Acute kidney injury (AKI) is a sharp decrease in glomerular filtration function over a short period of time due to various aetiologies (Hoste et al., 2018). AKI is associated with considerable morbidity and mortality and has a high risk of development of chronic kidney disease (CKD) or end-stage kidney disease (ESKD) (Lameire et al., 2013). Cisplatin [cis-diamminedichloroplatinum (II)], an inorganic platinum derivative, is widely used to treat solid tumors including those of the head, neck, lung, breast, ovary, and testis (Dasari and Tchounwou, 2014; Zhang et al., 2021). Cisplatin is mainly cleared by the kidneys and therefore has a high risk of causing acute and chronic nephrotoxicity. It was reported that AKI occurred in 31.5% of patients with cancer who received cisplatin for treatment across multiple tumor types (Latcha et al., 2016). AKI is the dose-limiting side effect of cisplatin that limits its clinical application in cancer patients (Karasawa and Steyger, 2015). Therefore, it is urgent to further explore the underlying mechanism and effective therapeutic regimens of cisplatin-induced AKI.

In recent years, accumulating evidence elucidates the reno-protective effect of Chinese herbal medicine (CHM) in multiple AKI models via different mechanisms of inhibiting inflammation, cell apoptosis, necroptosis, ferroptosis, and restraining oxidative stress etc., (Li H. D. et al., 2019; Li J. et al., 2023). Chen et al. included 15 randomized controlled trials to evaluate the efficacy of CHM as an adjunctive therapy for patients with AKI. The results showed that patients randomly assigned to CHM plus Western treatment had a statistically significant reduction in in-hospital mortality compared with those randomly assigned to Western treatment alone (Chen et al., 2016). In traditional Chinese medicine (TCM) theory, AKI is caused by qi deficiency, blood stasis, and toxin accumulation. Huangqi-Danshen decoction (HDD) is composed of Astragalus membranaceus (Huangqi) and Salvia miltiorrhiza (Danshen) and has the effect of invigorating qi and promoting blood circulation. According to TCM theory, HDD would be beneficial in treating AKI. Our previous studies have found that HDD had reno-protective effect in adenine-induced CKD mice and db/db diabetic mice (Liu et al., 2019a; Liu et al., 2019b; Liu et al., 2020). However, the protective effects and underlying mechanisms of HDD against cisplatin-induced AKI are unknown.

Metabolomics provides a tool to qualitatively and quantitatively analyze small molecular metabolites (typically <1,500 Da) in specific tissues, organs or biological fluids (German et al., 2005). Metabolomics, with its high-throughput and high-resolution characteristics, allows accurate analysis of metabolite changes in body fluids or tissues, thus helping to understand the development and progression of diseases (Abbiss et al., 2019). Several studies have used metabolomics analysis to identify novel biomarkers for the development, progression and prognosis of AKI and to reveal specific mechanisms of AKI (Cui et al., 2021; Ping et al., 2021; Tan et al., 2021; Yuan et al., 2022). Metabolomics reflects the overall changes of the organism by studying the changes of metabolites, which is similar to the holistic view of TCM and provides new ideas and methods for the research of CHM. The application of metabonomics is helpful to further clarify the mechanism of action of CHM (Wang et al., 2017; Wang et al., 2021). In this study, we first evaluated the protective effect of HDD on cisplatin-induced AKI, and secondly, we used ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) to detect changes in renal metabolites to clarify the mechanism of action of HDD.

In the present study, we explored whether HDD had a reno-protective effect on cisplatin-induced AKI, as well as its underlying mechanism. The results showed that HDD protected against cisplatin-induced renal dysfunction and tubular injury and suppressed apoptosis, inflammation, and oxidative stress in the kidney of AKI mice. Non-target metabolomics revealed that HDD regulated renal metabolite profiles in AKI mice, with a primary focus on nicotinate and nicotinamide metabolism. Furthermore, HDD administration was found to effectively restore the levels of NAD⁺ biosynthesis-related metabolites and enzymes expression in the kidney of AKI mice.

