Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: Diabetes was induced in REDD1 +/+ and REDD1 −/− mice by streptozotocin (STZ) administration. Non-diabetic control mice received vehicle (Veh). A REDD1 protein was evaluated in kidney cortical tissue homogenates by western blotting. Representative blots are shown. Molecular mass in kDa is indicated at right of each blot. B Correlation between fasting blood glucose and urine ACR is shown for REDD1 +/+ mice ( blue ; Pearson r = 0.72; p < 0.0001) and REDD1 −/− mice ( red ; Pearson r = 0.55; p = 0.029). C Ccl2 mRNA expression was quantified in kidney homogenates by qPCR. D CCL2 protein abundance was quantified in kidney homogenates by western blotting. E Il1b mRNA expression was quantified in kidney homogenates by qPCR. F IL-1β protein levels were determined in kidney homogenates by western blotting and quantified by ELISA. Individual data points are plotted with values presented as means ± SD ( n = 4–6). Differences between groups were identified by two-way ANOVA. * p < 0.05 versus Veh; # p < 0.05 versus REDD1 +/+ . n.d., not detected.

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Control, Western Blot, Expressing, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: A , B Diabetes was induced in REDD1 +/+ and REDD1 −/− mice by administration of streptozotocin (STZ). Non-diabetic control mice received vehicle (Veh). A Nuclear isolates were prepared from kidney homogenates. NF-κB and Lamin B were examined in nuclear isolates by western blotting and NF-κB activity was quantified by DNA-binding ELISA. Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. B REDD1 ( red ) and Nephrin ( green ) were visualized in kidneys by immunofluorescence microscopy. White box indicates area shown at increased magnification. Representative micrographs are shown (scale bar 50 μm). C – I Wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol (OC) for 48 h. NF-κB phosphorylation at S536 and REDD1 protein abundance was determined in cell lysates by western blotting ( C ). Nuclear localization of NF-κB p65 ( white arrowheads ) was evaluated by immunofluorescence ( D ). Nuclei were visualized with DAPI (scale bar 25 μm). NF-κB activity was measured in lysates from cells expressing NF-κB firefly luciferase/ Renilla luciferase reporter plasmids by dual luciferase assay ( E ). Relative expression of IL1B and CCL2 mRNA were determined by qPCR ( F ). IL-1β secreted into culture media was determined by ELISA ( G ). Chromatin immunoprecipitation (ChIP)-PCR analysis was carried out in WT and REDD1 KO podocytes to determine binding of p65 NF-κB to the promoter region of the CCL2 gene ( H ). CCL2 protein levels were determined in cell lysates by western blotting ( I ). J NF-κB p65 phosphorylation and NF-κB luciferase reporter activity was evaluated in REDD1 KO cells expressing either an empty vector control (EV) or hemagglutinin (HA)-tagged REDD1. Individual data points are presented as means ± SD ( n = 4–6). Differences between groups were identified by two-way ANOVA. * p < 0.05 versus Veh or NG; # p < 0.05 versus REDD1 +/+ , WT, or EV.

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Control, Western Blot, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Knock-Out, Phospho-proteomics, Quantitative Proteomics, Expressing, Luciferase, Chromatin Immunoprecipitation, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: A , B Differentiated wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol as an osmotic control (OC) for 48 h. Transwell migration assay was used to evaluate chemotaxis in a co-culture model with CIHP-1 and THP-1 macrophages ( A ). Macrophages were stained with crystal violet and cells that migrated across the Transwell were counted ( B ). C Cre-lox recombination was used to achieve conditional podocyte-specific REDD1 knockout (REDD1 PodKO). D – H Diabetes was induced in REDD1 fl/fl and REDD1 PodKO mice by streptozotocin (STZ) administration. Non-diabetic groups were administered a vehicle (Veh) control. All assessments were performed after 16 weeks of diabetes. Urine albumin to creatinine ratio (ACR) was determined (D). Kidney sections from diabetic and non-diabetic mice were immunolabeled for REDD1 ( red ) and the podocyte marker Nephrin ( green ) ( E ). Protein abundance of CCL2 was determined in renal homogenates by western blotting ( F ). Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. Kidney sections were immunolabelled for F4/80 ( red ) and nuclei were counterstained with Hoechst 33342 ( blue ) ( G ). Representative micrographs (scale bar 50 µm) are shown. Immune cell populations of CD11b + F4/80+ macrophages ( H ) and CD86 + M1 macrophages ( I ) were determined by flow cytometry. Individual data points are plotted. Significance was analyzed by two-way ANOVA and pairwise comparisons were made using the Tukey’s test for multiple comparisons. * p < 0.05 versus OC or Veh; #, p < 0.05 versus WT or REDD1 fl/fl .

