Plasma 2-hydroxyglutarate, a promising prognostic biomarker candidate for skeletal muscle injury in Fischer 344 rats.

Previously, we have demonstrated the potential of plasma 2-hydroxyglutarate (2HG) as an easily detectable biomarker for skeletal muscle injury in rats. Here, we examined whether plasma 2HG was superior to conventional skeletal muscle damage biomarkers, including aspartate aminotransferase (AST), creatine kinase (CK), and skeletal muscle-type CK isoenzyme (CK-MM) levels, in rats. Skeletal muscle injury was induced in 4- or 9-week-old male Fischer 344 rats by cerivastatin (CER) or tetramethyl-p-phenylenediamine (TMPD) administration. Plasma 2HG levels were measured on days 4, 8, and 11 (CER group) and at 6 and 24 hr post-administration (TMPD group). Plasma AST, CK, and CK-MM activities and histopathological changes in the rectus femoris muscle were evaluated at the study endpoints. In the CER group, AST, CK, and CK-MM increased in 4- and 9-week-old rats, whereas increases in CK (4- and 9-week-old rats) and CK-MM (4-week-old rats) were not obvious in the TMPD group. In both 4- and 9-week-old rats, plasma 2HG increased on day 8 and at 24 hr post-administration in the CER and TMPD groups, respectively. Histopathological analysis revealed myofiber vacuolation and necrosis in both groups. The histopathological damage to the rectus femoris muscle was more severe in the CER than in the TMPD group. Increased plasma 2HG was associated with CER- and TMPD-induced skeletal muscle injuries in rats and was not affected by age differences or repeated blood collection. The results suggest that plasma 2HG is superior to CK and CK-MM as a biomarker for mild skeletal muscle injury.

Plasma 2-hydroxyglutarate and hexanoylcarnitine levels are potential biomarkers for skeletal muscle toxicity in male Fischer 344 rats.

To identify new candidate biomarkers for skeletal muscle toxicity, an unbiased metabolomic analysis was performed in rats treated with two distinct myotoxicants, cerivastatin (CER) and tetramethyl-p-phenylenediamine (TMPD). Skeletal muscle toxicity was induced in male Fischer 344 rats by administering CER or TMPD and monitored using established endpoints, such as increased plasma creatine kinase (CK) activity and histopathology, and a metabolomic analysis of skeletal muscle and plasma samples. Plasma CK levels in CER-treated rats were markedly elevated at Day 11; however, those in TMPD-treated rats showed a statistically significant decrease at 24 hr after dosing. Light microscopy revealed that vacuolated or necrotic fibers were evident in all CER-treated rats on Day 11, and slightly vacuolated fibers were observed in TMPD-treated rats at 6 and 24 hr after dosing. Metabolomic analysis of the rectus femoris indicated increases in 2-hydroxyglutarate (2HG) in CER-treated rats and hexanoylcarnitine in CER- and TMPD-treated rats. There were also increases in plasma 2HG in CER-treated rats on Days 8 and 11 and in TMPD-treated rats at 24 hr after dosing and increases in plasma hexanoylcarnitine in CER-treated rats on Day 11 and in TMPD-treated rats at 6 and 24 hr after dosing. These experiments demonstrated the potential of plasma 2HG and hexanoylcarnitine as specific and easily detectable biomarkers for skeletal muscle toxicity in rats and demonstrated the value of metabolomics for biomarker detection and identification in toxicological studies.

R-268712, an orally active transforming growth factor-ß type I receptor inhibitor, prevents glomerular sclerosis in a Thy1 nephritis model.

R-268712 is a novel and specific inhibitor of activin receptor-like kinase 5 (ALK5), a transforming growth factor ß (TGF-ß) type I receptor. Evaluation of in vitro inhibition indicated that R-268712 is a potent and selective inhibitor of ALK5 with an IC50 of 2.5nM, an approximately 5000-fold more selectivity for ALK5 than p38 mitogen-activated protein kinase (MAPK). Oral administration of R-268712 at doses of 1, 3 and 10mg/kg also inhibited the development of renal fibrosis in a dose-dependent manner in a unilateral ureteral obstruction (UUO) model. Additionally, we evaluated the efficacy of R-268712 in a heminephrectomized anti-Thy1 glomerulonephritis model at doses of 0.3 and 1mg/kg. R-268712 reduced proteinuria and glomerulosclerosis significantly with improvement of renal function. Collectively, these results suggested that R-268712 and other ALK5 inhibitors could suppress glomerulonephritis as well as glomerulosclerosis by an inhibitory mechanism that involves suppression of TGF-ß signaling.

Cerivastatin induces type-I fiber-, not type-II fiber-, predominant muscular toxicity in the young male F344 rats.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are associated with adverse skeletal muscle toxicity, but the underlying mechanism remains unclear. To investigate the pathological mechanism of statin-induced myotoxicity, cerivastatin (20 ppm; corresponding to 2 mg/kg/day) was dietarily administered to young male F344 rats for 10 days, and time-course clinical observations, measurement of plasma creatine kinase activity, and light and electron microscopy of type I fiber-predominant skeletal muscle (soleus) or type II fiber-predominant skeletal muscles (extensor digitorum longus and tibialis anterior), were performed. Clinical symptoms including weakness of hind limbs, staggering gait and body weight loss, accompanied by marked plasma creatinine kinase elevation in rats fed cerivastatin at around Day 6 to 8. Interestingly, microscopic examination revealed that cerivastatin-induced muscle damages characterized by hypercontraction (opaque) and necrosis of the fibers were of particular abundance in the soleus muscle at Day 8, whereas these histological lesions in the extensor digitorum longus and tibialis anterior were negligible, even at Day 9. Prior to manifestation of muscle damage, swollen mitochondria and autophagic vacuoles in the soleus were observed as the earliest ultra structural changes at Day 6; then activated lysosomes, disarray of myofibril and dilated sarcoplasmic reticulum vesicles became ubiquitous at Day 8. These results demonstrate that cerivastatin induces type I fiber-predominant muscles injury, which is associated with mitochondrial damage, in young male F344 rats. Since the rat exhibiting type I fiber-targeted injury is a unique animal model for statin-induced myotoxicity, it will be useful for gaining insight into mechanisms of statin-induced myotoxicity.