Fluorescence lifetime imaging uncovers quantitative metabolic alterations in proximal tubular epithelial cells in type 2 diabetes

Kidneys are highly susceptible to metabolic changes associated with diabetes, which contribute to the progression of chronic kidney disease (CKD). Among the various cell types in the kidney, proximal tubular epithelial cells (PTECs) are particularly affected by diabetes. However, accurate detection and quantification of essential energy coenzymes such as nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide (FAD) in PTECs have been challenging. In this study, we employed fluorescence lifetime imaging (FLIM) with a phasor analysis approach to quantitatively assess metabolic activity specific to PTECs in diabetic kidneys. We analyzed NADH and FAD lifetime in PTEC cells through FLIM analysis and also analyzed metabolic changes in human kidney tissue according to the CKD stage. Furthermore, we compared metabolic changes following glucagon-like peptide-1 receptor agonist (GLP-1RA) treatment using db/db mice. Our results demonstrated a significant reduction in NADH lifetime in both the mitochondria and cytoplasm of PTECs under high glucose conditions. Phasor analysis in db/db mice revealed shortened NADH and FAD lifetimes, which were quantitatively validated by increased NADH and decreased FAD production in db/db kidneys, indicating a high redox ratio in diabetic kidneys. Additionally, the phasor plot and lifetime measurements effectively reflected metabolic alterations in db/db kidneys in response to GLP-1RA treatment. In the kidneys of patients with type 2 diabetes, the peak region of the phasor plot shifted counterclockwise toward longer lifetimes as CKD progressed compared to normal kidneys. Quantitative imaging using FLIM in diabetic kidneys enables the detection and measurement of NADH and FAD with spatial information in PTECs, thereby providing insights into the progression of diabetic kidney disease and the response to treatment.