1. Progranulin protects against renal ischemia/reperfusion injury in mice
Meng Zhou, Wei Tang, Yi Fu, Xiaoying Xu, Ziying Wang, Yi Lu, Feng Liu, Xinying Yang, Xinbing Wei, Yan Zhang, Juan Liu, Xue Geng, Chun Zhang, Qiang Wan, Ningjun Li, Fan Yi 
Kidney International 
Volume 87, Issue 5, Pages (May 2015)
DOI: /ki
Copyright © 2015 International Society of Nephrology Terms and Conditions

2. Figure 1
Progranulin was significantly reduced in the kidney from a mouse model of renal ischemia/reperfusion (I/R) injury. (a) Relative mRNA levels of progranulin (PGRN) in the kidney after renal ischemia/reperfusion. (b) Representative western blot gel documents and summarized data showing the protein levels of progranulin in the kidney after renal ischemia/reperfusion. (c) Representative photomicrographs of progranulin immunohistochemical staining in the kidney from wild-type (WT) and granulin−/− mice. Negative control by omission of the corresponding primary antibodies demonstrated no nonspecific staining. (d) Coimmunofluorescence staining for progranulin and tubular segment-specific markers in the kidney after renal ischemia/reperfusion. The following segment-specific tubular markers were used: proximal tubule, aquaporin-1 (AQP1); distal tubule, calbindin D28k; and collecting duct, aquaporin-3 (AQP3). It was found that progranulin was mainly expressed in proximal tubules and distal tubules. *P<0.05 vs. sham-operated WT mice (n=12).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

3. Figure 2
Progranulin deficiency exacerbated renal injury after ischemia/reperfusion. (a) Serum creatinine concentration (SCr) of different groups of mice after renal ischemia/reperfusion. (b) Blood urea nitrogen (BUN) levels of different groups of mice after renal ischemia/reperfusion. (c) Representative micrographs showing the morphology of kidneys from different groups of mice. (d) Quantitative assessment of tubular damage. (e) In situ terminal deoxynucleotidyl transferase–mediated uridine triphosphate nick-end labeling (TUNEL) assays were performed to assess renal cell death. Nuclei were revealed using 4',6-diamidino-2-phenylindole staining. (f) Quantitative assessment of the number of dead cells (numbers per high-power field). *P<0.05 vs. sham-operated wild-type mice, #P<0.05 vs. ischemic wild-type mice at the same experimental conditions (n=8).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

4. Figure 3
Progranulin deficiency increased inflammatory responses in the kidney after ischemia/reperfusion injury. (a) The levels of proinflammatory mediators including interleukin (IL)-1β, interleukin-6, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1 measured by enzyme-linked immunoabsorbent assay. (b) Representative sections of kidney stained for neutrophils from different groups of mice. (c) Representative sections of kidney stained for macrophages from different groups of mice. (d) Data analysis of neutrophil infiltrates in the kidney (numbers/high-power field). (e) Data analysis of macrophage infiltrates in the kidney (numbers/HPF).*P<0.05 vs. sham-operated wild-type mice, #P<0.05 vs. ischemic wild-type mice at the same experimental conditions (n=8).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

5. Figure 4
Progranulin decreased hypoxia-induced inflammatory responses and apoptosis in proximal tubule epithelial (HK-2) cells. (a) Representative western blot gel documents and summarized data showing the protein levels of progranulin in HK-2 cells under chemical anoxia/recovery condition, which was induced by incubating cells in glucose-free medium with antimycin A (AA, 10 μM)/2-deoxyglucose (2-DG, 25 mM) for 90 min (anoxia) to inhibit aerobic and substrate-dependent adenosine triphosphate generation, and then in vitro reperfusion was achieved by incubating cells in glucose-replete complete growth medium for 6, 12, 24, or 48 h (recovery), individually. (b) Representative western blot gel documents and summarized data showing the protein levels of progranulin in HK-2 cells cultured in a hypoxic environment for 2 h (0.1% O2), followed by reoxygenation at different time points (6, 12, 24, or 48 h). (c) Representative western blot gel documents and summarized data showing the protein levels of progranulin in HK-2 cells treated with different CoCl2 concentrations for 12 h. (d) The effect of recombinant human progranulin (rPGRN) on the mRNA levels of proinflammatory mediators in HK-2 cells treated with AA/2-DG (90 min), followed by recovery. (e) Summarized data showing cell apoptosis determined by flow cytometric analysis in HK-2 cells treated with AA/2-DG (90 min), followed by recovery (24 h). (f) Summarized data showing caspase-3 activity in HK-2 cells treated with AA/2-DG (90 min), followed by recovery (24 h). (g) Representative western blot gel documents and summarized data showing the expression levels of Bax and Bcl-2 in HK-2 cells treated with AA/2-DG (90 min), followed by recovery (24 h). *P<0.05 vs. control, #P<0.05 vs. vehicle of AA/2-DG treatment (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

