Positive effects of a novel non-peptidyl low molecular weight radical scavenger in renal ischemia/reperfusion: a preliminary report

Roberto Bassi 0 1 2 3 Andrea Vergani 1 3 Francesca D'Addio 0 1 3 Moufida Ben Nasr 1 3 Alessio Mocci 1 3 6 Maria Pia Rastaldi 5 Takaharu Ichimura 4 Joseph Vincent Bonventre 4 Paolo Fiorina 0 1 3 4 0 Medicine, San Raffaele Scientific Institute , Milan, Italy 1 Boston Children's Hospital, Harvard Medical School, Nephrology Division , Boston, MA, USA 2 DiSTeBA, Universita' del Salento , Lecce, Italy 3 Boston Children's Hospital, Harvard Medical School, Nephrology Division , Boston, MA, USA 4 Brigham and Women's Hospital, Harvard Medical School, Renal Division , Boston, MA, USA 5 Renal Research Laboratory, Fondazione IRCCS Ospedale Maggiore Policlinico & Fondazione D'Amico per la Ricerca sulle Malattie Renali , Milan, Italy 6 Department of Accident and Emergency , ASL, Bologna, Italy Ischemia/reperfusion (I/R) is one of the most common causes of acute kidney injury. Reactive oxygen species have been recognized to be an important contributor to the pathogenesis of I/R injury. We hypothesize that a non-peptidyl low molecular weight radical scavenger (IAC) therapy may counteract this factor, ultimately providing some protection after acute phase renal I/R injury. The aim of this preliminary study was to assess the ability of IAC to reduce acute kidney injury in C57BL/6 mice after 30-minute of bilateral ischemia followed by reperfusion. The rise in serum creatinine level was higher in C57BL/6 control mice after I/R when compared to IAC (1 mg)-treated mice. Control mice showed greater body weight loss compared to IAC-treated mice, and at pathology, reduced signs of tubular necrosis were also evident in IAC-treated mice. These preliminary evidences lay the basis for more comprehensive studies on the positive effects of IAC as a complementary therapeutic approach for acute phase renal I/R injury. - Introduction Kidney global or regional ischemia/reperfusion (I/R) is one of the most common causes of acute kidney injury (McCord 1985). During the peritransplant period, kidney transplanted patients are prone in 2-7% of the cases to experience I/R (Bonventre and Yang 2011), which can render the allograft more likely to develop acute rejection, and to progress towards long-term chronic allograft nephropathy (Thadhani et al. 1996; Cavaille-Coll et al. 2013). I/R injury is also a common event in a variety of pathological conditions such as diabetes and cardiovascular diseases (Luitse et al. 2012). Tissue hypoperfusion/ hypoxia leads to depletion of cellular ATP and cytoskeleton damage (Singh et al. 2013). The restoration of blood flow with production of reactive oxygen species (ROS), and activation of leukocytes and endothelial cells (Rabb 2012; Ko et al. 2011) contribute to reperfusion injury. Although many experimental studies show a decreased injury and preserved renal function after dampening ROS production, efficient treatments are still limited (Cavaille-Coll et al. 2013; Leung et al. 2013; Venturini et al. 2006; Fiorina et al. 2006). Currently, the therapy for I/R injury is mainly based on supportive care and fluid administration (Cavaille-Coll et al. 2013; Luitse et al. 2012; Fiorina et al. 2005, 2006) and I/R injury remains a major cause of morbidity and mortality (Cavaille-Coll et al. 2013; Luitse et al. 2012; Fiorina et al. 2005, 2006). Non-peptidyl low molecular weight radical scavenger (IAC), a clinically available drug (DAleo et al. 2009), has been shown to have antioxidant properties in different models of brain and islet induced ischemia (DAleo et al. 2009; Corsi et al. 2011). We studied the effect of a IAC-based therapy in a murine model of bilateral kidney I/R injury, aiming to establish a proof-of-concept for the use of IAC as novel complementary therapy for individuals at risk for renal acute ischemic injuries. Materials and methods Animals C57BL/6 (H-2Kb) mice were obtained from Jackson Laboratory (Bar Harbor, ME) and were housed in a pathogenfree environment; water and chow diet were provided ad libitum. Control (CTRL) and IAC-treated mice were weight (~20 grams), sex (male) and age (10 weeks-old) matched. Mice were cared for in accordance with institutional guidelines at the Harvard Medical School Facilities for Animal Care and Housing. Protocols were approved by the Harvard Animal Care and Use Committee. Interventional and functional studies Two groups of C57BL/6 mice (n = 10 each) were subjected to experimental kidney ischemia through bilateral simultaneous clamping of vascular pedicles for 30 minutes, followed by clamp removal to allow kidney reperfusion. Mice were then treated with a single intraperitoneal injection of IAC (1 mg) or saline solution at 60 minutes after ischemic injury induction (30 minutes after clamps removal). Blood samples were collected by retro-orbital vein puncture before kidney ischemia induction (baseline; BL) at day (D)1, D2 and D4. Renal function was assessed by serum creatinine measurement by Creatinine Reagent Kit (Pointe Scientific, Lincoln Park, MI). Mouse weight was measured using a Pesola Digital Platform Scale (Pesola AG, Baar, Switzerland). IAC was kindly provided by Medestea Research and Production (Turin, Italy). Murine kidney pathology Bilateral nephrectomy was performed at D4 in three mice per group for histological evaluation and acute tubular necrosis score computation (Fiorina et al. 2006). Kidney tissue was placed in 10% buffered formalin followed by paraffin embedding for haematoxylin and eosin staining. Histological slides for renal tissue damage evaluation, were examined by the operator without knowledge of the experimental design. Statistical analysis Data are expressed as mean SD. Unpaired t-test was used to compare difference between groups. Statistical significance was set as p value < 0.05. Analysis of data was performed using STATA v12 statistical package for Windows (StataCorp, TX, USA). Results Our preliminary results show that treatment with a single dose of IAC (1 mg) reduces kidney injuries in C57BL/6 mice during the first two days (acute phase) of experimentally induced bilateral I/R injury. In the untreated group (CTRL), one animal died after 48 hours, Figure 1 IAC treatment partially prevented serum creatinine levels increase, body weight loss and acute tubular necrosis after I/R induction. (A) Untreated control (CTRL) mice showed at day (D) 1 and D2 a more significant increase in serum creatinine levels after the induction of ischemia/reperfusion compared to non-peptidyl low molecular weight radical scavenger (IAC)-treated mice (D1 and D2: CTRL vs. IAC-treated, *p < 0.05). (B) Untreated CTRL mice showed a progressive and more evident reduction of body weight after ischemia/reperfusion induction compared to IAC-treated mice (D1, D2 and D4: CTRL vs. IAC-treated, *p < 0.05). (C) Untreated CTRL mice showed acute tubular necrosis signs in kidneys outer medulla with hyaline and granular casts accumulation. (D) Conversely, the extent of acute tubular necrosis was reduced in IAC-treated mice. while (...truncated)