The main dose-limiting side effect of cisplatin is nephrotoxicity. The utilization of cisplatin is an issue of balancing tumour toxicity versus platinum-induced nephrotoxicity. In this study, we focused on intraorgan distribution of common essential trace elements zinc, copper, and iron in healthy mouse kidneys and distribution of platinum after cisplatin treatment. Renal distribution in 12 nontreated Nu-Nu mice (males) was assessed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Furthermore, 9 Nu-Nu mice were treated with cisplatin. The order of elements concentration in kidneys was as follows: Fe > Zn > Cu. All three metals showed the higher concentrations at the cortex and medulla (28.60, 3.35, and 93.83
Some metals are essential for cell homeostasis. Zinc, iron, and copper are common essential elements and participate in the regulation of numerous physiological processes such as protein synthesis, enzymatic reactions, and antioxidant defences. These metals are useful at very low concentrations but can be toxic in larger amounts or certain forms [
Compounds containing metals have been utilized in medicine for decades, and platinum-containing chemotherapeutic agents remain key components for the treatment of various types of cancer [
For the studies of the effect of metals on the kidney, it is important to know how the levels of metals vary within different histological regions [
The method of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a useful tool for determination of element distribution in various samples. It has been utilized for quantitative imaging of platinum group elements in tumor spheroids [
In this study, we focused on the intraorgan distribution of zinc, copper, and iron in healthy mouse kidneys and distribution of platinum after cisplatin treatment by using LA-ICP-MS combined with routine microscopic techniques. Metal distribution maps obtained using LA-ICP-MS and histological samples images of the organ were correlated via the utilization of image processing.
The use of the animals followed the European Commission Guidelines. The experiments were performed with the approval of the Ethics Commission at the Faculty of Medicine, Masaryk University, Brno, Czech Republic. Renal distribution of zinc, copper, and iron was assessed in 12 eight-week-old nontreated Nu-Nu male mice (weight 25–30 g). Furthermore, 9 Nu-Nu male mice (3 control and 6 with induced tumours) were treated with cisplatin. Tumours were induced by subcutaneous injection of suspension with 105 PC-3 cells. The PC-3 prostate cancer cell line was derived from bone metastasis of a 4-grade prostatic adenocarcinoma of a 62-year-old Caucasian male and was purchased from HPA Culture Collections (Salisbury, UK). Mice received four doses of cisplatin (Cisplatin 1 mg/mL concentrate for solution for infusion, Ebewe, Austria) intraperitoneally within two weeks with the concentration of 0.5 mg/mL in volume 40
The animals were terminated two days after the last dose of cisplatin. Next, kidneys were extracted and embedded in CryoGlue Embedding medium (SLEE Medical, Mainz, Germany) and put inside liquid nitrogen for 10 s. Consequently, samples were cut on a Cryostat MTC (SLEE Medical) with thickness of 25
The determination of the distribution of elements of interests in cuts of the kidney was performed using LA-ICP-MS. All ablation experiments were done using a commercial laser ablation system UP 213 (New Wave, USA) consisting of Q-Switch Nd:YAG laser source emitting radiation with a wavelength of 213 nm and a pulse width of 4.2 nm and a moveable ablation cell (SuperCell™, washout time 1.04 s). Ablated material in the ablation cell was transported by helium with a flow rate of 1.0 L min-1 and introduced into the ICP. A quadrupole ICP-MS 7500ce (Agilent Technologies, Japan) instrument equipped with a collision cell, operated in He-mode (2.5 mL min−1) for minimization of possible interferences by polyatomic ions, was employed. Argon (0.6 L min−1) was admixed with the sample aerosol before entering the ICP torch.
The following isotopes were measured with given integration times 12C (0.01 s), 28Si (0.1 s), 56Fe (0.1 s), 65Cu (0.1 s), 66Zn (0.1 s), and 195Pt (0.1 s). Laser ablation parameters influencing resolution (laser beam diameter and scan speed) were optimized according to reference [
0.1 g of agarose powder (Agarose MP, Roche) was mixed with 5 mL miliQ water and known addition of standard reference solution Astasol (Analytika, Ltd.). This solution was heated to form agarose gel and pipetted on the quartz slide and let to dry (at room temperature) to form a thin film. Agarose gels were treated with known amounts of elements of interest (Fe, Cu, Zn, and Pt) to obtain a set of standards with 0, 10, 50, and 100 mg kg-1 of Fe, Cu, and Zn and 0, 2, 5, and 10 mg kg-1 of Pt.
The standards were ablated under the same laser ablation conditions as the samples (laser spot diameters of 100
The different ablation rate was compensated by the ablation of the whole thickness of the sample; therefore, no normalization was necessary during imaging. As published in reference [
The determination of the total concentration of elements of interests in decomposed kidney samples was performed by solution nebulization ICP-MS (a 7500ce ICP-MS spectrometer from Agilent Technologies, Japan, was employed). For this purpose, 0.1 g of kidney sample was weighted; 5 mL of HNO3 (Analpure, Analytika, Ltd.) was added. Microwave-assisted digestion (ETHOS 1; Milestone, Italy) was used for decomposition. The procedure consisted of two steps: (i) gradual increase in temperature from the room temperature up to 200°C over 15 min and (ii) 20 minutes at 200°C with the maximum input power at 1000 W. Before the analysis, the samples were diluted by factor 10. The solution was nebulized by Babington nebulizer coupled to a Scott double-pass spray chamber. The ICP-MS operating parameters were optimized with respect to getting the highest signal/noise ratio, minimal formation of oxide, and double-charged ions; the carrier gas (argon) flow rate was set to 0.75 L min−1, and the makeup gas (argon) flow rate was 0.40 L min−1. The sample uptake rate was 0.33 mL·min−1. The signals of monitored isotopes (56Fe, 65Cu, 66Zn, and 195Pt) were measured with an integration time of 0.1 s in five replicates. The quantification was done in two steps. In the first one, external calibration was used for semiquantitative determination of Fe, Cu, Zn, and Pt. This semiquantitative step was performed to allow for subsequent compensation of the matrix effect. Consequently, quantitative determination by standard addition was performed to correct the sample matrix effect. The results obtained for standard addition calibration were in accordance with results of semiquantitative analysis.
