Journal: Regulatory peptides

Article Title: Increased circulating angiotensin-(1-7) protects white adipose tissue against development of a proinflammatory state stimulated by a high-fat diet.

doi: 10.1016/j.regpep.2012.06.009

Figure Lengend Snippet: Fig. 3. Adipose tissue inflammatory markers. Western blot for COX2-epididymal adipose tissue (A), RT-PCR expression of IL-1β‐epididymal adipose tissue (B), SD immunohisto- chemical reaction for IL-1β in epididymal adipose tissue (C), TGR immunohistochemical reaction for IL-1β in epididymal adipose tissue (D), RT-PCR expression of TNF-α‐ epididymal adipose tissue (E), SD immunohistochemical reaction for TNF-α in epididymal adipose tissue (F), TGR immunohistochemical reaction for TNF-α in epididymal adipose tissue (G). Data are presented as mean±SEM; * pb0.05.

Article Snippet: The blot was probed with COX2 enzyme antibody (Cell Signaling, USA; 1:1000), diluted in 1× blocking buffer, washed in 1× TBS with 1% of Tween 20, and then incubated with either IRDye 800 anti-goat secondary antibody (Rockland Immunochemicals®, Gilbertsville, Pa.) diluted to 1:10,000 in blocking buffer followed by further washes with PBS-Tween 20 and one wash in TBS.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemical staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease

doi: 10.1152/ajprenal.00614.2016

Figure Lengend Snippet: Effectiveness of Cre-mediated recombination in NPHS2-Cre mice. To examine Cre-medicated recombination in vivo, NPHS2-Cre mice were crossed with a transgenic (TG) reporter animal expressing a two-color fluorescent Cre reporter allele (32). The reporter allele contains a cell membrane-localized red fluorescence protein (tdTomato) with widespread expression in all tissues and cell types prior to Cre recombinase exposure. Expression of Cre recombinase induces a cell membrane-localized enhanced green fluorescence protein (EGFP). A: in TG mice expressing both transgenes, green fluorescence is induced specifically in podocytes with red fluorescence confined to other cell types. B: little EGFP expression was observed in mice expressing only the reporter construct. C: we next determined the effect of podocyte-specific knockout (KO) on COX2 mRNA expression using quantitative RT-PCR. The presence of diabetes significantly affected glomerular COX2 mRNA levels. The increase in glomerular COX2 mRNA in wild-type (WT) Akita mice was largely prevented by KO of podocyte COX2 in diabetic animals. D: representative glomerular images stained for COX2 (green) and the podocyte marker synaptopodin (SYN) (red) and examined by confocal microscopy. In nondiabetic COX2+/+ and COX2−/− mice, COX2 staining was faint and largely confined to mesangial areas. In WT Akita mice, COX2 staining was granular and increased in both mesangial areas and podocytes compared to nondiabetic mice. SYN was also granular in both groups of Akita mice, as we have seen in another model of podocyte injury (45). As shown in the merged images, podocyte COX2 expression (yellow) was decreased by ~70%, by KO of COX2 in Akita mice, although focal areas of COX2 expression were still detected in KO Akita amice (arrow). Mesangial COX2 expression was not affected KO of COX2 in podocytes of diabetic mice. For the immunofluorescence studies, three to six mice were examined per group. For the quantitative RT-PCR experiments, we studied 11 KO Akita, 10 WT Akita, 5 COX2+/+, and 5 COX2−/− mice. Statistical analyses were performed using a two-way ANOVA followed by a Bonferroni post-hoc analyses. *P < 0.05 vs nondiabetic mice and KO Akita mice.

Article Snippet: Expression of COX2 in podocytes was detected by indirect immunofluorescence using a rabbit monoclonal antibody to COX2 from Cell Signaling Technology (cat. no. 12282) and a mouse monoclonal antibody to the podocyte marker synaptopodin (Progen Biotechnik).

Techniques: In Vivo, Transgenic Assay, Expressing, Fluorescence, Construct, Knock-Out, Quantitative RT-PCR, Staining, Marker, Confocal Microscopy, Immunofluorescence

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease

doi: 10.1152/ajprenal.00614.2016

Figure Lengend Snippet: Effect of podocyte-specific COX2 KO on kidney histopathology. A: kidney weights were similar in nondiabetic COX2+/+ mice and nondiabetic COX2−/−POD mice. B: minimal mesangial expansion was observed in either group of nondiabetic mice. C: tubule dilation and casts were similar in nondiabetic COX2+/+ mice and nondiabetic COX2−/−POD mice. D and E: representative low-power views of kidneys from nondiabetic COX2+/+ mice and nondiabetic COX2−/−POD mice. F and G: albuminuria was similar in both groups of nondiabetic mice at 12- and 20-wk of age. For the histologic studies, kidneys from 10 COX2+/+ and 8 COX2−/− mice were examined. Statistical analyses of histologic data were performed using a Fishers Exact test. Statistical analyses of the albuminuria data were performed using an ANOVA followed by a Bonferroni post hoc analysis.

Article Snippet: Expression of COX2 in podocytes was detected by indirect immunofluorescence using a rabbit monoclonal antibody to COX2 from Cell Signaling Technology (cat. no. 12282) and a mouse monoclonal antibody to the podocyte marker synaptopodin (Progen Biotechnik).