NAD⁺ is referred to as an “electron carrier” due to its capacity for accepting and donating electrons in cellular oxidation-reduction reactions (White and Schenk, 2012). This process is necessary for adenosine triphosphate (ATP) synthesis involved in cellular energy metabolism (Morevati et al., 2022). The kidney necessitates a substantial amount of energy to execute reabsorption function, and it is inherently one of the organs with the highest demand for NAD⁺ content (Zapata-Pérez et al., 2021). Apart from its function as an essential cofactor in energy metabolism, NAD⁺ also acts as a co-substrate for NAD⁺-consuming enzymes (Ralto et al., 2020; Zapata-Pérez et al., 2021; Morevati et al., 2022). The sirtuins (SIRTs), a family of NAD⁺-dependent histone deacetylases, are implicated in various physiological and pathological processes, including energy metabolism, aging, mitochondrial biogenesis, inflammation, DNA repairment, and stress resistance, etc .,(Vachharajani et al., 2016; Majeed et al., 2021; Rasti et al., 2023; Xu et al., 2023). The protective effects of SIRT1, SIRT3, and SIRT5 against cisplatin-induced kidney injury have been reported (Hasegawa et al., 2010; Kim et al., 2011; Morigi et al., 2015; Li W. et al., 2019). SIRT1 indirectly modulates peroxisome proliferator-activated receptor-γ coactivator-1-α (PGC-1α), whose diminished activity in kidney increases susceptibility to AKI (Fontecha-Barriuso et al., 2020). Pathologically activated NAD⁺-consuming poly (ADP-ribose) polymerases (PARPs) are implicated in renal ischaemia-reperfusion injury (IRI) (Martin et al., 2000), as evidenced by the increased resistance to renal IRI observed in Parp1-knockout mice (Zheng et al., 2005) and the protective effect against IRI provided by a PARP1 inhibitor (Ralto et al., 2020). NAD⁺ depletion has been observed in both cisplatin-induced and IRI models (Guan et al., 2017; Katsyuba et al., 2018). The reduction of renal NAD⁺ appears to inhibit certain reno-protective enzymes and trigger a series of reactions in the initiation and progression of AKI. Evidence to date suggests that AKI prognosis may be related to the capacity of NAD⁺ replenishment to buffer against pathological stress induced by reduced NAD⁺ levels (Ralto et al., 2020). In mammals, NAD⁺ is synthesized through three pathways: the de novo pathway from tryptophan, the salvage pathway from nicotinamide (NAM) and nicotinamide riboside (NR), and the Preiss-Handler pathway from nicotinic acid (NA) (Zapata-Pérez et al., 2021). Of note, impaired de novo NAD⁺ biosynthesis pathway was found in AKI (Poyan Mehr et al., 2018). The research also proposed that elevated urinary quinolinate/tryptophan could serve as an indicator for predicting AKI and other adverse outcomes. Our findings were in line with these results, as we observed an increase in both QA and QA/tryptophan levels in the kidneys induced by cisplatin. Since QA only participates in NAD⁺ de novo synthesis after being catalyzed by QPRT and not in other metabolic reactions (Poyan Mehr et al., 2018), QA accumulation indicates a reduction in QPRT activity. Further results from Western blot confirmed that the expression of QPRT was reduced in kidney induced by cisplatin and HDD significantly restored QPRT expression, reversing the elevated QA and QA/tryptophan levels. Besides, reduced intracellular NAD⁺ contents and a decreased ratio of NAD⁺: NADH were observed in cisplatin-treated renal tissue, resulting in reactive oxygen species (ROS) accumulation, inflammation, and kidney injury (Oh et al., 2014). NAM, a precursor of NAD⁺, exhibits potent antioxidant properties and can significantly mitigate the damage caused by ROS production under oxidative stress (Mejía et al., 2018). In our study, we observed a significant reversal of reduced levels of NAM and NAD⁺ in the kidneys of AKI mice with HDD treatment. NMNAT1 and NAMPT, two important enzymes in NAD⁺ synthesis pathways, were also upregulated upon HDD treatment.

