1. Volume 85, Issue 6, Pages 1357-1368 (June 2014)
Ablation of proximal tubular suppressor of cytokine signaling 3 enhances tubular cell cycling and modifies macrophage phenotype during acute kidney injury 
Nathan Susnik, Inga Sörensen-Zender, Song Rong, Sibylle von Vietinghoff, Xia Lu, Isabelle Rubera, Michel Tauc, Christine S. Falk, Warren S. Alexander, Anette Melk, Herrmann Haller, Roland Schmitt 
Kidney International 
Volume 85, Issue 6, Pages (June 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Acute kidney injury induces suppressor of cytokine signaling 3 (SOCS-3) expression. (a) Representative images of immunofluorescence stainings for SOCS-3 (red) and proximal tubular marker lotus tetragonolobus lectin (LTL; green) in control kidney and at 72h after ischemia/reperfusion (IR) injury, original magnification × 400. Asterisk marks heavily damaged tubule containing luminal cell debris and arrow indicates proximal tubule with intact brush border. (b) Quantitative PCR (qPCR) for SOCS-3 in kidneys at 48 or 72h after unilateral renal clamping (n=6) or (c) 4-day heavy aristolochic acid nephropathy (AAN) damage (n=5) compared with nondamaged control kidneys. (d) qPCR for SOCS-3 expression in primary tubular epithelial cells (PTECs) after 24h of 25ng/ml mouse recombinant Oncostatin M (OSM) stimulation. Scale bars represent mean values±s.e.m. *P<0.05; **P<0.01; ***P<0.001.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Sglt2-cre mice crossed with floxed suppressor of cytokine signaling 3 (SOCS-3) mice create a conditional knockout (KO) of SOCS-3 in proximal tubules. Staining for β-galactosidase in (a, b) Cre-negative and (c, d) Cre-positive reporter mice, original magnifications × 200 (a, c) and × 400 (b, d). (e) Representative immunoblot for SOCS-3 expression in primary tubular epithelial cells (PTECs) after oncostatin M (OSM) stimulation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type. (f) Immunoblot and densitometric analysis for renal SOCS-3 at 72h after initial ischemia/reperfusion (IR) injury. (g–i) Quantitative PCR (qPCR) for SOCS-3 expression after IR (n=6), heavy (n=6), and mild (n=5) aristolochic acid nephropathy (AAN). Scale bars represent mean values±s.e.m. *P<0.05; ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

4. Figure 3
Knockout of suppressor of cytokine signaling 3 (SOCS-3) in proximal tubules improves renal function after acute kidney injury. (a) Serum creatinine and (b) urea values of SOCS-3sglt2Δ/sglt2Δ mice and wild-type littermates after 20min of bilateral renal clamping; samples were taken on days 1, 3, and 7 after initial injury. Serum creatinine and urea at 4 days after initial injection for (c, d) heavy aristolochic acid nephropathy (AAN; n=6) and 6 days and 10 days after initial injection for (e, f) mild AAN (n=5). Scale bars represent mean values±s.e.m. **P<0.01; ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

5. Figure 4
Suppressor of cytokine signaling 3 (SOCS-3) deficiency leads to an increase in tubular proliferation after acute kidney injury (AKI). Quantification of Ki67-positive tubular cells of 10 non-overlapping fields per mouse in the cortex after (a) ischemia/reperfusion (IR) injury (n=5), (b) heavy aristolochic acid nephropathy (AAN; n=6), and (c) mild AAN (n=5). (d) Representative immunohistochemical stainings of Ki67 after IR, original magnification × 400. KO, knockout; WT, wild type. Quantitative PCR (qPCR) of renal E2F2 after (e) IR injury (n=5) and (f) heavy AAN (n=6). Scale bars represent mean values±s.e.m. *P<0.05; **P<0.01; ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
There is a greater increase of signal transducer and activator of transcription 3 (STAT-3) phosphorylation in tubules of SOCS-3sglt2Δ/sglt2Δ mice after acute injury. Representative immunoblots for tyrosine phosphorylated STAT-3 (P-STAT-3) and phosphorylated p42/44 (extracellular signal–regulated kinase (ERK)-1/2) of total kidney homogenate from (a) heavy aristolochic acid nephropathy (AAN) damage group and (b) ischemia/reperfusion (IR) damage. KO, knockout; WT, wild type. (c) Representative image of immunohistochemistry of tyrosine phosphorylated STAT-3 after heavy AAN injury, original magnification × 400. Arrows indicate positive staining for phosphorylated STAT-3 in the nucleus. (d) Proliferation measured by 5-bromodeoxyuridine (BrdU) uptake enzyme-linked immunosorbent assay (ELISA) in mouse proximal tubular (mPT) cells 40h after transfection with green fluorescent protein (GFP) or suppressor of cytokine signaling 3 (SOCS-3) plasmids and treatment for 16h with 50μM with Janus kinase (JAK) inhibitor AG490. Scale bars represent mean values±s.e.m.; n=3 independent experiments. **P<0.01; ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

