Analysis of Renal Platinum Content as a Novel Approach to Protect against Cisplatin Nephrotoxicity: A Review

Cisplatin (cis-diamine-dichloroplatinum (II), CDDP) is a prominent member of the effective broadspectrum antitumor drugs. However, its clinical usage is restricted due to serious side effects particularly nephrotoxicity. The vulnerability of the kidney to CDDP is almost certainly related to its primary role in the excretion of the drug as intact CDDP and its platinum containing products are excreted mainly in the urine. There is a correlation between the level of platinum in urine and nephrotoxicity because of renal uptake of the drug. Some analytical methods were applied for the determination of platinum content in biological fluids such as plasma, urine, serum, and peritoneal fluid. Studies have not documented a strong correlation between the renoprotective mechanism and the diminution of renal platinum content.


INTRODUCTION
All molecules containing platinum produce a hazardous effect on health because of the heavy poisonous metal, it is accumulated and not biodegradable by the human body [1,2]. Cisplatin (cis-diamine-dichloroplatinum (II), CDDP) is a prominent member of the broad-spectrum antitumor drugs. However, it is blamed for its prominent nephrotoxicity [3,4]. Acute and cumulative renal toxicity associated with histological damage has been shown in-vivo [5,6], in humans [7], and rat kidney slices model [8]. CDDP produces nephrotoxicity shortly after initiation of therapy or after long-term administration. In general, renal toxicity can be divided into two phases, an acute phase and a chronic phase [9]. The acute toxicity of CDDP is characterized by hypomagnesemia, hypokalemia, acute reduction in glomerular filtration rate, and high serum creatinine [10]. The chronic phase of renal dysfunction is characterized by low creatinine clearance with or without increment in serum creatinine [11]. Several studies have reported the devastating effects of CDDP as one of the most effective antineoplastic agents [3,4,6,8]. Indeed, the role of platinum uptake by the kidneys has been documented, however, the implication of CDDPuptake in the renoprotective mechanism has not been fully clarified [12]. This review explored the main analytical methods which have been approved and used for CDDP analysis in biological fluids. In addition, the review aimed to elaborate the contribution of diminution of renal CDDP content as one of the mechanisms involved for the renoprotection of antioxidants and amino acids.

Mechanisms of Cisplatin Renal
Tubules Damage

Oxidative stress and deoxyribonucleic acid (DNA) adduct formation
Histologically, the renal damage is mainly situated in the S3 segment of the proximal tubules in the outer stripe of the outer medulla and occurring rarely in the glomeruli [13]. Several in-vitro and in-vivo studies have suggested that CDDP-induced kidney injury is linked to the production of oxidative stress [12,14], lipid peroxidation [15] and reduction in the concentration of protein thiol [16] play a role in this toxicity. Two serial studies found that high lipid peroxides level is not mediated by direct membrane lipids peroxidation but was attributed to a reduction of antioxidant enzymes [17,18].
CDDP induced depletion of renal reduced glutathione GSH [16] and inhibition of antioxidant enzymes activity such as glutathione-Stransferase, glutathione peroxidase, catalase, and Cu-, Zn-superoxide dismutase in renal tissue, which shifts the cellular redox status resulting in an imbalance between free radicals and endogenous antioxidants occur leading to oxidative damage of membrane lipids [15,19]. Also, selective inhibition of glutathione biosynthesis by buthionine sulfoximine is known to enhance CDDP nephrotoxicity [20], while glutathione ester coadministration along with CDDP protects against CDDP-induced nephrotoxicity [21,22].
Another theory is based on a correlation of nephrotoxicity with structural changes in nuclear DNA of tubular cells through CDDP-induced interstrand and intrastrand DNA adducts as well as the adduct level in the kidney cells [23,24]. CDDP-induced renal toxicity is known to be of tubular origin [25], because the highest levels of CDDP -DNA adducts were seen in the renal tubules [26]. In addition, there is a good correlation between DNA adduct levels and drug efficacy as well as sensitivity in-vitro and in-vivo [27].

