Abstract
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.