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

Area of Science:

• Nephrology
• Genetics
• Stem Cell Biology

Background:

• Steroid-resistant nephrotic syndrome (SRNS) in children is often caused by NPHS2 variants.
• Previous studies on NPHS2 variants used overexpression in non-podocyte cell lines, limiting conclusions about protein mistrafficking.

Purpose of the Study:

• To investigate the cellular effects of pathogenic NPHS2 missense variants using human induced pluripotent stem cells (iPSCs) differentiated into kidney organoids.
• To determine the impact of specific NPHS2 variants on podocin (PODOCIN) protein expression, localization, and interaction with nephrin.

Main Methods:

• Generated iPSC lines with pathogenic NPHS2 missense variants (p.G92C, p.P118L, p.R138Q, p.R168H, p.R291W) and control lines.
• Differentiated iPSC lines into kidney organoids and assessed PODOCIN expression and localization via immunofluorescence.
• Performed transcriptional profiling of podocytes and interrogated PODOCIN-NEPHRIN interaction.

Main Results:

• All NPHS2 variant lines showed reduced PODOCIN protein levels without transcriptional changes.
• Distinct pathogenic variants resulted in unique PODOCIN subcellular mislocalization patterns, including ER and Golgi retention.
• Variant PODOCIN proteins exhibited altered NEPHRIN association, affecting NEPHRIN trafficking and leading to podocyte-specific apoptosis.

Conclusions:

• Specific endogenous NPHS2 variants cause distinct PODOCIN mislocalization within organoid podocytes.
• Understanding variant-specific effects on PODOCIN and NEPHRIN provides crucial insights into the pathobiology of NPHS2-associated nephrotic syndrome.