RIPK3 promotes sepsis-induced acute kidney injury via mitochondrial dysfunction
Angara Sureshbabu, Edwin Patino, Kevin C. Ma, Kristian Laursen, Eli J. Finkelsztein, Oleh Akchurin, Thangamani Muthukumar, Stefan W. Ryter, Lorraine Gudas, Augustine M. K. Choi, Mary E. Choi
Angara Sureshbabu, Edwin Patino, Kevin C. Ma, Kristian Laursen, Eli J. Finkelsztein, Oleh Akchurin, Thangamani Muthukumar, Stefan W. Ryter, Lorraine Gudas, Augustine M. K. Choi, Mary E. Choi
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Research Article Cell biology Nephrology

RIPK3 promotes sepsis-induced acute kidney injury via mitochondrial dysfunction

Abstract

Sepsis causes acute kidney injury (AKI) in critically ill patients, although the pathophysiology remains unclear. The receptor-interacting protein kinase-3 (RIPK3), a cardinal regulator of necroptosis, has recently been implicated in the pathogenesis of human disease. In mice subjected to polymicrobial sepsis, we demonstrate that RIPK3 promotes sepsis-induced AKI. Utilizing genetic deletion and biochemical approaches in vitro and in vivo, we identify a potentially novel pathway by which RIPK3 aggravates kidney tubular injury independently of the classical mixed lineage kinase domain-like protein–dependent (MLKL-dependent) necroptosis pathway. In kidney tubular epithelial cells, we show that RIPK3 promotes oxidative stress and mitochondrial dysfunction involving upregulation of NADPH oxidase-4 (NOX4) and inhibition of mitochondrial complex I and –III, and that RIPK3 and NOX4 are critical for kidney tubular injury in vivo. Furthermore, we demonstrate that RIPK3 is required for increased mitochondrial translocation of NOX4 in response to proinflammatory stimuli, by a mechanism involving protein-protein interactions. Finally, we observed elevated urinary and plasma RIPK3 levels in human patients with sepsis-induced AKI, representing potential markers of this condition. In conclusion, we identify a pathway by which RIPK3 promotes kidney tubular injury via mitochondrial dysfunction, independently of MLKL, which may represent a promising therapeutic target in sepsis-induced AKI.

Authors

Angara Sureshbabu, Edwin Patino, Kevin C. Ma, Kristian Laursen, Eli J. Finkelsztein, Oleh Akchurin, Thangamani Muthukumar, Stefan W. Ryter, Lorraine Gudas, Augustine M. K. Choi, Mary E. Choi

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Figure 7

Ripk3 deficiency reduces mitochondrial dysfunction in vitro.

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Ripk3 deficiency reduces mitochondrial dysfunction in vitro. (A and B) ...
(A and B) NT gRNA– or RIPK3 gRNA–transfected HK-2 cells were incubated in the absence or presence of LPS (1 μg/ml) or CpG DNA (1 μg/ml) for 24 hours. (C and D) HK-2 cells were pretreated with RIPK3 inhibitor (GSK’872) (1 μM) and then incubated in the absence or presence of LPS (1 μg/ml) or CpG DNA (1 μg/ml). JC-1 fluorescence was determined at 24 hours. (E) NT gRNA– or RIPK3 gRNA–transfected HK-2 cells were treated with or without LPS (1 μg/ml) and mitochondrial fractions were isolated at 6 hours. Protein levels of mitochondrial complex I (NDUFB8), –II (SDHB), –III (UQCRC-1), –IV (MTCO1), and –V (ATP5A) subunit levels (normalized to TIM23) were quantified in the mitochondrial fractions. (F and G) Intracellular ATP levels were determined in the primary tubular epithelial cells obtained from Ripk3+/+ or Ripk3–/– (F) and Mlkl+/+ or Mlkl–/– (G) mice treated with LPS at 6 hours. Data are means ± SEM of 3 independent experiments (n = 4 mice/group). Data are means ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA with Bonferroni’s post-hoc test (E) or 1-way ANOVA with Tukey’s post-hoc test (A–D, F, and G).