Adenine-induced animal model of chronic kidney disease: current applications and future perspectives

Abstract

Chronic kidney disease (CKD) with high morbidity and mortality all over the world is characterized by decreased kidney function, a condition which can result from numerous risk factors, including diabetes, hypertension and obesity. Despite significant advances in our understanding of the pathogenesis of CKD, there are still no treatments that can effectively combat CKD, which underscores the urgent need for further study into the pathological mechanisms underlying this condition. In this regard, animal models of CKD are indispensable. This article reviews a widely used animal model of CKD, which is induced by adenine. While a physiologic dose of adenine is beneficial in terms of biological activity, a high dose of adenine is known to induce renal disease in the organism. Following a brief description of the procedure for disease induction by adenine, major mechanisms of adenine-induced CKD are then reviewed, including inflammation, oxidative stress, programmed cell death, metabolic disorders, and fibrillation. Finally, the application and future perspective of this adenine-induced CKD model as a platform for testing the efficacy of a variety of therapeutic approaches is also discussed. Given the simplicity and reproducibility of this animal model, it remains a valuable tool for studying the pathological mechanisms of CKD and identifying therapeutic targets to fight CKD.

Keywords:

• Chronic kidney disease
• adenine
• inflammations
• oxidative stress
• programmed cell death
• metabolic disorders
• fibroblast/myofibroblast activation

1. Introduction

Chronic kidney disease (CKD) is a progressive disease with no cure and high morbidity and mortality [1–3]. The causes of CKD are complex, including common and well-researched factors such as diabetes, hypertension, obesity and chronic inflammation [4,5]. The pathological features of CKD are characterized by fibrosis, which is a repair cascade initiated by several different stimuli and pathogenic factors, leading to excessive accumulation of matrix connective tissue components [6]. However, at present, there is no accepted systematic strategy for CKD standardized screening and treatment, stressing a continual need to elucidate the underlying pathological mechanisms of CKD. In this regard, animal models of CKD have been invaluable in that utilization of these animal models not only enhances our understanding of the pathogenesis of kidney disease, but also provides excellent platforms for disease intervention in which the efficacy of testing compounds or pharmacological agents can be quantitatively assessed [7–11].

Numerous animal models have been employed to elucidate the pathological mechanisms of CKD [8,12–14]. CKD animal models induced by 5/6 nephrectomy [15–17], diabetic nephropathy [18–23], Hypertension-induced renal injury [4,24], and primary glomerular nephropathy are widely utilized [25–27]. These models have also been used to test the therapeutic effect of various drugs or compound [28–33]. However, this review will specifically focus on the highly prevalent adenine-induced rodent model of CKD, which is implemented in both mice and rats [34–37]. Table 1 presents a comparative analysis of the adenine model and other CKD-induced animal models.

Table 1. Comparison of animal models of CKD induced by a variety of approaches.

