INTRODUCTION

Acute kidney injury (AKI) is a common and life-threatening condition, marked by a sharp loss of renal function. It affects ∼15% of hospitalized patients and over 50% of those in intensive care units (ICUs), with high morbidity and mortality [1,2]. Since the standard biomarkers of AKI, blood urea nitrogen (BUN) and serum creatinine (SCr), do not exhibit noticeable changes until the glomerular filtration rate (GFR) has decreased by at least 50%, current diagnostic approaches are accurate and effective only in the advanced stage [3,4]. The renal function has already suffered severe and irreversible damage, potentially progressing to kidney failure by the time AKI is diagnosed clinically. Studies have shown that kidney failure accounts for ∼2 million deaths annually [5]. Thus, effective diagnosis of early AKI is crucial for timely therapeutic intervention and alleviating the risk of mortality.

Noninvasive imaging techniques such as positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) have been widely employed to assess renal function by monitoring the clearance of contrast agents [6–8]. However, their widespread applications are limited by equipment inaccessibility, insufficient spatiotemporal resolution, nonspecific accumulation of ‘always-on’ signals, and potential radiation risk [7]. In contrast, activatable optical probes offer high sensitivity, rapid response, and modular design, making them attractive for molecular sensing and disease diagnosis [9–12]. Yet a key point to their rational design lies in identifying suitable activators. Specifically, reactive oxygen species (ROS), established as early indicators of AKI due to their abnormal fluctuations are more sensitive than proteins [13–16], serve as sensitive activators that have spurred the development of various ROS-activated optical probes for AKI diagnosis (see Table S1 in the Supporting Information (SI)) [17–23]. However, most ROS-activated probes developed for AKI diagnosis employ a single reporter for either in vivo optical imaging or in vitro urinalysis, lacking the capability for synergistic diagnosis (Fig. 1a). On the one hand, the single-reporter-based diagnostic strategy is vulnerable to interference from frequent urination, leading to poor in vivo diagnostic precision [24]. On the other hand, the diagnostic specificity of urinalysis remains limited due to the lack of direct correlation between urine signals and the kidney’s actual damage in situ [25]. Therefore, a two-pronged strategy based on a unimolecular dual-reporter design simultaneously combining in vivo monitoring and in vitro urinalysis is anticipated to greatly improve the accuracy and timeliness of early AKI detection. However, owing to the challenges of achieving stable signals under conditions of frequent urination and the difficulties in constructing multifunctional probes with crosstalk-free multiplexed output, the two-pronged strategy has remained unrealized.

Figure 1.: Design and mechanisms of unimolecular dual-reporter probes with crosstalk-free multiplexed signal output for precise diagnosis of early AKI. (a) The design strategy of unimolecular probes for diagnosing AKI. (b) The chemical structure of DHU-AKI-3 and its released two reporters activated by ROS. (c) Schematic illustration showing the integration of in situ and in vitro detection of DHU-AKI-3 by crosstalk-free multiplexed output for precise diagnosis of early kidney injury in a living mouse.

To overcome these challenges, we developed a unimolecular dual-reporter platform that enables crosstalk-free multiplexed signal output to allow for in situ long-term imaging and portable urinalysis of early AKI (Fig. 1a). Each probe is tailored by three key blocks: photoacoustic (PA) indicator block (reporter 1: croconic acid (CA), absorption bands at ∼800 nm) [26–28], ROS-activated near-infrared fluorescence (NIRF) block (reporter 2: methylene blue (MB)) [29,30], and hydrophilic block (polyethylene glycol, PEG), which is essential for renal clearance (Scheme S1) [31]. The representative probe DHU-AKI-3 exhibits a PA signal (CA, reporter 1) and can be activated rapidly by ROS to release the ‘turn-on’ NIRF signal (MB, reporter 2) because of the cleavage of the urea bond by ROS (Fig. 1b). Owing to its high hydrophilicity, DHU-AKI-3 is predominantly filtered through the glomerulus and reabsorbed by renal tubules, ensuring preferential accumulation in kidneys. Following systemic administration, DHU-AKI-3 is metabolized primarily by renal clearance and activated by ROS in injured kidneys, swiftly releasing the CA indicator and the blue-colored MB fluorophore (Fig. 1c). Subsequently, the water-soluble MB (reporter 2) is rapidly excreted into urine, enabling portable urinalysis in vitro, whereas the hydrophobic CA (reporter 1) precipitates in the damaged kidney for in situ PA imaging. The precipitation-induced retention effect prolongs the intrarenal signal duration while facilitating the validation of portable urinalysis results [32]. This unimolecular dual-reporter two-pronged strategy provides an accurate and portable approach for early diagnosis of AKI, highlighting its potential for real-time monitoring of AKI progression at the point of care.