Uric acid stones in the urinary bladder of aryl hydrocarbon receptor (AhR) knockout mice

Abstract

The aryl hydrocarbon receptor (AhR) knockout mice raised in the laboratory of Fujii-Kuriyama have been under investigation for several years because of the presence in their urinary bladder of large, yellowish stones. The stones are composed of uric acid and become apparent in the bladders as tiny stones when mice are 10 wk of age. By the time the mice are 6 mo of age, there are usually two or three stones with diameters of 3-4 mm. The urate concentration in the serum was normal but in the urine the concentration was 40-50 mg/dL, which is 10 times higher than that in the WT littermates. There were no apparent histological pathologies in the kidney or joints and the levels of enzymes involved in elimination of purines were normal. The source of the uric acid was therefore judged to be from degradation of nucleic acids due to a high turnover of cells in the bladder itself. The bladder was fibrotic and the luminal side of the bladder epithelium was filled with eosinophilic granules. There was loss of E-cadherin between some epithelial cells, with an enlarged submucosal area filled with immune cells and sometimes invading epithelial cells. We hypothesize that in the absence of AhR there is loss of detoxifying enzymes, which leads to accumulation of unconjugated cytotoxins and carcinogens in the bladder. The presence of bladder toxins may have led to the increased apoptosis and inflammation as well as invasion of epithelial cells in the bladders of older mice.

Fig. 1.

Urate stones in the urinary bladder of AhR knockout mice. Urinary bladder of a 10-wk-old AhR knockout mouse (A) with a urate stone indicated by the arrow. These stones are rough and pitted in appearance (B). Urate stones at 6 mo of age (C) with diameters of 3–4 mm. (Scale bar in A, 500 μm; B, 200 μm; and C, 1 mm.)

Fig. 2.

Urine solutes and volume and serum uric acid measurements of wild-type and AhR knockout mice at 3 mo of age. Urine creatinine (A), nitrate (D), volume (E), osmolality (F), potassium (G), chloride (H), calcium (I), and sodium (J) were not significantly different in the knockout mice compared with their wild-type littermates. Uric acid (B) was ∼10-fold higher in the urine of knockout mice (P < 0.001). There was a small decrease in the total protein (C) in the AhR knockout mice (P = 0.039). Uric acid levels in the serum were not significantly changed at 3 mo of age (K). Error bars in K represent SD.

Fig. 3.

Fibrosis in the submuscosal layer of the 10-wk-old AhR knockout bladders. Masson's trichrome stain of AhR^+/− (A and C) and AhR^−/− (B and D) bladders, demonstrating fibrosis of the knockout bladder with an increased amount of collagen (blue) and enlarged blood vessels. (Scale bars in A and B, 500 μm; C and D, 20 μm.)

Fig. 4.

Invading epithelial cells are observed in the submucosal and muscle layers of the 6-mo-old AhR^−/− bladder. Some epithelial cells are marked with arrows (B). Masson's trichrome stain was used to stain collagen blue, cytoplasm red, and nuclei purple. (Scale bar in A, 500 μm; B, 20 μm.)

Fig. 5.

Numerous round particles were visible in the luminal side of the urothelium of a 10-wk-old AhR knockout mouse (B and D), whereas no particles exist in the heterozygous control (A and C). Cytoplasm and particles are stained pink with eosin and nuclei are stained purple with Mayer's hematoxylin. These structures stain positive (brown-black color) for Grocott's methenamine silver stain, which can be a marker for uric acid (F), whereas the control bladder remains negative (E). After a 1-h treatment with uricase and hydrogen peroxide, these granules are not present in the epithelium of the 4-mo-old AhR^−/− bladder (H). The granules are still present in the control treatment of only hydrogen peroxide in PBS (G). (Scale bars in A, B, E, and F, 20 μm; C, D, G, and H, 10 μm.)

Fig. 6.

Apoptotic cells are present in some areas of the 4-mo-old AhR^−/− bladder (B, D, and F), whereas there are very few in the AhR^+/− mouse (A, C, and E). DAPI is used to stain the nuclei blue (A and B) and FITC stains TUNEL⁺ cells green (C and D). (Scale bars, 20 μm.)

Fig. 7.

E-cadherin and F4/80 staining in 10-wk-old AhR^−/− and AhR^+/− mice. There is loss of E-cadherin in some areas of the AhR^−/− urothelium (B), whereas the heterozygous mice appear to have normal E-cadherin expression throughout the bladder (A). There were many more cells that stained positive for the macrophage marker, F4/80, in the stroma of the knockout bladders (D) compared with the heterozygous controls (C). (Scale bars, 20 μm.)