Even though it has been proven that cisplatin is effective for cancer therapy, its clinical use is limited by the side effects of cisplatin such as ototoxicity, peripheral neuropathy, and nephrotoxicity. Several attempts have been made to reduce the side effects of cisplatin, for example, forced hydration and diuresis, but there are still limited data regarding standard regimens for forced hydration and diuresis so that they can vary for each health facility and this can lead to uncertainty in determining the most appropriate regimen. Nephrotoxicity occurs in 34.1% of patients who had undergone cisplatin therapy for four cycles and after six cycles occurred in 51.8% of patients, according to data obtained from the National Cancer Hospital Dharmais, Jakarta, Indonesia. Nephrotoxicity is characterized by an increase in serum creatinine, changes in renal histopathology, decreased glomerular filtration rate, and potassium and magnesium levels. The mechanism of cisplatin nephrotoxicity is mainly due to cisplatin biotransformation into conjugated thiol compounds that can induce apoptosis or necrosis and inflammation characterized by the release of pro-inflammatory cytokines and chemokines.
Curcumin is a polyphenol compound obtained from turmeric (Curcuma domestica) and its safety has been proven by chronic toxicity testing at a dose of 8g/kg/d. Curcumin has been studied for its usefulness, namely as an antioxidant, anti-inflammatory, anticancer, and hypoglycemic. Curcumin through its anti-inflammatory activity can act as a renoprotector against cisplatin nephrotoxicity. Curcumin is a potent compound, but to date its clinical use is limited because of low bioavailability of around 1% after oral administration. Low bioavailability is caused by the poor solubility of curcumin in water, low intestinal permeability, and extensive first pass metabolism. The strategy to improve the bioavailability of curcumin is by forming curcumin nanoparticles. The formation of nanoparticles is expected to increase the solubility of curcumin and permeability in the intestine, thereby increasing its activity as a renoprotector.
Nanoparticles are microscopic particles that have dimensions of less than 1000nm. In general, there are two ways of nanoparticle synthesis, namely bottom–up and top–down. In this study, bottom–up nanoparticle synthesis was carried out by ionic gelation technique. Ionic gelation methods with polymers are one of synthesizing nanoparticles that are easy to control, do not require high shear stress, and do not require harmful organic solvents. Some polymers that can be used to synthesize nanoparticles are chitosan and sodium tripolyphosphate. Chitosan is a cationic biopolymer obtained from deacetylation of chitin from crab and shrimp shells. Chitosan is inert and biocompatible, so it is safe to use as a drug delivery system. Sodium tripolyphosphate is an anionic molecule that is listed as a food additive so that it is safe to use orally within certain limits. Ionic gelation method is based on ionic interactions between polymers that are positively charged chitosan with negatively charged sodium tripolyphosphate so that cross-linking between polymers and nanoparticles will occur. On the basis of the description above, this study aimed to determine the potential of curcumin nanoparticles synthesized by ionic gelation in reducing nephrotoxicity caused by cisplatin administration based on changes in serum creatinine, serum albumin, blood urea nitrogen (BUN), and kidney histopathology.
MATERIALS AND METHODS
Curcumin (Plamed Green Science, Shaanxi, China), chitosan, glacial acetic acid, sodium tripolyphosphate (NaTPP), dimethyl sulfoxide P (DMSO), ethanol, propylene methylcellulose (Na CMC), cremophor (Sigma- Aldrich, St. Louis, Missouri, United States), Sprague–Dawley rats aged 6–8 weeks (obtained from National Institute of Research and Development, Ministry of Health of Republic Indonesia), cisplatin (Mylan N.V, U.K), reagent kit and creatinine standards, reagent kits and urea standards, reagent kits and standards albumin (ReiGed Diagnostics, Gaziemir, Turkey), and 10% formalin buffer solution.
