Volume 81, Issue 4, Pages 370-378 (February 2012) Resveratrol improves renal microcirculation, protects the tubular epithelium, and prolongs survival in a mouse model of sepsis-induced acute kidney injury  Joseph H. Holthoff, Zhen Wang, Kathryn A. Seely, Neriman Gokden, Philip R. Mayeux  Kidney International  Volume 81, Issue 4, Pages 370-378 (February 2012) DOI: 10.1038/ki.2011.347 Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 1 Acute effects of resveratrol (RES) on renal cortical microcirculation at 6h. Cecal ligation and puncture (CLP) surgery caused a significant reduction in the categorical perfusion (a) and mean red blood cell (RBC) velocity (b) of the peritubular capillaries at 6h. Resveratrol administration at 5.5h acutely improved both categorical perfusion and mean RBC velocity measured at 6h, with 10mg/kg (intraperitoneal, i.p.) being the most efficacious dose tested to restore RBC velocity. *P<0.05 compared with sham+vehicle; #P<0.05 compared with sham+vehicle and CLP+vehicle; n=6–7. Kidney International 2012 81, 370-378DOI: (10.1038/ki.2011.347) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 2 Acute effects of resveratrol (RES) on mean arterial pressure (MAP) and heart rate (HR) at 6h. Cecal ligation and puncture (CLP) surgery resulted in a significant decrease in MAP (a) and HR (b) at 6h. Resveratrol at any of the tested doses had no significant (NS) effect on MAP or HR in CLP mice. However, RES (30mg/kg, intraperitoneal, i.p.) slightly increased HR in sham. *P<0.05 compared with sham by analysis of variance; #P<0.05 compared with sham by Student's t-test; n=3–7. Kidney International 2012 81, 370-378DOI: (10.1038/ki.2011.347) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 3 Acute effects of resveratrol (RES) on renal blood flow (RBF) and glomerular filtration rate (GFR) at 6h. Cecal ligation and puncture (CLP) caused a significant reduction in both RBF (a) and GFR (b) at 6h. Resveratrol (10mg/kg, i.v.) administered at 5.5h restored RBF and improved GFR at 6h. *P<0.05 compared with sham and #P<0.05 compared with sham and CLP; n=7–11 for (a) and n=4–5 for (b). Kidney International 2012 81, 370-378DOI: (10.1038/ki.2011.347) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 4 Effect of delayed resveratrol (RES) administration on renal cortical microcirculation at 18h. At 18h post cecal ligation and puncture (CLP), categorical perfusion (a) and mean red blood cell (RBC) velocity (b) remained low compared with 6h (Figure 1. A single dose of RES (10mg/kg, intraperitoneal, i.p.) administered at 6h did not improve the renal microcirculation at 18h. However, administration of an additional dose of RES (10mg/kg, i.p.) at 12h resulted in a complete restoration of both categorical capillary perfusion (a) and mean RBC velocity (b) at 18h. *P<0.05 compared with sham and #P<0.05 compared with sham and CLP; n=6–7. Kidney International 2012 81, 370-378DOI: (10.1038/ki.2011.347) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 5 Effects of delayed resveratrol (RES) administration on reactive nitrogen species (RNS) generation in the renal tubules at 18h. Representative pseudocolored images of rhodamine fluorescence (active generation during the imaging period) for sham, cecal ligation and puncture (CLP), and CLP+RES (two doses) are shown in (a–c), respectively ( × 200 original magnification). The quantification of rhodamine fluorescence is shown in (d). A single dose of RES at 6h (10mg/kg, i.p.) partially, but significantly, reduced the increase in rhodamine fluorescence caused by CLP, whereas a second dose given at 12h completely blocked rhodamine fluorescence (d). Representative images of immunoreactive nitrotyrosine–protein adducts (cumulative generation of peroxynitrite over 18h) are shown in (e) and (f) ( × 400 original magnification). Preincubation of the anti-nitrotyrosine antibody with 10mmol/l nitrotyrosine was used as the nonspecific binding control (not shown). Tissue from sham mice (e) showed faint specific staining, whereas tissue from CLP mice showed strong specific staining localized to the tubules (f). Tissue from CLP mice treated with two doses of RES showed little specific staining (g) at 18h. *P<0.05 compared with all other groups; n=6–7. DHR, dihydrorhodamine-1,2,3. Kidney International 2012 81, 370-378DOI: (10.1038/ki.2011.347) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 6 Effects of delayed resveratrol (RES) administration on renal morphology at 18h. Representative periodic acid–Schiff-stained sections for sham, cecal ligation and puncture (CLP), and CLP+RES (two doses) are shown in (a–c), respectively ( × 200 original magnification), and tissue injury scores are shown in d. CLP caused mild morphological changes characterized by loss of brush border, cast formation, and vacuolization (indicated by arrows). Administration of two doses of RES significantly reduced the tubular injury score. *P<0.05 compared with sham; n=6–9. Kidney International 2012 81, 370-378DOI: (10.1038/ki.2011.347) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 7 Effects of delayed resveratrol (RES) administration on renal function at 18h. At 18h post cecal ligation and puncture (CLP), mice showed elevated levels of blood urea nitrogen (a) and serum creatinine (b), as well as significantly reduced renal blood flow (RBF) (c) and glomerular filtration rate (GFR) (d). Administration of two doses of RES significantly decreased blood urea nitrogen and serum creatinine levels, while improving RBF and GFR at 18h (a–d). *P<0.05 compared with sham and #P<0.05 compared with sham and CLP; n=4–5 for a and b, n=6–8 for c and d. Kidney International 2012 81, 370-378DOI: (10.1038/ki.2011.347) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 8 Effects of delayed resveratrol (RES) administration on survival. Administration of RES (10mg/kg, i.p) to cecal ligation and puncture (CLP) mice at 6, 12, and 18h resulted in a significant improvement in survival compared with vehicle-treated mice (*P<0.01; Mantel–Cox log-rank test; n=10/group). Kidney International 2012 81, 370-378DOI: (10.1038/ki.2011.347) Copyright © 2012 International Society of Nephrology Terms and Conditions