Abstract: TH-PO061

Targeting Podocyte Endoplasmic Reticulum Calcium Depletion to Treat Nephrotic Syndrome

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Park, Sun-Ji, Washington University School of Medicine, St. Louis, Missouri, United States
  • Kim, Yeawon, Washington University School of Medicine, St. Louis, Missouri, United States
  • Miner, Jeffrey H., Washington University School of Medicine, St. Louis, Missouri, United States
  • Urano, Fumihiko, Washington University School of Medicine, Saint Louis, Missouri, United States
  • Chen, Ying Maggie, Washington University School of Medicine, St. Louis, Missouri, United States
Background

Emerging evidence has demonstrated that podocyte endoplasmic reticulum (ER) stress caused by gene mutations contributes to the pathogenesis of nephrotic syndrome (NS). ER stress-mediated calcium efflux from the ER to the cytosol can activate calcium-dependent protease calpain 2, which underlies the development of proteinuria. Here for the first time, we have shown that a novel ER calcium stabilizer can inhibit podocyte calpain activation.

Methods

We have developed a NS mouse model in which C321R mutation of the glomerular basement membrane constituent laminin β2 (LAMB2), a mutation identified in human patients, leads to podocyte ER stress. We isolated mouse glomeruli and cultured primary podocytes at the early stage of the disease to investigate the functional impact of ER stress. Meanwhile, we generated human podocytes stably expressing WT or C321R β2. Moreover, a Gaussia luciferase (GLuc)-based assay utilizing secreted ER calcium-monitoring proteins (SERCaMPs), and fluorescent calcium indicator Fluo-4 detected by flow cytometry were employed to monitor ER calcium leak in live cells and to measure cytosolic calcium levels, respectively.

Results

Podocyte ER stress triggered by the C321R mutation induced calpain activation, as indicated by the cleavage of its substrate spectrin. In addition, calpain 2 hyperactivation in mutant podocytes activated the caspase 12 apoptotic pathway, cleaved the podocyte cytoskeletal protein talin, and downregulated the podocyte slit diaphragm proteins nephrin and podocin. Furthermore, cytosolic calcium levels were increased in human podocytes expressing C321R-LAMB2 compare to WT-LAMB2, directly demonstrating ER calcium depletion in the mutant podocytes. RNAseq further highlighted dysregulated ER calcium signaling pathways in C321R podocytes compared to controls. Most excitingly, by utilizing GLuc-SERCaMP as a high throughput drug screening tool, a novel ER calcium stabilizer was identified to suppress calpain 2 activation in C321R podocytes.

Conclusion

Currently there is no treatment for most genetic forms of NS. Our study may open up a new avenue for treatment of NS caused by podocyte ER calcium depletion.

Funding

  • NIDDK Support