Redirection of sphingolipid metabolism drives cytoskeletal defects in SPLIS and reveals ROCK inhibition as therapy
Adam Majcher, Ranjha Khan, Kathrin Buder, Florence Bourquin, Julie D. Saba, Thorsten Hornemann
Adam Majcher, Ranjha Khan, Kathrin Buder, Florence Bourquin, Julie D. Saba, Thorsten Hornemann
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Research Article Genetics Metabolism Nephrology

Redirection of sphingolipid metabolism drives cytoskeletal defects in SPLIS and reveals ROCK inhibition as therapy

Abstract

Sphingosine-1-phosphate lyase (SPL) insufficiency syndrome (SPLIS), also known as nephrotic syndrome type 14, is an autosomal recessive multisystem disorder caused by loss-of-function mutations in SGPL1, encoding the enzyme responsible for the terminal degradation of sphingosine-1-phosphate (S1P). We investigated a patient carrying a previously undescribed c.1084T>A (p.Ser362Thr) SGPL1 variant and analyzed the metabolic and cellular consequences of SPL deficiency, using patient fibroblasts, SGPL1-KO HEK293T cells, and Sgpl1–/– and Sgpl1rosa+fl/fl mice. Metabolic stable isotope labeling revealed that SPL deficiency does not invariably result in S1P accumulation. Instead, SPL-deficient cells maintain near-normal S1P levels through (a) feedback regulation of de novo sphingolipid synthesis via the ORMDL–ceramide axis and (b) increased diversion of excess ceramides into glycosphingolipids. However, perturbation of sphingolipid homeostasis, either by exogenous sphingolipid load or disruption of compensatory regulation, induces pathological intracellular S1P accumulation. In vivo, Sgpl1–/– mice had pronounced urinary S1P excretion and renal S1P enrichment, accompanied by cytoskeletal disorganization and impaired epithelial morphogenesis. Mechanistically, we identify aberrant Rho/ROCK signaling as a key mediator of S1P-driven cytoskeletal dysregulation. Pharmacological ROCK inhibition with fasudil mitigated renal cytoskeletal defects in Sgpl1–/– and Sgpl1rosa+fl/fl mice and partially restored epithelial architecture. These findings redefine the metabolic consequences of SPL deficiency and identify S1P-driven Rho/ROCK hyperactivation as a tractable therapeutic target in SPLIS.

Authors

Adam Majcher, Ranjha Khan, Kathrin Buder, Florence Bourquin, Julie D. Saba, Thorsten Hornemann

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Figure 5

Analysis of S1P levels and kidney pathology in Sgpl1 mice.

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Analysis of S1P levels and kidney pathology in Sgpl1 mice. (A) Schematic...
(A) Schematic representation of tissue and body fluid sample collection from Sgpl1 WT, Sgpl1+/–, and Sgpl1–/– mice. (B) Total S1P levels in tissues from Sgpl1 WT (n = 3), Sgpl1+/– (n = 3), and Sgpl1–/– (n = 3) mice. (C) Total plasma S1P levels in Sgpl1 WT (n = 3), Sgpl1+/– (n = 3), and Sgpl1–/– (n = 3) mice. (D) Total urinary S1P levels in Sgpl1 WT (n = 3), Sgpl1+/– (n = 3), and Sgpl1–/– (n = 3) mice. Bar plots represent mean ± SD (n = 3). S1P levels were measured using LC-MS/MS. Statistical significance was calculated using a 2-tailed t test. Differences between WT and Sgpl1–/– were highly significant (P < 0.0001). (E) Kidney cortex histology of WT and SGPL1 KO mice stained with PAS stain. (Left, with inset detail) WT kidney cortex shows uniform glomeruli with normal size and cellularity. (Right, with inset detail) SGPL1 KO kidney cortex displays protein casts and glomeruli with heterogeneity in size and appearance, mesangial expansion, hypercellularity, and collagen deposition. Percent glomerulosclerosis: WT = 0%; KO = 37%; 50 or more glomeruli were analyzed per genotype. Scale bar: 100 μm. Created in BioRender.