Background

Chronic kidney disease (CKD) is the major public health burden worldwide. Three major etiologies of CKD leading to end-stage renal disease include diabetic nephropathy, glomerulonephritis, and nephrosclerosis. Although these etiologies can be identified by clinical presentation and laboratory tests in typical cases, some cases require histological assessment to clearly differentiate these diseases, especially in patients with diabetes mellitus. Renal biopsy is the most important procedure to assess the underlying pathologies in CKD. However, it has potential complications such as bleeding, blood transfusion, and loss of renal function. Therefore, a convenient and noninvasive method is warranted for the assessment of kidney disease. Several urinary biomarkers have been suggested to be useful in assessing kidney diseases (Joachim et al., 2021; Takata et al., 2023); however, no biomarkers have been proved to be directly linked to the diagnosis of diabetic kidney disease. Recent advancements in the field of light-emitting substances have opened new approaches for the diagnosis of kidney disease (Huang et al., 2017; Yan et al., 2019). An enzyme-activatable fluorescent probe was recently developed for photodynamic diagnosis (Urano et al., 2011). This technique is based on the fluorescent emittance from hydroxymethyl rhodamine green (HMRG) upon enzymatic reaction with target aminopeptidases, such as gamma-glutamyl transpeptidase (GGTP) and dipeptidyl-peptidase (DPP). While this technique was originally developed for detecting cancer, it can be applied to renal biopsy specimens (Iyama et al., 2020; Takata et al., 2022). Our recent work has focused on investigating the feasibility of the activatable fluorescent probe for noninvasive assessment of the underlying kidney disease (Yamada et al., 2022). Urinary fluorescence was significantly stronger in patients with diabetic nephropathy than those with glomerulonephritis upon DPP-HMRG incubation. Besides, it was stronger in patients with nephrosclerosis compared to those with glomerulonephritis after GGTP-HMRG. Underlying kidney disease can be differentiated by urinary fluorescence imaging in combination with GGTP-HMRG and DPP-HMRG. This study has some limitations regarding the applicability to clinical settings: the results require further confirmation with larger studies, and a quantitative threshold needs to be determined. However, since urinary fluorescence imaging can be easily and quickly obtained, this technique would help to detect kidney diseases, particularly during health checks. We herein present a protocol for the induction of fluorescence by activatable fluorescent probe with some modifications for better clarity of the images.