Abstract: FR-PO762
Podocytes Derived from Patients with Proteinuria Associated with Truncating Mutation in CLCN5 Display Increased Apoptosis and Defective Endocytosis
Session Information
- Glomerular Diseases: Mechanisms and Podocyte Biology
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Lane, Brandon M., Duke University, Durham, North Carolina, United States
- Chryst-Stangl, Megan, Duke University, Durham, North Carolina, United States
- Zhang, Sue, Duke University, Durham, North Carolina, United States
- Wu, Guanghong, Duke University, Durham, North Carolina, United States
- Gbadegesin, Rasheed A., Duke University, Durham, North Carolina, United States
Background
Pathogenic mutations in the gene encoding Chloride Voltage gated channel 5 (CLCN5) are generally associated with Dent Disease, a tubular disorder characterized by nephrolithiasis. However, a few CLCN5 variants have also been reported to cause nephrotic syndrome (NS) phenotypes. We previously identified a novel segregating hemizygote truncating mutation (c. 1199del, p.Gly400ValfsTer29) in CLCN5 in a family with four affected boys with NS phenotypes and biopsy proven focal segmental glomerulosclerosis (FSGS). CLCN5 encodes a member of the chloride ion channels and ion transporters that regulate endosomal acidification and receptor-mediated endocytosis in tubules but is also expressed in the podocyte. However, it is unknown if pathogenic mutations that produce NS phenotypes can affect podocyte homeostasis.
Methods
To determine if pathogenic mutations in CLCN5 can alter podocyte homeostasis, we examined endocytosis and apoptosis phenotypes in iPSC-derived podocytes from patients with CLCN5 mutations and their unaffected family members. We also examined the effect of CLCN5 gene deficiency in multiple immortalized podocyte cell lines by using lentiviral based gene knockdown. Automated live-cell imaging of internalized fluorescent pHrodo labeled dextran molecules was used quantify the endocytosis of small molecules. Live-cell imaging of fluorescent reporters of caspase 3 activity as well as propidium iodide were used to measure podocyte viability when podocytes were exposed to serum starvation.
Results
Podocytes derived from the affected family members with the truncating CLCN5 variant displayed decreased endocytosis of fluorescent dextran molecules and increased apoptosis compared to podocytes from unaffected family members (p<0.001 for both assays). Immortalized podocytes with CLCN5 gene deficiency displayed similar phenotypes.
Conclusion
Pathogenic variants in CLCN5 capable of inducing NS phenotypes cause decreased podocyte endocytosis. These reductions in endocytosis likely contribute to the reduced viability associated with deficiencies in CLCN5. Further investigation is required to determine if additional processes contribute to the increased apoptosis in CLCN5 deficient podocytes.
Funding
- Private Foundation Support