Currently, no blood biomarker that specifically indicates injury to the proximal tubule of the kidney has been identified. Kidney injury molecule-1 (KIM-1) is highly upregulated in proximal tubular cells following kidney injury. The ectodomain of KIM-1 is shed into the lumen, and serves as a urinary biomarker of kidney injury. We report that shed KIM-1 also serves as a blood biomarker of kidney injury. Sensitive assays to measure plasma and serum KIM-1 in mice, rats, and humans were developed and validated in the current study. Plasma KIM-1 levels increased with increasing periods of ischemia (10, 20, or 30 minutes) in mice, as early as 3 hours after reperfusion; after unilateral ureteral obstruction (day 7) in mice; and after gentamicin treatment (50 or 200 mg/kg for 10 days) in rats. In humans, plasma KIM-1 levels were higher in patients with AKI than in healthy controls or post-cardiac surgery patients without AKI (area under the curve, 0.96). In patients undergoing cardiopulmonary bypass, plasma KIM-1 levels increased within 2 days after surgery only in patients who developed AKI (P,0.01). Blood KIM-1 levels were also elevated in patients with CKD of varous etiologies. In a cohort of patients with type 1 diabetes and proteinuria, serum KIM-1 level at baseline strongly predicted rate of eGFR loss and risk of ESRD during 5-15 years of follow-up, after adjustment for baseline urinary albuminto-creatinine ratio, eGFR, and Hb1Ac. These results identify KIM-1 as a blood biomarker that specifically reflects acute and chronic kidney injury.

4 There was no correlation between HI receptor occupancy by terfenadine and the plasma concentration of the active acid metabolite of terfenadine in each subject.5 PET data on human brain were essentially compatible with those on HI receptor occupancy in guinea-pig brain determined by in vivo binding techniques, although for the same HI receptor occupancy the dose was less in human subjects than in guinea-pigs.6 The PET studies demonstrated the usefulness of measuring HI receptor occupancy with classical and second-generation antihistamines in human brain to estimate their unwanted side effects such as sedation and drowsiness quantitatively.

The rat zitter (zi) mutation induces hypomyelination and vacuolation in the central nervous system (CNS), which result in early-onset tremor and progressive flaccid paresis. By positional cloning, we found a marked decrease in Attractin (Atrn) mRNA in the brain of the zi͞zi rat and identified zi as an 8-bp deletion at a splice donor site of Atrn. Atrn has been known to play multiple roles in regulating physiological processes that are involved in monocyte-T cell interaction, agouti-related hair pigmentation, and control of energy homeostasis. Rat Atrn gene encoded two isoforms, a secreted and a membrane form, as a result of alternative splicing. The zi mutation at the Atrn locus darkened coat color when introduced into agouti rats, as also described in mahogany (mg) mice, carrying the homozygous mutation at the Atrn locus. Transgenic rescue experiments showed that the membrane-type Atrn complemented both neurological alteration and abnormal pigmentation in zi͞zi rats, but that the secreted-type Atrn complemented neither mutant phenotype. Furthermore, we discovered that mg mice exhibited hypomyelination and vacuolation in the CNS associated with body tremor. We conclude from these results that the membrane Atrn has a critical role in normal myelination in the CNS and would provide insights into the physiology of myelination as well as the etiology of myelin diseases. T he zitter rat was found in a colony of Sprague-Dawley rats as a tremorous mutant, and subsequent genetic analysis showed that the abnormality was caused by an autosomal recessive gene, zitter (zi) (1, 2). The tremor develops spontaneously at 3 weeks of age, and flaccid paresis of the hind limb is observed at around 6 months of age (3). The main pathological findings are progressive hypomyelination and vacuolation in the central nervous system (CNS) (4). Hypomyelination is characterized by a significant decrease in the density of myelinated fibers and the number of myelin lamellae and is accompanied by aberrant or elongated myelin sheath formation (4). Vacuoles consist mainly of swollen astrocytic processes and enlargement of extracellular space as well as periaxonal spaces. The vacuoles are first detected in the pons and the outer thalamus at 3 weeks of age. With increasing age, vacuoles extend into the deep cortex, hippocampus, cervical spinal gray matter, and the granular layer and white matter of the cerebellum (5). However, the initiation of myelination and the fundamental structures of myelin sheaths are normal in the zitter rat. The biochemical components of myelin, such as myelin basic protein, proteolipid protein, and myelin-associated glycoprotein, are also normally expressed (5, 6). Therefore, the zitter rat is expected to provide useful tools for the study of axon-glia interaction and the assembly of myelin sheaths in the complex process of CNS myelination.The zi gene has been mapped to a genomic region between IL-1␤ (Il1b) and prion protein (Prnp) on rat chromosome (Chr) 3q35 (3, 7). Prnp is known as a causative gene for spongiform ence...