7. Figure 6
Proximal tubular deficiency of suppressor of cytokine signaling 3 (SOCS-3) leads to an increase in leukocyte infiltration after acute injury. Representative immunofluorescence stainings for (a) CD45 and (b) F4/80 after acute kidney injury; original magnification × 400. DAPI, 4',6-diamidino-2-phenylindole; KO, knockout; WT, wild type. Quantifications of 10 non-overlapping fields per mouse for (c) CD45-positive cells after heavy aristolochic acid nephropathy (AAN; n=6), (d) CD45-positive cells, (e) F4/80-positive cell after ischemia/reperfusion (IR) injury (n=5), and (f) F4/80-positive cells after mild AAN (n=5). Scale bars represent mean values±s.e.m. *P<0.05; **P<0.01.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

8. Figure 7
Proximal tubular expression of suppressor of cytokine signaling 3 (SOCS-3) leads to a greater number of intrarenal classically activated macrophages. (a) Representative images of double staining for arginase I (Arg1; green) and F4/80 in wild-type and SOCS-3sglt2Δ/sglt2Δ mice; original magnification × 400. DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (b) Quantification of arginase I+ F4/80+ cells during acute kidney injury (AKI) in 8 non-overlapping fields per mouse (n=6 ischemia/reperfusion (IR), 9 wild-type (WT), 5 knockout (KO) in heavy aristolochic acid nephropathy (AAN), and n=6 mild AAN). (c) Representative immunoblot and densitometry for renal arginase I protein levels after heavy AAN. (d) Representative histograms of mean fluorescence intensity (MFI) of flow cytometry analysis of CD11b, F4/80, and major histocompatibility complex class II (MHC-II) and average MFI of MHC-II on CD11b+ F4/80+ cells in SOCS-3sglt2Δ/sglt2Δ mice and wild-type counterparts after heavy AAN, n=9 WT, n=5 KO. (e) Representative populations of flow cytometry analysis of CD11b+ F4/80+ MHC-II+ cells based on F4/80 and MHC-II signal intensity and average populations in SOCS-3sglt2Δ/sglt2Δ mice and wild-type counterparts after heavy AAN, n=9 WT, n=5 KO. (f) Quantitative PCR (qPCR) for renal inducible nitric oxide synthase (iNOS), Ym1, Fizz1, LIGHT, and Sphk1 after heavy AAN, n=14 WT, n=11 KO. (g) qPCR for renal interleukin (IL)-6, IL-10, tumor necrosis factor-α (TNF-α), and IL-12p40 after heavy AAN, n=14, WT n=11 KO. Scale bars represent mean values±s.e.m. *P<0.05; ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

9. Figure 8
Overexpression of suppressor of cytokine signaling 3 (SOCS-3) in tubular epithelial cells induces classically activated macrophages in coculture. (a) Diagram of experimental procedure in which primary tubular epithelial cells (PTECs) were cultured for 5 days before transfection with SOCS-3 or green fluorescent protein (GFP)–expressing plasmids. After 24h, medium was changed, PTECs were washed with phosphate-buffered saline (PBS), and elicited peritoneal macrophages were added. Cells were cocultured for 48h and then lysed for mRNA or protein, or fixed for staining. (b) Quantitative PCR (qPCR) for inducible nitric oxide synthase (iNOS), Ym1, Fizz1, LIGHT, and Sphk1 in transfected PTECs and PTEC/macrophage cocultures. (c) Representative immunoblot for arginase I in transfected PTECs and PTEC/macrophage coculture. (d) Representative immunostaining for iNOS (green) and F4/80 (red) in PTEC/macrophage coculture, original magnification × 400. Arrows indicate iNOS+ F4/80+ cells. DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (e) Quantification of 8 non-overlapping fields per sample of iNOS+ F4/80+ cells in PTEC/macrophage coculture. (f) qPCR for iNOS in transfected PTECs and PTEC/macrophage cocultures. N, PTECs from six separate mice, elicited peritoneal macrophages from six separate mice. Scale bars represent mean values±s.e.m. *P<0.05; ***P<0.01.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions