Combined kidney‑liver perfusion enhances the proliferation effects of hypothermic perfusion on liver grafts via upregulation of IL‑6/Stat3 signaling

Abstract

A limited number of studies have revealed that adding kidneys to liver perfusion may maintain an improved physiological balance; however, the underlying mechanism remains to be elucidated. The preset study confirmed the protective role of this new model and investigated the underlying mechanisms. Methods: A total of 12 rats were randomly assigned into two groups (n=6 for each group): The kidney‑liver perfusion (KL) group and liver perfusion (LP) group. Perfusate samples were collected during the perfusion process for the analysis of pH, K+ and liver function. Liver tissues were obtained for the evaluation of adenosine triphosphate (ATP), terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labelling and immunohistochemistry of Ki67. Cell cycle inhibitors, apoptosis‑associated genes and signal transducer and activator of transcription 3 (Stat3) were analyzed using quantitative polymerase chain reaction and western blot analysis. Results: Overall pH and K+ values of the KL group were significantly different from the LP group and more stable; aspartate aminotransferase, alanine transaminase and lactate dehydrogenase levels increased progressively over time in the LP group and were significantly different at different time points compared with pre‑perfusion levels and the KL group, which suggested the KL group was superior to the LP group. In addition, KL reduced portal vein resistance and was associated with lower ATP consumption compared with the LP group. Furthermore, liver proliferation was upregulated with the upregulation of the interleukin 6 (IL‑6)/Stat3 signaling pathway in KL compared with LP. The present study revealed for the first time that KL and hypothermic machine perfusion demonstrated a more proactive repair capability by maintaining liver regeneration via the upregulation of the IL‑6/Stat3 signaling pathway.

Figure 1.

LP and KL model of the study. LP, Liver perfusion; KL, Kidney liver perfusion.

Figure 2.

Schematic diagram of kidney-liver hypothermic machine perfusion. In this study, single vessel (portal vein) liver perfusion was performed and urine was collected. No significant results were indicated.

Figure 3.

pH, K⁺ and liver function tests are performed over time. The level of (A) pH, (B) K⁺, (C) ALT, (D) AST and (E) LDH in KL and LP groups over time. Circles or boxes represented the mean values, whereas bars indicated the mean ± standard error of mean. *P<0.05 vs. LP group at the same time point; ^#P<0.05 vs. pre-perfusion levels (0 h). LP, liver perfusion; KL, kidney liver perfusion; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine transaminase.

Figure 4.

Portal vein resistance and ATP expression in KL and LP groups. (A) The classical pressure record during perfusion from each subgroup. (B) The results of statistical analysis. (C) ATP levels in the KL and LP groups. Data represent mean ± standard error of the mean. *P<0.05 vs. LP group. ATP, adenosine triphosphate; LP, liver perfusion; KL, kidney liver perfusion; VR, vessel resistance.

Figure 5.

Effects of KL on hepatocyte apoptosis. (A) Identification of hepatocyte apoptosis was conducted by TUNEL assay (n=6, original magnification, ×200). The percentage of TUNEL-labeled nuclei was quantified. (B) Quantitative polymerase chain reaction and (C) western blot analysis of apoptosis. The quantitative data of cleaved caspase-3/caspase-3 and Bax/Bcl-2 are indicated. Data represent the mean ± standard error of the mean. LP, liver perfusion; KL, kidney liver perfusion; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling; B-cell lymphoma-2-associated X, Bax.

Figure 6.

Effects of KL on hepatocyte proliferation. (A) Immunofluorescence of the percentage of Ki67-labeled nuclei of the KL and LP groups (n=6, original magnification, ×200). Sections of liver were stained with DAPI (blue) and counterstaining with anti-Ki67 (red). (B) The mRNA expression levels of P21 and (C) P27 were indicated. Data represent the mean ± standard error of the mean. *P<0.05 vs. LP group. LP, liver perfusion; KL, kidney liver perfusion.

Figure 7.

Effects of KL on hepatocyte proliferation signaling. (A) IL-6, (B) HGF and (C) TNF-α were analyzed at the end of perfusion. (D) Protein expression and quantitative data of p-Stat3/t-Stat3 were indicated. Data represent the mean ± standard error of the mean. *P<0.05 vs. LP group. IL, interleukin; HGF, hepatocyte growth factor; TNF, tumor necrosis factor; LP, liver perfusion; KL, kidney liver perfusion; p-stat3, phosphorylated-signal transducer and activator of transcription; t, total.

Figure 8.

Kidney function tests were indicated over time. (A) CR amd (B) BUN were analyzed over time. Circles or boxes represented the mean values whereas the bars represented the standard error of mean. BUN, blood urea nitrogen; CR, creatinine.