Volume 83, Issue 4, Pages 647-661 (April 2013) Endogenous and exogenous pentraxin-3 limits postischemic acute and chronic kidney injury  Maciej Lech, Christoph Römmele, Regina Gröbmayr, Heni Eka Susanti, Onkar P. Kulkarni, Shijun Wang, Hermann-Josef Gröne, Bernd Uhl, Christoph Reichel, Fritz Krombach, Cecilia Garlanda, Alberto Mantovani, Hans-Joachim Anders  Kidney International  Volume 83, Issue 4, Pages 647-661 (April 2013) DOI: 10.1038/ki.2012.463 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Oxidative stress induces renal pentraxin 3 (PTX3) expression. Wild-type mice underwent unilateral or bilateral renal pedicle clamping. (a) Serum samples were obtained at different time points for PTX3 enzyme-linked immunosorbent assay (ELISA). Ptx3-deficient mice served as a negative control for ELISA specificity. Data are means±s.e.m. from 5 mice in each group. *P<0.05, **P<0.01. (b) Kidney protein isolates were prepared for PTX3 ELISA. Note that as early as 24h after surgery PTX3 protein expression was increased in the postischemic (IR) kidney as compared with the sham control kidney (Co). Ptx3-deficient mice served as a negative control for ELISA specificity. Data are means±s.e.m. from five mice in each group. *P<0.05, **P<0.01. (c) Wild-type mice underwent unilateral or bilateral renal pedicle clamping. Renal-cell suspensions were prepared from healthy, sham control, or postischemic kidneys at 24h, and magnetic bead anaylsis was used to separate CD45/CD11c+ renal myeloid cells from the rest, mostly renal nonimmune cells. Note that PTX3 mRNA was mostly induced in renal CD45/CD11c+ cells of postischemic kidneys. This effect was absent when mice had been pretreated with antioxidants. Data are means±s.e.m. from five mice in each group. P<0.05 vs. sham. (d) Renal parenchymal cells, CD11b+, or CD11c+ primary cells were exposed to 20ng/ml tumor necrosis factor (TNF)-α or 2.5mmol/l hypoxanthine and 0.005U/ml xanthine oxidase; Ptx3 mRNA expression was determined after 6h of stimulation. Data are mean ratios of the PTX3 mRNA vs. the respective 18s rRNA level±s.e.m. from three independent experiments. (e) CD11b+, primary tubular epithelial cells, or CD11c+ cells were cultured in medium +/- 20ng/ml TNF-α for different time points as indicated. Data are means±s.e.m. from three identical experiments. P<0.05 vs. medium. (f) Renal tissue was obtained at 5 days after unilateral renal pedicle clamping and stained for 4′6-diamidino-2-phenylindole (DAPI; blue), PTX3 (green), and F4/80+ macrophages (red). Representative merge images are shown for both genotypes and each time point at an original magnification of × 400. Bar=200μm. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Lack of pentraxin 3 (PTX3) and postischemic tubular necrosis. (a) Renal tissue was obtained at 1, 5, and 10 days after unilateral renal pedicle clamping and was stained for periodic acid–Schiff (original magnification × 100). The tubular injury score was determined in postischemic (IR) and sham-operated contralateral kidneys (Co) as described in Materials and Methods. *P<0.05 vs. wild-type mice. Staining with tetragonolobus lectin identified (b) proximal tubules and (c) immunostaining for Tamm–Horsfall protein distal tubules as illustrated in representative images at a magnification of × 100. The quantitative analysis was performed using image software as described in Materials and Methods, and is expressed as mean±s.e.m. of positivity per high-power field (h.p.f.). ***P<0.001 vs. wild-type mice. Bar=200μm. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Lack of pentraxin 3 (PTX3) and leukocyte recruitment to postischemic kidneys. (a) Renal tissue was obtained at 1, 5, and 10 days after unilateral renal pedicle clamping and stained for (a) neutrophils or (b) F4/80+ macrophages. Representative images are shown for both genotypes and each time point at an original magnification of × 100. The quantitative analyses were performed using image software as described in Materials and Methods, and are expressed as means±s.e.m. of positivity per high-power field (h.p.f.). *P<0.05, **P<0.01 vs. wild-type mice of the same time point. Bar=200μm. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Renal cytokine and chemokine mRNA expression after IR. Total RNA was extracted from ischemic (IR) and contralateral (Co) kidneys of Ptx3-deficient (white bars) or wild-type mice (black bars) at different time intervals after IR as indicated. mRNA expression levels were determined for the indicated targets by real-time RT-PCR. Data are expressed as mean of the ratio vs. the respective 18s rRNA level±s.e.m.; *P<0.05 vs. wild-type kidneys of the same time point. