Deciphering the Impact of RAC1-SPTAN1 in ARPKD Cystogenesis Using Multifaceted Models

Autosomal recessive polycystic kidney disease (ARPKD) leads to severe renal cysts and progressive kidney dysfunction, with no approved treatments. The absence of such cystic phenotypes in Pkhd1^-/^- mice underscores the need for novel models that better recapitulate the human disease. We developed kidney organoid-on-chip models that mimic patients' distal-nephron cysts, identifying RAC1/c-FOS as potential therapeutic targets. However, critical questions remain regarding RAC1 activation during cyst formation, cyst origins, and underlying molecular mechanisms. Using a multifaceted approach, organoid-on-chip models, transgenic mice, and patient kidney samples, we identified reduced levels of SPTAN1, a cytoskeletal spectrin protein, as a key regulator of RAC1 activation and cystic pathology. SPTAN1-mutant kidney organoids and mice exhibited distal-nephron cysts, and elevated RAC1/c-FOS expression, consistent with ARPKD patients. Transcriptomics and live imaging revealed altered calcium signaling and increased intracellular calcium. Single-cell RNA-seq identified SLC8A1, a sodium/calcium exchanger, as a marker distinguishing distal/connecting tubules from collecting ducts in human kidneys, predominantly expressed in cystic epithelia in organoids and human ARPKD kidneys. Restoring SPTAN1 in PKHD1^-/^- organoids via CRISPR activation alleviated cystic phenotypes, normalized intracellular calcium, and reduced RAC1/c-FOS expression. These findings position SPTAN1 as a central player in ARPKD pathogenesis and highlight epigenome editing as a potential therapeutic strategy.