Mesencephalic Astrocyte-Derived Neurotrophic Factor Orchestrates Endoplasmic Reticulum (ER)-Mitochondrial Calcium Homeostasis in Genetic CKD

Background: Autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD), a leading hereditary nephropathy and monogenic chronic kidney disease, has no targeted therapies. UMOD is expressed in the thick ascending limb (TAL) tubules and mutant UMOD is retained in the endoplasmic reticulum (ER). Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel ER resident chaperone, and its biological role in ADTKD remains to be elucidated. Methods: The first Umod deletion mutation mouse model, resembling ADTKD patients harboring the most prevalent H177-R185 del mutation, was generated by CRISPR. Meanwhile, stable HEK293 cells expressing WT or the mutant UMOD were established. Patient-derived ADTKD and WT organoids were differentiated from human induced pluripotent stem cells. To investigate the role of MANF in ADTKD, TAL-specific MANF knockout mice were generated, and shRNA was used to knockdown MANF in vitro. ER calcium depletion was assessed by a novel secreted ER calcium-monitoring protein (SERCaMP), and mitochondrial calcium load was determined by confocal live cell imaging of Rhod-2AM. In addition, mitochondrial respiratory function was evaluated by OROBOROS in isolated TALs and Seahorse assays in vitro. Results: MANF is induced in the mutant TALs carrying the Umod deletion mutation, isolated from the ADTKD mice and kidney organoids. MANF depletion in the mutant TALs further inhibits phosphorylation of AMP-activated protein kinase (AMPK), resulting in exacerbated failure of mitochondrial biogenesis and mitophagy, both in vivo and invitro. Mechanistically, for the first time, we demonstrate that MANF deficiency in the mutant TALs enhances ER calcium leak and subsequent mitochondrial calcium overload, leading to augmented p-AMPK suppression. Consequently, loss of MANF in the mutant TALs activates mitochondrial DNA leak-induced STING (stimulator of interferon genes) immune response, accelerating renal fibrosis in ADTKD. Conclusion: We provide the first evidence that MANF regulates ER-mitochondrial calcium transfer, which is essential to maintain mitochondrial health in ADTKD. Our discovery paves the way for potential therapeutic strategies targeting MANF and ER-mitochondria crosstalk in ADTKD. Funding: NIDDK Support - NIDDK Support, NIDDK Support, Veterans Affairs Support, Private Foundation Support