Prevention of Rhabdomyolysis-Induced Acute Kidney Injury via Attenuation of Oxidative Injury and Inflammation by Cinnamic Acid Coated Gold Nanoparticles in Mice

Acute kidney injury (AKI) is defined as complete or partial detrition in renal functions which may repercuss from injury caused by any drug, inflammation, hindrance in renal flow or ischemia (Kumar et al., 2015). Rhabdomyolysis is an important pathological process which may account for approximately 50% of cases of AKI (Bosch et al., 2009; Kim et al., 2010). Rhabdomyolysis can be defined as destruction of skeletal muscle leading to an increase in the levels of myoglobin and/or hemoglobin, and its deposition in the kidneys, resulting in severe kidney damage. This damage to skeletal muscles can also result from infections such as malaria, crush injury, drug intoxication, seizures etc. (Bosch et al., 2009). This type of kidney failure was the major reason for death of the injured soldiers in both the 1st and 2nd World Wars.

Even with the recent advances in health and medicine, currently there are no specific therapeutics available for AKI other than supportive treatment or renal transplant (Colledge et al., 2010). Although limited, the tubular epithelial cells do have the ability to regenerate and resume normal physiological function if the noxious stimulus is removed (Colledge et al., 2010; Feather et al., 2020). However, renal tissues can be irreversibly damaged if the stimulus is severe or long-term. Irreversible kidney damage accounts for 50–70% of death rate which has not declined in last ten decades (Feather et al., 2020). Preventive therapeutics are therefore an important aspect to explore for AKI which affect in lowering the mortality rate of AKI.

There are many established animal models for AKI one of which is rhabdomyolysis-induced AKI. Glycerol administration at a dose of 10ml/kg body weight intramuscularly can cause rhabdomyolysis due to oxidative injury to the renal tubular cells (Baliga et al., 1997; Ustundag et al., 2005; Fishman et al., 2012; Park et al., 2012; Cil et al., 2012). Intramuscular (IM) glycerol along with damage to myoglobin can also cause wreckage in red blood cells leading to release of hemoglobin in circulation, ultimately playing an additional role in the sequalae of kidney injury (Kim et al., 2010). Furthermore, inflammatory mediators are also activated and released in the blood. These mediators recruit leukocytes at the injury site leading to production of reactive oxygen species eventually causing necrosis of renal tissues (Chander et al., 2003). The process of inflammation is reported to be initiated by activation of NF-κB which instigates the inflammatory process via expression of various pro-inflammatory genes. Moreover, iNOS and COX-2 have been described as major activators of the inflammatory pathway (Chander et al., 2003). Henceforth, renal protective players also play an important part in AKI where Kim-1 and HO-1 expression plays a vital part in the protection of kidney morphology. This can be assessed by increment in expression of these in response to any injury to kidney cells (Cil et al., 2012). Keeping in mind the physiological response of renal cells to any insult, it can be elucidated that increment in renal protective genes and inhibition of inflammatory genes with scavenging of free radicals may halt or delay the entire process of kidney injury.

Cinnamic acid (CA) is found abundantly in Cinnamon cassia as one of its active compounds and a major constituent. The plant is used in flavours, as a synthetic azure and in pharmaceutical companies as well. Recent studies showed that CA also possesses antidiabetic activity by enhancing insulin secretion and sensitivity (Hafizur et al., 2015). In this study the selection of this compound was based on its anti-oxidant and anti-inflammatory activities.

Delivering a drug to the site of injury is a key component in treating many diseases. Because nano-particle can easily pass through cell membranes, they can provide us with improved drug bioavailability at specific sites while at the same time reducing cross-toxicity due to low dose requirements (Martis et al., 2012). Therefore, in this study we also investigated gold conjugated nanoparticles of CA.

