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An Inducible hiPSC-Derived Human Podocyte Model for Functional Analysis of TRPC6 Variants Associated with FSGS
Lilas Batool1, Krithika Hariharan2, Gabriel Stölting3
1Hamburg Center for Kidney Health (HCKH), University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany.
Abstract:
Podocyte injury is a characteristic feature of focal segmental glomerulosclerosis (FSGS) that leads to the development of nephrosis as its loss causes proteinuria and progressive glomerulosclerosis. The physiological function of podocytes is critically dependent on proper intracellular calcium levels; an excess or shortage of calcium influx in these cells may result in foot process effacement, apoptosis, and nephron degeneration. A key protein responsible for the regulation of calcium flux is the canonical transient receptor potential 6 (TRPC6) expressed in podocytes. Several mutations in the TRPC6 gene have been associated with FSGS. Here we present a systematically optimized inducible FSGS model system in human induced pluripotent stem cells (hiPSCs). We generated and phenotypically characterized three transgenic hiPSC lines with regulatable overexpression of TRPC6 wild-type and FSGS-associated gain-of-function (GoF, P112Q) and loss-of-function (LoF, G757D) mutations. Moreover, these cell lines were differentiated into induced podocytes (ipodocytes). We assessed the impact of TRPC6 GoF and LoF mutants on calcium influx in combination with TRPC6 agonists and antagonists. Our data showed relative calcium responses consistent with the GoF and LoF phenotypes. Transgenic iPSC-based models, like the one presented here, are instrumental to studying disease mechanisms in vitro and investigating the outcomes of, and possible therapeutic interventions for, this complex disease.
Insights
Focal segmental glomerulosclerosis (FSGS) involves podocyte injury and altered calcium levels. This study developed a human induced pluripotent stem cell (hiPSC) model to investigate TRPC6 mutations in FSGS, revealing insights into disease mechanisms.
Area of Science:
- Nephrology
- Stem Cell Biology
- Molecular Genetics
Background:
- Podocyte injury is central to focal segmental glomerulosclerosis (FSGS), leading to nephrosis, proteinuria, and glomerulosclerosis.
- Intracellular calcium homeostasis in podocytes is crucial; dysregulation causes foot process effacement, apoptosis, and nephron degeneration.
- The canonical transient receptor potential 6 (TRPC6) channel regulates podocyte calcium flux, and its mutations are linked to FSGS.
Purpose of the Study:
- To develop and characterize an inducible human induced pluripotent stem cell (hiPSC) model for studying FSGS.
- To investigate the impact of TRPC6 gain-of-function (GoF) and loss-of-function (LoF) mutations on calcium influx in differentiated podocytes.
- To establish a platform for in vitro disease mechanism studies and therapeutic intervention screening for FSGS.
Main Methods:
- Generation and phenotypic characterization of three transgenic hiPSC lines with inducible TRPC6 overexpression (wild-type, GoF mutant P112Q, LoF mutant G757D).
- Differentiation of hiPSC lines into induced podocytes (ipodocytes).
- Assessment of calcium influx in ipodocytes using TRPC6 agonists and antagonists to evaluate mutant phenotypes.
Main Results:
- Successful generation of hiPSC lines with regulatable TRPC6 expression, including FSGS-associated mutants.
- Differentiated ipodocytes exhibited calcium responses consistent with the predicted GoF and LoF phenotypes of TRPC6 mutations.
- The developed model system effectively demonstrated the impact of TRPC6 mutations on calcium homeostasis in podocytes.
Conclusions:
- The developed inducible hiPSC-based model provides a robust system for studying FSGS in vitro.
- This model allows for detailed investigation of TRPC6 mutation effects on podocyte calcium signaling.
- The platform is valuable for advancing understanding of FSGS pathogenesis and exploring potential therapeutic strategies.