6. Figure 5
Progranulin negatively regulated NOD2-mediated signaling. (a) Representative western blot gel documents and summarized data showing the levels of NOD2 in the kidney from different groups of mice. *P<0.05 vs. sham-operated wild-type mice, #P<0.05 vs. ischemic wild-type mice (n=8). (b) Representative western blot gel documents showing the levels of NOD2 in HK-2 cells with AA/2-DG treatment. (c) Representative western blot gel documents and summarized data showing the levels of phospho-p65 (p-p65), p65, p-IκBα, and IκBα in HK-2 cells with muramyl dipeptide (MDP) treatment. (d) Subcellular location of p65 determined by immunofluorescence microscopy in HK-2 cells with muramyl dipeptide MDP treatment. Nuclei were revealed using 4’,6-diamidino-2 -phenylindole staining. (e) Summarized data showing the effect of recombinant human progranulin on the mRNA levels of proinflammatory mediators in HK-2 cells with MDP treatment. (f) Summarized data showing the effect of recombinant human progranulin on cell apoptosis in HK-2 cells with MDP treatment measured by flow cytometric analysis. *P<0.05 vs. control, #P<0.05 vs. vehicle of MDP treatment (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

7. Figure 6
Both pretreatment with and delayed administration of recombinant human progranulin protected against renal ischemia/reperfusion injury in mice. (a) A representative figure showing the procedure of recombinant human progranulin treatment in this study. (b) Serum creatinine concentration of ischemic wild-type and granulin−/− mice with recombinant human progranulin treatment. (c) Blood urea nitrogen levels of ischemic wild-type and granulin−/− mice with recombinant human progranulin treatment. (d) Representative micrographs showing the morphology of kidneys from different groups of mice and quantitative assessment of tubular damage. *P<0.05 vs. ischemic mice, #P<0.05 vs. ischemic wild-type mice with recombinant human progranulin treatment at the same experimental conditions (n=8).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

8. Figure 7
Progranulin reduced inflammatory responses and NOD2 expression in the kidney after renal ischemia/reperfusion. (a) Representative sections of kidney stained for neutrophils and macrophages in the kidney from ischemic wild-type mice with recombinant human progranulin treatment. (b) Summarized data showing that neutrophil and macrophage accumulation in the kidney from ischemic wild-type mice with recombinant human progranulin treatment. (c) The levels of proinflammatory mediators measured by enzyme-linked immunoabsorbent assay in the kidney from ischemic wild-type mice with recombinant human progranulin treatment. (d) Representative western blot gel documents and summarized data showing the protein levels of NOD2 in the kidney from ischemic wild-type mice with recombinant human progranulin treatment. *P<0.05 versus ischemic WT mice (n=8).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

9. Figure 8
Progranulin also protected against AKI induced by cisplatin. (a) Representative western blot gel documents and summarized data showing the levels of progranulin in the kidney from different groups of mice. (b) Serum creatinine and blood urea nitrogen levels of different groups of mice. (c) Representative micrographs showing the morphology of kidneys and quantitative assessment of tubular damage from different groups of mice. *P<0.05 vs. control group of wild-type mice, #P<0.05 vs. wild-type mice with cisplatin treatment (n=8). (d) Representative western blot gel documents and summarized data showing the levels of progranulin in HK-2 cells with cisplatin treatment. (e) The effect of recombinant human progranulin on the mRNA levels of proinflammatory mediators in HK-2 cells treated with cisplatin. (f) Summarized data showing the effect of recombinant human progranulin on cell apoptosis in HK-2 cells treated with cisplatin. *P<0.05 vs. control, #P<0.05 vs. vehicle of cisplatin treatment (n=8).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions