Rational design of an activatable dual-color fluorogenic probe for revealing the interaction of adenosine-5′-triphosphate and peroxynitrite in pyroptosis associated with acute kidney injury

Author links open overlay panelYuxia Zou 【邹玉霞】a b 1,Tingting Duan【段婷婷】 a 1,Yan Wang b,Qing Ye b,Yiying Li a,Xiaofeng Wang 【王晓凤】a,Xuan Liu a,Xuejun Zhou【周学军】 a,Fabiao Yu【于法标】 a c,Heng Liu 【刘恒】a c

• aKey Laboratory of Emergency and Trauma of Ministry of Education, Department of Otolaryngology, Head and Neck Surgery, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 571199, China

• bShengli Clinical Medical College of Fujian Medical University, Department of Otolaryngology, Head and Neck Surgery, Fujian Provincial Hospital, Fuzhou 350001, China

• cKey Laboratory of Hainan Trauma and Disaster Rescue, Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China

Received 11 April 2024, Revised 17 June 2024, Accepted 24 July 2024, Available online 25 July 2024, Version of Record 26 July 2024.

https://doi.org/10.1016/j.snb.2024.136367

Highlights

• •An ideal NIRF probe P2 with dual-color fluorescence emission harnessed for the simultaneous detection of ATP and ONOO-.

• •An interrelationship between ATP and ONOO- during pyroptosis was verified.

• •P2 was successfully applied to monitor ATP and ONOO- levels in the AKI mice model.

Abstract

ATP and ONOO- play unique roles in various biological events and exhibit notable interactions. To date, there is no available chemical tool for investigating the correlation between ATP and ONOO- concentrations in pyroptosis associated with acute kidney injury (AKI). Herein, we designed a novel dual-color near-infrared fluorescent (NIRF) probe P2 for simultaneous visualization of ATP and ONOO- both in vitro and in vivo. Unlike previously reported single-site fluorescent probes, P2 enabled concurrent imaging of ATP and ONOO- in two distinct fluorescence channels, with emission wavelengths centered at 585 and 690 nm, which greatly reduced spectral cross-talk. Employing a HK-2 pyroptosis model, a significant interaction between ATP and ONOO- was unveiled. Elevated ONOO- production was found to correlate with decreased ATP levels; conversely, an increase in ATP levels was associated with rapid ONOO- scavenging. Remarkably, P2 allowed the assessment of cellular hypoxia by monitoring ATP and ONOO- concentrations. The commercial ONOO--scavenger uric acid showcased a protective effect on HK-2 cells via inhibition of the cellular pyroptosis pathway. Furthermore, P2 was successfully employed for imaging of ATP and ONOO- in the AKI mice model. Our findings confirmed that renal ischemia-reperfusion triggered a rise in ONOO- levels, concurrent with a decline in ATP levels. Surprisingly, the cells exhibited a compensatory recovery of ATP levels as the reperfusion time was prolonged. These results suggested the newly devised P2, as a pivotal chemical tool for the simultaneous monitoring of ATP and ONOO-, might open new avenues for disease diagnosis and treatment.

Graphical Abstract

Introduction

Adenosine-5′-triphosphate (ATP), primarily synthesized through cellular respiration, stands as a vital energy source for organisms. Disruption of ATP homeostasis is closely related to oxidative stress, which arises from the production of reactive oxygen species (ROS) [1], [2], [3]. Notably, peroxynitrite (ONOO-), an important ROS, is produced in response to stressful inflammation in vivo, further exacerbating the inflammatory response and causing cellular and tissue damage [4], [5]. In recent years, the emergence of pyroptosis, a novel form of programmed cell death observed in inflammatory cells, has attracted considerable attention. Often referred to as cellular inflammatory necrosis, pyroptosis triggers the activation of multiple caspases through inflammatory vesicles, leading to the cleavage of gasdermin family members, including GSDMD, culminating in cell death [6], [7]. This process plays a significant role in inflammatory-related diseases, such as atherosclerotic, neurological, and urological diseases [8], [9], [10]. Pyroptosis can be initiated by various pathological conditions, including oxidative stress. ATP is a key molecule in the non-classical pyroptosis pathway, and intracellular ATP levels tend to decrease during pyroptosis, which may be linked to cellular energy metabolism and oxidative stress [11], [12], [13]. As such, investigating changes in ATP and ONOO- levels in pyroptosis is essential to elucidate their mechanisms of action and relationships with various diseases.

The involvement of pyroptosis in the progression of acute kidney injury (AKI) has been reported [14], [15], [16]. AKI encompasses a group of clinical syndromes characterized by a sudden and profound deterioration in renal function, resulting in increased serum creatinine, decreased urine output, vascular dysfunction, intense inflammatory response, and tubular epithelial cell injury [17], [18]. Early detection and elimination of risk factors for acute tubular necrosis are crucial in preventing AKI, considering its diverse etiologies, with acute ischemia being one of the most common. Renal ischemia can damage vascular endothelial cells through an inflammatory response or inflammatory mediators produced by renal tubular cells, making ischemic AKI a stress-inflammatory disease. The release of superoxide anion and nitric oxide from glomerular capillary endothelial cells upregulates the levels of ONOO- through diffusion reactions in AKI. ONOO-'s potent oxidative properties can induce apoptosis and necrosis of renal tubular epithelial cells and activate inflammatory reactions, leading to oxidative damage of the glomerular filtration membrane [19]. Normal renal cells require high levels of ATP to maintain physiological functions. However, in the context of AKI, the concentration of ATP decreases significantly due to the ischemic and hypoxic state of renal tissues, affecting intracellular metabolism and functions [20], [21], [22]. Previous studies have shown that ONOO- decreases ATPase activity, inhibits ATP synthesis, and ultimately downregulates ATP levels in renal tissue [23], [24]. Consequently, ATP and ONOO- are implicated in the onset and progression of AKI, and a detailed study of their interaction mechanisms will be beneficial for the early diagnosis and treatment of AKI [25].

For the past few years, numerous single-site near-infrared fluorescent (NIRF) probes have been developed for the specific monitoring of ATP or ONOO- levels in cells or in vivo [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43]. Nevertheless, NIRF probes capable of imaging both ATP and ONOO- with minimal emission spectra crosstalk are rare [44]. Addressing this challenge, we engineered two structurally novel dual-color readout NIRF probes by integrating rhodamine and methylene blue into a molecular backbone via diethylenetriamine or 1-(2-aminoethyl) piperazine linker. Among them, P2 offered superior anti-interference performance. Even in the simultaneous presence of ATP and ONOO-, P2 was able to differentiate between ATP and ONOO- with minimal spectral overlap in two distinct fluorescence channels, which greatly reduced the output of false-positive fluorescence signals in the detection process. The reaction of ATP or ONOO- with P2 triggered rhodamine ring-opening or methylene blue deformylation, which correspondingly showed intense fluorescence signals at 585 and 690 nm. This spectral change provided an intuitive and sensitive means of detecting ATP and ONOO- in cells and mice. Leveraging P2, it was not only possible to distinguish normal from cancer cells but also verified the existence of intracellular ATP and ONOO- interactions. Importantly, through dynamic monitoring of ATP and ONOO- level fluctuations in pyroptosis, uric acid (UA) was found to be a potential inhibitor of pyroptosis. For the first time, P2 was employed to demonstrate a negative correlation between the expression levels of ATP and ONOO- in AKI, characterized by increased ONOO- levels and decreased ATP levels. Overall, this innovative dual-color activated NIRF probe P2 afforded an indispensable chemical tool for elucidating the complex roles of ATP and ONOO- in pyroptosis associated with AKI.

Scheme 1. (a) The molecular structure of P1 and P2. (b) The strategy for designing dual-color fluorogenic probe P2 for ATP and ONOO-.

Fig. 1. Spectral characterization of 10 μM P1 or P2. (a-f) UV–vis and fluorescence spectra of P1 or P2 in the absence or presence of ONOO- (25 μM), ATP (15 mM), 15 mM ATP + 25 μM ONOO-, 25 μM ONOO- + 15 mM ATP. (g) Fluorescence responses of P2 to increase concentrations of ATP from 0 to 15 mM. (h) Time-dependent fluorescence intensity of P2 in the presence of ATP (0, 5, 10, 15 mM). (i) Fluorescence enhancement at 585 nm of P2 upon treatment with different potential interfering species: 1) blank; 2) ADP (10 mM); 3) AMP (10 mM); 4) H2PO4- (500 μM); 5) HPO42- (500 μM); 6) PO43- (500 μM); 7) CO32- (500 μM); 8) SO42- (500 μM); 9) NO3- (500 μM); 10) Cl- (500 μM); 11) Na+ (500 μM); 12) K+ (500 μM); 13) Mg2+ (200 μM); 14) Ca2+ (200 μM); 15) Zn2+ (200 μM); 16) GSH (1 mM); 17) D-glucose (1 mM); 18) ATP (15 mM). (j) Fluorescence responses of P2 to increase concentrations of ONOO- from 0 to 25 μM. (k) pH influence on fluorescence intensity at 690 nm of P2 before and after the addition of 25 μM ONOO-. (m) Fluorescence enhancement at 690 nm of P2 upon treatment with different potential interfering species: 1) blank; 2) H2PO4- (500 μM); 3) HPO42- (500 μM); 4) PO43- (500 μM); 5) CO32- (500 μM); 6) SO42- (500 μM); 7) NO3- (500 μM); 8) Na+ (500 μM); 9) K+ (500 μM); 10) Mg2+ (200 μM); 11) Ca2+ (200 μM); 12) Zn2+ (200 μM); 13) GSH (1 mM); 14) H2O2 (100 μM); 15) ClO- (100 μM); 16) ·OH (100 μM); 17) 1O2 (100 μM); 18) ONOO- (25 μM). λex/em = 530/585 nm (ATP channel), λex/em = 630/690 nm (ONOO- channel).

Fig. 2. Fluorescence imaging of ATP and ONOO- interactions in CNE1 cells. (a) The cells were treated with SIN-1 (1.0 mM), SIN-1 (1.0 mM) and UA (500 μM), Omy A (25 μM), Omy A (25 μM), and ATP (10 mM) for 1.0 h each, respectively, and then stained with P2 (20 μM) for 20 min. (b) Average fluorescence intensity of P2 labeled cells in images A (Ch 1, ATP). (c) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 2, ONOO-). The data were shown as mean ± S.D. (* P < 0.05, ** P < 0.01, **** P < 0.0001, n = 3). Scale bar: 20 μm.

Fig. 3. Fluorescence imaging of ATP and ONOO- in normal and cancer cells. (a) NP69, CNE1, CNE2, and 5–8 F cells were incubated with P2 (20 μM) for 20 min. (b) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 1, ATP). (c) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 2, ONOO-). The data were shown as mean ± S.D. (*** P < 0.001, **** P < 0.0001, n = 3). Scale bar: 20 μm.

Fig. 4. Oxygen deprivation-induced pyroptosis of HK-2 cells. (a) The cells were pretreated with different concentrations of CoCl2·6 H2O (0.1 mM, 0.3 mM, 0.6 mM) for 24 h, and then stained with P2 (20 μM) for 20 min. (b) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 1, ATP). (c) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 2, ONOO-). (d) Cell viability of HK-2 cells upon treatment with different concentrations of CoCl2·6 H2O for 24 h. (e) Various concentrations of CoCl2·6 H2O pretreated HK-2 cells for 24 h induced the protein levels of C-caspase 1, N-GSDMD, and HIF-1α. (f - h) Relative expression of C-caspase 1, N-GSDMD, and HIF-1α proteins in HK-2 cells pretreated with different concentrations of CoCl2·6 H2O for 24 h. The data were shown as mean ± S.D. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 3). Scale bar: 20 μm.

