Animals and Tl administration

All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (approval No. A2023-080C) and were performed in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Eight-week-old male Wistar rats weighing 220–250 g (Oriental Yeast Co. Ltd., Tokyo, Japan) were maintained in standardized conditions (12-h light/dark cycles, 25 °C) with ad libitum access to water and food. The rats were randomly divided into control and Tl groups. Each rat received a single intraperitoneal injection of either double-distilled water or 30 mg/kg thallium sulfate (Tl2SO4) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) dissolved in double-distilled water, and samples were collected 2 or 5 days later. For the 2-day protocol, each group included four male rats. For the 5-day protocol, 15 and 8 male rats were included in the Tl and control groups, respectively. Three or four rats per group were used for each analysis. The 30 mg/kg Tl dose was selected based on the dose used in a previous study of acute Tl toxicity (Danilewicz et al. 1979; Leung and Ooi 2000). Tl distribution and production of reactive oxygen species (ROS) in the kidneys were also analyzed in another group of rats 4 h after injection of 120 mg/kg Tl2SO4 with or without pre-administration of 20 mg/kg of intraperitoneal furosemide as a Na–K–Cl cotransporter 2 (NKCC2) inhibitor (Dagan et al. 2009). The rats were euthanized at the specified time-point by intraperitoneal sodium pentobarbital (100 mg/kg) administration and their kidneys were excised (Fig. 1). Body and kidney weights were recorded for each rat.

Fig. 1

Severely abnormal kidney function 5 days after Tl administration. Timeline of the experimental procedure (a). Eight-week-old Wistar rats received a single administration of 30 mg/kg or the same volume of double-distilled water and were sacrificed 2 or 5 days later, with kidneys collected for subsequent analysis. Kidney weight 5 days after Tl administration (b). Analysis of blood (c) and urine (d) samples collected 5 days after Tl administration of rats. Each bar represents the mean and standard deviation. Ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001. ALB albumin; BUN blood urea nitrogen; Ca calcium; Cl chloride; Cre creatinine; IP inorganic phosphorus; K potassium; Mg magnesium; Na sodium; NAG N-acetyl-β-D-glucosaminidase; TP total protein; U- urinary level of; UA uric acid; UN urea nitrogen

Blood/urine analysis and kidney ultrasonography

Blood samples (anticoagulant in the syringe: 1.5 mg/mL K3-EDTA) and urine samples (12-h metabolic cage at 25 °C) were collected from the rats 5 days after administering Tl (30 mg/kg). The plasma and/or urine levels of albumin (Alb), blood urea nitrogen (BUN), creatinine (Cre), inorganic phosphorus (IP), uric acid (UA), sodium (Na), K, chlorine (Cl), calcium (Ca), total protein (TP), magnesium (Mg), and N-acetyl-β-D-glucosaminidase (NAG) were then measured according to the standard methods of Oriental Yeast Co. Ltd. (Tokyo, Japan). Urine proteins were fractionated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), with the loading sample corrected for specific gravity.

Ultrasound (Noblus, Hitachi-Aloka Medical Ltd., Tokyo, Japan) was used to observe kidney stones on days 2 and 5 after administering Tl.

Detection of mitochondrial production of ROS

Rats in the 4-h group were euthanized 4 h after administering Tl2SO4. Their kidneys were removed and sliced into 10-μm-thick cryosections. Mitochondrial ROS production was assessed using a fluorometric mitochondria superoxide assay. Cryosections were incubated with MitoROS 520 (AATBioquest Inc, Cosmo Bio Co., Tokyo, Japan) in assay buffer for 1 h at 37 °C and observed under a fluorescence microscope (Keyence BZ-9000, Keyence, Osaka, Japan) with an excitation wavelength of 540 nm and emission wavelength of 590 nm.

