Histology Atlas of the Developing Mouse Urinary System With Emphasis on Prenatal Days E10.5-E18.5.

Congenital abnormalities of the urinary tract are some of the most common human developmental abnormalities. Several genetically engineered mouse models have been developed to mimic these abnormalities and aim to better understand the molecular mechanisms of disease. This atlas has been developed as an aid to pathologists and other biomedical scientists for identification of abnormalities in the developing murine urinary tract by cataloguing normal structures at each stage of development. Hematoxylin and eosin- and immunohistochemical-stained sections are provided, with a focus on E10.5-E18.5, as well as a brief discussion of postnatal events in urinary tract development. A section on abnormalities in the development of the urinary tract is also provided, and molecular mechanisms are presented as supplementary material. Additionally, overviews of the 2 key processes of kidney development, branching morphogenesis and nephrogenesis, are provided to aid in the understanding of the complex organogenesis of the kidney. One of the key findings of this atlas is the histological identification of the ureteric bud at E10.5, as previous literature has provided conflicting reports on the initial point of budding. Furthermore, attention is paid to points where murine development is significantly distinct from human development, namely, in the cessation of nephrogenesis.

Mouse population-based evaluation of urinary protein and miRNA biomarker performance associated with cisplatin renal injury.

Discovery and qualification of novel biomarkers with improved specificity and sensitivity for detection of xenobiotic-induced injuries is an area of active research across multiple sectors. However, the majority of efforts in this arena have used genetically limited rodent stocks that lack variability in xenobiotic responses inherent to genetically heterogeneous human populations. In this study, genetically diverse Diversity Outbred (DO) mice were used as a surrogate for human clinical populations to investigate performance of urinary kidney biomarkers against classical preclinical kidney injury biomarkers (blood urea nitrogen [BUN] and serum creatinine). In this study, cisplatin was used as a paradigm kidney toxicant, with female adult DO mice exposed to a single injection (5 mg/kg) of cisplatin or vehicle and necropsied 72 h post-exposure and 18 h following overnight urine collection. Interindividual variability in kidney toxicity was observed, with DO mice experiencing either no tubule cell necrosis or minimal-mild necrosis. A panel of urinary protein biomarkers and profiled miRNAs were assessed by receiver-operating characteristic curves as to their ability to distinguish non-responder versus responder animals, as defined by histopathological evidence of renal tubule cell necrosis. A surprising outcome of these studies was that BUN was elevated alongside of urinary miRNA and protein biomarkers in animals with grade 2 proximal tubular cell necrosis; but not elevated with grade 1 necrosis. These studies demonstrate a novel approach for using a rodent population to better assess sensitivity of candidate biomarkers, especially for proposed clinical applications. Impact statement Recent studies have indicated that several urinary proteins and miRNA species may be suitable as biomarkers for acute kidney injury. A major focus on biomarker qualification is demonstrating improved specificity and sensitivity over gold standard tests. In this study, a mouse population model, Diversity Outbred mice, was used to demonstrate that neither the urinary protein markers nor the miRNA species assayed in urine could reliably detect low severity kidney injury better than blood urea nitrogen. This study has implications for use of these biomarkers in the clinic, where interindividual heterogeneity is present within patient populations and for which the underlying tissue pathology may not be known.

Renal Tumors in 26-Week Tg.Rash2 Mice Carcinogenicity Studies.

We report renal tubular adenomas and a carcinoma in 26-week Tg.rasH2 mouse carcinogenicity studies, which have not been reported to date either at our facility or in other published data. However, during the year 2014, renal tubular tumors were present in 4 studies conducted at our facility. Because of their morphological similarity to the amphophilic-vacuolar (AV) phenotypic variant of renal tubule tumors noted in Sprague-Dawley and Fischer 344 rats, which are thought to be familial, as well as the genetic homogeneity of Tg.rasH2 mice, we tracked the parents of these mice with tumors in each study. The origin of these tumors could not be traced back to any of the parents or even an animal barrier, and these tumors were not attributed to the vehicle or test article. Although the exact mechanism of tumorigenesis was not known, based on the available information, the development of renal tumors in these mice was considered random and spontaneous.

Evaluation of hydroxyatrazine in the endocrine disruptor screening and testing program's male and female pubertal protocols.

Two critical components of the validation of any in vivo screening assay are to demonstrate sensitivity and specificity. Although the Endocrine Disruptor Screening Program's Tier 1 Male and Female Pubertal Protocols have been shown to be sensitive assays for the detection of weak endocrine disrupting chemicals (EDCs), there are concerns that the assays lack specificity for EDC effects when a chemical induces systemic toxicity. A lack of specificity, or the ability to correctly identify an inactive or "negative" chemical, would increase the probability of identifying false positives. Here, we orally exposed rats to hydroxyatrazine (OH-ATR), a biotransformation by-product of the chlorotriazine herbicides that produced nephrotoxicity following a 13-week dietary exposure. Based on a previous study in our laboratory, males were dosed with 11.4 to 183.4 mg/kg OH-ATR and females were dosed with 45.75 to 183.4 mg/kg OH-ATR. Following exposure in both sexes, there was a dose-response increase in mean kidney weights and the incidence and severity of kidney lesions. These lesions included the deposition of mineralized renal tubule concretions, hydronephrosis, renal tubule dilatation, and pyelonephritis. However, no differences in body weight, liver weight, or reproductive tissue weights, reproductive or thyroid histology, hormone concentrations or the age of pubertal onset were observed. Therefore, the results demonstrate that the endpoints included in the pubertal assay are useful for nonendocrine (systemic) effects that define an no observable effect level (NOEL) or lowest observable effect level (LOEL) and provide one example where an impact on kidney function does not alter any of the endocrine-specific endpoints of the assay.

