Protection of Coenzyme Q-10 Against Contrast-induced Acute Kidney Injury in Diabetic Rats

Background: Diabetes Mellitus (DM) is a important risk factor for Contrast-induced acute kidney injury (CI-AKI). DM and CI-AKI share oxidative damage and inammation mechanisms that induction of protective and cellular adaptation enzymes as coenzyme Q-10 (COQ-10). The aim of this study was to investigate the therapeutic potential of COQ-10 in renal function, renal hemodynamics, oxidative prole and renal histology in diabetic rats submitted to the CI-AKI model. Methods: Wistar rats, male, randomized into four groups: Citrate- control animals, received citrate buffer (streptozotocin vehicle, 0.4 ml); DM- animals that received streptozotocin (60 mg/kg); DM+IC: DM animals, treated with iodinated contrast (IC, 6 ml/kg); DM+IC+COQ-10: DM animals treated with COQ-10 (10 mg/kg) and that received with IC (6 ml/kg). The protocols lasted 4 weeks. Were evaluated the renal function by inulin clearance and serum creatinine, renal hemodynamics by renal blood ow (RBF) and renal vascular resistance (RVR), markers of oxidative stress such as urinary peroxides and nitrate, lipid peroxidation, thiols in renal tissue and renal histological analysis. Results: DM animals showed reduced renal function which was reected with an increase of serum creatinine and signicant reduced of inulin clearance, as well as a reduction on RBF, increased RVR and redox imbalance with a higher urinary peroxides, nitrate lipid peroxidation levels and depletion of thiols in renal tissue. IC treatment exacerbated theses changes in DM + IC. COQ-10 administration ameliorates renal function, prevented hemodynamic changes, neutralize oxidative damage and progression of the histologic damage in the DM+IC+COQ-10 group. Conclusion: This study is the rst that demonstrated a renoprotection of COQ-10 in experimental model of


Background
Contrast-induced acute kidney injury (CI-AKI) has been the third most common cause of acute renal injury in hospitalized patients become a signi cant source of hospital morbidity and mortality, length of hospitalization, and healthcare costs [1,2]. Diabetes Mellitus (DM) is one of the world's most common chronic metabolic disorders and it is associated with loss of kidney function, increasing the risk of chronic kidney disease (CKD) [3,4]. DM is considered an important risk factor for CI-AKI. Almost 28,2% of patients that developed CI-AKI was associated with preprocedural hyperglycemia and 26,6% was related have severe level of glomerular ltration rate [5,6].
Chronic hyperglycemia contributes to increase endothelin and angiotensin levels, causing intrarenal vasoconstriction, change of intrarenal blood ow, reducing pH and oxygen delivery, increasing reactive oxygen species (ROS) and In ammatory cytokines [3,7]. The increase in the ROS in DM may be associated the development of CI-AKI, whose the pathogenesis is due of endothelial dysfunction, defective nitrovasodilation, cellular toxicity from the contrast media and tubular apoptosis resulting in hypoxia [2,8]. Therefore, DM and CI-AKI share oxidative damage and in ammation mechanisms that favors oxidative stress and cytokines liberation [2,3].
Oxidative stress is de ned with an imbalance between the ROS and antioxidant defenses, and is responsable by increase of formation mutagenic compounds, atherogenic activity, and in ammatory processes [9,10].
Coenzyme Q-10 (COQ-10), a protein of the mitochondrial respiratory and has been highlighted by plays an antioxidant and anti-in ammation [4,11]. As an intracellular antioxidant, COQ-10 is able to protects phospholipids and proteins of membrane from oxidative damage and also demonstrates antiin ammatory effect participating in the modulation of in ammatory cytokines and transcription factors [12,13]. COQ-10 induction has already evidenced renoprotective activity in an animal models of diabetic nephropathy and cisplatin nephrotoxicity [14,15].
Thus, the aim of this study was to investigate the therapeutic potential of COQ-10 in renal function, renal hemodynamics, oxidative pro le and renal histology in diabetic rats submitted to the CI-AKI model.

Animals
Adult male Wistar rats (weighing 250-290 g) were used. The animals obtained from the Institute of Biomedical Sciences at the University of Sao Paulo, were housed at Experimental Laboratory of Animal Models (LEMA) at the School of Nursing, University of Sao Paulo, in a room at a controlled temperature (25ºC/77 ºF) on alternating light/dark cycles, and had free access to water and rat chow. The study was approved by the Ethical Committee of Experimental Animals, University of Sao Paulo (CEEA -protocol nº 055/15).

Iodinated contrast induced CI-AKI model
The animals received 6 mL/kg of iodinated contrast (IC, meglumine ioxithalamate and sodium) intraperitoneal (i.p.), single dose [17]. Diabetes Mellitus + iodinated contrast + CoenzymeQ-10 (DM + IC + COQ-10, n = 7): DM rats receiving received COQ-10, i.p., diluted in 80% Tween, 1%. Treatment with COQ-10 was started on the 22th day of the experimental protocol. There were four preconditioning days, followed by a dose administered on the same day as CI administration and the last dose on the following day.

