Figure 1.
Uric acid induces renal infiltration of T cells and macrophages.
(a) Representative micrographs show kidney morphology by H/E staining. (b) Immunohistochemical staining revealed an increased infiltration of CD3+ T cells in kidney of hyperuricemia mice after continuous injection of 7d and 14d, respectively. (c) Immunohistochemical staining revealed an increased infiltration of CD68+ macrophage in kidney of hyperuricemia mice after continuous injection of uric acid for 7d and 14d, respectively. (d) Mice were daily injected with uric acid intraperitoneally. At different time points, as indicated, serum was collected and serum urate levels (μmol/L) were determined. Data are presented as mean ± SEM from five animals per group at each time point, *P<0.05 versus sham (e and f) Graphic presentations of quantitative data. Positive cell numbers per high-power field (×400) were counted. Ten randomly selected field of each kidney were counted. Data are mean ± SEM of five animals per group. *P<0.05 versus sham-control.
Figure 2.
Uric acid induces RANTES, MCP-1 and TNF-α expression in hyperuricemia mice kidneys.
(a) Q-PCR results showed that renal RANTES, MCP-1 and TNF-α mRNA expression were increased in kidney of hyperuricemia mice after continuous injection of uric acid for 7d and 14d, respectively. *P<0.05 versus sham-control. (b) Western blot results showed that renal RANTES protein expression was increased in hyperuricemia mice kidneys. (c) Graphic presentation of relative RANTES protein abundance normalized to actin. *P<0.05 versus control (n = 5).
Figure 3.
Uric acid induces RANTES, MCP-1 and TNF-α expression in tubular epithelial cells.
(a through c) Q-PCR results showed that RANTES, MCP-1 and TNF-α mRNA expression were increased in NRK-52E cells after uric acid treatment for different time periods as indicated. *P<0.05 versus control. (b) Western blot results showed that RANTES protein expression was increased in NRK-52E cells after uric acid treatment. (c) Graphic presentation of relative RANTES protein abundance normalized to actin. *P<0.05 versus control (n = 3).
Figure 4.
Blockage of urate transporter by probenecid inhibits uric acid-induced RANTES, MCP-1 and TNF-α expression in tubular epithelial cells.
(a through c) Q-PCR results showed that probenecid inhibits uric acid-induced RANTES (a), MCP-1 (b) and TNF-α (c) mRNA expression in NRK-52E cells. *P<0.05 versus control. #P<0.05 versus uric acid treated cells without probenecid incubation (n = 3). (d) Western blot results showed that probenecid inhibits uric acid-induced RANTES protein expression in NRK-52E cells. (e) Graphic presentation of relative RANTES protein abundance normalized to actin. *P<0.05 versus control. #P<0.05 versus uric acid treated cells without probenecid incubation (n = 3).
Figure 5.
Modulation of urate transporter regulates tubular express of RANTES, MCP-1 and TNF-α in response to uric acid treatment.
(a) RT-PCR analysis showed the URAT1 expression level after plasmid transfection for 24h. (b) Graphic presentation of relative mRNA abundance normalized to actin. *P<0.05 versus pcDNA3 transfected group (n = 3). (c) Q-PCR results showed that upregulation of URAT1 by plasmid transfection sensitized tubular express of RANTES, MCP-1 and TNF-α mRNA after 6 h of uric acid treatment. *P<0.05 versus control. #P<0.05 versus cells transfected with pcDNA3 (n = 3). (d) Western blot results showed that upregulation of URAT1 by plasmid transfection sensitized tubular express of RANTES protein after 24h of uric acid treatment. (e) Graphic presentation of relative RANTES protein abundance normalized to actin. *P<0.05 versus control. #P<0.05 versus uric acid treated cells transfected with pcDNA3 (n = 3). (f) RT-PCR analysis showed the URAT1 expression level after transfection of URAT1 siRNA for 24h. (g) Graphic presentation of relative mRNA abundance normalized to actin. *P<0.05 versus negative control (N.C.) siRNA transfected group (n = 3). (h) Q-PCR results showed that downregulation of URAT1 by RNA interference partially decreased tubular express of RANTES, MCP-1 and TNF-α mRNA after 6h of uric acid treatment in NRK-52E cells. *P<0.05 versus control. #P<0.05 versus uric acid treated cells transfected with negative control siRNA (n = 3).
Figure 6.
Culture media from uric acid treated tubular cells induce the recruitment of macrophage in vitro.
Probenecid reduces the media-induced recruitment of macrophage. (a) Schematic depiction of the chemotaxis assay. Macrophage monolayer on transwell filters was incubated conditioned media from NRK-52E cells treated with or without uric acid for 4 hours, and macrophage migration was then determined. (b) Representative pictures show the migrated macrophage in the bottom chambers of transwell plates in various groups as indicated. (c) For quantification, positive stained nuclei per field (×200) were counted. Ten randomly selected fields were counted. As for the total cell number, ten randomly chose fields (×200) were counted before the chemotaxis assay. Data are expressed as the percentage of migrated cells in total cells added and presented as means ± SEM of three independent experiments. *P<0.05 versus control.
Figure 7.
Uric acid activates NF-κB signaling in tubular epithelial cells.
(a) Western blot analysis showed that uric acid induced p65 NF-κB phosphorylation and activation in NRK-52E cells. (b) Western blot showed that uric acid induced IκB phosphorylation in NRK-52E cells. (c) Graphic presentation of relative phosphs-p65 NF-κB protein abundance normalized to p65 NF-κB. *P<0.05 versus control (n = 3). (d) Graphic presentation of relative phosphs-IκB protein abundance normalized to IκB. *P<0.05 versus control (n = 3). (e) Immunofluorescence staining demonstrated that p65 NF-κB underwent nuclear translocation upon uric acid stimulation in NRK-52E cells.
Figure 8.
NF-κB signaling is critical for mediating RANTES expression in tubular cells.
NRK-52E cells were treated with 200μmol/L of uric acid in the absence or presence of NF-κB inhibitor (NF-κB SN50). (A) Immunofluorescence staining demonstrated that incubation with NF-κB SN50 inhibited nuclear translocation of p65 NF-κB upon uric acid stimulation in NRK-52E cells. (b) Inhibition of NF-κB signaling abrogated RANTES expression stimulated by uric acid in NRK-52E cells.
Figure 9.
Uric acid activates NF-κB signaling pathway in kidney of hyperuricemia mice.
(a) Western blot demonstrated that uric acid induced p65 NF-κB phosphorylation and activation in kidney of hyperuricemia mice. (b) Western blot showed that uric acid induced IκB phosphorylation in vivo. (c) Graphic presentation of relative phospho-p65 NF-κB and phospho-IκB protein abundance normalized to p65 NF-κB and IκB, respectively. *P<0.05 versus control (n = 3). (d) Immunofluorescent staining demonstrated that p65 NF-κB underwent nuclear translocation in kidney of hyperuricemia mice. Kidney sections were immunostained for total p65 (red) and the nuclei (blue). Arrowheads indicate nuclear staining of p65 NF-κB. (e) Enlarged image shows nuclear translocation of p65 NF-κB in tubules of hyperuricemia mice (day 7) demonstrated by immunofluorescent staining.