Effects of the Inducible Nitric-Oxide Synthase Inhibitor

The mortality rate for septic patients with acute renal failure is approximately doubled compared with patients with sepsis alone. Unfortunately, the treatment for sepsis-induced renal failure has advanced little during the last several decades. Because sepsis is often caused by lipopolysaccharide (LPS), a mouse model of LPS challenge was used to study the development of kidney injury. We hypothesized that inducible nitricoxide synthase (iNOS)-catalyzed nitric oxide production and that generation of reactive nitrogen species (RNS) might play a role in the microcirculatory defect and resulting tubular injury associated with LPS administration. Fluorescent intravital videomicroscopy was used to assess renal peritubular capillary perfusion and document RNS generation by renal tubules in real time. As early as 6 h after LPS administration (10 mg/kg i.p.), RNS generation (rhodamine fluorescence), redox stress [NAD(P)H autofluorescence], and the percentage of capillaries without flow were each significantly increased compared with saline-treated mice (p 0.05). The generation of RNS was supported by the detection of nitrotyrosine-protein adducts in the kidney using immunohistochemistry. The iNOS inhibitor L-N-(1-iminoethyl)-lysine (L-NIL; 3 mg/kg i.p.) completely blocked the increase in rhodamine fluorescence and NAD(P)H autofluorescence and prevented the capillary defects at 6 h after LPS administration. These results suggest that iNOSderived RNS is an important contributor to the peritubular capillary perfusion defects and RNS generation that occur during sepsis and emphasize that pharmacological inhibition of iNOS may provide beneficial effects during sepsis by improving renal capillary perfusion and reducing RNS generation in the kidney. Approximately 750,000 cases of sepsis are reported each year in the United States with an estimated cost incurred nationally at $17 billion annually (Dal Nogare, 1991). Acute renal failure (ARF) is a complication in approximately 20 to 50% of septic patients diagnosed by positive blood culture (Schrier and Wang, 2004), and the mortality rate for septic patients with ARF is approximately doubled compared with patients with sepsis alone (Angus and Wax, 2001). Therefore, renal failure is considered a critical prognostic factor in sepsis (Russell, 2006). Unfortunately, treatment of sepsis-induced ARF has advanced little during the last several decades (De Vriese and Bourgeois, 2003; Wan et al., 2003). Thus, a better understanding of the mechanism of kidney injury initiated by sepsis could lead to the uncovering of new therapeutic targets and more effective treatments for this serious condition. Because sepsis is often caused by Gram-negative bacterial lipopolysaccharide (LPS), murine models of LPS challenge are frequently used to study the mechanisms of sepsis-induced renal failure. Studies suggest that the effects of LPS appear, at least in part, to be mediated by Toll-like receptor 4 and the initiation of the release of inflammatory cytokines, such as TNF (Cunningham et al., 2002, 2004). Notably, in septic patients with ARF, cytokine levels are positively correlated with mortality (Simmons et al., 2004), supporting the importance of the inflammatory response in the prognosis of sepsis-induced ARF. Moreover, the levels of circulating adhesion molecules are increased in the septic patient, suggesting that vascular inflammation and endothelial activation may play a role in the development of organ failure (Boldt et This work was supported by a UAMS Pilot Study Grant (P. R. Mayeux) and by the University of Arkansas for Medical Sciences Graduate Student Research Fund (L. W.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.117184. ABBREVIATIONS: ARF, acute renal failure; LPS, lipopolysaccharide; NO, nitric oxide; iNOS, inducible nitric-oxide synthase; RNS, reactive nitrogen species; ROS, reactive oxygen species; DHR, dihydrorhodamine 123; RBC, red blood cell; IVVM, intravital videomicroscopy; L-NIL, L-N-(1-iminoethyl)-lysine; FITC-dextran, fluorescein isothiocyanate-dextran. 0022-3565/07/3203-1061–1067$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 3 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 117184/3185863 JPET 320:1061–1067, 2007 Printed in U.S.A. 1061 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from al., 1996). Cytokines and adhesion molecules are also elevated in the kidney following LPS challenge in the mouse, as is the generation of NO via another recognized inflammatory response, induction of inducible nitric-oxide synthase (iNOS) (Cunningham et al., 2004; Guo et al., 2004; Wu et al., 2007). Collectively, these inflammatory responses are probably contributors to renal injury during sepsis. In models of ARF associated with iNOS induction, the contributions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are emerging as important mediators, particularly in ischemia/reperfusion injury (Noiri et al., 1996, 2001; Walker et al., 2000; Vinas et al., 2006). Recent studies also suggest that iNOS-derived NO/RNS may play a role in the pathogenesis of LPS-induced renal injury (Wang et al., 2003; Cuzzocrea et al., 2006). However, the dynamics of RNS generation in the kidney has never been examined. The development of sepsis-induced renal injury is a complex process. Recently, we reported that peritubular capillary perfusion is severely disrupted following LPS administration in mice (Tiwari et al., 2005; Wu et al., 2007). Furthermore, this microcirculatory defect was positively correlated to the development of tubular cell stress (Wu et al., 2007). The aim of the present study was to examine the relationship between the decrease in peritubular capillary perfusion and the generation of RNS. We used intravital videomicroscopy (IVVM) to monitor peritubular capillary perfusion and RNS generation following LPS administration. The iNOS inhibitor L-N(1-iminoethyl)-lysine (L-NIL) was used to examine the role of iNOS. This study is the first to link early changes