Due to the potent nephrotoxicity of cisplatin, its clinical application as an anticancer agent for solid tumors is considerably restricted (Tang et al., 2023). Given that cisplatin is excreted through the kidneys, high doses or prolonged exposure to cisplatin leads to its accumulation in renal tubular cells (Zhang et al., 2021). This accumulation triggers diverse cellular stress responses, including apoptosis, inflammation, oxidative stress, DNA damage, and mitochondrial pathology (Wei et al., 2007; Basu and Krishnamurthy, 2010; Tang et al., 2021; Tang et al., 2023). Macrophages have critical roles in renal inflammation and repair. Our results found obvious macrophage infiltration in the kidneys of AKI mice and could be alleviated by HDD treatment (Figure 3). Macrophages are highly heterogeneous and can differentiate into two phenotypes, pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. Previous studies have indicated that macrophage polarization played essential roles in renal fibrosis and folic acid nephropathy (Jiao et al., 2021a; Jiao et al., 2021b; An et al., 2023). However, the phenotype of macrophages and their response to HDD treatment in the present study need further investigation. NAD⁺ plays a crucial role in anti-aging, anti-inflammation, anti-oxidation, and DNA repair processes (Hershberger et al., 2017; Zapata-Pérez et al., 2021). The NAD⁺ precursor, NAM, has been reported to enhance mitochondrial metabolism and reduce ROS, implying that NAM supplementation may be a potential reno-protective approach against cisplatin nephrotoxicity (Tran et al., 2016). Combined with the results of our experiment, we have discovered that HDD could significantly restore NAM levels in cisplatin-induced kidney. Taken together, HDD may exert reno-protective effects against cisplatin-induced AKI through restoring NAD⁺ biosynthesis and thus replenish NAD⁺ content, thereby promoting a cascade of responses.

Although targeting NAD⁺ biosynthesis has been found to confer reno-protection (Allison, 2019) and improve health (Katsyuba et al., 2018), its effectiveness is limited due to the lack of renal targeting ability (Duan et al., 2023). It has been reported that natural products can modulate NAD⁺ homeostasis and exhibit various biological activities, including anti-apoptosis, anti-inflammation, anti-oxidation, improving energy metabolism, and neuroprotection. And most of them increase the content of NAD⁺, which is for preventing and treating diseases (Guo et al., 2023). Huangqi-Danshen, a classic combination of CHM, exerts invigorating effects on qi, promotes blood circulation, and facilitates detoxification. A plethora of studies have revealed that both Huangqi and Danshen possess beneficial improvements on diverse diseases by inhibiting inflammatory response, reducing cell apoptosis, alleviating oxidative stress, and activating immunomodulation (Ko et al., 2005; Qin et al., 2012; Shahrajabian et al., 2019; Li R. et al., 2023; Luo et al., 2023; Yang et al., 2023). Several components of Danshen have been reported to target NAD⁺ metabolism for therapeutic effects. Salvianolic acid B reversed the depletion of cellular NAD⁺ induced by angiotensin II to protect cardiomyocyte (Liu et al., 2014). Apigenin could increase NAD⁺ level and suppress neurodegeneration (Roboon et al., 2021) and protect against obesity and metabolic syndrome (Escande et al., 2013). The regulation of NAD⁺ metabolism by these components in models of kidney disease has not been reported. TCM adheres to the concept of disease prevention should take priority over disease onset. The results of our present study implied that HDD pretreatment could effectively counteract the nephrotoxic side effects induced by cisplatin to a certain extent, which was in line with the concept of disease prevention. However, the downstream effects of enhancing NAD⁺ availability and precise mechanisms by which HDD modulates NAD⁺ metabolism remain unclear and necessitate further investigation.

In summary, HDD exerted a protective effect against cisplatin-induced AKI and suppressed apoptosis, inflammation, and oxidative stress in the kidney of AKI mice, which may be attributed to the modulation of NAD⁺ biosynthesis.