Renal uptake of cisplatin
The exact mechanism of CDDP nephrotoxicity is unclear. The vulnerability of the kidney to CDDP is linked to its crucial role in the excretion of both the intact drug and its platinum containing products [28]. CDDP is cleared by both glomerular filtration and tubular secretion [3], and the lower urinary excretion of CDDP might be due to a tubular reabsorptive process [29]. Thus, CDDP concentrations within the kidney exceed those in blood, which indicates accumulation of drug by renal parenchymal cells. In 1999, a strong correlation between the level of platinum in urine and nephrotoxicity because of reabsorption of the drug into the nephron was reported [

Mass Spectrometry Technique
Several analytical methods were applied for the determination of platinum content in biological fluids such as plasma, urine, serum, and peritoneal fluid. One of these methods used is the inductively coupled plasma mass spectrometry technique [42]. The detection of platinum was achieved in the linear range of 0.01-100 ng/mL, with inter-and intraday precision and accuracy (≤15%), recovery, robustness and stability. It was found that the quantification limit was 18.0 ng/mL platinum in plasma, 8.0 ng/mL platinum in ultrafiltrate and 6.1 ng/mL in urine as well as the peritoneal fluid. The spectroscopic method was proposed for the detection of CDDP in the urine, but it has some disadvantages as the time required for derivatizing is 24 hours [43]. Cisplatin was determined in the urine and plasma by quenched phosphorescence in the range 5×10−7 to 5×10−5M. [44], while the interaction between CDDP and G-quadruplex DNA was used to detect CDDP by using the fluorescence method [45].

Atomic Absorption Spectrometry
The atomic absorption spectrometry technique was applied to assess CDDP and monohydrated form in plasma. The linearity was 60-600 and 87.5-700 nM for CDDP and monohydrated CDDP in deproteinized plasma, respectively. The lower limits of quantification of both CDDP and the monohydrated CDDP were 60 and 87.5 nM, respectively. The samples were taken from the patient who received 75mgm −2 cisplatin as a 1-h intravenous infusion. [46]. Accurate and sensitive Atomic Absorption Spectrometry (AAS) methods coated with graphite tube were developed by [47,48] for analysis of CDDP in tissue and serum. Cisplatin was quantified using AAS by measuring the complex formed from its reaction with diethyldithiocarbamic acid, the complex was extracted into methylene chloride then mixed with acetonitrile to release the platinum which determined by Zeeman atomic absorption (AA) spectrophotometer [49].
An in vitro experiment to investigate the distribution of liposomal encapsulated CDDP in blood was developed by Meerum and coworkers [50]. In this method, total platinum concentration including the liposomal encapsulated platinum, protein-bound platinum released from the liposomes as well as the free platinum were assessed in plasma. In addition, a fraction of CDDP released from the liposomal carrier and the free platinum was measured in plasma ultrafiltrate by graphite furnace atomic absorption spectrometry (GF-AAS). As well CDDP was detected in the liposome and other biological fluids by (CE-ICP-MS) method [51] and by using HPLC [52] while the separation of the free CDDP from liposomal encapsulated and protein-bound was achieved by using a capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) [53]. The electro analytical method was created for analysis of CDDP, the method based upon the replacement of mercury electrode by metallothionein and the determination of cisplatin were performed by adsorptive transfer stripping technique and differential pulse voltammetry [54].
Coupled plasma atomic emission spectrometric method was applied for the analysis of both CDDP and its hydrolysis products in addition to the two methionine-platinum complexes in aqueous solutions [55]. Moreover, A number of inductively coupled plasma-mass spectrometric methods were reported for the determination of CDDP [56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71]. From the aforementioned studies, modifications have been done to increase the accuracy of CDDP analysis in the biological fluids as well as purification of the platinum being analyzed.

High-performance Liquid Chromatography (HPLC)
HPLC methods are used for the separation of CDDP and its hydrolysis products using C18 column and a mobile phase composed of 3% (v/v) methanol, 0.05mMsodium dodecyl sulfate, and pH 2.5 (adjusted with triflic acid) [72]. Two methods were reported. One needs a pretreatment procedure and the detection wavelength is 210 nm [73]. The other needs an automated column switching technique [74]. Liquid chromatography post-column derivatization assay in plasma was proposed by Farrish et al., [75] who suggested that in order to increase the CDDP stability before being analyzed on a chemically generated anion exchange column, samples were treated with acetonitrile and a citrate buffer. The reaction forms a complex which is used for isolated of CDDP on an anion-exchange column using 0.125M succinic acid-sodium hydroxide buffer pH 5.2 and methanol (2:3, v/v) as a mobile phase at 344 nm [28] or its reaction with sodium bisulfite to give products which have enhanced absorptivity at 280-300 nm.

