The Ginkgo biloba Extract Reverses the Renal Effects of Titanium Dioxide Nanoparticles in Adult Male Rats

The Ginkgo biloba extract (GbE) is a commercial product used as a nutraceutic herbal remedy in Europe and US. It contains 27% of the polyphenols isorhamnetin, kaempferol, and quercetin, as antioxidants. We used male adult Wistar rats (200–300 g), divided into four groups: control group (treated with 5.0 mg/kg of sodium chloride, intravenous), titanium dioxide nanoparticles (TiO2-NPs) group (5.0 mg/kg, intravenous), GbE group (10 mg/kg, intraperitoneal), and GbE + TiO2-NPs group (treated 24 h before with 10 mg/kg of GbE, intraperitoneal), followed, 24 h later, by 5.0 mg/kg of TiO2-NPs intravenously. The statistical analysis was performed using Student's t-test for grouped data with ANOVA posttest. The GbE protected renal cells against the effects of TiO2-NPs because it reversed the increased activity of γ-glutamyltranspeptidase and the enzymatic activity of dipeptidylaminopeptidase IV at all times tested (0–5, 5–24, 24–48, and 48–72 h). Also it reversed the glucosuria, hypernatriuria, and urine osmolarity at three times tested (5–24, 24–48, and 48–72). Thus, we conclude that GbE has a beneficial activity in the cytoplasmic membranes of brush border cells on the renal tubules, against the adverse effects that can be produced by some xenobiotics in this case the TiO2-NPs, in experimental rats.


Introduction
The main function of kidneys, in mammals, is the excretion of metabolic end products from the body and the regulation of extracellular fluid volume and electrolyte composition [1]. Their high blood flow, combined with their ability to concentrate solutes, exposes them to high concentration of xenobiotics present in the systemic circulation. Because of the rich blood supply of the kidneys, in relation to their mass, this organ is particularly liable to damage by toxic substances.
Once into the body, the NPs can interact with cell structures like the plasmatic membrane and cause disruption of it. Some of the biological effects of TiO 2 -NPs in nanomedicine, after intravenous injection, deliver TiO 2 -NPs into the human body: they induce pathological lesions in liver, spleen, kidneys, and brain [9]. Also, the intravenous administration of TiO 2 -NPs (5 mg/kg) in rats causes an accumulation of nanoparticles in the kidneys with the highest burden on a day 1 after exposure and remains until day 14 [10]. Furthermore, after a single oral administration (5 g/kg) in mice, these nanoparticles change some serum biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDH, and BUN), and pathology of the kidneys indicated that renal injury was induced after exposure [11].

Biochemistry Research International
One of the first ultrastructural changes seen after treatment with many nephrotoxins is sloughing of proximal tubule brush borders [12]. The enzymes -glutamyltranspeptidase ( -GTP) and dipeptidylaminopeptidase IV (DAP-IV) are predominantly located on the apical membrane (brush border) of proximal cells [13,14]. Thus, xenobiotics (in this case TiO 2 -NPs) can produce release of -GTP and DAP-IV from their site in the brush border membrane of renal tubules, causing their urinary excretion (enzymuria). Moreover, along the nephron, the reabsorption and secretion of solutes (sodium and glucose) are done by different cotransporters such as sodium-glucose sodium-phosphate, sodiumamino, Na-K-2Cl, and Na-Cl. Therefore, the damage of the cytoplasmic membrane would disrupt the function of these cotransporters. These transporters are the major participants in urine osmolarity [15].
Our hypothesis was that pretreatment with GbE as a single dose (10 mg/kg of body weight), administered intraperitoneally, would reverse the renal effects of the intravenous administration of a single dose of TiO 2 -NPs, in the kidneys of adult male rats.

Animals and Reagents.
Male adult Wistar rats (200-300 g) were used and maintained in stainless steel cages with a 12 h light/dark regime. The rats were handled according to the Guiding Principles in the Use of Animals in Toxicology.
The rats were kept with food and water ad libitum and at room temperature (24 ± 1 ∘ C). The urine was continuously collected, in vessels attached to the metabolic cages, from 0 to 5 h, from 5 to 24 h, from 24 to 48 h, and from 48 to 72 h.
Biochemical assays were as follows. The specific activity of -glutamyltranspeptidase was determined in 50 mM Tris-Cl, pH 9.0, 10 mM MgCl 2 , with 20 mM glycylglycine and -glutamyl p-nitroanilide as substrate, in a spectrophotometer at 405 nm [41]. The specific activity of dipeptidylaminopeptidase-IV was assayed in 50 mM Tris-Cl, pH 8.0, with gly-pro p-nitroanilide as substrate, also at 405 nm [42] in a spectrophotometer. The enzymatic activities were carried out at room temperature (25 ± 1 ∘ C), in 0.5 mL final incubation volume. The initial enzymatic rates were calculated from continuous recording, usually in duplicates, in a Varian UV/VIS spectrophotometer (Varian DMS 80).
Protein was measured with the Folin phenol reagent using bovine serum albumin as standard [43]. We also measured in urine the volume, the concentration of creatinine [44], the pH in a pH meter, the osmolality in a microosmometer (Osmette, Precision Systems Model 5004), and the concentration of sodium in a flame photometer (Corning M410).