Techniques: Histopathology

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease

doi: 10.1152/ajprenal.00614.2016

Figure Lengend Snippet: Glomerular ultrastructure in nondiabetic mice. A and B: examination of kidneys by transmission electron microscopy (TEM) revealed no qualitative differences in glomerular ultrastructure in nondiabetic COX2+/+ mice or nondiabetic COX2−/−POD mice. C: number of filtration slits per micrometer (µm) of glomerular basement membrane (GBM) was similar in both groups. For the studies, two mice were studied in each group. Statistical analysis was performed using an unpaired t-test.

Article Snippet: Expression of COX2 in podocytes was detected by indirect immunofluorescence using a rabbit monoclonal antibody to COX2 from Cell Signaling Technology (cat. no. 12282) and a mouse monoclonal antibody to the podocyte marker synaptopodin (Progen Biotechnik).

Techniques: Transmission Assay, Electron Microscopy, Filtration

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease

doi: 10.1152/ajprenal.00614.2016

Figure Lengend Snippet: Effect of COX2 KO on SBP and cystatin C levels

Article Snippet: Expression of COX2 in podocytes was detected by indirect immunofluorescence using a rabbit monoclonal antibody to COX2 from Cell Signaling Technology (cat. no. 12282) and a mouse monoclonal antibody to the podocyte marker synaptopodin (Progen Biotechnik).

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease

doi: 10.1152/ajprenal.00614.2016

Figure Lengend Snippet: Effect of podocyte-specific COX2 KO on albuminuria and kidney histopathology in diabetic mice. A: as an index of glomerular albuminuria, urinary protein samples were separated by protein electrophoresis, and the gels were stained with Coomassie Brilliant Blue R-250, as described in materials and methods. Prominent bands of the molecular weight of albumin (~66 kDa) were detected in diabetic mice, but these bands were difficult to detect in nondiabetic controls. B and C: both diabetes and genotype (COX2+/+ or COX2−/−) significantly influenced the albuminuria results. Albuminuria was increased in both groups of diabetic mice compared to controls at 16 and 20 wk of age. Podocyte-specific KO of COX2 further enhanced albuminuria at the 16- and 20-wk time points. D–G: mesangial expansion was significantly increased in diabetic WT and KO Akita mice compared to nondiabetic animals, but the extent of mesangial expansion was similar in both groups of Akita mice. H: tubule dilation and casts were observed only in diabetic mice, but there were no significant differences between groups. For the histologic studies, kidneys from seven nondiabetic controls, 14 WT Akita mice, and seven KO Akita mice were examined in each group. For the albuminuria studies, seven nondiabetic controls, 12 WT Akita mice and 8 KO Akita mice were studied in each group. Statistical analysis of the albuminuria data was performed using a two-way ANOVA followed by a Bonferroni post hoc analysis. Categorical data were analyzed using a Fishers exact test. *P < 0.05 or †P < 0.01 vs. nondiabetic mice. ‡P < 0.05 or §P < 0.01 vs. WT Akita mice.

Article Snippet: Expression of COX2 in podocytes was detected by indirect immunofluorescence using a rabbit monoclonal antibody to COX2 from Cell Signaling Technology (cat. no. 12282) and a mouse monoclonal antibody to the podocyte marker synaptopodin (Progen Biotechnik).

Techniques: Histopathology, Protein Electrophoresis, Staining, Molecular Weight

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease

doi: 10.1152/ajprenal.00614.2016

Figure Lengend Snippet: Effect of COX2 KO on glomerular structure in Akita mice

Article Snippet: Expression of COX2 in podocytes was detected by indirect immunofluorescence using a rabbit monoclonal antibody to COX2 from Cell Signaling Technology (cat. no. 12282) and a mouse monoclonal antibody to the podocyte marker synaptopodin (Progen Biotechnik).

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease

doi: 10.1152/ajprenal.00614.2016

Figure Lengend Snippet: Effect of podocyte-specific COX2 KO on urinary eicosanoid excretion and glomerular renin expression. A and B: presence of diabetes significantly enhanced urinary eicosanoid excretion. Both urinary PGE2 metabolites and the urinary thromboxane 2 (TxA2) metabolite TxB2 were increased in both groups of diabetic mice compared to nondiabetic controls. This increase in urinary eicosanoid excretion was statistically significant for PGE2 in WT Akita mice compared to nondiabetic animals. C and D: similarly, the presence of diabetes significantly reduced expression of renin mRNA. Relative expression of renin mRNA was decreased in both groups of diabetic mice compared to nondiabetic controls. For the urinary eicosanoid studies, eight nondiabetic controls, 15 WT Akita, and 11 KO Akita mice were studied. For the renin mRNA studies, eight mice were studied in each group. For the immunoblotting studies, eight nondiabetic, six WT Akita, and six KO Akita mice were studied. For the quantitative RT-PCR studies, eight mice were examined in each group. Statistical analyses were performed using a two-way ANOVA followed by a Bonferroni post hoc analysis. *P < 0.05 or †P < 0.01 vs. nondiabetic mice.

Article Snippet: Expression of COX2 in podocytes was detected by indirect immunofluorescence using a rabbit monoclonal antibody to COX2 from Cell Signaling Technology (cat. no. 12282) and a mouse monoclonal antibody to the podocyte marker synaptopodin (Progen Biotechnik).

Techniques: Expressing, Western Blot, Quantitative RT-PCR