Fig 1.
Body and kidney parameters and renal morphology.
Allopurinol 75 μg/ml causes a reduction of body weight (a) and an increase of kidney/body weight ratio (b) only in KO mice. Representative macroscopic images of dissected fresh renal tissue after treatment with allopurinol 75 μg/ml or vehicle (c). Allopurinol does not cause macroscopic changes in HPRT+/- and WT mice. The vehicle-treated HPRT-/- kidneys have a normal appearance as well. Instead, allopurinol administered to HPRT-/- animals produces profound modifications of the renal structure, with pale and yellowish appearance. This is shown in more details in panel (d), where yellow deposits can be observed in the cortex (black arrows) and yellow streaks in the medulla (white arrows). Analysis of the fresh tissue by polarized light microscopy demonstrates that the deposits are of crystalloid nature (e). *p<0.05 as compared to vehicle-treated HPRT-/-. Scale bars: 1 mm.
Fig 2.
(a) Representative images of ethanol-fixed eosin-stained kidney sections observed by light (left panels) and polarized microscopy (right panels). Independent of the drug dosage, renal morphology is preserved and analysis by polarized microscopy is completely negative in sections from allopurinol-treated HPRT+/- and WT animals, whereas HPRT-/- kidneys have altered structure with numerous crystals filling the tubular lumens. Scale bars = 50 μm. (b) Reversed-phase HPLC analysis of solubilized crystals collected from the tubular lumina of a HPRT-/- kidney section showing a main peak (upper panel) identified as xanthine by comparison with a commercial standard (lower panel).
Fig 3.
Histological images of kidney sections from mice treated with 75 μg/ml allopurinol (a). Normal renal morphology is detected in WT-treated mice (upper panels), whereas profound changes can be seen in HPRT-/- animals (lower line of each panel), where tubules look dilated and filled by casts and cells, and abundant matrix deposition can be observed in the interstitium, where reticulin fibers are highlighted by Gordon and Sweet GW staining and collagen I/III is demonstrated PR staining. Matrix deposition affects also the glomerular structures. Scale bars = 50 μm.A statistically higher percentage of positively stained interstitial areas was observed in allopurinol-treated HPRT-/- mice than WT animals, as shown in panels b (GW), c (MT), and d (PR). Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. Results are expressed as mean ± SE. ***p< 0.001 versus HPRT+/+. Abbreviations: EE, hematoxylin eosin; PAS, periodic acid-schiff; GW, Gordon and Sweetstaining; MT, Masson's Trichrome, PR, Picrosirus-red.
Fig 4.
Transmission electron microscopy.
Tubules from allopurinol-treated HPRT-/- animals appear altered at the subcellular level, showing mitochondrial swelling (yellow arrows, a, b), numerous lysosomes (red arrow, a), shrinking of the tubular basement membrane (blue arrow, b), and loss of the brush border (white arrow, b). Scale bar = 2 μm.
Fig 5.
Renal function evaluation and oxidized purines /urate/inosine serum concentration.
(a) Macroscopic examination of collected urine shows that, as compared to the yellow color of urine characterizing all vehicle-treated animals and allopurinol-treated WT and HPRT+/- mice, a transparent urine is produced by HPRT-/- mice after allopurinol administration. (b) The image is an example of the urinary sediment from an allopurinol-treated HPRT-/- mouse, showing numerous cells and crystals. Scale bars = 50 μm. (c) Reversed phase HPLC analysis of blood concentration of hypoxanthine (black), xanthine (gray), urate (light gray), and inosine (white) in HPRT-/- (left panel) and WT (right panel) mice. Error bars are standard deviation of the mean. *p < 0.05; **p<0.01 versus vehicle.
Fig 6.
Renal lipids and inflammatory changes in kidney of HPRT-/- treated mice.
(a) Red Oil O staining images from allopurinol-treated WT and HPRT-/- animals. The WT tissue is completely negative, whereas kidneys from HPRT-/- mice show numerous positive areas in the interstitium, mainly within and around the lumen of dilated tubules. Scale bars = 50 μm. (b) Gene expression analysis of adipogenesis-related molecules C-EBP alpha and beta and PPAR alpha and gamma shows no differences based on the genotype in vehicle-treated mice, whereas a significant increase of C-EBP alpha and beta and a significant decrease of PPAR alpha are detected in allopurinol-treated HPRT-/- animals. Results are expressed as mean ± standard deviation. ***p< 0.001 versus HPRT-/- treated with vehicle alone. (c) The macrophage marker CD68 is present in numerous areas of the cortex and the medulla in kidney sections of allopurinol-treated HPRT-/- mice (right panels), whereas it appears almost completely negative in corresponding sections of allopurinol-treated WT mice (left panels). Scale bars = 50 μm. (d) Quantification of the number of CD68 positive macrophages in the kidney sections. Results are expressed as mean ± SE. *** p < 0.001 versus HPRT+/+ allopurinol treated mice. (e) Gene expression analysis of the pro-inflammatory molecules gp91phox, MCP-1 and TNF-α indicates no differences based on the genotype in mice administered vehicle (white bars), whereas a statistically significant increase of all three molecules is present in allopurinol HPRT-/- kidneys. mRNA level normalized to the value of HPRT +/+ mice treated with vehicle alone Results are expressed as mean ± standard deviation. ***p< 0.001 versus mice of the same genotype treated with vehicle alone.
Fig 7.
(a) Representative images of E-cadherin staining, showing diffuse positivity in tubular cells in kidney sections from allopurinol-treated WT mice (left panels). The right panels show corresponding tissue sections from allopurinol-treated HPRT-/- animals, showing profoundly decreased E-cadherin expression in the dilated tubuli. Scale bars = 50 μm. (b) Immunohistochemical staining for α-SMA. Kidneys from allopurinol-treated WT mice (left panels) show only scattered expression of the molecule. At higher magnification (lower panels), positivity is found in vascular structures, such as peritubular capillaries and glomerular arterioles (arrows), whereas the staining is completely negative in tubules. A diffusely increased expression in the tubulointerstitium is instead observed in kidney sections from allopurinol-treated HPRT-/- mice (right panels). At higher magnification, the molecule appears localized in tubular cells and around the tubules (lower panels). α-SMA, α-smooth muscle actin. Scale bars = 50 μm. Quantification of E-cadherin (c) and α-SMA (d) shows a statistically significant difference in allopurinol-treated WT and HPRT-/- animals Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. *** p < 0.001 versus WT. (e) Gene expression analysis of pro-fibrotic molecules TGF-β α-SMA and PAI-1 shows no differences based on the genotype in mice administered vehicle (white bars), whereas a statistically significant increase of all three molecules is present in allopurinol HPRT-/- kidneys (gray bars). mRNA level normalized to the value of HPRT+/+ mice treated with vehicle alone. Results are expressed as mean ± standard deviation.*p< 0.05; *** p< 0.001 versus mice of the same genotype treated with vehicle alone.
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).