Statistical Analysis.
We calculated the significance of the differences between group means with the two-tailed Student's -test for grouped data with ANOVA posttest of the urinary parameters, using the software Prism 4 (GraphPad Software Inc.); graphs were produced using Slide Write Plus version 4.0 for Windows (Advanced Graphics Software Inc.).

Results
The pretreatment with a single and intraperitoneal dose of the Ginkgo biloba extract (GbE) reversed the renal effects of a single dose of TiO 2 -NPs (5 mg/kg, intravenous), studied in the urine of adult male rats.

The Effects of GbE on the Renal Effects of Titanium Dioxide
in the Urine Osmolarity. The GbE diminished significantly ( < 0.05) the urinary hyperosmolarity produced by TiO 2 only from 5-24 h (580 ± 19 to 272 ± 69) and again from 48 to 72 h (516 ± 7 to 357 ± 20 mOsmol/L), as depicted in Figure 5. The values from 24 to 48 h were not significantly different that control or GbE-treated rats.

The Effects of GbE and Titanium Dioxide on Other Urinary
Parameters. Finally, the GbE alone did not significantly modify the concentrations of protein and creatinine, the volume, or the pH of urine, respectively, compared to control or GbE-pretreated rats, as shown in Table 1. Likewise, the GbE did not significantly modify the water and food intakes or the body weight, respectively, also compared to control or GbEpretreated rats, as depicted in Table 2.
In Figure 6, we show a scheme about the possible mechanisms of the GbE polyphenols to protect the cytoplasmic membrane from renal effects produced by the titanium dioxide nanoparticles, at the luminal side of the brush border cells, all along the renal tubules of rat kidneys.

Discussion
The administration of a single and intraperitoneal dose of the Ginkgo biloba extract (GbE, 10 mg/kg of body weight) significantly (>0.05) reversed the renal effects of a single and intravenous dose (5 mg/kg) of titanium dioxide (TiO 2 ) nanoparticles, studied in the urine of adult male rats. The dose of TiO 2 that we used is 11.8 times lower than its LD 50 [45]. TiO 2 -NPs are a fine white powder, often used as pigments or additives for ceramics, paints, paper, plastics, food, sunscreens, and toothpaste [46]. Living organisms are exposed to TiO 2 -NPs and may develop toxic effects. The toxicity of TiO 2 -NPs has mainly been studied in vitro [47][48][49][50] but with fewer studies in vivo [51] and a growing need for in vivo research on the effects of nanoparticles [52,53]. Ginkgo biloba is one of the most widely used herbal remedies in Europe and US [54]. It is well known that the GbE contains 27% of the polyphenols isorhamnetin, kaempferol, and quercetin [24][25][26][27].
We believe that the renoprotective effects of the GbE, against the effects of TiO 2 nanoparticles, are mainly due to the higher content and the interaction of the three GbE polyphenols with the cytoplasmic membrane of the brush border cells on the renal tubules and perhaps also to a synergistic effect among them.
In relation to the specific activity of -glutamyltranspeptidase ( -GTP) and dipeptidylaminopeptidase IV (DAP-IV), because they are in contact with the lumen of the renal tubules, and due to their location in the brush border membrane of renal proximal tubules [13,14], they are the most susceptible parameters to the direct adverse effect by TiO 2 -NPs, and thus, they are the most benefited by the GbE at all times tested. This can be explained because the polyphenols exert a screen effect combined with its chelating and antioxidant activity. These properties have been well studied in quercetin [71,72]. According to the other parameters, the time-related effects of TiO 2 -NPs were detected after the enzymuria, as increased concentration of the urinary glucose (glucosuria) and increased concentration of the urinary sodium (hypernatriuria), along with increased of urine osmolality. Similarly, with its polyphenols, the GbE exerted its beneficial mentioned properties [71,72], protecting and preventing a dysregulation in the function of nephron cotransporters involved in the regulation of sodium, glucose, and therefore the osmolarity, produced by the TiO 2 -NPs.
Likewise, it is possible that the GbE renoprotection is potentiated by its components as reported in vivo for their pharmacokinetics in rats [73] and for the treatment of cancer [74][75][76]. We also believe that the polyphenols of the GbE participate, as antioxidants [77], in the renoprotective effects against TiO 2 , mainly at later times [78].
We do not rule out the participation of the antioxidant effects of GbE components on the renal effects like oxidative stress, generated by nanoparticles, reported by several authors [55,[79][80][81][82].
Finally, we could not find significant differences among the control, the GbE, and the TiO 2 -treated rats for the following urinary parameters: the volume, the concentration of protein, the concentration of creatinine, or the pH. Likewise, the water and food intakes as well as the gain in body weight were not statistically different in control rats, compared with the GbE, and the TiO 2 -treated rats.
due to the interaction and protection of the GbE polyphenols with the cytoplasmic membrane on renal tubules of the male adult rats. Thus, we conclude that GbE has a beneficial activity in the cytoplasmic membranes of brush border cells on the renal tubules, against the adverse effects that can be produced by some xenobiotics, in this case the TiO 2 -NPs, in experimental rats. Therefore, our research highlights the pharmacological activity of the Ginkgo biloba extract, which can be used as an alternative treatment to protect renal cells against the toxicity produced by TiO 2 -NPs.