The effect of Cyclophilin D depletion on liver regeneration following associating liver partition and portal vein ligation for staged hepatectomy

Abstract

Aim: Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) is a modification of two-stage hepatectomy profitable for patients with inoperable hepatic tumors by standard techniques. Unfortunately, initially poor postoperative outcome was associated with ALPPS, in which mitochondrial dysfunction played an essential role. Inhibition of cyclophilins has been already proposed to be efficient as a mitochondrial therapy in liver diseases. To investigate the effect of Cyclophilin D (CypD) depletion on mitochondrial function, biogenesis and liver regeneration following ALPPS a CypD knockout (KO) mice model was created.

Methods: Male wild type (WT) (n = 30) and CypD KO (n = 30) mice underwent ALPPS procedure. Animals were terminated pre-operatively and 24, 48, 72 or 168 h after the operation. Mitochondrial functional studies and proteomic analysis were performed. Regeneration rate and mitotic activity were assessed.

Results: The CypD KO group displayed improved mitochondrial function, as both ATP production (P < 0.001) and oxygen consumption (P < 0.05) were increased compared to the WT group. The level of mitochondrial biogenesis coordinator peroxisome proliferator-activated receptor γ co-activator 1-α (PGC1-α) was also elevated in the CypD KO group (P < 0.001), which resulted in the induction of the mitochondrial oxidative phosphorylation system. Liver growth increased in the CypD KO group compared to the WT group (P < 0.001).

Conclusions: Our study demonstrates the beneficial effect of CypD depletion on the mitochondrial vulnerability following ALPPS. Based on our results we propose that CypD inhibition should be further investigated as a possible mitochondrial therapy following ALPPS.

Fig 1. Alterations of adenosine 5′ -triphosphate (ATP) production.

Endogenous (A) and exogenous substrate (complex I: Glutamate-malate complex II: Succinate) stimulated (B and C) ATP production preoperatively (preop., 0 h), and at 24 h, 48 h, 72 h, and 168 h after Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) (N = 6 per time point per group). * P < 0.050, ** P < 0.0010 versus wild type (WT); # P < 0.050, ## P < 0.001 WT versus corresponding controls (preop.); § P < 0.050, §§ P < 0.0010 CypD KO versus corresponding controls (preop.). Statistical analysis was performed with a two-way ANOVA and Tukey’s post hoc test.

Fig 2. Changes in oxygen consumption levels.

Basal (A and B) and substrate (complex I: Glutamate-malate complex II: Succinate) induced (C and D) oxygen consumption preoperatively (preop., 0 h), and at 24 h, 48 h, 72 h, and 168 h after Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) (N = 6 per time point per group). * P < 0.050, ** P < 0.0010 versus WT; # P < 0.050, ## P < 0.001 wild type (WT) versus corresponding controls (preop.); § P < 0.050, §§ P < 0.0010 CypD KO versus corresponding controls (preop.). Statistical analysis was performed with a two-way ANOVA and Tukey’s post hoc test.

Fig 3. Changes in reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) content.

Basal (A and B) and substrate (complex I: Glutamate-malate complex II: Succinate) induced (C and D) NAD(P)H preoperatively (preop., 0 h), and at 24 h, 48 h, 72 h, and 168 h after Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) (N = 6 per time point per group). * P < 0.050, ** P < 0.0010 versus wild type (WT); # P < 0.050, ## P < 0.001 WT versus corresponding controls (preop.); § P < 0.050, §§ P < 0.0010 CypD KO versus corresponding controls (preop.). Statistical analysis was performed with a two-way ANOVA and Tukey’s post hoc test.

Fig 4. Expression of gene regulatory proteins participating in mitochodrial biogenesis.

Peroxisome proliferator-activated receptor γ coactivator (PGC) 1-α (A), nuclear respiratory factor 1 (B) preoperatively (preop., 0 h), and at 24 h, 48 h, 72 h, and 168 h after Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) (N = 6 per time point per group). * P < 0.050, ** P < 0.0010 versus wild type (WT); # P < 0.050, ## P < 0.001 WT versus corresponding controls (preop.); § P < 0.050, §§ P < 0.0010 CypD KO versus corresponding controls (preop.). Statistical analysis was performed with a two-way ANOVA and Tukey’s post hoc test.

Fig 5. Changes in the expression of the respiratory chain complexes.

Complex I (A), complex II (B), complex III (C), complex IV (D) total cell lysate protein concentrations preoperatively (preop., 0 h), and at 24 h, 48 h, 72 h, and 168 h after Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) (N = 6 per time point per group). * P < 0.050, ** P < 0.0010 versus wild type (WT); # P < 0.050, ## P < 0.001 WT versus corresponding controls (preop.); § P < 0.050, §§ P < 0.0010 CypD KO versus corresponding controls (preop.). Statistical analysis was performed with a two-way ANOVA and Tukey’s post hoc test.

Fig 6. Expression of caspase-3.

Uncleaved (A) and cleaved (B) caspase-3 total cell lysate protein concentrations preoperatively (preop., 0 h), and at 24 h, 48 h, 72 h, and 168 h after Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) (N = 6 per time point per group). * P < 0.050, ** P < 0.0010 versus wild type (WT); # P < 0.050, ## P < 0.001 WT versus corresponding controls (preop.); § P < 0.050, §§ P < 0.0010 CypD KO versus corresponding controls (preop.). Statistical analysis was performed with a two-way ANOVA and Tukey’s post hoc test.

Fig 7. Liver regeneration following Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) in the ligated right medial lobe.

Increase in liver mass of non-ligated lobe (A) and mitotic rate (B) and ki67 index (C) preoperatively (preop., 0 h), and at 24 h, 48 h, 72 h, and 168 h after ALPPS (N = 6 per time point per group). Histological structure on the left (hematoxylin and eosin stain; original magnification: ×150) and ki67 immunohistochemistry (on the right) of the regenerating right median (RM) lobe (D) preoperatively (preop.) and at 24 h, 48 h, 72 h, and 168 h after ALPPS in the Cyclopihilin D knockout (CypD KO) and wild type (WT) group. * P < 0.050, ** P < 0.0010 versus wild type (WT); # P < 0.050, ## P < 0.001 WT versus corresponding controls (preop.); § P < 0.050, §§ P < 0.0010 CypD KO versus corresponding controls (preop.). Statistical analysis was performed with a two-way ANOVA and Tukey’s post hoc test.