Preparation of curcumin nanoparticles. Preparation of curcumin nanoparticle was carried out at the Laboratory of Pharmaceutical Technology Formulation of Solid Preparations, Faculty of Pharmacy, University of Pancasila, Jakarta, Indonesia. One gram of curcumin was added to mixed solvent (4 mL tween 80, 20 mL glycerin, 10 mL ethanol 70%, 20 mL propylene glycol, 2 mL DMSO, and 14 mL cremophor). Then, 1% chitosan solution was added in the mixture. The mixture was stirred using a magnetic stirrer for 10min. Subsequently, 0.3% NaTPP was dripped at a speed of 1 drop per 3sec in a 300rpm magnetic stirrer to form nanoparticles characterized by homogeneous turbidity. The solution remained stirred for 15min to get a stable curcumin nanoparticle suspension.
Nephroprotection effect of curcumin and nanocurcumin
The test was carried out in the Pharmacology Laboratory, Faculty of Pharmacy, University of Pancasila, Jakarta, Indonesia after obtaining ethical approval from the Health Research Ethics Committee of the Faculty of Medicine, Universitas Indonesia (Protocol no. 0531/UN2.F/ETIK/2018). All the actions were taken by minimizing pain and suffering in experimental animals. Rats that will be used are acclimatized for 1 week in a cage so they can adapt to the new environment. During acclimatization, rats were given uniform food and drink. A total of 24 rats were divided into four groups: Group I as normal control without any treatment; Group II as negative control by administering 0.5% Na CMC for 9 days and later on 9th day cisplatin 7 mg/kg body weight (BW) was given intraperitoneally (i.p.); Group III was given nanocurcumin at a dose of 100 mg/kg BW orally for 9 days and later on the 9th day cisplatin 7 mg/kg BW was administered i.p.; (iv) Group IV was given curcumin at a dose of 100 mg/kg BW orally for 9 days and later on the 9th day cisplatin 7 mg/kg BW was administered i.p.. On the 11th day, all rats were decapitation. The blood and kidney were collected for morphologhy observed and measurement serum creatinine, blood urea nitrogen, albumin, histopathology features.
Kidney weight ratio
Weighing kidney was carried out for each group on the 11th day and then the ratio of renal weight and rat body weight was calculated.
Measurement of serum creatinine, blood urea nitrogen, albumin and histopathological features of the kidneys
50 μL of the test serum was reacted with 1000 μL of the reagent for creatinine examination in the eppendorf tube, and then homogenized. Creatinine levels were measured by Microlab 300 (Shenzhen, China). Sipper was dipped into an eppendorf tube containing test serum and the reagent. Then, the creatinine level would be measured by the device.
Blood urea nitrogen
10 μL of the test serum was reacted with 1000 μL of the reagent to examine urea in the eppendorf tube, and then homogenized. Creatinine levels were measured by Microlab 300. Sipper was dipped into an eppendorf tube containing test serum and the reagent. Then the urea content would be measured by the device.
10 μL of the test serum was reacted with 1000 μL of the reagent to examine albumin in an eppendorf tube, and then homogenized and incubated at a room temperature for 5min. The albumin level was measured by Microlab 300. Sipper was dipped into an eppendorf tube containing the serum and the reagent. Then the albumin level was measured by the device.
Kidney and ovarian organ were fixation with 10% formalin buffer for 24-48 hrs. Tissue preparations that have been processed are then stained with Hematoxylin-Eosin (HE) staining. Histopathological analysis was carried out by anatomical pathologists.
Data is presented in the form of mean ± standard deviation (SD). The homogeneity test (Levene test) with a significance value of homogeneity P > 0.05. Comparison of the variable distribution between the groups was carried out using the One Way ANOVA with a significance limit (α) 0.05. The analysis is continued by least significant difference test (LSD).
Kidney weight ratio
Kidney weight ratio was calculated by comparing kidney weight with rat body weight on day 11. The calculation results of renal weight ratio are shown in Table 1.
The LSD test results showed a significant difference in the ratio of renal weight of Group I (normal) to other groups, indicating that kidney damage occurred in Groups II (given cisplatin), III (given cisplatin + nanocurcumin), and IV (given cisplatin + curcumin). But, there were significant differences between group that only get cisplatin and group that receive nanocurcumin and curcumin. These findings suggest that the administration of nanocurcumin and curcumin as a pretreatment can improve the condition of the kidneys, but have not been able to achieve normal conditions. In addition, there was a decrease in renal weight ratio in the group that received nanocurcumin and curcumin, but there were significant differences between the two groups. Nanocurcumin is more effective in reducing the decrease renal weight ratio.