Two-generation reproductive toxicity study of dietary bisphenol A in CD-1 (Swiss) mice.

Dietary bisphenol A (BPA) was evaluated in a mouse two-generation study at 0, 0.018, 0.18, 1.8, 30, 300, or 3500 ppm (0, 0.003, 0.03, 0.3, 5, 50, or 600 mg BPA/kg/day, 28 per sex per group). A concurrent positive control group of dietary 17beta-estradiol (0.5 ppm; 28 per sex) confirmed the sensitivity of CD-1 mice to an endogenous estrogen. There were no BPA-related effects on adult mating, fertility or gestational indices, ovarian primordial follicle counts, estrous cyclicity, precoital interval, offspring sex ratios or postnatal survival, sperm parameters or reproductive organ weights or histopathology (including the testes and prostate). Adult systemic effects: at 300 ppm, only centrilobular hepatocyte hypertrophy; at 3500 ppm, reduced body weight, increased kidney and liver weights, centrilobular hepatocyte hypertrophy, and renal nephropathy in males. At 3500 ppm, BPA also reduced F1/F2 weanling body weight, reduced weanling spleen and testes weights (with seminiferous tubule hypoplasia), slightly delayed preputial separation (PPS), and apparently increased the incidence of treatment-related, undescended testes only in weanlings, which did not result in adverse effects on adult reproductive structures or functions; this last finding is considered a developmental delay in the normal process of testes descent. It is likely that these transient effects were secondary to (and caused by) systemic toxicity. Gestational length was increased by 0.3 days in F1/F2 generations; the toxicological significance, if any, of this marginal difference is unknown. At lower doses (0.018-30 ppm), there were no treatment-related effects and no evidence of nonmonotonic dose-response curves for any parameter. The systemic no observable effect level (NOEL) was 30 ppm BPA (approximately 5 mg/kg/day); the reproductive/developmental NOEL was 300 ppm (approximately 50 mg/kg/day). Therefore, BPA is not considered a selective reproductive or developmental toxicant in mice.

Two-generation reproductive toxicity evaluation of dietary 17beta-estradiol (E2; CAS No. 50-28-2) in CD-1 (Swiss) mice.

No information exists on reproductive/developmental effects in mice exposed to dietary 17beta-estradiol (E2) over multiple generations. Therefore, under OECD Test Guideline 416 with enhancements, CD-1 mice (F0 generation, 25 mice/sex/group) were exposed to dietary E2 at 0, 0.001, 0.005, 0.05, 0.15, or 0.5 ppm ( approximately 0, 0.2, 1, 10, 30, or 100 mug E2/kg body weight/day) for 8 weeks prebreed, 2 weeks mating, approximately 3 weeks gestation, and 3 weeks lactation. At weaning, selected F1 offspring (F1 parents; 25/sex/group) and extra retained F1 males (one per litter) were exposed to the same dietary concentrations and durations as the F0 generation; study termination occurred at F2 weaning; F1/F2 weanlings (up to three per sex per litter) were necropsied with organs weighed. At 0.5 ppm, effects were increased F1/F2 perinatal loss, prolonged F0/F1 gestational length, reduced numbers of F2 (but not F1) litters/group, reduced F1/F2 litter sizes, accelerated vaginal patency (VP) and delayed preputial separation (PPS), increased uterus + cervix + vagina weights (UCVW) in F0/F1 adults and F1/F2 weanlings, and decreased testes and epididymides weights (TEW) in F1/F2 weanlings. At 0.15 ppm, effects were increased UCVW in F0/F1 adults and F1/F2 weanlings, accelerated VP, delayed PPS, and reduced TEW in F1/F2 weanlings. At 0.05 ppm, UCVW were increased in F1/F2 weanlings, and PPS was delayed only in extra retained F1 males. There were no biologically significant or treatment-related effects on F0/F1 parental body weights, feed consumption, or clinical observations, or on F0/F1 estrous cyclicity, F0/F1 andrology, or F1/F2 anogenital distance at any dose. The no observable effect level was 0.005 ppm E2 ( approximately 1 mug/kg/day). Therefore, the mouse model is sensitive to E2 by oral administration, with effects on reproductive development at doses of 10- 100 mug/kg/day.

Three-generation evaluation of dietary para-nonylphenol in CD (Sprague-Dawley) rats.

