Strain-related differences in urine composition of male rats of potential relevance to urolithiasis.

In carcinogenicity studies with PPAR gamma and alpha/gamma agonists, urinary bladder tumors have been reported in Harlan Sprague-Dawley (HSD) and Charles River Sprague-Dawley (SD) but not Wistar (WI) rats, with urolithiasis purported to be the inciting event. In two 3-month studies, the authors investigated strain-related differences in urine composition by sampling urine multiple times daily. Urine pH, electrolytes, creatinine, protein, citrate and oxalate levels, and serum citrate were assessed; urine sediment was analyzed by scanning electron microscopy and energy dispersive x-ray spectroscopy. HSD rats had significantly higher urine calcium than SD or WI rats, primarily as calcium phosphate-containing precipitate. When compared to SD rats, HSD rats had lower urine volume, higher urine protein, and a comparable (week 4) to lower (week 13) burden of MgNH(4)PO(4) aggregates. Relative to WI rats, HSD rats had higher urine protein and magnesium and lower serum and urine citrate. Overall, the susceptibility to urolithiasis in male rats was HSD > SD > WI; this was likely due to strain-related differences in the amount of urine protein (a nidus for crystal formation), lithogenic ions, citrate (an inhibitor of lithogenesis), and/or volume. Strain-related differences in urine composition need to be considered when interpreting the outcome of studies with compounds that alter urine composition.

Subchronic urinary bladder effects of muraglitazar in male rats.

Muraglitazar, a PPARalpha/gamma dual agonist, was dosed orally to rats once daily for 13 weeks to evaluate urinary and urothelial changes of potential relevance to urinary bladder tumorigenesis. Groups of 17 young or aged rats per sex were fed a normal or 1% NH4Cl-supplemented diet and were dosed with 0, 1, or 50 mg/kg muraglitazar. Lithogenic ions and sediment were profiled from freshly voided urine samples collected 24 h after dosing, and drug exposures were measured. Urinary citrate, oxalate, and epidermal growth factor (EGF) were assayed from 18-h urine collections. Urothelium was assessed by light microscopy, scanning electron microscopy, and BrdU and TUNEL immunohistochemistry. When fed a normal diet, urine pH was higher in males (above 6.5). Urine volume/body weight was greater in females. Urine soluble/total calcium and magnesium and phosphorus/creatinine ratios were lower in male rats fed a normal diet. Urine citrate levels were decreased and oxalate was increased in young male rats treated with 50 mg/kg muraglitazar compared to age/sex/diet-matched controls. No changes in urine sediment were detected 24 h after dosing. In young male rats treated with 50 mg/kg on normal diet, multifocal urothelial necrosis and proliferation were observed, whereas urothelial apoptosis and urine EGF levels were unchanged compared to age/sex/diet-matched controls. Urothelial necrosis and proliferation were not correlated to systemic or urinary drug exposures and were prevented by dietary acidification. These data suggest that muraglitazar-associated changes in urine composition predispose to urothelial cytotoxicity and proliferation in the urinary bladder of young male rats and that urine sediment must be profiled at multiple daily timepoints to fully qualify drug-induced changes in urine composition.

Rodent carcinogenicity profile of the antidiabetic dual PPAR alpha and gamma agonist muraglitazar.

The carcinogenic potential of muraglitazar, a dual human peroxisome proliferator-activated receptor alpha/gamma agonist, was evaluated in 2-year studies in mice (1, 5, 20, and 40 mg/kg) and rats (1, 5, 30, and 50 mg/kg). Benign gallbladder adenomas occurred at low incidences in male mice at 20 and 40 mg/kg (area under the curve [AUC] exposures > or = 62 times human exposure at 5 mg/day) and were considered drug related due to an increased incidence of gallbladder mucosal hyperplasia at these doses. There was a dose-related increased incidence of transitional cell papilloma and carcinoma of the urinary bladder in male rats at 5, 30, and 50 mg/kg (AUC exposures > or = 8 times human exposure at 5 mg/day). At 30 and 50 mg/kg, the urinary bladder tumors were accompanied by evidence of increased urine solids. Subsequent investigative studies established that the urinary bladder carcinogenic effect was mediated by urolithiasis rather than a direct pharmacologic effect on urothelium. Incidences of subcutaneous liposarcoma in male rats and subcutaneous lipoma in female rats were increased at 50 mg/kg (AUC exposures > or = 48 times human exposure at 5 mg/day) and attributed, in part, to persistent pharmacologic stimulation of preadipocytes. Toxicologically relevant nonneoplastic changes in target tissues included thinning of cortical bone in mice and hyperplastic and metaplastic adipocyte changes in mice and rats. Considering that muraglitazar is nongenotoxic, the observed tumorigenic effects in mice and rats have no established clinical relevance since they occurred at either clinically nonrelevant exposures (gallbladder and adipose tumors) or by a species-specific mechanism (urinary bladder tumors).

Dietary ammonium chloride for the acidification of mouse urine.

A novel therapeutic compound was found to induce bladder tumors in male rats. Given the location of the tumors and the increased amounts of calcium- and magnesium-containing solids found in the urine of treated animals, we hypothesized that tumorigenesis was secondary to urine crystal formation rather than a direct effect of the drug on urothelium. To investigate the basis for the response, a method of acidifying rodent urine was needed. This study tested the efficacy of 1% dietary NH(4)Cl in reducing the urinary pH of male mice. After 1 wk, urinary pH (mean +/- SD) at 1 h after light onset was 7.51 +/- 0.32 among controls compared with 6.21 +/- 0.31 for the NH(4)Cl-fed group. After 2 wk of supplementation, urinary pH was 7.78 +/- 0.41 for controls and 6.20 +/- 0.30 for the NH(4)Cl-fed group. To investigate whether the time of collection altered urinary pH, samples also were collected 8 h after the start of the light cycle on the day of the 2-wk collection. Urinary pH was 7.12 +/- 0.28 for the control group and 5.80 +/- 0.23 for the NH(4)Cl-fed mice. The pH differences between control and NH(4)Cl-fed groups and the differences in pH within groups at 1 and 8 h were statistically significant. Dietary NH(4)Cl is an effective urinary acidifier for mice. When evaluating the pH of mouse urine, care should be taken to compare samples collected at the same time after the start of the light cycle.

Comparison of technicians' ability to detect clinical signs in rats housed in wire-bottom versus solid-bottom cages with bedding.

Rodent toxicology studies have historically been performed in wire-bottom cages, but the 1996 Guide for the Care and Use of Laboratory Animals recommends solid-bottom caging with bedding. Some investigators have expressed concern that changing to solid-bottom cages would interfere with technicians' ability to detect clinical signs. To test this hypothesis, rats were housed in both types of caging and given compounds to induce a variety of subtle clinical signs common to toxicology studies including chromodacryorrhea, soft stool, stereotypic behaviors, mild hypoactivity, abnormal postures, and discolored urine. For one comparison, fecal pellets were removed to simulate decreased production of feces. Technicians, blinded from knowing which animals had been treated, observed the rats and recorded the clinical signs they detected. The technicians who administered the treatments verified that clinical signs were present before and after the blinded technicians made their observations. The number of animals observed with clinical signs divided by the number of animals verified with signs was calculated for each compound and compared between the cage types by using the Fisher Exact Test. The only statistically significant difference observed was a diminished ability to detect discolored, dark urine from rats in wire-bottom cages. These results suggest that concerns about technical staff's inability to detect clinical signs in toxicity tests should not prevent investigators from using solid-bottom cages with bedding.