Blood urea nitrogen levels
BUN level measurements were performed on rat blood taken on the 11th day. The results of BUN measurements are shown in Table 2.
Cisplatin-treated rats showed a significant increase in the BUN level, leading to a decrease in kidney function. Rats (Groups III and IV) treated with nanocurcumin and curcumin showed a decrease in the BUN level leading to an increase in kidney function. The group that received nanocurcumin treatment showed a greater decrease in the BUN level as compared with the group receiving curcumin.
Serum creatinine levels
Measurement of serum creatinine level was carried out on rat blood taken on the 11th day. The results of serum creatinine measurements are shown in Table 3.
Cisplatin-treated rats showed a significant increase in the creatinine levels leading to a decrease in the kidney function. Rats (Groups III and IV) treated with nanocurcumin and curcumin showed a decrease in the creatinine levels leading to an increase in the kidney function. The group that received nanocurcumin showed a greater decrease in creatinine levels as compared with the group receiving curcumin.
Level of albumin serum
Measurement of serum albumin level was carried out on rat blood taken on the 11th day. The results of serum albumin measurements are shown in Table 4.
Cisplatin-treated rats showed a significant decrease in albumin values leading to a decrease in kidney function. Rats (Groups III and IV) treated with nanocurcumin and curcumin showed a greater increase in the serum albumin level leading to an increase in kidney function. The group that received nanocurcumin pretreatement showed a greater increase in the serum albumin level as compared with the group receiving curcumin.
Figure 1A and D shows a histological representation of kidneys from each group, namely the control group (A), the group that administered cisplatin (B), the group that administered cisplatin and nanocurcumin (C), and the group that administered cisplatin and curcumin (D). In the normal group, the kidneys of the rats were found in the normal shape, whereas in the group that administered cisplatin, necrosis was seen. In the group that received nanocurcumin had a renal histology structure that was almost the same as the group that received curcumin. The two groups are no different from the normal group. This shows that nanocurcumin and curcumin can reduce damage to renal histology caused by cisplatin. Nanocurcumin can reduce necrosis in epithelial cells more significantly (P < 0.01) as compared with curcumin (P < 0.05) [Figure 2].
Curcumin is a polyphenol compound obtained from turmeric (C. domestica). Curcumin has been widely studied for its benefits such as an antioxidant, anti-inflammatory, anticancer, and hypoglycemic. Curcumin through its anti-inflammatory effect can act as a renoprotector against cisplatin nephrotoxicity. Curcumin at a dose of 100 mg/kg body weight i.p. given shortly after cisplatin administration at a dose of 20 mg/kg BW i.p. showed a significant increase in kidney function parameters and kidney histopathology. In a study by Soetikno et al., curcumin at a dose of 100 mg/kg BW p.o. administered for 9 days showed an increase in the kidney function induced by cisplatin at a dose of 7 mg/ kg BW i.p. Curcumin has a low bioavailability of around 1% after oral administration. Low bioavailability is caused by the solubility of curcumin, which is very low in water, low intestinal permeability, and extensive first cross metabolism. After nano-formulation, the concentration of curcumin in the kidney was significantly increased. AUCs of curcumin powder in kidney is 168.4 µg min/g and nanoparticulate curcumin was 286.4 µg min/g. which were larger than curcumin powder. These pharmacokinetics data suggest that nanoparticulate curcumin might offer greater therapeutic effect than conventional curcumin from a pharmacodynamics perspective. The formation of curcumin nanoparticles has been able to increase the bioavailability of curcumin, thereby increasing renoprotector activity. Curcumin nanoparticles can reduce nephrotoxicity by decreasing serum creatinine, BUN, and increasing serum albumin greater than conventional curcumin in rats induced by cisplatin. Cisplatin injection damages the glomeruli by an inflammatory mechanism, which results in the increased permeability of the glomerulus and podocytes (highly specialized cells) and this is responsible for the reduced level of albumin in the blood. Pretreatment with curcumin and nanoparticle curcumin for 9th day before cisplatin treatment shows a significant increase in the plasma albumin level (P < 0.05).
