Liver proteomics for therapeutic drug discovery: inhibition of the cyclophilin receptor CD147 attenuates sepsis-induced acute renal failure

Abstract

Objective: Sepsis-induced multi-organ failure continues to have a high mortality. The liver is an organ central to the disease pathogenesis. The objective of this study was to identify the liver proteins that change in abundance with sepsis and subsequently identify new drug targets.

Design: Proteomic discovery study and drug target validation. For the proteomics study, three biological replicate mice were used per group.

Setting: Research institute laboratory.

Subjects: Three-month-old C57BL/6 mice.

Interventions: We used a mouse model of sepsis based on cecal ligation and puncture, but with fluid and antibiotic resuscitation. Liver proteins that changed in abundance were identified by difference in gel electrophoresis. We compared liver proteins from 6-hr post-cecal ligation and puncture to sham-operated mice ("early proteins") and 24-hr post-cecal ligation and puncture with 6-hr post-cecal ligation and puncture ("late proteins"). Proteins that changed in abundance were identified by tandem mass spectrometry. We then inhibited the receptor for one protein and determined the effect on sepsis-induced organ dysfunction.

Results: The liver proteins that changed in abundance after sepsis had a range of functions such as acute phase response, coagulation, endoplasmic reticulum stress, oxidative stress, apoptosis, mitochondrial electron transfer proteins, and nitric oxide metabolism. We found that cyclophilin increased in abundance after cecal ligation and puncture. When the receptor for this protein, CD147, was inhibited, sepsis-induced renal dysfunction was reduced. There was also a significant reduction in serum cytokine production when CD147 was inhibited.

Conclusion: By applying proteomics to a clinically relevant mouse model of sepsis, we identified a number of novel proteins that changed in abundance. The inhibition of the receptor for one of these proteins, cyclophilin, attenuated sepsis-induced acute renal failure. The application of proteomics to sepsis research can facilitate the discovery of new therapeutic targets.

Figure 1

Serum transaminase levels 6 and 24 hr after CLP. A) Serum aspartate transaminase (AST) concentration 6 hr after sham surgery and 6 and 24 hr after CLP surgery n=3 per group. B) Serum alanine transaminase (ALT) concentration 6 hr after sham surgery and 6 and 24 hr after CLP surgery. (n=3 per group).

Figure 2

Histological liver injury after CLP. Typical histology is presented 6 hr post sham surgery, 6 hr post CLP and 24 hr post CLP. The upper panels are 10× and lower panels are 40× original magnification. PAS stained sections (Bar = 50μM)

Figure 3

Typical two-dimensional electrophoresis gel. Proteins increased in abundance 6 hr after CLP are red. Proteins decreased in abundance 6 hr after CLP are green. An example of proteins which increased (A) and decreased (B) in abundance are shown.

Figure 4

Liver proteins identified by DIGE which increased or decreased in abundance 6 hr after CLP (compared with sham). The fold change is expressed as a ratio of protein abundance 6 hr post-CLP / protein abundance 6 hr post-sham surgery. Error bars represent S.E.M.

Figure 5

Liver proteins identified by DIGE which increased or decreased in abundance 24 hr after CLP (compared with 6 hrs post-CLP). The fold change is expressed as a ratio of protein abundance 24 hr post-CLP / protein abundance 6 hr post-CLP. Error bars represent S.E.M.

Figure 6

The sepsis-induced fold change in liver protein abundance as measured by western blot and DIGE.

Figure 7

Western blot for cyclophilin in liver tissue after sham and 24 hr post-CLP surgery.

Figure 8

Anti CD147 antibody significantly reduces renal injury 24 hr post-CLP. A) Renal injury measured by blood urea nitrogen. B) Renal injury measured by HPLC serum creatinine (n=18-20 per group; * p<0.05).

Figure 9

Anti CD147 antibody significantly reduces serum cytokine levels 24 hr post-CLP. Serum TNF-α (A), IL-6 (B), and IL-10 (C) concentrations after vehicle or anti CD147 antibody treatment at 0, 6, and 18 hrs post surgery (n= 5-9 per group; * p<0.05).

Figure 10

Anti CD147 antibody significantly reduces pancreatic injury but does not significantly reduce liver injury 24 hr post-CLP. A) Pancreatic injury measured by serum amylase concentration. B) Liver injury measured by serum aspartate transaminase C) Liver injury measured by serum alanine transaminase (n=13-20 per group; * p<0.05).