The renal phenotype of allopurinol-treated HPRT-deficient mouse
Fig 8
Xanthine effects in vitro and tubular cell phenotype.
(a) Phase contrast (upper panels) and polarized microscopy (lower panels) show the deposition of xanthine after incubation for 48 h (right panels) as compared to vehicle alone (left panels). (b) Oil Red O staining demonstrates increased intracellular lipids in cells exposed to xanthine (right panel) as compared to vehicle (left panel) and uric acid (lower panel). Scale bars = 50 μm. (c) At 48 h incubation, cells exposed to vehicle (left panel) demonstrate the typical cobblestone appearance, whereas a more elongated shape (white arrows) can be observed after xanthine (middle panel) and, to a lesser extent, uric acid (right panel) incubation. Scale bars = 50 μm. (d) At 96 h incubation, light microscopy (upper panels) confirms a more diffuse appearance of elongated cells after xanthine and uric acid exposure. This associates with loss of E-cadherin expression (lower panels) particularly in xanthine-treated cells. Scale bars = 50 μm. (e) Western blot analysis and (f) densitometric quantification of α-SMA in vehicle-, xanthine- and uric acid-treated cells at 96 h. (g) Quantification results of E-cadherin staining in 96h-incubated cells. *p < 0.05 versus cells treated with vehicle alone and versus uric acid treated cells. At 96h, treatment with xanthine reduces cell number (h) and viability (i), more than vehicle and uric acid. Cytotoxicity of xanthine is confirmed by increased LDH release (j) in the medium, which is higher compared to vehicle and uric acid. The supernatant of Triton X-100-treated cells is used as the positive control. Results are expressed as mean ± SEM. *p< 0.05; § p< 0.001 versus vehicle treated cells in (h) and (i) and versus Triton treated cells in (k).