To synthesize the CA-conjugated AuNPs, CA (1 mM solution) was mixed with HAuCl4.3H2O (1 mM solution) and stirred. After 30 minutes, 0.4 mL of 40 mM NaBH4 was mixed, resulting in a complete reduction of gold ions to form the CA-conjugated AuNPs. The CA-conjugated AuNPs were obtained after freeze drying. Their UV–Visible spectra were taken by Thermo Scientific Evolution 300 UV-Visible spectrophotometer. The shape and size of the CA-conjugated AuNPs was also determined by using Atomic Force Microscope (AFM, Agilent 5500) operating in tapping mode.

International guidelines were followed for all animal procedures and institutional ethical committee approval was obtained for proper animal use (Protocol No.: 2016-0002). Animal house facility at International Center for Chemical and Biological Sciences, University of Karachi, Karachi was used to breed and maintain male albino mice. Experimental mice were kept with free access to rodent chow and water in conventional cages water at 22-23°C with a 12-h light-dark cycle. All animals were acclimatized prior to the start of experiments.

Animals were segregated into four groups with six mice in each. Animals in test groups were pretreated with CA (50 mg/Kg) and CA-AuNPs (30 mg/Kg) for 4 days. On the fourth day, all the mice were water deprived for 24-h followed by IM injection of glycerol. 24 h later, all animals were sacrificed humanly under anesthesia (60 mg/Kg pentobarbital sodium) for further studies.

After sacrificing the animals, blood was drawn through cardiac puncture. Serum was isolated and urea-creatinine levels were measured by Reflotron® Dry Chemistry Analyzer (Roche, Switzerland) to assess renal functions in all four animal groups.

Kidney tissues were fixed in Bouin’s fixative for four h followed by dehydration in graded isopropyl alcohol. The tissues were then paraffin embedded. Six micrometer sections were cut followed by hematoxylin and eosin (H and E) and periodic acid Shiff’s (PAS) staining. For immunohistochemistry of actin and COX-2, we followed our previous protocol (Siddiqui et al., 2019).

Mouse kidney tissues were homogenized with TRIzol reagent (Life Technologies, USA) and mRNA was isolated following standard protocol. The cDNA was synthesized using the RevertAid first strand cDNA synthesis kit (Fermentas, USA). Real-time PCR studies were carried out using the Agilent Technologies (USA) for mice iNOS, NFkB p50, Kim-1 and HO-1. The primer sequences and Real Time RT-PCR protocol was followed according to our previous study (Siddiqui et al., 2019).

Statistical analysis of data was performed using SPSS version 20. All values were expressed as mean ± SEM. The data obtained from results were compared among or within groups by t-test. Significant difference was considered if the P-value was found to be of less than 0.05.

Synthesized CA-conjugated AuNPs were characterized by UV-Visible spectroscopy. The AuNPs showed characteristics surface Plasmon resonance at around 520-550 nm. The UV-Visible spectrum of CA-AuNPs (Fig. 1A) showed an absorption maximum at 527 nm indicating the formation of CA conjugated AuNPs. Size determination and morphological studies of the synthesized CA-AuNPs were carried out using AFM technique. The AFM results showed that the CA conjugated AuNPs were sphere-shaped having average size ranges from 10-30 nm (Fig. 1B).

Figure 2A and E shows normal mice kidney cortex and medulla. In animals treated with 50% glycerol at a dose of 10 mL/kg body weight, an increase in proximal convoluted tubular necrosis was observed (Fig. 2B), and the casts were found to be deposited in the loop of Henle (LH). Furthermore, damage to the LH cells was also seen (Fig. 2F). However, the animals pre-treated with CA at a dose of 50 mg/kg body weight demonstrated remarkable much less in tubular damage and protein cast depositions (Fig. 2C, G). The CA-AuNPs pre-treated (30 mg/kg) animals revealed almost complete protection against damage as shown in Figure 2D, H. The extent of tubular necrosis was also measured by the software NIS-Elements AR 3.2 (Nikon, Japan) and data showed a significant decrease (p <0.001) in tubular necrosis in both treatment groups (Fig. 3). However, it is seen that there is a notable reduction in the damage treated by CA-AuNPs at a lower dose as compared to CA alone.

