TGF-β1 induces podocyte injury through Smad3-ERK-NF-κB pathway and Fyn-dependent TRPC6 phosphorylation

Area of Science:

• Nephrology
• Molecular Biology
• Cell Biology

Background:

• Transforming growth factor-beta 1 (TGF-β1) is implicated in podocyte injury and glomerular diseases.
• The precise molecular mechanisms underlying TGF-β1-induced podocyte damage remain unclear.

Purpose of the Study:

• To investigate the role of the ion channel TRPC6 and its associated signaling pathways in TGF-β1-treated mouse podocytes.
• To elucidate the molecular mechanisms by which TGF-β1 induces podocyte injury.

Main Methods:

• Primary mouse podocyte culture treated with TGF-β1.
• Analysis of podocyte apoptosis, actin filament organization, TRPC6 expression and phosphorylation, intracellular calcium levels, and Fyn kinase activity.
• Immunoprecipitation assays to assess protein interactions.
• Western blotting for signaling pathway components (Smad3, ERK, NF-κB).
• Inhibition studies using specific kinase inhibitors and gene knockdown.

Main Results:

• TGF-β1 increased podocyte apoptosis and actin disorganization in a time-dependent manner.
• TGF-β1 upregulated TRPC6 protein, phosphorylated TRPC6, and increased cytosolic free Ca(2+) levels, effects attenuated by TRPC6 knockdown.
• Fyn kinase interacted with TRPC6, and Fyn knockdown inhibited TGF-β1-induced TRPC6 phosphorylation and Ca(2+) influx.
• TGF-β1 activated Smad3, ERK, and NF-κB (RelA/p65), with ERK and NF-κB translocating to the nucleus.
• Inhibition of ERK and NF-κB pathways reduced TGF-β1-induced TRPC6 protein upregulation and Ca(2+) flux.

Conclusions:

• TGF-β1 induces podocyte damage primarily by upregulating TRPC6 protein expression.
• The Smad3-ERK-NF-κB signaling pathway is crucial for TGF-β1-induced TRPC6 upregulation.
• Fyn-dependent tyrosine phosphorylation of TRPC6 plays a significant role in activating its channel function and mediating TGF-β1 podocyte injury.