Open Access

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

  • Roberto Bassi1, 2, 3,
  • Andrea Vergani1,
  • Francesca D’Addio1, 3,
  • Moufida Ben Nasr1,
  • Alessio Mocci1, 4,
  • Maria Pia Rastaldi5,
  • Takaharu Ichimura6,
  • Joseph Vincent Bonventre6 and
  • Paolo Fiorina1, 3, 6Email author
SpringerPlus20143:158

https://doi.org/10.1186/2193-1801-3-158

Received: 28 November 2013

Accepted: 11 March 2014

Published: 24 March 2014

Abstract

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.

Keywords

Ischemia/reperfusion Kidney disease Kidney transplantation Radical oxygen species Inflammation

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 (D’Aleo et al. 2009), has been shown to have anti-oxidant properties in different models of brain and islet induced ischemia (D’Aleo 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 pathogen-free 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, while none died in the IAC-treated group (p = ns, data not shown). The mouse from the CTRL group that did not survive, showed the highest serum creatinine value (2.60 mg/dl at D1). We intentionally used an interventional protocol for I/R with a low mortality rate, to gain better insight on the effect of IAC on kidney function rather than on a survival effect.

At baseline, serum creatinine levels were similar between CTRL and IAC-treated mice (BL: CTRL = 0.30 ± 0.40 vs. IAC-treated = 0.22 ± 0.30 mg/dl, p = ns), however a rapid increase in serum creatinine was observed in both groups soon after kidney I/R (Figure 1A). Serum creatinine levels in CTRL mice were significantly higher at D1 and D2 when compared to levels observed in IAC (1 mg)-treated mice (D1: CTRL = 1.59 ± 0.17 vs. IAC-treated = 1.31 ± 0.19 mg/dl, p = 0.02; D2: CTRL = 1.02 ± 0.21 vs. IAC-treated = 0.79 ± 0.13 mg/dl, p = 0.01), (Figure 1A). At D4 serum creatinine levels showed no difference between CTRL and IAC-treated mice, (D4: CTRL = 0.71 ± 0.13 vs. IAC-treated = 0.57 ± 0.10 mg/dl, p = ns), (Figure 1A).
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.

Body weight was comparable between CTRL and IAC-treated mice at baseline (BL: CTRL = 24.1 ± 0.2 vs. IAC-treated = 24.9 ± 0.4 gr, p = ns), (Figure 1B). CTRL mice showed a progressive and more significant reduction of body mass during the follow-up period as compared to IAC-treated mice (D1: CTRL = 22.5 ± 0.2 vs. IAC-treated = 23.4 ± 0.3 gr, p = 0.02; D2: CTRL = 21.8 ± 0.1 vs. IAC-treated = 22.7 ± 0.3 gr, p = 0.02; D4: CTRL = 20.8 ± 0.2 vs. IAC-treated = 22.2 ± 0.4 gr, p = 0.01), (Figure 1B).

We finally examined the extent of kidney damage at D4 post I/R induction, particularly acute tubular necrosis (a hallmark of I/R injury). In CTRL mice, acute tubular necrosis was preferentially localized to the outer medulla of kidneys, with evident amounts of hyaline and granular casts (Figure 1C). In contrast, at D4 after I/R induction, in IAC-treated mice a marked reduction of acute tubular necrosis was evident as compared to untreated CTRL mice (Figure 1D). No signs of acute tubular necrosis were detectable in kidneys of sham-operated animals (data not shown).

Discussion

I/R injury is an important contributor in acute kidney injury (Thadhani et al. 1996). After the ischemic event, organ reperfusion is accompanied by a cascade of inflammatory responses boosted by the local recruitment of peripheral leukocytes and the release of ROS (Rabb 2012). Together they lead to a common downstream pathway that results in the activation of pro-apoptotic genes such as caspase-3 and ultimately to acute kidney injury (Yang et al. 2005). Moreover, oxidative stress, occurring when ROS generation exceeds the capacity of anti-oxidant defenses, may cause indiscriminate damage to lipids, proteins and DNA, leading to future cell dysfunction and tissue damage (Yang et al. 2005). We here showed that non-peptidyl low molecular weight radical scavenger treatment, prevented serum creatinine increase, body weight loss and kidney tubular damage in C57BL/6 mice in the first two days after experimental kidney I/R injury. Thus, our preliminary results suggest that a non-peptidyl low molecular weight radical scavenger, having anti-oxidant properties, may warrant consideration as a complementary therapeutic strategy for the treatment of acute phase renal I/R injury in kidney transplanted patients (possibly reducing the risk for immediate acute rejection after transplant) or in individuals at risk for oxidative stress-related organ/tissue damage.

Abbreviations

(S): 

Serum

(BL): 

Baseline.

Declarations

Acknowledgments

P.F. was consultant for Medestea Research and Production. We thank Melissa Chin for manuscript editing.

Funding

Paolo Fiorina is the recipient of a JDRF Career Development Award, an ASN Career Development Award, and an ADA Mentor-based Fellowship grant. P.F. is also supported by a Translational Research Program (TRP) grant from Boston Children’s Hospital; Harvard Stem Cell Institute grant (“Diabetes Program” DP-0123-12-00); Italian Ministry of Health grant RF- 2010-2303119. Roberto Bassi is supported by an ADA Mentor-based Fellowship grant to P.F and by an AST Genentech Clinical Science Fellowship grant. P.F. is the recipient of an Italian Ministry of Health grant: (“Staminali” RF-FSR-2008-1213704). A.V. has been supported by an NIH-Research Training grant to Boston Children’s Hospital in Pediatric Nephrology (T32DK007726-28).

