METHODS

Materials, reagents and characterization methods

The source of materials and reagents, and characterization methods used can be found in Supplementary Information.

Cell culture and confocal laser scanning microscopy (CLSM) imaging

Human proximal tubular epithelial cell line (HK-2) was cultured in basic Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. The cells were incubated at 37°C under 5% CO2 and split with trypsin/EDTA solution (0.25%) as recommended by the manufacturer.

Cells (5 × 108 per mL) were separately plated on 14 mm glass coverslips and allowed to adhere for 12 h. The stock solution of reporters in DMF (5 mM) was diluted with phosphate buffered saline (PBS, 10 mM, pH 7.4) with final concentration of 10 μM. The cells were then incubated with different analytes for a pre-set time at 37°C. After incubation, the cells were washed two times with PBS. CLSM imaging was performed on ZEISS LSM 880 Confocal Laser Scanning Microscope with a 60 × oil-immersion objective lens for cells. Red channel: 700 ± 50 nm, _λ_ex = 633 nm.

In vivo fluorescence imaging

Healthy mice and cisplatin-induced AKI mice model were all given an i.v. injection of DHU-AKI-3 (10 mg/kg body weight, 200 μL) and saline (control). Then, all of the mice were anaesthetized and the imaging experiments were implemented on a self-constructed small animal in vivo imaging system with 635 nm excitation laser and Andor EMCCD 597. The bladder (ventral side) of mice images were recorded at 0, 0.5, 1, 2 and 3 h.

Urinalysis in vitro

The individual differences of mice urine may be large, and therefore normalization between urine samples is required. In this study, we normalized the urine volume and injected dose as far as possible. The urine samples were collected from different experimental groups of living mice at 3 h post-treatment with various compounds. On the one hand, the collected urine samples were detected by fluorescence imaging with 635 nm excitation laser. On the other hand, urine samples were dropped onto the test strips followed by taking pictures of the test strip using the camera of smartphone. Then, the Color Recognition software immediately readout R (Red), G (Green), B (Blue) values of urine pictures. Each group has six parallel replicates.

Ethical statements

All animal experiments were conducted in accordance with the Chinese Regulations for the Administration of Affairs Concerning Experimental Animals (SYXK-2020–0018) and were approved by the Institutional Animal Care and Use Committee of Donghua University (DHUEC-NSFC-2024–44). All procedures complied with the Guide for the Care and Use of Laboratory Animals and conformed to internationally accepted guidelines (National Institutes of Health Guide for the Care and Use of Laboratory Animals, Publication No. 85–23, revised 1996). Efforts were made to minimize animal suffering and to reduce the number of animals used.