Mass Spectroscopy
The use of the mass spectroscopy is one of the most selective and sensitive techniques used for the analysis of platinum-containing drugs, CDDP was separated with its mono and dehydrated complexes using high-field asymmetric waveform ion mobility spectrometry (FAIMS) [97]. A combination of both size exclusion chromatography-ICP-MS (SEC-ICP-MS) and ESI-MS technique have been used to detect structural information of CDDP metabolites which react with metallothionein and GSH resulting in CDDP-mediated side effects [98]. Peleg-Shulman studied the interaction between platinum and the protein by using either ubiquitin or myoglobin as model protein and identified platinum-protein adducts [99], while other studies reported the binding of CDDP to transferrin [100,101]. Moreover, the same technique was used for quantitative analysis of phospholipid alteration in resistant and sensitive cancer cells to CDDP [102]. One of the methods was applied for comparison of different methods for determination of platinum -DNA interaction and study the advantages and disadvantages of these methods [103].
Determination of platinum was established by the colorimetric method. It was based on the change of the red color result from the binding of platinum with gold nanoparticles (AuNPs) to blue, this binding prevents aggregation of AuNPs in the presence of cationic polymer. The absorbances were measured at 610 and 520 nm and the linearity was 0.24-2 μM [104].

Miscellaneous Methods
Because of the low solubility and non-volatile nature of CDDP, it is very difficult to determine it by the usual methods, and so it needs to use ESI-MS especially when it is used with the HPLC technique. Cui and his colleagues [105] applied this method to test the characteristics of CDDP and identify three hydrolysis products. Determination of CDDP in the pharmaceutical preparation and in blood samples of patients with cancer was carried out by using gas chromatography [106], the method based on the complex formation between platinum and bis(isovalerylacetone) ethylenediimine then extraction with chloroform.
Many methods were reported for analysis of CDDP by capillary electrophoresis, one of them applied to examine the behaviors of CDDP in sodium chloride solution, The reagent used for detection was 4-nitrosodimethylaniline [107], some of these methods used for separation the hydrolysis products of CDDP originated because physiological stimulation [108] as well two methods used micellar electrokinetic capillary chromatography were applied for separation of platinum in aqueous solution [109,110], and in tumor tissues [111]. Capillary electrophoresis is either used to validate the interaction between CDDP and human serum albumin [112] or to investigate the interaction between CDDP and other anticancer drugs and nucleotides [113][114][115], the absorption bands of the formed adducts were shifted compared to unmodified nucleotides.

NOVEL RENOPROTECTIVE STRA-TEGIES
As the anti-tumor activity and renal toxicity in CDDP-based chemotherapy are mediated in part by different mechanisms, selective inhibition of its nephrotoxicity might be achieved while retaining the antineoplastic activity [116]. Ibrahim and coworkers have stated that "continued aggressive high-dose CDDP necessitates investigating newer measures of preventing dose-limiting nephrotoxicity, that inhibit the administration of CDDP at tumoricidal doses" [117]. In recent years, newer therapeutic strategies are being investigated aimed at minimizing CDDP-induced nephrotoxicity while increasing its antitumor efficacy through the simultaneous supplementation of preventive agents. Such strategies may include 1) inhibition of pathways leading to activation of CDDP to a nephrotoxin, 2) reduction of renal uptake of platinum, 3) use of antioxidants to counter the effect of reactive oxygen molecules, 4) inhibition of CDDP-induced cell injury, MAPKs inhibitors, 5) inhibition of the inflammatory response by IL-10 and specific suppression of TNF-α can, 6) target inhibition of apoptotic mechanism activated by CDDP specifically in kidney cells, 7) uses of cytoprotective agents that can protect normal cells, but not tumor cells, from CDDP, 8) uses of agents that enhance cell proliferation and differentiation and finally, uses of novel therapies like serum thymic factor and amino acids.  [125]. Thus, several studies have investigated the antiapoptotic effect of many antioxidants such as dimethylthiourea (DMTU), Indole 3 carbinol, N-acetly cysteine (NAC), sodiumthiosulfate (STS), carvedilol and coenzyme Q specifically provide partial protection against CDDP-induced apoptosis [126][127][128][129][130]. The question with many studies concerning the antiapoptotic effect of antioxidants is if the antiapoptotic effect is secondary to antioxidative stress and/or has an independent mechanism. Another question to consider is can the antioxidants reduce the platinum uptake by the kidney and how much the effect will be? It was found by El Naga and Mahran that Indole-3-carbinol ameliorated the CDDP induced-nephrotoxicity through antioxidant effect without altering the cellular uptake of CDDP [130], another older study by Hannemann and coworkers reported that the antioxidant N,N'diphenyl-p-phenylenediamine (DPPD) did not reduce the uptake of platinum compounds in rat renal cortical slices [131]. Conclusively, we suggested that there was no strong evidence that the antioxidants might have their renoprotection through suppression of renal platinum uptake. Therefore, further investigation is warranted to elaborate on the involvement of renal platinum uptake in the nephroprotection mechanism.

