AA causes mtDNA damage of cultured podocytes.

<p>(A and B) Representative micrographs show the comet assay of mtDNA isolated from control (A) and 12h of AA-treated (B) podocytes. (C) Graphic presentation of Tail moment in control and AA treated podocytes. Tail moment was measured using tail DNA percent x tail moment length. Tail DNA percent was calculated by 100 x tail DNA immunofluorescent intensity / total DNA immunofluorescent intensity. * P < 0.05 versus control (n = 3). (D and E) Q-PCR analysis of ERCC1 (D) and ERCC2 (E) expression levels normalized to GAPDH in podocytes at different time points of AA treatment. * <i>P</i> < 0.05 versus control (<i>n</i> = 3).</p

AA causes mtDNA damage in mice kidneys.

<p>(A and B) Representative micrographs show the comet assay of mtDNA isolated from mice kidneys of sham (A) and day 3 after AA (B). (C) Graphic presentation of Tail moment of mtDNA in kidneys of sham and day 3 after AA. * P < 0.05 versus sham (n = 7). (D-G) Q-PCR analysis of ERCC1 (D), ERCC2 (E), Mgmt (F) and PARP (G) expression levels normalized to GAPDH in sham and different time points after AA administration. * <i>P</i> < 0.05 versus sham (<i>n</i> = 4).</p

AA causes podocyte foot-process effacement in mice.

<p>(A-F) Representative micrographs show the morphology in mice at different time points after AA administration. Kidney sections from sham (A and D), day 3 after AA (B and E) and day 7 after AA (C and F) were subjected to hematoxylin-eosin (HE) (A-C) and periodic acid-schiff (PAS) (D-F) (magnification: ×400). (G-I) Representative electron micrographs show podocytes foot process structures. Scale bar, 1μm. Arrows indicate infiltrated cells. Stars show podocyte foot-process effacement.</p

AA administration leads to albuminuria in mice.

<p>(A) Schematic illustration of experimental protocols. Arrows indicate the timing of AA injections. Arrowheads depict the time points when mice were killed after AA administration. (B) Urinary albumin levels at different time points after 6mg/kg body weight of AA administration. Urinary albumin data after correction to creatinine are presented as mean ± SEM. * P < 0.05 versus sham (n = 7). (C) SDS-PAGE demonstrates urine proteins at different time points after AA administration. Urine samples (pooled from 7 mice) were separated on 10% SDS-PAGE after corrected to creatinine. BSA (1µg) was loaded as a positive control. (D) AA was administered at different doses as indicated. Urinary albumin levels at different time points after different doses of AA administration. Urinary albumin data after correction to creatinine are presented as mean ± SEM. * P < 0.05 versus sham, # P < 0.05 verse day 3 after AA (n = 7).</p

AA causes podocyte depletion in mice.

<p>(A) Western blot analysis of WT1 expression levels in mice received AA. Whole kidney lysates were immunobloted with antibodies against WT1 and Tubulin, respectively. Samples from two individual animals per group were used. (B) Graphic presentation of relative WT1 abundance normalized to Tubulin. * <i>P</i><0.05 versus sham (<i>n</i> = 3). (C-E) Representative micrographs show the podocyte abundance at sham (C), day 3 after AA (D) and day 7 after AA (E). Kidney sections were stained with antibodies against WT1. (magnification: ×400) (F) Quantitative determination of the glomerular podocyte numbers at different time points after AA administration. * P < 0.05 versus sham (n = 4). </p

AA impairs the filtration barrier function of cultured podocytes.

<p>(A) Schematic depiction of the paracellular permeability influx assay. Podocyte monolayer on collagen-coated transwell filters was incubated without or with AA for 48 hours, and albumin permeability across podocyte monolayer was determined. (B) Graphic presentation of the albumin influx across podocyte monolayer. Duration of albumin incubation is shown on x-axis. Data are presented as mean ± SEM. * P < 0.05 versus control (n = 3).</p

Autophagy Attenuates Diabetic Glomerular Damage through Protection of Hyperglycemia-Induced Podocyte Injury

<div><p>Despite the recent attention focused on the important role of autophagy in maintaining podocyte homeostasis, little is known about the changes and mechanisms of autophagy in podocyte dysfunction under diabetic condition. In this study, we investigated the role of autophagy in podocyte biology and its involvement in the pathogenesis of diabetic nephropathy. Podocytes had a high basal level of autophagy. And basal autophagy inhibition either by 3-methyladenenine (3-MA) or by Beclin-1 siRNA was detrimental to its architectural structure. However, under diabetic condition in vivo and under high glucose conditions in vitro, high basal level of autophagy in podocytes became defective and defective autophagy facilitated the podocyte injury. Since the dynamics of endoplasmic reticulum(ER) seemed to play a vital role in regulating the autophagic flux, the results that Salubrinal/Tauroursodeoxycholic acid (TUDCA) could restore defective autophagy further indicated that the evolution of autophagy may be mediated by the changes of cytoprotective output in the ER stress. Finally, we demonstrated in vivo that the autophagy of podocyte was inhibited under diabetic status and TUDCA could improve defective autophagy. Taken together, these data suggested that autophagy might be interrupted due to the failure of ER cytoprotective capacity upon high glucose induced unmitigated stress, and the defective autophagy might accelerate the irreparable progression of diabetic nephropathy.</p> </div

Uric acid induces RANTES, MCP-1 and TNF-α expression in tubular epithelial cells.

<p>(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. *<i>P</i><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. *<i>P</i><0.05 versus control (<i>n</i> = 3).</p

Uric acid induces RANTES, MCP-1 and TNF-α expression in hyperuricemia mice kidneys.

<p>(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. *<i>P</i><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. *<i>P</i><0.05 versus control (<i>n</i> = 5).</p

The tendencies of Treg cells in PBMC after a single DFPP.

<p>A-D. Treg cell was expressed as CD4+CD25+ CD127^low/−cells in PBMC. E, F. The frequency of CD4+CD25+ Treg cells in MHD patients with CHC during the DFPP. G, H. The frequencies of CD4+CD25+ CD127^low/− Treg cells in MHD patients with CHC during the DFPP</p