Procedures and timing of experimental protocols
All protocols of experimental groups lasted four week. Animals were allocated in individual metabolic cages on 27th day, for 24 hours, for collection of urine and determination of urinary ow. On the 28th day of the protocol, the rats were anesthetized with 10 mg/kg xylazine and 90 mg/kg ketamine i.p., and submitted surgical procedure for renal function and hemodynamics measurement. After, a blood sample was collected through a puncture of the abdominal aorta. Finally, animals were submitted euthanasia according to guidelines for animal experimentation and removal of kidneys for thiols assay and histological analysis.

Renal Function Measurement
Inulin clearance (ml/min): renal function was evaluated based on inulin clearance. Inulin was injected in right jugular vein by catheter (polyethylene tube PE-60), as a loading dose (100 mg/kg), followed by a continuous infusion of 0.04 ml/min. After a 30 min equilibration period, three urine collections were made through the bladder catheter and two blood samples were then obtained at the carotid catheter. The serum and urinary inulin were measured using the Anthrone method [19,20].
Renal vascular resistance: Mean arterial blood pressure (MAP) by catheter inserted into the left carotid (polyethylene tube PE-60) and renal blood ow (RBF) were measured and the renal vascular resistance (RVR) e was calculated with the formula: RVR = MAP/RBF [19].
Oxidative pro le Urinary peroxides: Were determined by method of ferrous oxidation of xylenol orange version 2 (FOX-2). Results were expressed as nmol/g urinary Cr [22].
Urinary nitrate: Was measured using the Griess method. Results were expressed as nmol/g urinary Cr [23].
Urinary thiobarbituric acid reactive substances (TBARS): Urinary TBARS is an indirect measure of lipid peroxidation. This assay is based on the reaction of urine samples with 17.5% trichloroacetic acid (TCA) and 0.6% thiobarbituric acid at 95ºC for 20 min, after cooling, 70% TCA is added and incubated for 20 min. The amount of TBARS was expressed as nmol / g urinary Cr [24].
Soluble non-protein thiols in renal tissue: The thiol antioxidant assay was by Ellman method. The amount of soluble thiols was corrected for total protein measured by Bradford method and results was expressed as nmol / mg total protein [25,26].
Urinary Cr by Jaffé method was used to correct oxidative parameters [21].

Histological Analysis
Tubulointerstitial damage: kidney tissue was sectioned, stained with hematoxylin and eosin, and examined under light microscopy (magni cation ×400). Tubulointerstitial damage was examined for extent of cortical and outer medullae involvement of tubule interstitial damage of tubules that displayed: tubular epithelial swelling, vacuolar degeneration, necrosis, and desquamation, presence of an in ammatory cell in ltrate, tubular lumen dilatation or tubular atrophy [19].

Statistical Analysis
The results are reported as the mean ± standard error (SEM). Used the analysis of variance by One Way ANOVA and post test Newman-Keuls to comparisons of groups. Statistical signi cance was de ned at p < 0.05. All statistical analyses were performed Graph-Pad Prism version-7 for Windows®.

Effect of COQ-10 treatment on renal function
The results show the effect of COQ-10 on renal function after injury is demonstrated in Table 1. Rats submitted to DM showed a signi cant increase in urinary ow, serum Cr and decrease in inulin clearance. DM + IC group resulted additional elevation in serum Cr and a reduction in inulin clearance compared to the DM group, whereas these parameters were changed by the COQ-10 treatment that signi cantly decreased serum Cr and improved inulin clearance in the DM + IC + COQ-10 group.

Effect of COQ-10 treatment on hemodynamic parameters
Data illustrate in Table 2 the effect of COQ-10 in renal hemodynamic. Was observed a signi cant reduction in RVR and elevation in RBF on the DM group, this changes were exacerbated in DM + IC group, whereas, COQ-10 prevented hemodynamic changes. As showed, the treatment with COQ-10 signi cantly increased RBF and decreased RVR in DM + IC + COQ-10 group. Effects of COQ-10 treatment on oxidative pro le The Table 3 summarize the oxidative pro le. DM group show signi cantly increased oxidative metabolites and reduction antioxidant activities of soluble non-protein thiols in renal tissue. The redox imbalance ndings were more pronounced in the diabetic group that was treated with IC, DM + IC, compared to DM. Treatment with COQ-10 reduced oxidative stress demonstrated by decreased urinary peroxides, nitrite and TBARS excretion in DM + IC + COQ-10 group, furthermore, COQ-10 signi cantly preserved antioxidant capacity, con rmed by increased of soluble non-protein thiols in renal tissue.

Histological analysis
As shown in Table 4 and Fig. 1, all groups showed slight changes with impairment of less than 5% of the tissue focal areas. Histological changes in DM group was signi cantly higher compared to the citrate group. Figure 1B shows DM resulted in a discrete edema and increased interstitial area. The DM + IC group showed a signi cant increase in the tubulointerstitial lesion area compared to the DM group. After IC (Fig. 1C), kidneys presented tubulointerstitial injury characterized by edema, attening of tubular cells and diffuse in ammatory interstitial in ltration (Fig. 1B). Treatment with COQ-10 show signi cantly reduced in extension area of the tubulointerstitial lesion compared to DM + IC as illustrate images in Fig. 1D of renal histological analysis.

Discussion
The present study evaluated the participation of oxidative stress in the pathophysiology of CI-AKI with DM as a risk factor and investigated the role of COQ-10 as a possible treatment for this pathology. Oxidative stress and in ammation status are correlated in the prognosis of CI-AKI in DM, therefore, the investigation of antioxidants alternatives that promote renoprotection is of great importance.
The development of CI-AKI in DM was evidenced in this study with dysfunctions in renal function, renal hemodynamics and the installation of oxidative damage. Clinically CI-AKI is de ned an increase in serum creatinine ≥ 0.5 mg/dL or 25% increase of serum creatinine from the baseline value at 48 h after of contrast media administration [27].
The COQ-10 has demonstrated high therapeutic potential due to its antioxidant and anti-in ammatory activities in many injury models, including studies of nephrotoxicity by cisplatin and cyclosporine [15,28,29,30]. Our results highlighted the role of COQ-10 in the modulation of pathophysiological processes induced by nephrotoxicity of IC, showing that the treatment with COQ-10 exerted a protective effect on the renal function of diabetic animals submitted to CI-AKI. The renoprotective effect was evidenced by the increase in inulin clearance and decrease in serum creatinine in DM animals that received IC and treatment with COQ-10.
Increased RVR and the decrease in RBF in DM after IC, as observed in this study, can be attributed to vasoconstriction due viscosity and osmolarity of contrast media. The vasoconstriction contributes to hypoxia and development of oxidative injury that culminate in endothelial dysfunction [4,25].
Additionally, DM is associated with development of hypoxia inducible factors (HIF) that enhanced activity of renin-angiotensin system may also intensify the vasoconstriction via endothelin synthesis and increased effect of adenosine [2,3]. In this study, treatment with COQ-10 demonstrated improvement in renal hemodynamics with reduced RVR and elevated RBF. Studies suggest that COQ-10 stimulates the production of prostaglandin-1 and prostacyclin, which aid in vasodilation, and reduce peripheral resistance by preserving the vasodilator nitric oxide, promoting the reduction of nitrogen dioxide to nitric oxide, helping to maintain this bioregulatory agent [11,12].
In the present study, it was observed a signi cant increase of oxidative stress via TBARS, FOX and nitrate elevation and a reduction of thiols levels. Hyperglycemia increases oxidative stress in CI-AKI by activating stress-activated proteins kinase, functional proteins glycosylation, glucose autoxidation and the formation of reactive nitrogen species, such peroxynitrite, that has been related in the enhanced in ammation in diabetes by decreased nitric oxide bioavailability [10,17,31]. ROS production in DM has been linked to vasoconstriction, vascular cell hypertrophy and migration, endothelial dysfunction, modi cation of extracellular matrix proteins, and increased renal sodium reabsorption [3,31]. Enhanced macrophage migration induces the release of in ammatory and pro brotic cytokines, stimulating greater ROS production. Thus, the oxidative stress induced by cytokine production in DM associated of contrast injury increase ROS establishing a vicious cycle [2,32,33,34].
Despite its primary role in the production of ATP, COQ-10 is considered a substance of great antioxidant and anti-in ammatory activity, due capable of stabilize two free radicals to each molecule of COQ-10 in its redox cycle, is quickly recovered, may inhibit NF-kB and protein kinases, reduce free radical delivering them to recovery of antioxidants cycle, such vitamin E, e ciency in interrupting radical chain reactions such as lipid peroxidation, also to avoid nitrosative stress reacting [35,36].
In this study, the treatment of diabetic animals with COQ-10 demonstrated the ability to preserve the reserve of systemic thiol anti-oxidant after IC administration. Intracellular antioxidants mechanisms such glutathione, a non-protein thiol, exercise role in neutralization of ROS, protecting against oxidative damage while their decrease contributes to the oxidative attack on cells. COQ-10 demonstrated to preserve glutiona in an animal model of cisplatin-induced nephrotoxicity by increase of selenium, necessary for the composition of glutathione [29,37,38].
In the present study, diabetic animals demonstrated mild tubulointerstitial changes typical of the development of diabetic nephropathy [39]. The histological changes observed in animals that received IC were due to the association of the insult caused by hyperglycemia and IC, reinforcing that the mechanism involved in the reduction of renal function is mainly related to renal hemodynamic changes and oxidative damage that favor the installation of IC -AKI. Our ndings indicate that the administration of COQ-10 prevented the progression of the extension area with tissue damage after the use of IC.
Considering that DM is a modi able risk factor for IC nephrotoxicity, the implementation of preventive strategies with innovative pharmacological interventions, such as COQ-10, can establish a promising scenario and e ciently reverse the adverse effects of pathophysiology of the NIC.

Conclusion
This study is the rst that demonstrated a renoprotection of COQ-10 in experimental model of risk factor of DM for CI-AKI. COQ-10 presented an antioxidant effect on the CI-AKI in diabetic rats, by improving function and renal hemodynamics, preserving morphology and reducing oxidative stress.