to cell death caused by environmental triggers. These abnormalities might translate into the development of proteinuria in vivo, with a progressive decline in renal function, leading to FSGS.

Highlights

Familial steroid-resistant nephrotic syndrome (SRNS) is a subgroup of nephrotic syndromes classified by genetic mapping studies that progress to end stage renal disease (ESRD) [1]
Real time PCR analysis revealed that PAX2 peaked in the first stage of differentiation, confirming the proper induction of intermediate mesoderm-like cells (Figure 1)
At day 6 of the differentiation protocol, SIX2, a marker that characterizes a population of self-renewing multipotent nephron progenitors (NP), was half expressed in PAX2G189R/+ NP than in control cells without compromising cell differentiation (Supplementary Figure S1)

Summary

Introduction

Familial steroid-resistant nephrotic syndrome (SRNS) is a subgroup of nephrotic syndromes classified by genetic mapping studies that progress to end stage renal disease (ESRD) [1]. Dysfunction of the glomerular podocyte, a specialized cell that forms the glomerular filtration barrier, is pivotal in the paradigm for the pathogenesis of FSGS [2]. The discovery of mutations in various genes encoding podocyte proteins is one of the most exciting advances in the understanding of the pathogenesis of SRNS and has been made possible by studying familial forms [4]. The FSGSassociated genes identified so far encode proteins located in the podocyte slit diaphragm, cell membrane, cytosol, actin cytoskeleton, nucleus, mitochondria and lysosomes. A disease-segregating missense heterozygous mutation (c.565G > A, p.G189R) in the PAX2 gene has recently been identified in a large family with adult onset autosomal dominant FSGS [5]. Human iPSC were induced to differentiate toward UB-like cells as previously described [13]. Cells were grown for 2 days in a chemically defined basal medium [DMEM/F12 + GlutaMAX, 17.5 mg/mL BSA fraction V (Merck Millipore, Burlington, MA, USA), 17.5 μg/mL human insulin (Sigma-Aldrich), 275 μg/mL human holo-transferrin (Sigma-Aldrich), 450 μM 1-thioglycerol (Sigma-Aldrich), 0.1 mM non-essential amino acids (Gibco), 1% Pen-Strep] supplemented with 50 ng/mL human fibroblast growth factor

Methods

Results

Conclusion