Histological analysis and immunohistochemistry

Central short-axis cross-sections of the rat kidney were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 2.5-µm-thick sections. For histological analysis, the sections were stained with hematoxylin and eosin (H&E), von Kossa, and Alizarin red for Ca crystals and with De Galantha for UA crystals. Immunohistochemical (IHC) analysis was performed as previously described (Taal et al. 2012; Unuma et al. 2013a, b). Paraffin-embedded sections were deparaffinized, and antigenicity was retrieved by microwave heating. The sections were incubated with a 1:100 dilution of rabbit polyclonal anti-Ca2+-ATPase antibody (Cosmo Bio Co. Ltd., Tokyo, Japan), followed by a 1:400 dilution of horseradish peroxidase (HRP)-labeled anti-rabbit Ig antibody (Dako, Glostrup, Denmark). HRP labeling was detected using a peroxidase substrate solution with 0.8 mM diaminobenzidine and 0.01% H2O2 (Tojo et al. 2008).

Electron microscopy

Electron microscopy of the rat kidney was performed as previously described (Tojo et al. 2008; Unuma et al. 2013a, b). Briefly, central short-axis cross-sections of the kidney were fixed with 2.5% glutaraldehyde, followed by 1% osmium tetroxide, and embedded in epoxy resin. Ultra-thin sections were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (HT7800, Hitachi High-Tech Co., Tokyo, Japan). Epoxy resin blocks or 200-μm vibratome slices from 2.5% glutaraldehyde-fixed kidneys were directly observed using low-vacuum scanning electron microscopy (LVSEM, TM4000Plus, Hitachi High-Tech Co., Tokyo, Japan) with an acceleration voltage of 10 kV (Tojo et al. 2023).

Elemental analysis of the kidney, urinary crystals, and Tl distribution and imaging

After Tl administration, elemental analysis of the kidney and urinary crystals on 200-μm vibratome kidney slices and urinary sediment was performed using LVSEM (TM4000Plus or TM3030) equipped with an energy dispersive X-ray spectroscopy system (AZtecOne software, Oxford Instruments Nanoanalysis, High Wycombe, UK) at an accelerating voltage of 15 kV in the highest beam current mode (Lens mode 4) in a vacuum (< 50 Pa). Tl distribution in the kidneys of Wistar rats was assessed 10-μm-thick cryosections of the kidneys harvested 4 h after 120 mg/kg Tl2SO4 administration using particle-induced X-ray emission (PIXE).

Ca, Cl, K, phosphorus (P), sulfur (S), and silicon (Si) distributions were determined by micro-PIXE (μPIXE) analysis using a Model OM-2000 microbeam scanning PIXE system (Oxford Microbeams Ltd., Oxford, UK) with an Si (Li) X-ray detector (Gresham Sirius80, active area: 80 mm2; Gresham Power Electronics, Salisbury, UK) (Ishikawa et al. 2009). Tl, zinc (Zn), and iron (Fe) distributions were determined by μPIXE analysis using a CdTe X-ray detector (XR-100 T-CdTe, active area: 25 mm2; Amptek, Bedford, MA, USA), with high X-ray absorbency for detecting elements with high Z atomic numbers and high density (5.85 g/mL) for efficient detection of X-rays (10–20 keV) emitted by heavy elements (Homma-Takeda et al. 2010). Elemental images were constructed using the intensity data of the Kα (laser ablation for Tl) lines at each point by scanning the specimens under the following conditions: proton energy, 3.0 MeV; integrated current, 0.2 μC; and beam size, 1 × 1 μm. Dissolution studies were conducted on unstained paraffin-embedded rat kidney sections to identify the composition of Ca-containing crystals in the outer medulla using 10% acetic acid, 10% hydrochloric acid, and 10% sodium hydroxide.

DNA microarray and qPCR analysis

RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and purified using an RNeasy RNA Purification Kit (Qiagen, Hilden, Germany) for DNA microarray analysis. The RNA integrity was assessed using a BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and hybridized using a Clariom S array (Thermo Fisher Scientific). The DNA microarray results were analyzed using Transcriptome Analysis Console software (Thermo Fisher Scientific). cDNA was synthesized for quantitative reverse transcription-mediated real-time polymerase chain reaction (qPCR) analysis using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). A StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) was used for qPCR. The relative abundance of RNA was computed, and the amplification products were detected using the comparative cycle threshold (Ct) method and SYBR Green fluorescence dye. Supplementary Table 1 lists the primers used.

Statistical analysis

Dunnett’s post-hoc test was used for comparisons between groups. Two-tailed P values < 0.05 were considered statistically significant. Statistical analyses were performing using GraphPad Prism (Version 9.0.0, GraphPad Software, San Diego, CA, USA).