Exploiting Copper Redox for ¹⁹F Magnetic Resonance-Based Detection of Cellular Hypoxia

We report a pair of fluorinated, redox-active copper complexes for potential use as ¹⁹F MRI contrast agents for detecting cellular hypoxia. Trifluorinated Cu­(II) ATSM-F₃ displays the appropriate redox potential for selective accumulation in hypoxic cells and a completely quenched ¹⁹F NMR signal that is “turned on” following reduction to Cu­(I). Incubation of cancer cells with CuATSM-F₃ resulted in a selective detection of ¹⁹F signal in cells grown under hypoxic conditions

Involvement of Endoplasmic Reticulum Stress in Albuminuria Induced Inflammasome Activation in Renal Proximal Tubular Cells

<div><p>Albuminuria contributes to the progression of tubulointerstitial fibrosis. Although it has been demonstrated that ongoing albuminuria leads to tubular injury manifested by the overexpression of numerous proinflammatory cytokines, the mechanism remains largely unknown. In this study, we found that the inflammasome activation which has been recognized as one of the cornerstones of intracellular surveillance system was associated with the severity of albuminuria in the renal biopsies specimens. In vitro, bovine serum albumin (BSA) could also induce the activation of NLRP3 inflammasome in the cultured kidney epithelial cells (NRK-52E). Since there was a significant overlap of NLRP3 with the ER marker calreticulin, the ER stress provoked by BSA seemed to play a crucial role in the activation of inflammasome. Here, we demonstrated that the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA) which was proved to be an enhancer for the adaptive capacity of ER could attenuate the inflammasome activation induced by albuminuria not only in vitro but also in diabetic nephropathy. Taken together, these data suggested that ER stress seemed to play an important role in albuminuria-induced inflammasome activation, elimination of ER stress via TUDCA might hold promise as a novel avenue for preventing inflammasome activation ameliorating kidney epithelial cells injury induced by albuminuria.</p></div

Exploiting Copper Redox for ¹⁹F Magnetic Resonance-Based Detection of Cellular Hypoxia

We report a pair of fluorinated, redox-active copper complexes for potential use as ¹⁹F MRI contrast agents for detecting cellular hypoxia. Trifluorinated Cu­(II) ATSM-F₃ displays the appropriate redox potential for selective accumulation in hypoxic cells and a completely quenched ¹⁹F NMR signal that is “turned on” following reduction to Cu­(I). Incubation of cancer cells with CuATSM-F₃ resulted in a selective detection of ¹⁹F signal in cells grown under hypoxic conditions

Expression of the inflammasome markers in renal tubular epithelia is associated with the severity of proteinuria.

<p>(The information and histological diagnoses are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072344#pone.0072344.s002" target="_blank">Table S1</a>). And samples were divided into the following categories according to the severity of proteinuria: mild (proteinuria<0.5 g/24 h); moderate (proteinuria 1.0–3.5 g/24 h); and severe (proteinuria>5 g/24 h). A. Representative micrographs by immunofluorescence staining of caspase-1 from patients with proteinuria, demonstrating the predominance of tubular staining for active caspase-1 and the correlation between intensity of tubular staining and proteinuria level. B. Representative micrographs by immunofluorescence staining of IL-1β. C. Representative micrographs by immunohistochemical staining of IL-18. D. Scatter diagram demonstrated the relative abundance of the semiquantitative histomorphometric analysis of the immunostaining of caspase-1, IL-1β, and IL-18 (n = 5). *<i>P</i><0.05 vs. mild (proteinuria<0.5 g/24 h); # P<0.05 vs. moderate (proteinuria 1.0–3.5 g/24 h).</p

TUDCA attenuates the inflammasome activation induced by BSA in NRK-52E cells in vitro.

<p>A, B and C: Western blot analysis shows TUDCA attenuates the caspase-1 activation and maturation of IL-1β and IL-18 protein induced by BSA in NRK-52E cells. Growth arrested NRK-52E cells were pretreated without (same amount of DMSO) or with 100 umol/L TUDCA for 0.5 hours, and then followed by incubation without or with 5 mg/ml BSA for 12 hours as indicated. C, D and E: Graphic presentation showed the relative abundance of caspase-1, IL-1β and IL-18 protein after normalization with α-tubulin in various groups. Data are presented as mean±SEM of three independent experiments. *<i>P</i><0.05 vs. normal control; # <i>P</i><0.05 vs. group with BSA treatment.</p

Bovine serum albumin induced endoplasmic reticulum stress in NRK-52E cells.