Fig. 5. Oxygen-glucose deprivation-induced pyroptosis of HK-2 cells. (a) The cells treated with oxygen-glucose deprivation for different times (1 h, 2 h) were then stained with P2 (20 μM) for 20 min. (b) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 1, ATP). (c) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 2, ONOO-). (d) Cell viability of HK-2 cells under different times of oxygen-glucose deprivation. (e) Different oxygen-glucose deprivation times induced protein levels of C-caspase 1, N-GSDMD, and HIF-1α in HK-2 cells. (f - h) Relative expression of C-caspase 1, N-GSDMD, and HIF-1α proteins in HK-2 cells under various oxygen-glucose deprivation times. The data were shown as mean ± S.D. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 3). Scale bar: 20 μm.

Fig. 6. Protective effect of UA on HK-2 cells post-I/H. Control group: no treatment; 2 h + DMSO: 2 h of I/H while adding 5 μL of DMSO per ml of the medium; 2 h + UA: 2 h of I/H with simultaneous addition of UA (500 μM). (a) The cells treated with oxygen-glucose deprivation for different times (1 h, 2 h) were then stained with P2 (20 μM) for 20 min. (b) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 1, ATP). (c) Average fluorescence intensity of P2 labeled cells in images (a) (Ch 2, ONOO-). (d) Cell viability of HK-2 cells under 2 h + DMSO, 2 h + UA treatments. (e) 2 h + DMSO, 2 h + UA induced protein levels of C-caspase 1, N-GSDMD, and HIF-1α in HK-2 cells. (f - h) Relative expression of C-caspase 1, N-GSDMD, and HIF-1α proteins in HK-2 cells under 2 h + DMSO, 2 h + UA treatments. The data were shown as mean ± S.D. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 3). Scale bar: 20 μm.

Fig. 7. (a) An illustration of the experiment design for the mice model. (b) Imaging of ATP and ONOO- in the kidney of unilateral ischemic AKI mice. Acute ischemia of the left kidney for 15 min followed by reperfusion for 24 h, 48 h, 72 h; no treatment of the right kidney. (c) Average fluorescence intensity of Ch 1 images in panel (b). (d) Average fluorescence intensity of Ch 2 images in panel (b). (e, f) H&E staining results of the left and right kidneys (72 h of reperfusion). (g) Imaging of ATP and ONOO- in the kidney of bilateral ischemic AKI mice. Acute ischemia of the right and left kidney for 15 min followed by reperfusion for 24 h and 48 h. (h) Average fluorescence intensity of Ch 1 images in panel (g). (i) Average fluorescence intensity of Ch 2 images in panel (g). The data were shown as mean ± S.D. (n = 3). (j, k) H&E staining results of the left and right kidneys, respectively. The illustration (a) was created with the help of BioRender.com

4. Conclusion

In summary, we have disclosed a novel NIRF probe P2 with dual-color fluorescence emission and harnessed it for the simultaneous detection of ATP and ONOO- in vitro and in vivo. P2 possessed outstanding optical properties for imaging intracellularly endogenous and exogenous ATP and ONOO- and distinguishes between cancerous and normal cells. The research unveiled an interplay between ATP and ONOO-, with an imbalance closely related to pyroptosis and AKI. Utilizing the dual-color imaging capability of P2, the dynamics of ATP and ONOO- during pyroptosis were sensitively monitored. In the CoCl2·6 H2O and oxygen-glucose deprivation-stimulated HK-2 cell experiments, not only did WB analysis reveal that cellular pyroptosis was triggered via the caspase-1-dependent classical pathway, but also a significant increase in the level of ONOO- and a decrease in the level of ATP were observed. Moreover, UA was found to have a prominent cytoprotective effect, suggesting its potential as a therapeutic agent through the inhibition of pyroptosis. Benefitting from P2, we successfully tracked changes in ATP and ONOO- levels in the AKI mice model. Specifically, ONOO- levels were dramatically elevated in the kidneys of AKI mice, while ATP levels initially declined and then gradually recovered with the prolongation of reperfusion time. These findings further demonstrated the detection and diagnostic potential of P2 in the process of AKI. We believed that an in-depth exploration of ATP and ONOO- interactions would offer valuable insights into the development of innovative diagnostic and therapeutic approaches for related diseases.