Stabilization of Podocyte Actin Cytoskeleton by SMPDL3b Attributed to Inhibition of Store-Operated Ca²⁺Entry by Choline

Our recent studies demonstrate that deletion of the Smpdl3b gene, encoding sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), induces minimal change disease-like podocytopathy in mice. However, the underlying molecular mechanisms remain unclear. While SMPDL3b has been traditionally regarded as a lipid-modifying enzyme regulating sphingolipid metabolism, emerging evidence challenges this view, showing that SMPDL3b lacks enzymatic activity against sphingolipids. High-resolution structural analysis indicates that SMPDL3b preferentially cleaves CDP-choline, leading to subsequent production of choline, an intracellular messenger. Based on these findings, we hypothesize that SMPDL3b-dependent CDP-choline cleavage and subsequent choline production play a pivotal role in regulating podocyte actin cytoskeleton. To test our hypothesis, we isolated podocytes from WT/WT and Smpdl3b -/- mice for primary culture. Using LC-MS/MS, we observed significantly reduced CDP-choline levels and remarkably elevated choline concentrations in Smpdl3b -/- podocytes compared to WT/WT podocytes. Immunofluorescent staining of F-actin and paxillin (focal adhesion marker) revealed that Smpdl3b gene deletion disrupted actin cytoskeleton and markedly reduced focal adhesion in podocytes, which was reversed by choline and phosphocholine, but not CDP-choline treatment. Atomic force microscopy confirmed that reduced cellular elasticity in Smpdl3b -/- podocytes was restored by choline and phosphocholine, but not CDP-choline. The therapeutic effects of phosphocholine were blocked by pre-treatment with Lansoprazole (phosphocholine phosphatase inhibitor), implicating choline as the active product. Mechanistically, Smpdl3b deletion increased stromal interaction molecule 1 (STIM1)-Orai1 interaction and store-operated Ca 2+ entry (SOCE) in podocytes, which were substantially attenuated by choline and phosphocholine but unaffected by CDP-choline. Lansoprazole pre-treatment similarly blocked phosphocholine’s effects, further supporting the role of choline. These findings suggest that SMPDL3b regulates podocyte actin cytoskeleton stability via CDP-choline cleavage and choline production, which suppress Orai1 channel-mediated SOCE and stabilize the actin cytoskeleton. This study is supported by NIH grants DK054927 and DK120491. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.