<p>A. Western blot analysis shows the expression of GRP78 and the phosphorylation of eIF2a in NRK-52E cells after treatment without or with 5 mg/ml BSA for various time periods in serum-free medium. The whole cell lysate was immunoblotted with GRP78, phosphorylated- eIF2α and eIF2α antibody, respectively. The same blot was reprobed with α-tubulin to confirm equal loading of each lane. B. Western blot analysis shows the expression of GRP78 and the phosphorylation of eIF2a in NRK-52E cells without or with different amounts of BSA for 12 h in serum-free medium. C and D: Graphical presentation shows the relative abundances of GRP78 after normalization with α-tubulin and the phosphorylation of eIF2α after normalization with eIF2α. Data are presented as mean±SEM of three independent experiments. *<i>P</i><0.05 vs. normal control (the relative abundance of GRP78 protein level); # P<0.05 vs. normal control (the phosphorylation of eIF2α).</p

Bovine serum albumin induced the inflammasome activation in NRK-52E cells in vitro.

<p>A,C and E: Western blot analysis shows the caspase-1 activation and maturation of IL-1β and IL-18 protein in NRK-52E cells after treatment without or with 5 mg/ml BSA for various time periods in serum-free medium. The whole cell lysate was immunoblotted with caspase-1, IL-1β and IL-18 antibody, respectively. The same blot was reprobed with α-tubulin to confirm equal loading of each lane. B, D and F: Western blot analysis shows the caspase-1 activation and maturation of IL-1β and IL-18 protein in NRK-52E cells without or with different amounts of BSA for 12 h in serum-free medium. The whole cell lysate was also immunoblotted with caspase-1, IL-1β and IL-18 antibody, respectively. G through I: The inflammasome markers were detected by an indirect immunostaining in NRK-52E cells. NRK-52E cells were treated without (left column) or with 5 mg/ml BSA (right column) for 24 hours in serum-free medium. G: Immunostaining of caspase-1; H: Immunostaining of IL-1β; I: Immunostaining of IL-18.</p

The inflammasome markers were activated in the renal tubular epithelia of diabetic nephropathy in the renal biopsies.

<p>A. Representative micrographs by immunofluorescence staining of caspase-1 (red) and collagen IV (green) in nondiabetic patients and diabetic patients, demonstrating the predominance of tubular staining for active caspase-1 in diabetic renal biopsies. B. Representative micrographs by immunofluorescence staining of IL-1β (red) and collagen IV (green). C. Representative micrographs by immunohistochemical staining of IL-18. D. The histogram demonstrated the relative abundance of the semiquantitative histomorphometric analysis of the immunostaining of caspase-1, IL-1β, and IL-18 (n = 5). *<i>P</i><0.05 vs. the patients with mild mesangium proliferative glomerulonephritis.</p

Bovine serum albumin induced NLRP3-inflammasome activation in NRK-52E cells.

<p>A. Western blot analysis showed the expression of NLRP3 and ASC proteins in NRK-52E cells after treatment without or with 5 mg/ml BSA for various time periods in serum-free medium. The whole cell lysate was immunoblotted with NLRP3 and ASC antibody, respectively. The same blot was reprobed with α-tubulin to confirm equal loading of each lane. B. Western blot analysis shows the expression of NLRP3 and ASC proteins in NRK-52E cells without or with different amounts of BSA for 12 h in serum-free medium. C and D: Graphical presentation shows the relative abundances of NLRP3 and ASC after normalization with α-tubulin. Data are presented as mean±SEM of three independent experiments. *<i>P</i><0.05 vs. normal control (the relative abundance of NLRP3 protein level); # P<0.05 vs. normal control (the relative abundance of ASC protein level).</p

Hypoxia-Responsive ¹⁹F MRI Probes with Improved Redox Properties and Biocompatibility

¹⁹F magnetic resonance imaging (MRI), an emerging modality in biomedical imaging, has shown promise for in vitro and in vivo preclinical studies. Here we present a series of fluorinated Cu­(II)­ATSM derivatives for potential use as ¹⁹F magnetic resonance agents for sensing cellular hypoxia. The synthesized complexes feature a hypoxia-targeting Cu²⁺ coordination core, nine equivalent fluorine atoms connected via a variable-length poly­(ethylene glycol) linker. Introduction of the fluorine moiety maintains the planar coordination geometry of the Cu²⁺ center, while the linker length modulates the Cu^2+/+ reduction potential, ¹⁹F NMR relaxation properties, and lipophilicity. In particular, the ¹⁹F NMR relaxation properties were quantitatively evaluated by the Solomon–Bloembergen model, revealing a regular pattern of relaxation enhancement tuned by the distance between Cu²⁺ and F atoms. Finally, the potential utility of these complexes for sensing reductive environments was demonstrated using both ¹⁹F MR phantom imaging and ¹⁹F NMR, including experiments in intact live cells