RIPK3 promotes kidney fibrosis via AKT-dependent ATP citrate lyase
Mitsuru Imamura, Jong-Seok Moon, Kuei-Pin Chung, Kiichi Nakahira, Thangamani Muthukumar, Roman Shingarev, Stefan W. Ryter, Augustine M.K. Choi, Mary E. Choi
Mitsuru Imamura, Jong-Seok Moon, Kuei-Pin Chung, Kiichi Nakahira, Thangamani Muthukumar, Roman Shingarev, Stefan W. Ryter, Augustine M.K. Choi, Mary E. Choi
View: Text | PDF
Research Article Cell biology Nephrology

RIPK3 promotes kidney fibrosis via AKT-dependent ATP citrate lyase

Abstract

Renal fibrosis is a common pathogenic response to injury in chronic kidney disease (CKD). The receptor-interacting protein kinase-3 (RIPK3), a regulator of necroptosis, has been implicated in disease pathogenesis. In mice subjected to unilateral ureteral obstruction–induced (UUO-induced) or adenine diet–induced (AD-induced) renal fibrosis, models of progressive kidney fibrosis, we demonstrate increased kidney expression of RIPK3. Mice genetically deficient in RIPK3 displayed decreased kidney fibrosis and improved kidney function relative to WT mice when challenged with UUO or AD. In contrast, mice genetically deficient in mixed-lineage kinase domain-like protein (MLKL), a downstream RIPK3 target, were not protected from UUO-induced kidney fibrosis. We demonstrate a pathway by which RIPK3 promotes fibrogenesis through the AKT-dependent activation of ATP citrate lyase (ACL). Genetic or chemical inhibition of RIPK3 suppressed the phosphorylation of AKT and ACL in response to TGF-β1 in fibroblasts. Inhibition of AKT or ACL suppressed TGF-β1–dependent extracellular matrix production and myofibroblast differentiation in fibroblasts. Pharmacological inhibition of ACL suppressed UUO-induced kidney fibrosis. RIPK3 expression was highly regulated in human CKD kidney. In conclusion, we identify a pathway by which RIPK3 promotes kidney fibrosis independently of MLKL-dependent necroptosis as a promising therapeutic target in CKD.

Authors

Mitsuru Imamura, Jong-Seok Moon, Kuei-Pin Chung, Kiichi Nakahira, Thangamani Muthukumar, Roman Shingarev, Stefan W. Ryter, Augustine M.K. Choi, Mary E. Choi

×

Figure 5

RIPK3 regulates TGF-β1–induced ECM production via the AKT/ACL pathway in fibroblasts.

Options: View larger image (or click on image) Download as PowerPoint
RIPK3 regulates TGF-β1–induced ECM production via the AKT/ACL pathway in...
NIH 3T3 fibroblasts were stimulated with (A) TGF-β1 (0.1–10 ng/ml) for 24 hours or (B) TGF-β1 (5 ng/ml) for 6, 12, and 24 hours. Cell lysates were subjected to Western blot analysis for Col-I, α-SMA, phospho-AKT (Ser473) (p-AKT), AKT, phospho-ATP citrate lyase (p-ACL), ACL, RIPK3, RIPK1, and β-actin. (C–F) NIH 3T3 fibroblasts were treated with (C) nontargeting siRNA (control siRNA) or a mixture of RIPK3 siRNA (SMART Pool) (siRIPK3) for 24 hours and then stimulated with or without TGF-β1 (5 ng/ml, or 10 ng/ml) for 30 minutes to 2 hours. Additionally, NIH 3T3 fibroblasts were treated with (D) control siRNA or 3 independent siRNA targeting RIPK3 (RIPK3 siRNA #1, #2, and #3) for 6 hours and then stimulated with or without TGF-β1 (5 ng/ml) for 15 hours. NIH 3T3 fibroblasts were also treated with (E) RIP3K inhibitor (RIP3K-I) (10 μM) or DMSO (Control) for 12 hours and then stimulated with or without TGF-β1 (5 ng/ml) for 12 hours. Otherwise, NIH 3T3 fibroblasts were treated with (F) necrostatin-1 (Nec-1) (60 μM) or DMSO (Control) for 1 hour, followed by stimulation with or without TGF-β1 (5 ng/ml) for 24 hours. Western blot analysis for expression of RIPK3, Col-I, α-SMA, FN, p-AKT, AKT, p-ACL, ACL, and β-actin is shown.