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

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.