SEMI-QUANTITATIVE IMMUNOHISTOCHEMICAL ANALYSIS OF MALE RAT-SPECIFIC α2u-GLOBULIN ACCUMULATION FOR CHEMICAL TOXICITY EVALUATION

Hamamura, Masao; Hirose, Akihiko; Kamata, Eiichi; Katoku, Koshiro; Kuwasaki, Emiko; Oshikata, Takafumi; Nakahara, Yutaka; Ema, Makoto; Hasegawa, Ryuichi

doi:10.2131/jts.31.35

Cited by 10 publications

(4 citation statements)

References 22 publications

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“…In addition, the standard deviation of over 30% of the average urine volume in both genders suggests great individual differences. Gender differences in urine protein are reportedly caused by differences in sex hormones [16,17]. …”

Section: Discussionmentioning

confidence: 99%

Historical control data from 13-week repeated toxicity studies in Crj:CD (SD) rats

Lee¹,

Lee²,

Do³

et al. 2012

Lab Anim Res

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Reference ranges of standard experimental parameters are useful for comparisons in toxicology. The aim of this study was to collect data from 13-week repeated toxicity studies in Crl:CD (SD) rats, a strain widely used for toxicity and efficacy research, for establishing domestic reference values. Data on body weight, food consumption; urinalysis, hematological, and blood biochemical parameters; and organ weights were collected from 11 toxicity studies in 220 Crl:CD (SD) rats (110 males and 110 females). The studies had been performed at a single testing facility over the last 5 years and involved animals sourced from a single breeder. The findings were collated as means, standard deviations, percentages, and ranges. Urine volume, uterus weight, eosinophil, and basophil counts, and triglyceride, total bilirubin, and gamma-glutamyl transpeptidase levels showed standard deviations of 30% or more. These historical control data would help to interpret the effects of test substances in routine toxicity and efficacy studies.

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Section: Discussionmentioning

confidence: 99%

Historical control data from 13-week repeated toxicity studies in Crj:CD (SD) rats

Lee¹,

Lee²,

Do³

et al. 2012

Lab Anim Res

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“…It is a precursor of a kidney fatty acid-binding protein, involved in mechanism of binding and transport of fatty acids ( http://www.uniprot.org/ ) and it is overexpressed in renal cortex rather than medulla [ 59 ]. This protein can strongly bind toxic agents like alkans [ 58 , 60 ] and is associated with the accumulation of hyaline droplet that leads to damage of the proximal tubular epithelium and renal cancer [ 61 , 62 ].…”

Section: Discussionmentioning

confidence: 99%

Decreased Tissue COX5B Expression and Mitochondrial Dysfunction during Sepsis‐Induced Kidney Injury in Rats

Hinkelbein

Böhm

Braunecker

et al. 2017

Oxidative Medicine and Cellular Longevity

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Background. Sepsis is defined as a life-threatening organ dysfunction due to a dysregulated host response to infection. Sepsis is the dominant cause of acute kidney injury (AKI), accounting for nearly 50% of episodes of acute renal failure. Signaling cascades and pathways within the kidney are largely unknown and analysis of these molecular mechanisms may enhance knowledge on pathophysiology and possible therapeutic options. Material and Methods. 26 male Wistar rats were assigned to either a sham group (control, N = 6) or sepsis group (N = 20; cecal ligature and puncture model, 24 and 48 hours after CLP). Surviving rats (n = 12) were decapitated at 24 hours (early phase; n = 6) or 48 hours (late phase; n = 6) after CLP and kidneys removed for proteomic analysis. 2D-DIGE and DeCyder 2D software (t-test, P < 0.01) were used for analysis of significantly regulated protein spots. MALDI-TOF in combination with peptide mass fingerprinting (PMF) as well as Western Blot analysis was used for protein identification. Bioinformatic network analyses (STRING, GeneMania, and PCViz) were used to describe protein-protein interactions. Results. 12 spots were identified with significantly altered proteins (P < 0.01) in the three analyzed groups. Two spots could not be identified. Four different proteins were found significantly changed among the groups: major urinary protein (MUP5), cytochrome c oxidase subunit B (COX5b), myosin-6 (MYH6), and myosin-7 (MYH7). A significant correlation with the proteins was found for mitochondrial energy production and electron transport. Conclusions. COX5B could be a promising biomarker candidate since a significant association was found during experimental sepsis in the present study. For future research, COX5B should be evaluated as a biomarker in both human urine and serum to identify sepsis.

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“…As for immunostaining, the sections were deparaffinized, hydrated, and quenched for endogenous peroxidase with hydrogen peroxide, and nonspecific reactions were blocked with a normal serum. Epitope retrieval was performed using pronase treatment for 20 minutes (for all animals) or for 5 or 10 minutes (for selected animals) at 37 to 40°C for anti-α 2u -globulin antibody (our in-house antibody 15 ) and was performed in a microwave for 5 minutes for anti-LAMP (LAMP1, Abcam plc, Cambridge, UK). Anti-α 2u -globulin antibody was incubated overnight at 4°C at a 1:80,000 dilution, and anti-LAMP antibody was incubated for 60 minutes at room temperature at a 1:400 dilution.…”

Section: Methodsmentioning

confidence: 99%

Two types of deposits, hyaline droplets and eosinophilic bodies, associated with α<sub>2u</sub>-globulin accumulation in the rat kidney

Hamamura¹,

Oshikata²,

Katoku³

et al. 2017

J Toxicol Pathol

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Alpha2u-globulin is an adult male rat-specific protein that accumulates spontaneously or inductively in the renal proximal tubular epithelium and forms microscopically observable deposits, which are generally referred to as “hyaline droplets,” whereas a specific type of deposits is referred to as “eosinophilic bodies” by Japanese toxicologic pathologists. We compared hyaline droplets and eosinophilic bodies using special stains including immunostaining for α2u-globulin and lysosome-associated membrane protein in spontaneously occurring and d-limonene-induced cases. Eosinophilic bodies appeared simultaneously and increased in parallel with the hyaline droplets in the induced case. In both of the spontaneous and induced cases, hyaline droplets and eosinophilic bodies were associated with α2u-globulin and lysosomes, although there were differences in the forms and staining properties that probably reflected the purity or density of α2u-globulin. According to the results, it is not necessary for eosinophilic bodies to be strictly distinguished from hyaline droplets, and it is reasonable to identify eosinophilic bodies as hyaline droplets in α2u-globulin nephropathy in routine toxicity studies, as they have been recognized to be a sequence of changes associated with accumulation of α2u-globulin.

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SEMI-QUANTITATIVE IMMUNOHISTOCHEMICAL ANALYSIS OF MALE RAT-SPECIFIC α2u-GLOBULIN ACCUMULATION FOR CHEMICAL TOXICITY EVALUATION

Cited by 10 publications

References 22 publications

Historical control data from 13-week repeated toxicity studies in Crj:CD (SD) rats

Historical control data from 13-week repeated toxicity studies in Crj:CD (SD) rats

Decreased Tissue COX5B Expression and Mitochondrial Dysfunction during Sepsis‐Induced Kidney Injury in Rats

Two types of deposits, hyaline droplets and eosinophilic bodies, associated with α<sub>2u</sub>-globulin accumulation in the rat kidney

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