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Knock-Out, Control, Transwell Migration Assay, Chemotaxis Assay, Co-Culture Assay, Staining, Immunolabeling, Marker, Quantitative Proteomics, Western Blot, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Minocycline, a microglial inhibitor, blocks spinal CCL2-induced heat hyperalgesia and augmentation of glutamatergic transmission in substantia gelatinosa neurons

doi: 10.1186/1742-2094-11-7

Figure Lengend Snippet: Intrathecal administration of CCL2 induces heat hyperalgesia and increases the magnitude of evoked glutamatergic EPSCs of substantia gelatinosa neurons. (A) CCL2 (1 μg) was intrathecally injected into 2-month-old rats. One day after the injection, the hot plate assay was used to measure hind-paw withdrawal latency. As a control, the vehicle was intrathecally injected into rats. Compared to wild-type (WT) rats and rats intrathecally administrated with the vehicle, the latency of hind-paw withdrawal caused by the heat stimulus was significantly reduced in the rats intrathecally injected with CCL2. Note that co-administration of CCR2 antagonist BMS CCR2 22 with CCL2 completely blocked CCL2-induced thermal hyperalgesia. Each bar shows the mean ± standard error for 12 or 20 rats. * P < 0.01 compared to wild-type rats. (B) One day after intrathecally injecting the vehicle or CCL2 (1 μg) into the rats, glutamatergic EPSCs were evoked by a stimulating electrode placed at the dorsal root entry zone and recorded from an outer lamina II neuron in a spinal cord slice prepared from a vehicle- or CCL2-treated rat. Compared to representative EPSCs evoked by various magnitudes of stimulation currents for control rats, the amplitude of representative EPSCs was significantly higher for lamina II neurons of CCL2-treated rats. (C) Slope of input–output curve for evoked EPSCs was significantly increased in substantia gelatinosa neurons of CCL2-injected rats. Each point represents the mean ± standard error for 13 to 15 neurons from seven rats. Holding potential ( V H ) was -60 mV. * P < 0.01 compared to control neurons.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Injection, Control

Journal: Journal of Neuroinflammation

Article Title: Minocycline, a microglial inhibitor, blocks spinal CCL2-induced heat hyperalgesia and augmentation of glutamatergic transmission in substantia gelatinosa neurons

doi: 10.1186/1742-2094-11-7

Figure Lengend Snippet: Intrathecal injection of CCL2 strengthens glutamatergic transmission in lamina II neurons via a presynaptic mechanism. (A) One day after intrathecally injecting vehicle or CCL2 (1 μg) into the rats, spontaneous mEPSCs were recorded from outer lamina II neurons in spinal cord slices. Compared to a control lamina II neuron, the frequency of spontaneous mEPSCs was significantly increased in a lamina II neuron from a CCL2-injected rat. V H = -60 mV. (B) Intrathecal administration of CCL2 (1 μg) greatly increased the frequency of mEPSCs in substantia gelatinosa neurons without affecting the mean amplitude. Each bar shows mean ± standard error for 12 neurons from 6 rats. * P < 0.01 compared to control neurons. Con, control; mEPSC, miniature excitatory postsynaptic current.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Injection, Transmission Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Minocycline, a microglial inhibitor, blocks spinal CCL2-induced heat hyperalgesia and augmentation of glutamatergic transmission in substantia gelatinosa neurons

doi: 10.1186/1742-2094-11-7

Figure Lengend Snippet: Minocycline inhibits CCL2-induced heat hyperalgesia and blocks CCL2-induced enhancement of glutamatergic transmission in substantia gelatinosa neurons. (A) Intrathecal administration of CCL2 (1 μg) decreased hind-paw withdrawal latency and caused heat hyperalgesia. Intrathecal co-injection of minocycline (100 μg) and CCL2 (1 μg) completely blocked the CCL2-induced reduction in the latency of hind-paw withdrawal and thermal hyperalgesia. Each bar shows mean ± standard error for 10 rats. * P < 0.01 compared to control rats. (B) One day after intrathecal administration of CCL2 (1 μg), the frequency of spontaneous mEPSCs was greatly increased a lamina II neuron. Intrathecal co-injection of CCL2 (1 μg) and minocycline (100 μg) inhibited the CCL2-induced increase in the frequency of mEPSCs in a lamina II neuron. V H = -60 mV. (C) After intrathecal co-administration with minocycline, CCL2 did not significantly increase the frequency of mEPSCs in substantia gelatinosa neurons. Each bar shows mean ± standard error for 12 neurons from 6 rats. * P < 0.01 compared to control neurons. # P < 0.01 compared to CCL2-treated neurons. Con, control; mEPSC, miniature excitatory postsynaptic current.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Transmission Assay, Injection, Control

Journal: Journal of Neuroinflammation

Article Title: Minocycline, a microglial inhibitor, blocks spinal CCL2-induced heat hyperalgesia and augmentation of glutamatergic transmission in substantia gelatinosa neurons

doi: 10.1186/1742-2094-11-7

Figure Lengend Snippet: TNF-α antagonist WP9QY blocks CCL2-induced enhancement of excitatory synaptic transmission in substantia gelatinosa neurons and inhibits CCL2-induced heat hyperalgesia. (A) Intrathecal administration of CCL2 increased the frequency of spontaneous mEPSCs in a lamina II neuron. Intrathecal co-injection of WP9QY and CCL2 completely inhibited the CCL2-induced increase in the frequency of mEPSCs in a lamina II neuron. V H = -60 mV. (B) After co-administration with WP9QY, CCL2 failed to increase the frequency of mEPSCs in substantia gelatinosa neurons. Each bar shows mean ± standard error for 12 neurons from 6 rats. * P < 0.01 compared to control neurons. # P < 0.01 compared to CCL2-treated neurons. (C) Intrathecal injection of CCL2 (1 μg) decreased hind-paw withdrawal latency and caused thermal hyperalgesia. Intrathecal co-administration of WP9QY and CCL2 completely inhibited CCL-induced heat hyperalgesia. Each bar represents mean ± standard error for ten rats. * P < 0.01 compared to control rats. Con, control; mEPSC, miniature excitatory postsynaptic current.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Transmission Assay, Injection, Control