Authors’ Affiliations

(1)
Harvard Medical School, Nephrology Division, Boston Children’s Hospital
(2)
DiSTeBA, Universita’ del Salento
(3)
Medicine, San Raffaele Scientific Institute
(4)
Department of Accident and Emergency, ASL
(5)
Renal Research Laboratory, Fondazione IRCCS Ospedale Maggiore Policlinico & Fondazione D’Amico per la Ricerca sulle Malattie Renali
(6)
Harvard Medical School, Renal Division, Brigham and Women’s Hospital

References

  1. Bonventre JV, Yang L: Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest 2011, 121: 4210-4221. 10.1172/JCI45161View ArticleGoogle Scholar
  2. Cavaillé-Coll M, Bala S, Velidedeoglu E, Hernandez A, Archdeacon P, Gonzalez G, Neuland C, Meyer J, Albrecht R: Summary of FDA workshop on ischemia reperfusion injury in kidney transplantation. Am J Transplant 2013, 13: 1134-48. 10.1111/ajt.12210View ArticleGoogle Scholar
  3. Corsi L, Zavatti M, Geminiani E, Zanoli P, Baraldi M: Anti-inflammatory activity of the non-peptidyl low molecular weight radical scavenger IAC in carrageenan-induced oedema in rats. J Pharm Pharmacol 2011, 63: 417-422. 10.1111/j.2042-7158.2010.01233.xView ArticleGoogle Scholar
  4. D'Aleo V, Del Guerra S, Martano M, Bonamassa B, Canistro D, Soleti A, Valgimigli L, Paolini M, Filipponi F, Boggi U, Del Prato S, Lupi R: The non-peptidyl low molecular weight radical scavenger IAC protects human pancreatic islets from lipotoxicity. Mol Cell Endocrinol 2009, 309: 63-6. 10.1016/j.mce.2009.05.010View ArticleGoogle Scholar
  5. Fiorina P, Venturini M, Folli F, Losio C, Maffi P, Placidi C, La Rosa S, Orsenigo E, Socci C, Capella C, Del Maschio A, Secchi A: Natural history of kidney graft survival, hypertrophy, and vascular function in end-stage renal disease type 1 diabetic kidney-transplanted patients: beneficial impact of pancreas and successful islet cotransplantation. Diabetes Care 2005, 28: 1303-10. 10.2337/diacare.28.6.1303View ArticleGoogle Scholar
  6. Fiorina P, Ansari MJ, Jurewicz M, Barry M, Ricchiuti V, Smith RN, Shea S, Means TK, Auchincloss H Jr, Luster AD, Sayegh MH, Abdi R: Role of CXC chemokine receptor 3 pathway in renal ischemic injury. J Am Soc Nephrol 2006, 17: 716-23. 10.1681/ASN.2005090954View ArticleGoogle Scholar
  7. Ko GJ, Jang HR, Huang Y, Womer KL, Liu M, Higbee E, Xiao Z, Yagita H, Racusen L, Hamad AR, Rabb H: Blocking Fas ligand on leukocytes attenuates kidney ischemia-reperfusion injury. J Am Soc Nephrol 2011, 22: 732-42. 10.1681/ASN.2010010121View ArticleGoogle Scholar
  8. Leung KC, Tonelli M, James MT: Chronic kidney disease following acute kidney injury-risk and outcomes. Nat Rev Nephrol 2013, 9: 77-85.View ArticleGoogle Scholar
  9. Luitse MJ, Biessels GJ, Rutten GE, Kappelle LJ: Diabetes, hyperglycaemia, and acute ischaemic stroke. Lancet Neurol 2012, 11: 261-271. 10.1016/S1474-4422(12)70005-4View ArticleGoogle Scholar
  10. McCord JM: Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985, 312: 159-163. 10.1056/NEJM198501173120305View ArticleGoogle Scholar
  11. Rabb H: The promise of immune cell therapy for acute kidney injury. J Clin Invest 2012, 122: 3852-3854. 10.1172/JCI66455View ArticleGoogle Scholar
  12. Singh P, Ricksten SE, Bragadottir G, Redfors B, Nordquist L: Renal oxygenation and haemodynamics in acute kidney injury and chronic kidney disease. Clin Exp Pharmacol Physiol 2013, 40: 138-147. 10.1111/1440-1681.12036View ArticleGoogle Scholar
  13. Thadhani R, Pascual M, Bonventre JV: Acute renal failure. N Engl J Med 1996, 334: 1448-1460. 10.1056/NEJM199605303342207View ArticleGoogle Scholar
  14. Venturini M, Fiorina P, Maffi P, Losio C, Vergani A, Secchi A, Del Maschio A: Early increase of retinal arterial and venous blood flow velocities at color Doppler imaging in brittle type 1 diabetes after islet transplant alone. Transplantation 2006, 81: 1274-7. 10.1097/01.tp.0000208631.63235.6aView ArticleGoogle Scholar
  15. Yang B, Jain S, Pawluczyk IZ, Imtiaz S, Bowley L, Ashra SY, Nicholson ML: Inflammation and caspase activation in long-term renal ischemia/reperfusion injury and immunosuppression in rats. Kidney Int 2005, 68: 2050-67. 10.1111/j.1523-1755.2005.00662.xView ArticleGoogle Scholar

Copyright

© Bassi et al.; licensee Springer. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.