in renal microcirculation and tubular cell stress to iNOS-derived RNS generation by tubular epithelium. Materials and Methods Lipopolysaccharide (Escherichia coli 055:B5 strain) and fluorescein isothiocyanate-dextran (FITC-dextran; 150,000 Da) were purchased from Sigma-Aldrich (St. Louis, MO). Dihydrorhodamine 123 (DHR) was purchased from Invitrogen (Carlsbad, CA). Rabbit polyclonal antinitrotyrosine antibody was purchased from Upstate Cell Signaling Solutions (Lake Placid, NY). DakoCytomation labeled streptavidin biotin System-horseradish peroxidase kit was purchased from Dako North America Inc. (Carpinteria, CA). L-N-(1Iminoethyl)-lysine 2HCI was purchased from Axxora (San Diego, CA). Mouse Model of Endotoxin-Induced Renal Injury. All animals were housed and killed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval of the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. Male C57BL/6 mice (8 weeks of age) were acclimated for 1 week with free access to food and water. At the start of the experiment, mice were injected with saline or LPS (10 mg/kg i.p.). At the indicated time after IVVM analysis, blood was collected via cardiac puncture under isoflurane anesthesia. This was followed immediately by cervical dislocation. Kidneys were then rapidly harvested and fixed in 10% phosphatebuffered formalin before embedded in paraffin. Serum creatinine concentration was determined using a Roche Cobas Mira Clinical Analyzer (Roche Diagnostic Systems, Inc., Branchburg, NJ). Evaluation of Peritubular Capillary Dysfunction with IVVM. At 15 min before IVVM, mice were administered FITCdextran (150,000 Da, 2 mol/kg) and DHR (4 mol/kg) via the tail vein. Mice were prepared for IVVM as described elsewhere (Tiwari et al., 2005). In brief, mice were anesthetized with isoflurane and underwent laparotomy to expose the left kidney. The kidney was positioned on a glass stage above an inverted fluorescent microscope (Zeiss Axiovert 200; Carl Zeiss GmbH, Jena, Germany) equipped with a digitizing camera (Hamamatsu, Bridgewater, NJ) and kept moist with saline and covered. Core temperature was monitored and maintained at 36–37°C using an infrared heat lamp. During IVVM, the renal intravascular space and red blood cell (RBC) movement were visualized with FITC-dextran using an excitation of 470 nm and an emission of 520 nm. From the left kidney of each animal, videos of 10 s each were captured at approximately 15 frames/s from five randomly selected fields of view (200 ). Capillary function was analyzed as described previously (Tiwari et al., 2005; Wu et al., 2007). In brief, randomly selected vessels (approximately 150 per kidney) were classified into three categories of blood perfusion: “Continuous Flow”, where RBC movement in the vessel was not interrupted during the video; “Intermittent Flow”, where RBC movement stopped or reversed any time during the video; and “No Flow”, where no RBC movement was detected. Data were expressed as the percentage of vessels in each of the three categories. Evaluation of Renal Tubular Epithelial Cell Stress Using IVVM. IVVM can be used to assess cellular redox stress by monitoring [NAD(P)H] autofluorescence (Paxian et al., 2004; Wunder et al., 2005). NAD(P)H autofluorescence was visualized at an excitation of 365 nm and an emission of 420 nm. To minimize photobleaching, a 3-s exposure was used to capture videos of five randomly selected fields of view per animal. Imaging settings were identical for all fields of view. The intensity of NAD(P)H autofluorescence was quantified using AxioVision Imaging Software (Carl Zeiss GmbH). Data were expressed as arbitrary units per micromolars. Detection of ROS/RNS Using IVVM. DHR is oxidized to fluorescent rhodamine by hydroxyl radical, nitrogen dioxide, peroxynitrite, and peroxidase-derived species (Halliwell and Whiteman, 2004). For each field of view captured for assessing capillary perfusion, additional images of rhodamine fluorescence were captured using an emission of 507 nm and an excitation of 530 nm. A 3-s exposure was used to minimize photobleaching. Imaging settings were identical for all fields of view. The intensity of rhodamine fluorescence was quantified using AxioVision Imaging Software (Carl Zeiss GmbH). Data were expressed as arbitrary units per M. Immunohistochemistry. Immunohistochemistry was used to identify the presence of nitrotyrosine-protein adducts, a biomarker of peroxynitrite generation (Beckman et al., 1990). Paraffinembedded sections (3 m) from the right kidney (not used for IVVM) were cleared in xylene and rehydrated with ethanol. Endogenous peroxidase activity and nonspecific protein binding were blocked using reagents supplied in the DakoCytomation labeled streptavidin biotin System-horseradish peroxidase kit. Sections were incubated with rabbit antinitrotyrosine antibody (1:400 dilution) overnight at 4°C. Sections were then incubated with the second antibody (biotinylated link supplied by the kit) at room temperature for 30 min. Sections were incubated with streptavidin peroxidase for 30 min, washed with phosphate-buffered saline, and stained with a chromagen solution supplied by the manufacturer. Gill’s hematoxylin II was used as a counterstain. To determine nonspecific binding, antinitrotyrosine was incubated with 3-nitrotyrosine (10 mM) for 1 h before use. Treatment with L-NIL. The role of iNOS was examined using the iNOS inhibitor L-NIL (Moore et al., 1994). The dose of L-NIL (3 mg/kg i.p.) was chosen based on our previous studies in the mouse and rat (Thompson et al., 1991; Zhang et al., 2000) and was administered at the same time as LPS. Statistical Analysis. Data were analyzed with Prism 4.0 software for Mac (GraphPad Software, San Diego, CA). Each “n” represents data or tissue obtained from one mouse. Data are expressed as means S.E.M. A one-way ANOVA followed by the Student-Newman-Keuls post test was used to determine differences between groups. A p value 0.05 was considered significant. 1062 Wu and Mayeux at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from