In order to determine the metal distribution in different regions of histological samples, manually drawn regions of interest have been identified in histological images. For computation of metals in each region, it is necessary to register (i.e., align) images of the distribution of metals to the histological image. Real scale of both distribution of metals and the histological image is known, but simple rescaling to the same scale provides unsatisfactory results, thanks to different translation and rotation during sample placement. Additionally, compensation for compression of the sample during slicing is needed. For compensation of these deformations, we have used image registration techniques in order to align the images.
Because of multimodal nature of the images, mutual information [
Finally, a manually drawn mask of regions in histological images was used to determine concentration values of the investigated metals to build their distribution images. Due to nonnormal distributions of these metals in the mask regions, which is caused by inaccurate labelling, the median of metal concentration values was rather used for the statistical analysis. Data were analysed using paired tests ANOVA in Matlab 2017a software (MathWorks, Natick, MA, USA).
The Fe, Zn, and Cu concentrations in homogenized kidney tissue samples was assessed by solution nebulization ICP-MS. The concentration order of these elements in the homogenized kidney samples was as follows: Fe > Zn > Cu.
In order to obtain the spatial distribution of these metals, LA-ICP-MS was performed and combined with light microscopy images of hematoxylin-eosin samples. Image processing has been utilized to colocalize LA-ICP-MS maps with histological sample image. Cortex, medulla, pelvis, and the urinary tract were assessed separately. According to Figures
Distribution of Fe, Zn, and Cu in kidney. (a) Distribution of metals in mineralised (decomposed) regions of kidney, (b) metal distribution maps obtained using LA-ICP-MS, and light microscopy image of a representative sample.
After treatment with cisplatin, the concentration of platinum in the mouse kidneys was enhanced more than 60-times (
Platinum distribution in kidneys of mouse untreated or treated with cisplatin. (a) Concentration of Pt in mineralized (decomposed) kidney samples before and after treatment with cisplatin. (b) Distribution of Pt in histological and mineralised regions of kidney, (c) metal distribution maps obtained using LA-IC/-MS and light microscopy image of a representative sample.
The comparison between individual samples of kidney was done based on the ratio of elements of interest concentrations. The main focus was on Pt as a chemotherapeutic agent and Cu, because copper transporter Ctr1 is responsible for Pt uptake [
When the Zn/Cu ratio values are compared, in Table
Summarization of Pt/Cu, Pt/Zn, and Zn/Cu median concentration ratios in homogenized and mineralized kidney samples and concentration range found using LA-ICP-MS (in parenthesis).
Treatment | Pt/Cu | Pt/Zn | Zn/Cu |
---|---|---|---|
Nonreceiving PC-3 and nontreated with cisplatin | — | — | 7 (5-20) |
Nonreceiving PC-3 and treated with cisplatin | 0.6 (0.25-2) | 0.2 (0.08-0.4) | 4 (2-18) |
Receiving PC-3 and nontreated with cisplatin | — | — | 1 (4-20) |
Receiving PC-3 and treated with cisplatin | 0.9 (0.4-4) | 0.2 (0.05-0.5) | 7 (3-13) |
The Pt/Cu, Pt/Zn, and Zn/Cu concentration ratios were also measured by scanning the sample using LA-ICP, and an increased ratio of Pt/Cu and Pt/Zn was found in the outermost part of kidneys. In case of Pt/Cu the ratio raised up to 4, whereas the Pt/Zn ratio is significantly lower (up to 0.5).
Nephrotoxicity is one of the major side effects of chemotherapy with metal-based drugs. It is associated with considerable morbidity and mortality. The renal accumulation of metals is greater than in other organs, because kidneys are a major route for their excretion [
With the use of image processing, it was possible to colocalize element distribution maps obtained by LA-ICP-MS with corresponding histological sample images for precise localization of elements in regions of the kidney.
After platinum treatment, the concentration of platinum in kidneys was enhanced more than 60 times. Platinum accumulation was as follows: cortex > medulla > pelvis > urinary tract. The prevailing cortical localization of platinum after cisplatin treatment is in good accordance with previous studies [
In conclusion, based on the results of the present study and the facts about cisplatin treatment, it can be assumed that structures present in cortex such as proximal convoluted tubule, glomerulus, and distal convoluted tubule could be severely damaged by cisplatin during cancer treatment. Furthermore, the heterogeneous distribution of Fe, Zn, Cu, and Pt in the kidney indicated that cautious sampling and precise localization of measured elements within the organ is needed in any comparative study about elements distribution in kidney. More precise localization can be achieved by combining the use of LA-ICP-MS with light microscopy and image processing.
The data that support the findings of this study are available upon reasonable request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This work was supported by funds from the Specific University Research Grant, as provided by the Ministry of Education, Youth and Sports of the Czech Republic in the year 2021 (MU-NI/A/1246/2020 and MUNI/A/1698/2020). The Czech Science Foundation supported this work (grant no. 20-02203S).
Supplementary Figure 1: metal distribution maps obtained using LA-IC/-MS and light microscopy image of a representative sample.