The blood urea and creatinine were significantly elevated (p <0.001) in AKI in comparison with normal controls. However, the administration of CA and CA-AuNPs led to a significant decrease in the serum urea and creatinine levels (p <0.001) (Fig. 4A and B).

PAS stained sections of the kidney (Fig. 5) shows normal pattern of PCT and LH brush borders is shown in Figure 5A and E. Remarkable damage was seen in the brush borders of the AKI group (Fig. 5B and F). CA treatment decreased the damage to the brush border induced by glycerol (Fig. 5C and G). Conversely, almost thorough protection of the brush borders was seen in the animals treated with CA-AuNPs (Fig. 5D and H).

The test compound and its nanoformulation were also investigated for actin cytoskeleton immunohistochemistry (Fig. 6A-D). Figure 6A shows normal actin cytoskeleton pattern while a massive destruction of actin was observed in the animals treated with glycerol (Fig. 6B). CA was observed to reduction in actin disruption (Fig. 6C). However, the treatment of CA-AuNPs almost entirely prevented the actin cytoskeleton (Fig. 7D).

Figure 6E shows no immunohistochemical staining of COX-2 in sections of the normal group. As expected, increased expression was seen in the AKI group (Fig. 6F). As compared to AKI group, animals in CA (Fig. 6G) and CA-AuNPs (Fig. 6H) treated groups exhibited marked decrease in the expression of COX-2.

mRNA expressions of iNOS, NFκB p50, HO-1 and Kim-1 are shown in Figure 7. A significant increase in iNOS (p <0.001) and NFκB p50 (p <0.001) mRNA expression was observed in AKI control animals. In comparison to the control group, a significant decrease of iNOS (p <0.001) and NFκB p50 (p <0.001) was seen in CA and CA-AuNPs treated groups (Fig. 7A and B) despite the glycerol injection. The HO-1 mRNA level was not significantly higher in AKI group compared to the normal controls. However, the level of HO-1 was significantly higher (p <0.05) in the animals treated with CA and CA-AuNPs as compared to the glycerol treated group (Fig. 7C). The Kim-1 expression was significantly higher (p <0.05) in AKI treated group as compared to the normal controls, whereas the level of Kim-1 in CA and CA-AuNPs treated groups were noted to be significantly higher (p <0.05) than in the AKI group.

Natural or synthetic products that have been proposed to be active against AKI have been discovered using the rhabdomyolysis- induced AKI which establishes the fact of this model being widely accepted. One of the major factors for this is the adjacency with the human pathogenesis of AKI. Inflammatory cascades that are cause of necrosis in the renal cells, are consequence of oxidative injury caused to the proximal convoluted tubules by glycerol. In our study, we not only found necrotic cells but also observed the presence of condensed nuclei, thus showing the process of apoptosis in the cells. Therefore, this model was used to study the protective effects of CA and CA-AuNP on mice.

Potent anti-oxidant activity of CA has been reported previously in diabetic models of rat (Rahimi et al., 2005). Anti-oxidant property was the prime reason for selection of this compound as oxidative injury is a major factor in rhabdomyolysis-induced AKI. This anti-oxidant activity was also presented in our study with the evidence of less injury to the kidney from glycerol-induced AKI. The test compound also prevented damage to tubular brush borders concomitantly reducing serum urea and creatinine levels in comparison to AKI group. CA preserved the architecture of actin cytoskeleton present in the extracellular matrix and brush borders. Protein expression for COX-2, an initiator of inflammatory response was also inhibited by CA and CA-AuNPs. This inhibition of the inflammatory cascade further enhances the protective effect of CA and CA-AuNPs to the kidney cells.