Amino Acids and Renal Uptake of Platinum
From two decades, it had been proved that amino acids and protein-derived peptides possess vasodilatory effects on renal vessels and improve glomerular filtration rate (GFR) [132,133]. They increase RBF and GFR through an important renal vasodilator which is NO. Alanine [134], glycine, and glutathione [135] are reported to protect renal proximal tubules from hypoxic/anoxic injury.
The mechanisms involved in the cytoprotective effect of amino acids are not clarified. A study conducted by Weinberg & coworkers reported the cytoprotective effect of small amino acid including; glycine, D-alanine, L-alanine, and βalanine [136]. It was proposed that the cytoprotective effect of neutral small amino acids is attributed to their ability to influence the tertiary protein structure of renal cell membranes. They could be accumulated within the cell without disrupting pH or binding to reactive sites on intracellular proteins, and so they would correct the membrane-damaging actions of cytotoxic agents [137].
In the last years, certain amino acids have been shown to prevent CDDP-induced nephrotoxicity in vivo: L-cysteine [138], L-methionine [139]), Nacetylcysteine [126], glycine [140], L-arginine [141], N-benzoyl-b-alanine [137] and glutamine [142]. However, the nephroprotective mechanisms are not well understood. Modulation of CDDP uptake in renal tissues by sulfur-amino acids had been suggested [143], Kroning and coworkers suggested that these amino acids such as N-acetylcysteine, cysteine, methionine, and DL-homocysteine might have prevented the CDDP-induced cytotoxicity in the kidney because of their inhibition of CDDP uptake in the cultured S1, S3, and DCT cells. They also suggested the structural element R-CH(NH2)-[CH2]1 2-S-R that might play a significant role in blocking the transport of CDDP.
Besides the sulfur-containing amino acids, few studies investigated the nephroprotective effect of glycine and L-arginine through a hemodynamic and non-hemodynamic nephroprotective mechanism that involves NO production [15,141]. A study by Mahran et al. [8] has suggested for the first time the nonhemodynamic mechanisms of glycine and Larginine nephroprotection against CDDP in rat renal cortical slices through the restoration of the antioxidant cellular defense mechanism. In addition, they added another new mechanism for L-arginine nephroprotection through lowering the platinum uptake by the kidney tissue.
Furthermore, the role of organic cation transporter 2 (OCT2) has been known in the nephroprotective mechanism of some amino acids and their derivatives. The human organic cation transporter 2 (hOCT2) is highly expressed in the renal proximal tubules and plays a crucial role in the secretion of platinum cation molecules. It was discovered that a single nucleotide polymorphism in hOCT2 gene (Ala270Ser) significantly reduced the platinum transport as well as the CDDP-induced toxicity compared to the wild-type hOCT2 [144]. A study by Kim and colleagues documented that glutamine inhibited the CDDP-induced expression of OCT2 which in turn inhibiting the CDDP accumulation and thus nephrotoxicity [145].

CONCLUSION
Several studies have reported the devastating effects of CDDP as one of the most effective antineoplastic agents. Indeed, the role of platinum uptake by the kidneys has been documented, however, the implication of CDDPuptake in the renoprotective mechanism has not been fully clarified. Moreover, a number of analytical methods have been approved and used for CDDP analysis in biological fluids while studying the nephroprotective effect of several agents. In this context, we did not find a strong correlation between the renoprotection and the diminution of renal platinum content.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.