Materials and Reagents

Pipettes (M&S Instruments, catalog numbers: F144059M, F144058, and F144055M) and pipette tips (VIOLAMO, catalog numbers: V-1000, V-200, and V-10)
1.5 mL Eppendorf centrifuge tubes
Phosphate buffer saline (PBS) (FUJIFILM, catalog number: 166-23555)
Dimethyl sulfoxide (FUJIFILM, catalog number: 041-29351)
EP-HMRG (GORYO Chemical, catalog number: GC811), which targets DPP. Dissolve in dimethyl sulfoxide at 1 mM and store at -20 °C before use (not necessary to filter)
ProteoGREEN^TM-gGlu (GORYO Chemical, catalog number: GC801), which targets GGTP. Dissolve in dimethyl sulfoxide at 1 mM and store at -20 °C before use (not necessary to filter)
Aqua-auto Kainos CRE-III plus (KAINOS, catalog number: TKA7500), for creatinine measurement

Equipment

Stereomicroscope (BioTools, catalog number: BS-3048BT)
Fluorescent unit (BioTools, catalog number: BT-ExSM)
Band pass filter (FUJIFILM, catalog number: BPB-45). This filter should be set at the light source of the fluorescent unit
Sharp cut filter (FUJIFILM, catalog number: SC-52). This filter should be set at the lens side of the fluorescent unit
Labnet C-1200 mini centrifuge (Marshall Scientific, product code: C-1200)

Procedure

Preparation of the fluorescent probe
1. Thaw EP-HMRG/ProteoGREEN^TM-gGlu stock solution at room temperature (protect from light).
2. Dilute EP-HMRG/ProteoGREEN^TM-gGlu stock solution into PBS to prepare working solution at a final concentration of 10 μM.
3. Scheme representing how the probe works is showed in Figure 1.
  
  Figure 1. Scheme of the reaction of fluorescent probes. Figure adapted from Yamada et al. (2022).
Fluorescence imaging of the urine
1. Collect a urine sample from human subjects (1 mL is enough).
2. Briefly (a few seconds) spin down to remove sediment and transfer supernatant to another tube for testing.
3. Dilute urine sample in PBS to a creatinine concentration of 1 μg/μL.
4. Mix 10 μL of diluted urine sample and 10 μL of EP-HMRG/ProteoGREEN^TM-gGlu stock solution by pipetting to prepare fluorescent working solution. The final concentration of creatinine should be 0.5 μg/μL and fluorescent probes should be 5 μM (20 μL is enough for fluorescent imaging).
5. Incubate for one minute under dark conditions at room temperature.
6. Illuminate and observe the fluorescence. An excitation wavelength of 450 nm can pass through the band pass filter; then, the fluorescent working solution is excited and emits fluorescence. The sharp cut filter eliminates redundant wavelengths except for fluorescence (~520 nm).
7. Capture images of the samples at 2.0× magnification.
8. Scheme of the fluorescent imaging is showed in Figure 2.
  
  Figure 2. Scheme of the fluorescent unit for the imaging

Notes

Prepare fluorescent working solution each time just before usage.

Acknowledgments

Protocol was based on “Fluorescence imaging using enzyme-activatable probes for detecting diabetic kidney disease and glomerular diseases”, in International Journal of Molecular Sciences (Yamada et al., 2022).

Competing interests

This research was founded by JSPS KAKENHI, grant number JP22K16242 (T.I.).

Ethics

This study was conducted in accordance with the Declaration of Helsinki and approved by the ethical committee of Tottori University Hospital (approval number: 18A135 and 20A001). Informed consent was obtained from all subjects.

References

Joachim, H. and Shilpak, M. G. (2021). The promise of tubule biomarkers in kidney disease: A review. Am J Kidney Dis 78(5): 719-727.
Takata, T. and Isomoto, H. (2023). The versatile role of uromodulin in renal homeostasis and its relevance in chronic kidney disease. Intern Med doi: 10.2169/internalmedicine.1342-22.
Yan, F., Tian, X., Luan, Z., Feng, L., Ma, X. and James, T. D. (2019). NAG-targeting fluorescence based probe for precision diagnosis of kidney injury. Chem Commun (Camb) 55(13): 1955-1958.
Huang, J., and Gretz, N. (2017). Light-emitting agents for noninvasive assessment of kidney function. ChemistryOpen 6(4); 456-471.
Urano, Y., Sakabe, M., Kosaka, N., Ogawa, M., Mitsunaga, M., Asanuma, D., Kamiya, M., Young, M. R., Nagano, T., Choyke, P. L. and Kobayashi, H. (2011). Rapid cancer detection by topically spraying a gamma-glutamyltranspeptidase-activated fluorescent probe. Sci Transl Med 3(110): 110ra119.
Iyama, T., Takata, T., Yamada, K., Mae, Y., Taniguchi, S., Ida, A., Ogawa, M., Yamamoto, M., Hamada, S., Fukuda, S., Kanda, T., Sugihara, T., Isomoto, H. and Urano, Y. (2020). A novel method for assessing the renal biopsy specimens using an activatable fluorescent probe. Sci Rep 10(1): 12094.
Takata, T., Isomoto, H., Iyama, T. and Yamada, K. (2022). A novel imaging technique for the on-site assessment of renal biopsy specimens. Bio Protoc 12: e4517.
Yamada, K., Takata, T., Iyama, T., Hamada, S., Mae, Y., Sugihara, T. and Isomoto, H. (2022). Fluorescence imaging using enzyme-activatable probes for detecting diabetic kidney disease and glomerular diseases. Int J Mol Sci 23: 8150.