Bioavailability has always been a major factor for exhibition of clinical activity for any moiety for a probable drug candidate. Hence, drug delivery is an important concern in clinical research practice. Many drugs have inefficient bioavailability to their specific organ or site of activity. Furthermore, the toxicities of some drugs are very high, specifically towards the liver and kidney. To address these important issues, gold nanoparticles conjugated with CA were used in this study. The gold nanoparticles appear to be safe as no apparent cytotoxicity in animals treated with gold nanoparticles alone were observed (data not shown). CA-AuNPs showed protective activity on AKI better than CA alone. Next, we also elucidated the molecular pathways of protection by CA and its gold nanoparticles and investigated the role of iNOS, NF-κB, HO-1 and Kim-1 following CA treatment in animals with AKI.

We know that nitric oxide species promote production of other inflammatory cytokines (Mungrue et al., 2004). In accordance to this, we also observed a significant rise in iNOS mRNA expression in the glycerol treated animals. Conversely, it was down regulated in CA and CA-AuNPs treated animals. This implicates that CA and CA-AuNPs treatment negatively modulates the high oxidative environment in AKI group.

NF-κB regulates transcriptions of various genes by complex mechanisms (Gilmore, 2006). It also contributes in the production of cytokines and survival of cells (Perkins, 2007; Sana et al., 2010). NF-κB is mainly responsible for inflammatory feedback mechanisms through various pathways affecting the duration and degree of the inflammation (Hayden and Ghosh, 2008; Lawrence, 2009). Findings in this study are in accordance with these reports as we also observed raised NF-κB expression in the AKI group in comparison to normal controls. Conversely, the NF-κB expression levels were noticeably decreased in CA and CA-AuNPs treated animals.

Heme oxygenase (HO) enzyme catalyzes the degradation of heme and produces iron, biliverdin and carbon monoxide (Kikuchi et al., 2005). HO-1 is induced in stress such as during oxidative burst. Protective role of HO-1 is reported during oxidative stress. It also plays an anti-inflammatory role by up-regulating interleukin-1R and interleukin-10 antagonist expression (Piantadosi et al., 2011). The cellular preventative effects of HO-1 are exerted by inhibition of a variety of immune system mediated inflammatory pathways. In this study, we observed significantly increased levels of HO-1 in CA and CA-Au-NPs treated animals as compared to normal and glycerol treated animals. This explains the protective role of the compound through HO-1 modulation.

Kidney injury molecule-1 (Kim-1) is a new kidney injury marker (Lim et al., 2013; Yang et al., 2015). The Kim-1 expression is increased in inflammation. In accordance to these reports, we also observed increased Kim-1 expression in glycerol treated animals in comparison to normal controls. All treatment groups revealed much higher mRNA expression of Kim-1. Consequently, it may have a crucial role in the inhibition of inflammation. Thus, we can conclude that CA and CA-Au-NPs have anti-inflammatory actions with protective activity against rhabdomyolysis-induced AKI.

CA prevents rhabdomyolysis-induced AKI shown by significantly lower blood urea and creatinine levels. The nano-formulations of CA (CA-AuNPs) can shield kidney architecture such as kidney tubular brush borders and actin cytoskeleton very well at a much less dose compared to CA alone. The kidney protective mechanisms of these compounds could involve downregulation of the COX-2 protein, and thus, reducing the effect of oxidant injury and inflammation. Moreover, they inhibit mRNA expressions of NF-κB, and iNOS and escalate HO-1 and Kim-1 expressions.

The authors are grateful to Dr. Panjwani Center for Molecular Medicine and Drug Research, ICCBS, University of Karachi, for providing the facility and funds to conduct the research work.

The International Center for Chemical and Biological Sciences Institutional Animal Care and Use Committee (IACUC) approved the use of animals on the ASP referenced above for one year period (04/08/2014 through 13/08/2015). The animals were taken care of for the entire study as per the guidelines of the committee for the purpose of control and supervision of of experimental animals.

The authors have declared no conflict of interest.