This study evaluated the potential for dietary para-nonylphenol (NP; CAS No. 84852-15-3) to affect parental fertility and growth and development of three offspring generations in CD (Sprague-Dawley [SD]) rats, including sperm counts across generations to determine the validity of equivocal reductions observed in the F2 generation by R. E. Chapin et al. (1999, Toxicol. Sci. 52, 80-91). Male rat kidney toxicity was also examined based on inconsistent observations in NP-exposed rats at 2000 ppm but not at 200 or 650 ppm in Purina 5002 (H. C. Cunny et al., 1997, Regul. Toxicol. Pharmacol. 26, 172-178) and at all of these NP concentrations in NIH-07 diet (R. E. Chapin et al., 1999, Toxicol. Sci. 52, 80-91). Concentrations were 0, 20, 200, 650, and 2000 ppm NP in Purina 5002 diet and 0 and 650 ppm NP in NIH-07 diet. 17beta-estradiol (E2) was used as a positive control at 2.5 ppm in Purina 5002 diet. There were no NP effects on any reproductive parameters in any generation, including sperm counts. Kidney toxicity (histopathology) occurred at 650 and 2000 ppm with no clear difference for the two diets. Ovarian weight was decreased at 2000 ppm NP in all generations, with no effect on reproduction. Dietary E2 at 2.5 ppm caused renal, reproductive, and developmental (lactational and peripubertal) toxicity in all generations. This study confirmed that dietary NP is not a selective reproductive toxicant with an no observable adverse effect level (NOAEL) of > 2000 ppm ( approximately > 150 mg/kg/day) and provided an NOAEL for male rat kidney toxicity of 200 ppm NP (approximately 15 mg/kg/day).

Spontaneous and irradiation-induced tumor susceptibility in BRCA2 germline mutant mice and cooperative effects with a p53 germline mutation.

Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the loss of both genes enhances tumor development. To elucidate this interaction in an animal model, mice lacking the carboxy terminal domain of Brca2 were crossed with p53 heterozygous mice. Females from this intercross were then irradiated with an acute dose of 5 Gy ionizing radiation at 5 weeks of age and compared to nonirradiated controls. We found decreased survival and timing of tumor onsets, and significantly higher overall tumor incidences and prevalence of particular tumors, including stomach tumors and squamous cell carcinomas, associated with the homozygous loss of Brca2, independent of p53 status. The addition of a p53 mutation had a further impact on overall survival, incidence of osteosarcomas and stomach tumors, and tumor latency. The spectrum of tumors observed for this Brca2 germline mouse model suggest that it faithfully recapitulates some human disease phenotypes associated with BRCA2 loss. In addition, these findings include extensive in vivo data demonstrating that germline Brca2 and p53 mutations cooperatively affect animal survivals, tumor susceptibilities, and tumor onsets.

Reproductive toxicity evaluation of dietary butyl benzyl phthalate (BBP) in rats.

Butyl benzyl phthalate (BBP) was administered in the diet at 0, 750, 3750, and 11,250 ppm ad libitum to 30 rats per sex per dose for two offspring generations, one litter/breeding pair/generation, through weaning of F2 litters. Adult F0 systemic toxicity and adult F1 systemic and reproductive toxicity were present at 11,250 ppm (750 mg/kg per day). At 11,250 ppm, there were reduced F1 and F2 male anogenital distance (AGD) and body weights/litter during lactation, delayed acquisition of puberty in F1 males and females, retention of nipples and areolae in F1 and F2 males, and male reproductive system malformations. At 3750 ppm (250 mg/kg per day), only reduced F1 and F2 offspring male AGD was present. There were no effects on parents or offspring at 750 ppm (50 mg/kg per day). The F1 parental systemic and reproductive toxicity no observable adverse effect level (NOAEL) was 3750 ppm. The offspring toxicity NOAEL was 3750 ppm. The offspring toxicity no observable effect level (NOEL) was 750 ppm, based on the presence of reduced AGD in F1 and F2 males at birth at 3750 ppm, but no effects on reproductive development, structures, or functions.

The effect of chronic progressive nephropathy on the incidence of renal tubule cell neoplasms in control male F344 rats.

Chronic progressive nephropathy (CPN) is the most frequently diagnosed lesion in the rat kidney. It has many components including degeneration and regeneration of renal tubule (RT) epithelium, glomerular lesions and interstitial inflammation and fibrosis. The incidence and severity of CPN is strain, age, and sex dependent and may be altered by a number of factors including exposure to xenobiotics. In National Toxicology Program (NTP) 2-year bioassays, xenobiotic-associated increased severity (exacerbation) of CPN often occurs in association with a marginal increased incidence of renal tubule cell neoplasms (RTCN). The relationship between CPN and RTCN development has not been definitively determined. The present study evaluated the association between severity of CPN and the occurrence of RTCN in control male F344 rats. A slight but statistically significant increase in CPN severity was present in those animals with RTCN compared to aged-matched controls without RTCN. Although these data suggest there is a positive correlation between CPN and RTCN, cause and effect were not determined. This marginal association suggests that the number of RTCNs that may develop secondary to chemically exacerbated nephropathy would be few.