Identification of novel translational urinary biomarkers for acetaminophen-induced acute liver injury using proteomic profiling in mice.

Drug-induced liver injury (DILI) is the leading cause of acute liver failure. Currently, no adequate predictive biomarkers for DILI are available. This study describes a translational approach using proteomic profiling for the identification of urinary proteins related to acute liver injury induced by acetaminophen (APAP). Mice were given a single intraperitoneal dose of APAP (0-350 mg/kg bw) followed by 24 h urine collection. Doses of ≥275 mg/kg bw APAP resulted in hepatic centrilobular necrosis and significantly elevated plasma alanine aminotransferase (ALT) values (p<0.0001). Proteomic profiling resulted in the identification of 12 differentially excreted proteins in urine of mice with acute liver injury (p<0.001), including superoxide dismutase 1 (SOD1), carbonic anhydrase 3 (CA3) and calmodulin (CaM), as novel biomarkers for APAP-induced liver injury. Urinary levels of SOD1 and CA3 increased with rising plasma ALT levels, but urinary CaM was already present in mice treated with high dose of APAP without elevated plasma ALT levels. Importantly, we showed in human urine after APAP intoxication the presence of SOD1 and CA3, whereas both proteins were absent in control urine samples. Urinary concentrations of CaM were significantly increased and correlated well with plasma APAP concentrations (r = 0.97; p<0.0001) in human APAP intoxicants, who did not present with elevated plasma ALT levels. In conclusion, using this urinary proteomics approach we demonstrate CA3, SOD1 and, most importantly, CaM as potential human biomarkers for APAP-induced liver injury.

Investigation of calmodulin and basic fibroblast growth factor (bFGF) in idiopathic myelofibrosis: evidence for a role of extracellular calmodulin in fibroblast proliferation.

The urinary concentration of calmodulin and basic fibroblast growth factor (bFGF) was determined in a total of 53 patients with various chronic myeloproliferative disorders (CMPD), including 22 patients with idiopathic myelofibrosis (IMF). Calmodulin excretion was significantly elevated in IMF (0.29 +/- 0.04 microgram/mmol creatinine) (P < 0.001), when compared to polycythaemia vera (PV) (0.14 +/- 0.02), essential thrombocythaemia (ET) (0.13 +/- 0.04), chronic myeloid leukaemia (CML) (0.16 +/- 0.02), unclassified myeloproliferative disorders (UMPD) (0.11 +/- 0.02) and age-matched controls (0.1 +/- 0.02) (P < 0.001). In contrast, bFGF was slightly elevated in all CMPD conditions when compared to age-matched controls. A neutralizing antibody to calmodulin was demonstrated to significantly influence the in vitro proliferation of normal human fibroblasts, an effect dependent on both cell density and the presence of fetal calf serum (FCS). Essentially, the antibody reduced FCS-induced proliferation of low-density fibroblasts but had little or no inhibitory effect on high-density fibroblasts in the absence of FCS. In addition, extracellular calmodulin was shown not to interact with known fibroblast mitogens, namely, IFG-1, EGF, bDGF and PDGF. We conclude that extracellular calmodulin should be considered, in addition to PDGF, TFG-beta and EGF, as a potential mitogen involved in the stromal reaction of idiopathic myelofibrosis.

Raised urinary calmodulin levels in idiopathic myelofibrosis: possible implications for the aetiology of fibrosis.

Platelet growth factors (e.g. PDGF and TGF-beta) are thought to be pathogenetically important in the stromal reaction characteristic of idiopathic myelofibrosis (IM). We have investigated a possible pathogenetic role for a further platelet mitogen, calmodulin. Platelets are rich in calmodulin, of which 30-40% is releasable with a time course that differs from alpha-granule proteins. In IM urinary calmodulin concentrations were 3-fold those of the normals controls. We suggest that an abnormal release of calmodulin may occur from platelets/megakaryocytes in patients with IM, and that calmodulin should be considered, along with other growth factors, in the pathogenesis of marrow fibrosis.