A Novel Non-invasive Qualitative Assay Using Urinary Fluorescence Imaging to Assess Kidney Disease

Materials and Reagents

1. Pipettes (M&S Instruments, catalog numbers: F144059M, F144058, and F144055M) and pipette tips (VIOLAMO, catalog numbers: V-1000, V-200, and V-10)

2. 1.5 mL Eppendorf centrifuge tubes

3. Phosphate buffer saline (PBS) (FUJIFILM, catalog number: 166-23555)

4. Dimethyl sulfoxide (FUJIFILM, catalog number: 041-29351)

5. 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)

6. 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)

7. Aqua-auto Kainos CRE-III plus (KAINOS, catalog number: TKA7500), for creatinine measurement

Equipment

1. Stereomicroscope (BioTools, catalog number: BS-3048BT)

2. Fluorescent unit (BioTools, catalog number: BT-ExSM)

3. Band pass filter (FUJIFILM, catalog number: BPB-45). This filter should be set at the light source of the fluorescent unit

4. Sharp cut filter (FUJIFILM, catalog number: SC-52). This filter should be set at the lens side of the fluorescent unit

5. Labnet C-1200 mini centrifuge (Marshall Scientific, product code: C-1200)

Procedure

1. 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).

      
      

2. 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

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