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Renal selectin, Toll-like receptor, and macrophage marker mRNA expression after ischemia–reperfusion. (a) Total RNA was extracted from ischemic (IR) and contralateral (Co) kidneys of Ptx3-deficient (white bars) or wild-type mice (black bars) at different time intervals after IR as indicated. (b) mRNA expression levels were determined for the indicated targets by real-time RT-PCR. Data are expressed as mean of the ratio vs. the respective 18s rRNA level±s.e.m.; *P<0.05, **P<0.01 vs. wild-type kidneys of the same time point. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Lack of pentraxin 3 (PTX3) and bilateral ischemia–reperfusion (IR) injury. (a) Serum creatinine levels were determined 24h after bilateral renal pedicle clamping in Ptx3-deficient and wild-type mice. In addition, Ptx3-deficient mice were treated either with anti-P-selectin or control IgG immediately after renal reperfusion. (b) Total RNA was extracted from ischemic kidneys of Ptx3-deficient mice treated either with anti-selectin P or isotype control IgG at 24h after reperfusion. Tumor necrosis factor (TNF), IL-6, and P-selectin mRNA expression levels were determined for the indicated targets by real-time RT-PCR. Data are expressed as mean of the ratio vs. the respective 18s rRNA level±s.e.m.; *P<0.05 vs. isotype control. (c) Renal tissue was obtained and periodic acid–Schiff-stained sections were assessed for tubular injury as described in Materials and Methods. (d) Neutrophil immunostaining was quantified by digital image analysis as described in Materials and Methods. Data are expressed as means±s.e.m. of positivity per high-power field (h.p.f.). *P<0.05 vs. Ptx3-deficient mice treated with isotype control IgG. Representative images are shown at an original magnification of × 100. Iso.Ab., isotype antibody; SeIP.Ab., P-selectin antibody. Bar=200μm. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 In vivo microscopy from postischemic cremaster muscles of wild-type and Ptx3-deficient mice.In vivo microscopy was performed on cremaster muscle postcapillary venules of wild-type and Ptx3-deficient mice as described in Materials and Methods. Six mice in each group underwent scrotal ischemia–reperfusion, and recordings of (a) leukocyte rolling, (b) firm adhesion, and (c) transendothelial migration were taken after 60 and 120min, respectively. Data are means±s.e.m. *P<0.05 vs. wild-type control of the same time point. (d) Microvascular fluorescein isothiocyanate (FITC)-dextran leakage was determined 120min after injection. Data are means±s.e.m. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Treatment of postischemic acute kidney injury (AKI) with recombinant pentraxin 3 (PTX3). Wild-type mice underwent bilateral renal pedicle clamping as described in Materials and Methods. Groups of mice (n=5) received vehicle (phosphate-buffered saline, PBS) or 100μg of recombinant PTX3 as a single intravenous injection immediately after reperfusion or PTX3 was injected either 6 or 12h after reperfusion. Serum and kidney samples were harvested from all mice 24h after reperfusion. (a) Serum creatinine levels, (b) tubular injury score, and (c) interstitial neutrophil infiltrates were quantified as described in Materials and Methods. Data are means±s.e.m. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle treatment. Representative images are shown at an original magnification of × 100. Bar=200μm. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 9 Lack of pentraxin 3 (PTX3) and postischemic chronic kidney injury. (a) Kidneys were obtained at 10 weeks after unilateral renal pedicle clamping; loss of weight of ischemic kidney (IR) compared with contralateral kidney (Co) was presented as delta IR-Co kidney. (b) Total RNA was extracted from ischemic (IR) and contralateral (Co) kidneys of Ptx3-deficient (white bars) or wild-type mice (black bars) at 10 weeks after IR as indicated. mRNA expression levels were determined for the indicated targets by real-time RT-PCR. Data are expressed as mean of the ratio vs. the respective 18s rRNA level±s.e.m.; *P<0.05 vs. wild-type kidneys of the same time point. (c) Staining with tetragonolobus lectin identified proximal tubules, and (d) immunostaining for SMA was used as fibrosis marker, as illustrated in representative images at a magnification of × 100. The quantitative analysis was performed using image software as described in Materials and Methods, and is expressed as mean±s.e.m. of positivity per high-power field (h.p.f.). **P<0.01 vs. wild-type mice. Bar=200μm. Kidney International 2013 83, 647-661DOI: (10.1038/ki.2012.463) Copyright © 2013 International Society of Nephrology Terms and Conditions