The ratio of kidney weight is one of the parameters that can be used to determine the toxicity that occurs in the kidney organs. Under the conditions of kidney toxicity, kidney weight will increase in proportion to the toxicity that occurs in the kidneys. In rats exposed to cisplatin, there was a decrease in ability to concentrate urine and also in papillary hypertonicity, which was known to be the main cause of rat weight loss. Thus, the increase in kidney weight is not only due to an increase in BW but also due to the enlargement of tubular cells in the kidneys exposed to cisplatin. The increase in renal weight correlates with an increase in serum creatinine and BUN levels.
The results of measurements of urea nitrogen levels in this study showed that rats that administered cisplatin showed a significant increase in the urea nitrogen level leading to a decrease in kidney function. In the group that administered nanocurcumin and curcumin pretreatment showed a significant decrease in the urea nitrogen level leading to an increase in kidney function. Urea production mainly occurs in the liver through the urea cycle or also known as the ornitine cycle. Urea can be found in the blood and excreted through the renal tubules so that the urea nitrogen level in the blood can describe the excretory function of the kidneys. Rats that received cisplatin showed an increase in the urea nitrogen level due to a decrease in renal function and the reabsorption that occurred in the henle arch. Under normal circumstances, urea is only reabsorbed in the distal tubule and proximal tubule, wherein the proximal tubule 40%–50% of filtered urea is reabsorbed.
Rats that received cisplatin showed a significant increase in the creatinine levels leading to a decrease in the kidney function. In groups that administered pretreatment nanocurcumin and curcumin showed a decrease in the creatinine levels leading to an increase in kidney function. Creatinine is a metabolic result of creatine and phosphocreatine. Creatinine has a molecular weight of 113Da. Creatinine is filtered in the glomerulus and reabsorbed in the tubular. Plasma creatinine is synthesized in the skeletal muscle so that levels depend on muscle mass and weight. The initial process of creatine biosynthesis takes place in the kidneys involving the amino-acid arginine and glycine. According to one in vitro study, creatine was converted to creatinine in the amount of 1.1% per day. In creatinine formation, there is no mechanism for reuptake by the body, so most creatinine is excreted through the kidneys. If renal dysfunction occurs, creatinine filtration ability will decrease and serum creatinine will increase. A doubling of serum creatinine levels indicates a decrease in kidney function by 50% as well as a threefold increase in serum creatinine reflecting a decline in kidney function by 75%.
In this study, it was found that rats given cisplatin showed a significant decrease in albumin values leading to a decrease in kidney function. In the group given nanocurcumin pretreatment and curcumin showed an increase in the albumin level leading to an increase in kidney function as compared with the group that only received cisplatin. Albumin is used to measure the nephrotoxicity of cisplatin, because cisplatin can cause necrosis of podocyte cells––cells that play a role in albumin filtration in the glomerulus. The presence of necrosis in podocyte cells will cause larger pores in the glomerulus, so that proteins with large molecular weights such as albumin can pass glomerular filtration, consequently the serum albumin value will decrease.
Pathology and anatomy of rat kidneys were examined by examining the morphology of the kidneys, and then followed by histopathological examination of the kidneys. The renal morphology of each group did not show any abnormalities. Histopathological examination was then performed on the kidneys taken on the 11th day, which had been preserved in a 10% formalin buffer solution. Changes in histopathological features mainly occur in the proximal tubule because cisplatin mostly accumulates in the proximal tubule because in this part the most cisplatin transporters are expressed, namely Ctr1 and OCT2. Necrosis in cells is characterized by nucleus loss and also brush border, which is microvilli found in the proximal tubule that functions to optimize reabsorption. This is one of the causes of changes in magnesium and potassium levels when cisplatin-induced nephrotoxicity occurs.
Curcumin nanoparticles can reduce cisplatin nephrotoxicity by decreasing serum creatinine, BUN, and can increase serum albumin more effectively than curcumin. Both curcumin nanoparticles and curcumin are able to reduce changes in histopathological features because of cisplatin nephrotoxicity.
Financial support and sponsorship
This work was supported by the Ministry of Research Technology and Higher Education, National Ministry of Republic Indonesia through research grant of Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) 2019.
Conflicts of interest
There are no conflicts of interest.
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