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Recently, it was reported that the intraperitoneal administration of 30 mg/kg/day troglitazone to heterozygous superoxide dismutase 2 gene knockout (Sod2þ/-) mice for twenty-eight days caused liver injury, manifested by increased serum ALT activity and hepatic necrosis. Therefore, we evaluated the reproducibility of troglitazone-induced liver injury in Sod2þ/-mice, as well as their validity as an animal model with higher sensitivity to mitochondrial toxicity by single-dose treatment with acetaminophen in Sod2þ/-mice. Although we conducted a repeated dose toxicity study in Sod2þ/-mice treated orally with 300 mg/kg/day troglitazone for twenty-eight days, no hepatocellular necrosis was observed in our study. On the other hand, six hours and twenty-four hours after an administration of 300 mg/kg acetaminophen, plasma ALT activity was significantly increased in Sod2þ/-mice, compared to wild-type mice. In particular, six hours after administration, hepatic centrilobular necrosis was observed only in Sod2þ/-mice. These results suggest that Sod2þ/-mice are valuable as an animal model with higher sensitivity to mitochondrial toxicity. On the other hand, it was suggested that the mitochondrial damage alone might not be the major cause of the troglitazone-induced idiosyncratic liver injury observed in humans.

For an understanding of the molecular basis of the marked decrease in catalase activity of various tumor cells, expression of the catalase gene was studied in rat and human hepatoma cell lines and in rat liver, which was used as a control with high activity. RNA blot hybridization profiles and run-on assays indicated that the decrease in catalase activity was due to depression of catalase gene transcription. Chloramphenicol acetyltransferase (CAT) assays for the fragments with various lengths of the 5'-flanking region (up to -4.5 kb from the ATG codon) of the catalase gene revealed the presence of several cis-acting elements involved in the negative regulation of transcription. The most-upstream element with the strongest activity (-3504 to -3364 bp), when linked to the catalase promoter region (-126 bp) of the CAT construct and subjected to an in vitro transcription assay, did not yield transcripts in experiments with the hepatoma nuclear extract, whereas the unlinked template did yield transcripts. A gel shift competition assay using hepatoma nuclear extract showed the core sequence of the silencer element to be 5'-TGGGGGGAG-3'. A homology search found that the same core sequence was also present in 5'-flanking regions of the albumin gene and of some other liver enzyme genes, the expression of which has been reported to be down regulated in some hepatoma cells. Southwestern (DNA-protein) analysis demonstrated that an approximately 35-kDa nuclear protein bound to the silencer element was present in hepatoma cells but not in rat liver cells.

Kidney injury molecule-1 (KIM-1) has been qualified by the Food and Drug Administration and European Medicines Agency as a urinary biomarker to monitor preclinical nephrotoxicity in rats and on a case-by-case basis for the translation of potentially nephrotoxic drugs into first-in human studies. Although mouse models are widely employed in preclinical studies, few urinary biomarker studies have been performed in mice due to limited urine availability and lack of sensitive assays. Here, we report the development and validation of two different assays for quantitative assessment of mouse urinary KIM-1 (uKIM-1) and compare the sensitivity of KIM-1 relative to other standard markers in ischemia reperfusion and aristolochic acid (AA)-induced kidney injury in mice. A sensitive, reproducible, and quantitative microbead-based KIM-1 ELISA was established, which requires only 10 μl urine for triplicate determination with an assay range of 12.21 pg/ml to 50 ng/ml. The second assay is a laminar flow dipstick assay, which has an assay range of 195 pg/ml to 50 ng/ml and provides quantitative assessment of KIM-1 in 15 min. uKIM-1 levels increased with increasing time of ischemia or time after AA administration. After only 10-min ischemia followed by 24-h reperfusion, uKIM-1 was significantly elevated by 13-fold, whereas serum creatinine (sCr), blood urea nitrogen, N-acetyl-β-glucosaminidase (NAG), and proteinuria levels did not change. After AA administration, uKIM-1 levels were significantly upregulated by greater than threefold within 12 h, whereas sCr and NAG levels were unchanged. Mouse KIM-1 was stable for multiple freeze-thaw cycles, for up to 5 days at room temperature and up to at least an year when stored at -80°C.

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