Abstract
Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase Cα (PKCα) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor β1 (TGFβ1) activates PKCα signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKCα signaling with the chemical inhibitor Go6976 or by transfection with PKCα siRNA largely reduced expression of the autophagy-associated protein lysosomal-associated membrane protein 2 (LAMP2) and also inhibited autophagosome-lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKCα siRNA transfection also markedly inhibited TGFβ1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKCα signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKCα activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.
Highlights
Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts
Our findings suggest that blocking protein kinase C (PKC)␣ activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis
We found that PKC␣ signaling activation mediates transforming growth factor 1 (TGF1)-induced fibroblast activation and contributes to kidney fibrosis by promoting autophagic flux
Summary
Inhibition of PKC␣ reduces LAMP2 expression and induces lysosomal dysfunction in NRK-49F cells. The Western blotting analyses revealed that LAMP2 abundance was significantly decreased after Go6976 treatment (Fig. 1E). Western blotting analyses revealed that PKC␣ siRNA transfection significantly increased LC3B-II and SQSTM1/p62 abundance in NRK-49F cells (Fig. 2C). To further clarify the role for autophagic flux retardation in fibroblast activation, we treated NRK-49F cells with CQ and 3-MA, and we found that CQ and 3-MA could remarkably inhibit TGF1-induced FN and ␣-SMA expression (Fig. 3, E and F). On day 7 after UUO, Go6976 treatment increased the abundance of SQSTM1/p62 and LC3B-II, and it decreased LAMP2 abundance compared with the abundance from mice treated with vehicle (Fig. 4C). These results suggest blocking PKC␣ signaling inhibits autophagic flux and lysosome function in the interstitial myofibroblasts from kidneys with UUO nephropathy, which confirmed the results from NRK-49F cells. Administration of Go6976 could significantly down-regulate RANTES, TNF␣, and MCP1 mRNA expression
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