1. Volume 85, Issue 1, Pages 82-93 (January 2014)
TIMP2 and TIMP3 have divergent roles in early renal tubulointerstitial injury 
Zuocheng Wang, Konrad Famulski, Jiwon Lee, Subhash K. Das, Xiuhua Wang, Philip Halloran, Gavin Y. Oudit, Zamaneh Kassiri 
Kidney International 
Volume 85, Issue 1, Pages (January 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Loss of tissue inhibitor of metalloproteinase-2 (TIMP2) or TIMP3 differentially alters gene expression in kidneys at 3 days after unilateral urethral obstruction (UUO). (a) Hierarchical clustering of interquartile range (IQR)–filtered transcripts showing global changes in gene expression. Similarity measure was the Euclidean distance and clustering algorithm was average linkage, as implemented in GeneSpring 7.3. Branch lengths represent the degree of similarity; blue, red, and green tiles indicate no changes, increase, and decrease in expression, respectively. UUO samples were normalized to their sham controls. (b) Total number of genes unique to TIMP2−/− and TIMP3−/− or overlapping with wild-type (WT) in sham (top) or 3 days after UUO (bottom). Averaged expression of (c) intermediate and late injury- and repair-induced transcripts (IRITD3 and IRITD5), and (d) kidney selective transcript (KT1) and solute carrier genes (KT2) in WT, TIMP2−/−, and TIMP3−/− kidneys after sham or UUO. n=8/genotype/group. *P<0.05 compared with corresponding sham; #P<0.05 compared with TIMP2−/− UUO. PBT, pathogenesis-based transcript set.
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Copyright © 2014 International Society of Nephrology Terms and Conditions

3. Figure 2
Loss of tissue inhibitor of metalloproteinase-2 (TIMP2) or TIMP3 affects early gene expression in kidneys at 3 days after unilateral urethral obstruction (UUO). Expression of (a) primary macrophage–associated transcripts (PMATs) and early injury- and repair-induced transcripts (IRITD1), and (b) cytotoxic T cell–associated transcripts (CAT1, formerly CAT) and interferon-γ and rejection-induced transcripts reflecting IFNG effect (GRIT1, formerly tGRIT) in wild-type (WT), TIMP2−/−, and TIMP3−/− kidneys after sham or UUO. n=8/genotype/group. *P<0.05 compared with the corresponding sham group. #P<0.05 compared with WT UUO. IFNG, interferon gamma; PBT, pathogenesis-based transcript set.
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4. Figure 3
Loss of tissue inhibitor of metalloproteinase-3 (TIMP3), but not of TIMP2, activates tumor necrosis factor-α (TNFα)–converting enzyme (TACE), caspase-3, and mitogen-activated protein kinase (MAPK) signaling pathways at 3 days after unilateral urethral obstruction (UUO). (a) Representative western blots and averaged protein content of phosphorylated (p-TACE) and total TACE in the membrane protein fraction. Na+/K+-ATPase was used as loading control for the membrane fraction. The quality of membrane fractionation can be further referred to in Supplementary Figure S1c online. (b) Representative western blots and averaged protein content of cleaved and total caspase-3. (c) Representative TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining. Arrows indicate apoptotic cells. Scale bar=50μm. (d) Representative western blots and averaged protein content of phospho- and total levels of extracellular signal–regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK1/2) in the kidneys of indicated groups after sham or UUO. n=5/genotype/group; *P<0.05 versus corresponding sham; #P<0.05 versus WT and TIMP2−/− UUO groups. AU, arbitrary unit.
Kidney International  , 82-93DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

5. Figure 4
Histological analysis and second harmonic generation (SHG) imaging showing increased tubulointerstitial fibrosis and disrupted extracellular matrix in tissue inhibitor of metalloproteinase-3 (TIMP3)–deficient kidneys after unilateral urethral obstruction (UUO). (a) Light microscopy of periodic acid–Schiff (PAS)–stained, and fluorescent microscopy of picrosirius red (PSR)–stained, formalin-fixed kidney sections after 3 days of UUO. Scale bar=50μm. (b) SHG and multiphoton excitation fluorescence (MPEF) imaging in unfixed and unstained kidneys showing the fibrillar collagen network (magenta) and tubular endogenous autofluorescence (green) in wild-type (WT) and TIMP3−/− kidneys at 3 days after UUO. Scale bar=80μm.
Kidney International  , 82-93DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
Differential impact of tissue inhibitor of metalloproteinase-3 (TIMP3) versus TIMP2 loss on mRNA expression of profibrotic mediators, and on matrix metalloproteinase-2 (MMP2) activation. mRNA expression levels of (a) collagen I and III, (b) connective tissue growth factor (CTGF), and transforming growth factor-β (TGFβ) by TaqMan real-time PCR in wild-type (WT), TIMP2−/−, and TIMP3−/− kidneys 3 days after sham or unilateral urethral obstruction (UUO). (c) Representative gelatin zymography (i) and averaged band intensities for MMP9 (90kDa) (ii), pro-MMP2 (72kDa) (iii), and cleaved MMP2 (64kDa) (iv), in the indicated groups. +ve Ctrl, positive control. Coomassie blue–stained gel is shown as the loading control. n=8/genotype/group; *P<0.05 versus corresponding sham; #P<0.05 versus WT-UUO group. AU, arbitrary unit.
Kidney International  , 82-93DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

7. Figure 6
Loss of tissue inhibitor of metalloproteinase-2 (TIMP2) suppressed the activation of the transforming growth factor-β (TGFβ) signaling pathway after unilateral urethral obstruction (UUO). (a) Representative western blots for (i) latent TGFβ (45kDa), mature TGFβ (25kDa), phosphorylated and total Smad2/3 (60/52kDa), and averaged protein content for (ii) latent TGFβ, (iii) mature TGFβ, and (iv) p-Smad-to-total Smad ratio in the indicated groups at 3 days after sham or UUO. (b) Representative western blots on nuclear protein fraction showing Smad4 levels. Averaged nuclear Smad4 level in the indicated group is shown in the bar graph. A nucleus-specific protein (Histone H3) and a cytosol-specific protein (caspase-3) were used to demonstrate protein fractionation efficacy. The quality of nuclear fractionation can be further referred to in Supplementary Figure S1c online. *P<0.05 versus corresponding sham; #P<0.05 versus WT-UUO. AU, arbitrary unit; WT, wild type. n=6/genotype/group.
Kidney International  , 82-93DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

8. Figure 7
Tissue inhibitor of metalloproteinase-2 (TIMP2) deficiency attenuates tubulointerstitial fibrosis after unilateral urethral obstruction (UUO). (a) Picrosirius red (PSR)–stained sections visualized by fluorescent microscopy, and averaged collagen volume fraction in TIMP2−/− kidneys at 14 days after UUO (14d-UUO) compared with parallel sham and wild-type (WT) group. Scale bar=50μm. (b) Trichrome-stained and light microscopy of WT and TIMP2−/− kidneys at 14 days after sham or UUO. Scale bar=100μm. (c) Representative western blot and averaged protein content of collagen I and collagen III in TIMP2−/−-UUO compared with WT-UUO kidneys at 14 days after UUO. n=5/genotype/group. *P<0.05 versus corresponding sham; #P<0.05 versus WT-UUO.
Kidney International  , 82-93DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions