Contrast-induced nephropathy and oxidative stress: mechanistic insights for better interventional approaches

Contrast-induced nephropathy (CIN) or contrast-induced acute kidney injury (CI-AKI) is an iatrogenic acute kidney injury observed after intravascular administration of contrast media for intravascular diagnostic procedures or therapeutic angiographic intervention. High risk patients including those with chronic kidney disease (CKD), diabetes mellitus with impaired renal function, congestive heart failure, intraarterial intervention, higher volume of contrast, volume depletion, old age, multiple myeloma, hypertension, and hyperuricemia had increased prevalence of CIN. Although CIN is reversible by itself, some patients suffer this condition without renal recovery leading to CKD or even end-stage renal disease which required long term renal replacement therapy. In addition, both CIN and CKD have been associated with increasing of mortality. Three pathophysiological mechanisms have been proposed including direct tubular toxicity, intrarenal vasoconstriction, and excessive production of reactive oxygen species (ROS), all of which lead to impaired renal function. Reports from basic and clinical studies showing potential preventive strategies for CIN pathophysiology including low- or iso-osmolar contrast media are summarized and discussed. In addition, reports on pharmacological interventions to reduce ROS and attenuate CIN are summarized, highlighting potential for use in clinical practice. Understanding this contributory mechanism could pave ways to improve therapeutic strategies in combating CIN.


Introduction
Contrast-induced nephropathy (CIN) or contrastinduced acute kidney injury (CI-AKI) is an iatrogenic acute kidney injury (AKI) observed after intravascular administration of contrast media (CM) for diagnostic procedures or therapeutic angiographic interventions [1][2][3][4]. Chemical hypersensitivity has also been reported as another side effect of CM [5]. According to the Kidney Disease Improving Global Outcomes (KDIGO) clinical practice guidelines for AKI, a serum creatinine (Cr) Patients with high risk of developing CIN include chronic kidney disease (CKD) and diabetes mellitus (DM) with impaired renal function. Other associated risks include congestive heart failure, volume depletion, old age, hypertension, and hyperuricemia increasing CIN prevalence by up to 25% [7].
The pathophysiological mechanisms of CIN have not been completely elucidated. Currently, several mechanisms including direct effect, indirect effect, and generation of reactive oxygen species (ROS) have been proposed (Fig. 1). In direct effects, CM with high osmolality can directly cause cytotoxicity in nephrons including renal tubular epithelial cells and endothelial cells, leading to mitochondrial dysfunction, cellular apoptosis or necrosis and interstitial inflammation [12]. In indirect effects, CM can alter renal hemodynamics, leading to intrarenal vasoconstriction contributing to medullary hypoxia [12]. Regarding ROS generation, CM can either cause excessive ROS production or reduce antioxidant enzyme activity, resulting in increased oxidative stress and leading to impaired renal function [13]. In addition, medullary hypoxia also leads to enhanced ROS formation, resulting in mitochondrial oxidative stress and mitochondrial dysfunction [13]. Overall, it can be seen that mitochondrial function and oxidative stress play important roles in the pathophysiology of CIN [13]. Therefore, strategies that reduce oxidative stress as well as protecting mitochondrial dysfunction are potential targets for CIN prevention.
KDIGO clinical practice guidelines for AKI state there is no definitive treatment available for established CIN [6]. Thus, the prevention of CIN is the best option. This review aims to comprehensively summarize the available in vitro, in vivo, and clinical reports regarding the pathophysiologic roles of mitochondria and ROS in CIN. Reports on pharmacological interventions to prevent CIN by targeting ROS and mitochondria are also presented and discussed with their potential for clinical use in in the future.

Searching methodology and selection criteria
A comprehensive search of the literature was performed using PubMed covering the period from database inception to September 2019. The search for literature included only articles written in English. An article was rejected if it was clearly a letter or case report. The search used the following keywords: contrast-induced nephropathy; oxidative stress; mitochondria; prevention; and statin either in the title, abstracts, or in the text. The relevance of the subject and eligibility of all publications detected was further evaluated, and data were then extracted from relevant papers to be included in this comprehensive review. Fig. 1 Pathophysiology of CIN. Pathogenesis of CIN consists of 3 mechanisms; direct effect, indirect effect, and generation of ROS. Direct effects include, direct cytotoxicity of CM to nephron leading to cellular apoptosis or necrosis and tubular injury. Indirect effects are that CM could alter renal hemodynamics, leading to intrarenal vasoconstriction, contributing to medullary hypoxia. This mechanism is mediated by the increase in vasoconstrictive mediators including renin, angiotensin II, and endothelin along with the decreasing of vasodilatory mediators including nitric oxide and PGI 2 . Lastly, CM can generate ROS and also reduce antioxidant enzyme activity as a result of various complex mechanisms which result in oxidative stress, leading to progression of impaired renal function. CIN, contrast-induced nephropathy; CM, contrast media; PGI 2 , prostaglandin I 2 ; ROS, reactive oxygen species

Pathogenesis of CIN via ROS generation: reports from in vitro studies
In vitro studies offer the unique opportunity to evaluate the activation of intracellular signaling pathways involved in cellular apoptosis or necrosis, which could pave ways for developing specific therapies to be used in in vivo and clinical studies. However, a major shortcoming of preclinical models of CIN relates to the fact that contrast administration alone does not cause AKI in animals. Multiple stressors are required to be utilized concomitantly to inflict CIN; such as inhibition of nitric oxide (NO), dehydration, and use of prostaglandin inhibitor. A summary of findings from in vitro reports is shown in Table 1.

Roles of mitogen-activated protein kinase (MAPK) pathways in CIN
ROS induced by CM could activate the MAPK signaling pathway through 4 cascades, including extracellular signal-related kinases (ERK) 1 and 2, c-JUN N-terminal kinase (JNK) 1, 2, and 3, p38-MAPK, and ERK5 [14]. These pathways contribute to the activation of caspase-9 and caspase-3, thus inducing apoptosis [15]. In HK-2 cells, CM increased cell injury and decreased cell viability, leading to severe mitochondrial vacuolar degeneration and nucleus fragmentation [16]. CM also increased ROS production via upregulation of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2), Nox4 and p22phox, [16] and triggered apoptosis via induction of caspase 3/7 activity, MAPK pathways (including p38, JNK and ERK pathways), and B-cell lymphoma 2-associated X protein (Bax) expression [17]. Transfection of Nox4 short interfering ribonucleic acid (siRNA) caused a reduction in ROS production and apoptosis [17]. These findings indicated that both Nox and MAPK pathways are involved in the CM-induced ROS production.
Different types of high-osmolar CM were studied in renal cortical cells isolated from male Fischer 344 rats (Table 1). CM was shown to induce renal cell injury in a dose-dependent manner regardless of type of high-osmolar CM [18]. In human embryonic kidney 293 T cells, CM activated JNK/activating transcriptional factor 2 (ATF2) signaling pathways and decreased cell viability. Transfection with ATF2 siRNA caused reduced apoptosis in those CM-treated cells [19]. These findings indicated that JNK/ ATF2 pathways are involved in CM-induced ROS production ( Table 1).

Roles of silent information regulator 1 (SIRT1) in CIN
SIRT1 is a histone deacetylase of nicotinamide adenine dinucleotide (NAD + ), which mainly exists in the nucleus [20]. In NRK-52E rodent tubular cells, CM caused oxidative stress and decreased cell viability by downregulation of SIRT1 [21]. Transfection with SIRT1 siRNA resulted in increased apoptosis in these cells treated with CM [21]. These findings indicated that CM downregulated SIRT1, leading to increased cell apoptosis ( Table 1).

Pathogenesis of CIN via ROS generation: reports from in vivo studies
Consistent with in vitro reports, data from in vivo studies demonstrated that CM increased ROS levels and apoptosis, leading to impaired renal function [19]. A summary of these in vivo reports is shown in Table 2.

Roles of MAPK and SIRT1 in CIN
In mice, CM administration activated the Nox4/Nox2 axis, resulting in increased ROS production, and involving the MAPK pathway (including p38, JNK and ERK pathways) resulting in apoptosis, leading to impaired renal function [17]. CM administration also downregulated SIRT1 and upregulated peroxisome proliferator-activated receptor gamma-assisted activating factor-1α-Forkhead-box transcription factor 1 (PGC-1α-FoxO1) signaling mediated oxidative stress and apoptosis, leading to impaired renal function (Table 2) [21].

Roles of Rho/Rho-kinase (Rho/ROCK) pathway in CIN
The Rho/ROCK pathway is an important regulator in vascular smooth muscle cell contraction, cell migration, proliferation and differentiation [22]. Administration of CM in mice increased Rho/ROCK pathway activity, contributing to increased nuclear factor-kB (NF-kB) transcriptional activity, oxidative stress, inflammation and apoptosis, finally resulting in impaired renal function (Table 2) [23].

Roles of nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf-2/HO-1) pathway in CIN
The Nrf-2/HO-1 pathway is involved in many functions including mitochondrial oxidative stress, autophagy, and programmed cell death [24]. Nrf-2, when translocated into the nucleus, stimulates transcription of genes that encode detoxifying and antioxidant enzymes, contributing to cellular protection by reducing oxidative stress [25]. In CM-treated rats, the Nrf-2/HO-1 pathway was upregulated to develop adaptive cytoprotective responses to counteract tissue damage, increased oxidative stress and apoptosis caused by CM (Table 2) [26][27][28]. Fig. 2 illustrates the mechanisms involved in the pathogenesis of CIN from in vitro and in vivo reports.

Effects of statins on the prevention of CIN
Statins have been shown to exert renoprotective effects in CIN via inhibition of uptake of contrast into renal tubular cells, attenuation of endothelial dysfunction and oxidative stress, anti-inflammation, anti-proliferation of mesangial cells, and protection of podocytes [9]. Clinical studies of statins on the prevention of CIN are summarized in Table 5 and Fig. 3.
The largest randomized-controlled trial in rosuvastatin was done in 2,998 patients with type 2 DM and CKD who underwent coronary or peripheral angiography, receiving either pre and post-intervention rosuvastatin or standard care. The rosuvastatin-treated group had lower incidence of CIN and high-sensitivity CRP (hsCRP) [74]. In the PRATO-ACS trial, the incidence of CIN in non-ST elevated ACS patients undergoing CAG who receive rosuvastatin in statin-naïve patients was lower than in control group [75]. With simvastatin, the prospective randomized-controlled trials in patients undergoing CAG demonstrated that short-term pretreatment of high-dose simvastatin reduced the incidence of CIN [76,77]. Simvastatin also reduced inflammation by decreasing hsCRP, P-selectin, and intracellular cell adhesion molecule 1 (ICAM-1) [77].
Despite these promising findings, inconsistent reports exist. The PROMISS trial failed to show a difference between simvastatin and placebo with respect to a primary end point based on the mean peak increase in plasma Cr within 48 h after CAG in patients with CKD [78]. Also, another randomized-controlled trial demonstrated that short-term administration of high-dose atorvastatin with oral NAC did not decrease incidence of CIN in pre-existing CKD patients, [79] and a retrospective study, statin given before non-emergent PCI increased the incidence of CIN [80]. Similarly, a prospective cohort study in patients with or without CKD undergoing CAG demonstrated that high plasma atorvastatin or rosuvastatin was associated with increased CIN risk [81]. Therefore, currently statins are not recommended in the guidelines for CIN prevention.

Effects of antioxidants on the prevention of CIN
Many clinical trials have investigated the effects of various antioxidants on the prevention of CIN. These include NAC, ascorbic acid, sodium bicarbonate, sodium-2-mercaptoethane sulphonate (MESNA), and nebivolol. A summary of these reports is shown in Table 6 and Fig. 3.    were similar renoprotective effects for 6 months after PCI [115] Prospective, audited, multicenter regional registry

N-Acetylcysteine (NAC)
For nearly two decades, many randomized-controlled trials have investigated the roles of oral NAC for CIN prevention in patients receiving PCI or diagnostic CAG undergoing computed tomography. They demonstrated the protective effect of NAC to CIN in both low and high risk patients compared with placebo [82][83][84][85][86][87][88][89][90][91]. The prospective studies demonstrated that NAC prevented the decline in urinary NO end-products [85,91]. However, many conflicting reports exist, with no significant evident benefits of oral NAC in CIN prevention [92][93][94][95][96][97][98][99][100][101][102][103][104][105][106]. Therefore, routine administration of NAC for the prevention of CIN or longer-term adverse events after angiographic procedures is not recommended [93]. Decisive factors in NAC for CIN prevention are dosage and treatment duration. NAC was commonly given only for two days prior to CM administration in those trials. It is possible that the duration of NAC treatment was too short to be effective in counteracting CINinduced ROS production. Furthermore, since NAC has a very short plasma half-life, dosing twice daily could be insufficient to achieve consistent renal protective effects [7]. Future studies are needed to test this hypothesis. A summary of these reports on the effects of oral administration of NAC to prevent CIN is shown in Table 6.
Effects of intravenous administration of NAC on CIN protection has been investigated in patients requiring emergent CAG. In a prospective randomized-controlled study in low risk patients, short-term intravenous NAC treatment could prevent CIN [107]. However, 7% of the patients developed anaphylactoid reactions in that report. In addition, conflicting reports exist [108,109]. Intravenous NAC reduced oxidative stress after reperfusion of myocardial infarction, however it did not provide additional clinical benefit to nephropathy [108,109]. Also, intravenous NAC at higher doses could be associated with significant side effects (anaphylactoid reaction, hypotension, bronchospasm) [12]. Despite equipoise on its efficacy, NAC has been widely used in clinical practice in high risk patients due to its low cost, ready availability, easy administration, and limited toxicity in an oral form.

Ascorbic acid, sodium bicarbonate and sodium-2-mercaptoethane sulphonate (MESNA)
In a randomized, double-blind, placebo-controlled trial of patients with Cr ≥ 1.2 mg/dL undergoing CAG, the use of ascorbic acid was associated with a significant reduction in the rate of CIN [110]. For sodium bicarbonate, a randomized trial in patients undergoing CAG demonstrated that bicarbonate supplementation prevented CIN when it was given 6 h prior to CAG and administered continuously for another 6 h after CAG [111]. MESNA has the potential to act as a ROS scavenger. In a pilot study in CKD patients undergoing CAG, renal function improved after MESNA pre and posttreatment [112]. These findings indicated that these antioxidants could prevent CIN in patients with renal impairment. However, future studies may add weight to these limited reports.

Nebivolol
Although in vivo studies report the benefits of antioxidant effects by β-receptor antagonists leading to prevention of CIN, [56,57] a report from a clinical study demonstrated otherwise. In a cross-sectional casecontrol study, the patients with risk factor for CIN that received nebivolol had no change in renal function before and after CAG, and did not prevent CIN in patients undergoing CAG [113]. These inconsistent reports could be due to a small sample size from a single center and different times of follow-up on Cr since a 1 month follow up might allow the development of a tolerance to the vasodilatory effects of the drug.
In summary, this review provides a comprehensive narrative review that summarizes findings of the pathogenesis of CIN from in vitro and in vivo studies and the novel interventions for prevention of CIN. Consistent as well as controversial reports regarding the clinical findings are also summarized for potential interventions to prevent CIN. This review provides fundamental knowledge for future basic and clinical studies to find novel interventions to prevent CIN in a clinical setting. However, since our review does not include the non-English original articles it is possible that other potential interventions to prevent CIN be missing.

Conclusions
CIN is associated with adverse outcomes. These include renal replacement therapy, prolonged hospitalization, increased mortality, and increased financial burden. For this reason, the appropriate prophylactic interventions before CM administration in high-risk patients are important in reducing CIN. Since hypoxic-toxic injury, including altered renal microcirculation, medullary hypoxia and ROS-mediated cellular injury is a fundamental pathogenesis of CIN, understanding these various complex pathways could lead to prevention. Although a variety of experimental studies and clinical trials have demonstrated potential pharmacological interventions to prevent CIN, inconclusive results exist. Future       [112] α-GST, α-glutathione S-transferase; BUN, blood urea nitrogen; CAD, coronary artery disease; CAG, coronary angiography; CHF, congestive heart failure; CIN, contrast-induced nephropathy; CKD, chronic kidney disease; CM, contrast media; Cr, creatinine; CrCl, creatinine clearance; CT, computed tomography; DM, diabetes mellitus; eGFR, estimated glomerular filtration rate; GFR, glomerular filtration rate; GSH, glutathione; IV, intravenously; LDL, low-density lipoprotein; LOOHs, lipid hydroperoxides; LVEF, left ventricular ejection fraction; MI, myocardial infarction; NAC, N-acetylcysteine; NGAL, neutrophil gelatinase-associated lipocalin; NO, nitric oxide; PCI, percutaneous coronary intervention; STEMI, ST-segment elevation myocardial infarction