Kidney Organoids Generated Using an Allelic Series of NPHS2 Point Variants Reveal Distinct Intracellular Podocin Mistrafficking

Abstract

Background:NPHS2 variants are the most common cause of steroid-resistant nephrotic syndrome in >1-month-old children. Missense NPHS2 variants were reported to cause mistrafficking of the encoded protein, PODOCIN, but this conclusion was based on overexpression in some non-podocyte cell lines. Methods: We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2, encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H and p.R291W, as well as control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. Results: All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. While wildtype PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal diseaseassociated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN co-localization elucidated the variantspecific impact on NEPHRIN association and hence NEPHRIN trafficking. Conclusion: Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the impact of each variant on protein levels and localization and impact on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants.

Elucidating the Proximal Tubule HNF4A Gene Regulatory Network in Human Kidney Organoids

“…[21][22][23][24][25][26] Previous studies used kidney organoids from geneedited PSCs or patient-derived PSCs to model developmental abnormalities and kidney diseases, such as polycystic kidney diseases [27][28][29][30][31][32][33] and nephrotic syndrome. [34][35][36][37][38] Kidney organoids thus provide a good model system to study gene function and elucidate pathophysiological mechanisms during human kidney development.…”

Organoids are not organs: Sources of variation and misinformation in organoid biology

“…These models have enabled insights into how specific cell types contribute to the development of specific tissue structures and have also elucidated how gradients of specific growth factors, cytokines, and morphogens influence cellular identity during organogenesis. Most notable, the ability to study morphogenesis in vitro with PSCs derived from patients, or via introduction of allelic series of mutations associated with congenital disorders, has provided a tool to investigate diseases, including microcephaly, autism spectrum disorder, cystic kidney disease, ciliopathies and glomerulopathies, congenital cardiac anomalies, and many other conditions ( Brandão et al., 2020 ; Cruz et al., 2017 ; Dorison et al., 2022 , 2023 ; Dvela-Levitt et al., 2019 ; Forbes et al., 2018 ; Freedman et al., 2015 ; Lancaster et al., 2013 ; Majmundar et al., 2021 ; Paulsen et al., 2022 ; Rowe and Daley, 2019 ; Tran et al., 2022 ). In addition, PSC-derived organoids have been used to generate tissue models to study microbial interactions including viral infection of SARS-CoV-2 ( Vanslambrouck et al., 2022 ; Yang et al., 2020 ) and Zika virus ( Tang et al., 2016 ), and bacteria such as Clostridium difficile ( Leslie et al., 2015 ) and Salmonella ( Forbester et al., 2015 ).…”

“…It will therefore be necessary to analyze multiple biological replicates, and/or multiple patient lines. In a recent example, we studied the impact of an allelic series of NPHS2 missense mutations on protein trafficking of the encoded protein, PODOCIN, within the podocytes of kidney organoids ( Dorison et al., 2023 ). This involved transcriptional profiling of age-matched isolated glomeruli from five distinct mutant lines, the parental control together with a patient (homozygous) and parent (heterozygous) line.…”

“…This entire experiment was done as a biological triplicate, such that all lines were simultaneously differentiated for isolation on three occasions for profiling. While any given mutant/control pair could identify transcriptional changes, there were no common disease-associated transcriptional changes identified ( Dorison et al., 2023 ). While this may appear unsurprising given that the mutated gene does not encode a transcription factor, what this rigorous design reveals is the danger of using differentially expressed genes from less powered experiments as the only readout when performing disease modeling.…”

Pinpointing Podocin Trafficking Defects in Kidney Organoids

“…To address this question, Dorison et al 9 used kidney organoids to model missense variants in NPHS2 . The organoids were differentiated from gene-edited and patient-derived induced pluripotent stem cells (iPSC) to create an allelic series for investigating the functional consequences of variants in NPHS2 (Figure 1).…”