Detecting drug-induced nephrotoxicity using simultaneous label-free autofluorescence multiharmonic microscopy.

Drug-induced nephrotoxicity (DIN) is a major cause of drug development failure, yet many cases are detected only late in clinical trials or after approval. Conventional methods to detect DIN often lack sensitivity and specificity, particularly for early or region-specific kidney injury. Here, we evaluate Simultaneous Label-free Autofluorescence Multiharmonic (SLAM) microscopy as a rapid, label-free imaging approach to quantify kidney microstructural and functional metabolic changes associated with DIN. SLAM simultaneously captures endogenous NAD(P)H and FAD autofluorescence, collagen-derived second harmonic generation, and third-harmonic generation from structural interfaces. SLAM images were acquired from the cortex and outer medulla (OM) of rat kidneys after cisplatin dosing at days 1, 6, and 29 post-administration. Imaging revealed region-specific injury, with the OM showing greater sensitivity to DIN than the cortex. The most prominent changes-tubular degeneration and hyaline casts-peaked at day 6, whereas tubular dilation and fibrosis-related features persisted to day 29. Feature-based classification captured these spatial and temporal patterns, achieving higher balanced accuracy in the OM (0.944) than in the cortex (0.860). Key model drivers included granularity and inter-channel correlations, underscoring the value of multi-channel SLAM data. Overall, these results demonstrate the potential of SLAM microscopy for sensitive, region-specific detection and characterization of DIN in preclinical safety studies. SLAM microscopy provides a rapid, label-free way to detect, localize, and classify drug-induced nephrotoxicity with improved sensitivity and regional specificity, potentially strengthening preclinical kidney safety screening and reducing late-stage development failures.