Models	Species	Advantages	Disadvantages	Reference
Adenine	Mouse/rat	Mimics renal crystallization and tubulointerstitial fibrosis, helps to study CKD progression mechanisms.	Some mortality (mice), inadequate for AKI-CKD transition research, impacts other organs	[38–45]
Folic acid	Mouse/rat	Simple, reproducible, useful for studying AKI-CKD transition	Some variability, lacking clinical relevance	[46–49]
Cisplatin	Mouse/rat	Used to study the mechanism of AKI due to its nephrotoxicity, widely used in chemotherapy.	More toxic, lacks specificity, time-consuming for CKD	[50–53]
Angiotensin II	Mouse	Replicates physiological and pathological features of malignant hypertension-related kidney damage.	May cause multi-system effects and is not specific to the kidneys alone.	[54–56]
Streptozocin	Mouse/rat	Commonly used to prepare models for diabetic nephropathy, good representativity for type 1 diabetes.	Primarily induces type 1 diabetes models, the induced nephropathy might not fully match the pathological process in patients, time-consuming for CKD, some mortality	[57–60]
Cyclosporine	Mouse/rat	Simple, clinically relevant	Requires very low salt diet, expensive, hepatotoxic	[61,62]
Adriamycin	Mouse/rat	Simple, clinically relevant	Highly histotoxic, some mortality	[62–65]
Aristolochic acid	Mouse/rat	Useful for studying AKI-CKD transition, clinically relevant	High toxicity, potential carcinogenic risks, may have off-target effects, complex, time-consuming and expensive for CKD	[66,67]
Gentamycin	Rat	Clinically relevant, reversible AKI	Audiotoxicity, not suitable for CKD studies, decrease in clinical reports	[68–70]
Mercury and its compounds	Mouse/rat	Well-suited for studying acute nephrotic effects	Environmental and health hazards, toxic to other organs	[71–73]
Unilateral ureteral obstruction	Mouse/rat	Useful for studying tubulointerstitial fibrosis, replicates obstructive CKD	Requires surgical intervention, functional compensation exists	[48,74]
5/6 nephrectomy	Mouse/rat	Gold standard for CKD modeling, mimics the progressive renal failure; after loss of renal mass in human	Highly invasive, potential for surgical complications	[75,76]
Ischemia-reperfusion	Mouse/rat	Clinically mimics ischemic injury in AKI, good for studying early CKD events	High variability, risk of acute death, requires careful surgical technique	[77,78]

Note. This table is not meant to catalog all the animal models of kidney injury in the literature. Instead, it lists only popular and widely used animal models are listed. It should also be noted that when rats or mice are used, most investigators choose to use young adult animals typically aged from 6 to 8 weeks.

Among all the CKD animal models induced by various approaches outlined in Table 1, the adenine-induced model provides certain advantages that are lacking in other models. Firstly, as a nucleobase (a purine derivative), adenine is environmentally nontoxic, presenting no hazard in routine laboratory handling. Secondly, in contrast to modalities such as 5/6 nephrectomy for modeling CKD, adenine induction is administered as a simple gavage or dietary feeding, which is non-surgical, noninvasive, and animal-friendly. Third, the method of adenine induction allows the production of mild, moderate and severe chronic kidney models with a stable disease course and a high success rate, which vary with the adenine dosage and duration of administration [38, 79]. Fourth, the adenine model is capable of effectively simulating the progression of renal disease in both male and female test animals, and has provided key insights into sexual dimorphism, reflecting the clinical reality of CKD that affects both genders [80–84]. Undeniably, the adenine-induced CKD model has its own disadvantages. One disadvantage is that the impact of adenine on reproductive and other organ systems remains speculative, as well as the fact that specific biomarkers of adenine-induced CKD have not been identified. Furthermore, while adenine-induced CKD primarily affects renal tubules, the intricate molecular and biochemical mechanisms leading to nephron injury by adenine have yet to be elucidated.

2. Adenine metabolism and renal implications

Adenine, a fundamental purine nucleobase, plays a crucial role in a variety of biochemical and physiological processes within cells. These processes including DNA and RNA synthesis, the generation of cellular energy through ATP and its related compounds, the modulation of extracellular signaling via ATP, ADP, and adenosine, the formation of the second messenger cAMP, and serving as vital cofactors for numerous enzymes, such as nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD) [85]. Adenine is a common dietary component present in foods like meats, fish, beer and legumes [86, 87], and while it is endogenously generated in the body, intake from these dietary sources also contributes to its overall pool [88]. Within the gastrointestinal tract, physiological levels of adenine are absorbed and then metabolized by adenine phosphoribosyl transferase (APRT) to yield AMP, a precursor to a variety of adenine derivatives including adenosine, inosine 5′-monophosphate, hypoxanthine, and xanthine [89]. Ultimately, xanthine is further metabolized into uric acid, which is excreted by the kidney [90–94]. Additionally, adenine undergoes metabolism through a distinct path at physiological doses, in a reaction catalyzed by xanthine oxidase (XO), which leads to the formation of 2,8-dihydroxyadenine (2,8-DHA) eventually excreted in urine (Figure 1) [95,96]. APRT deficiency is an uncommon autosomal recessive disorder of purine metabolism that can lead to kidney stones and CKD because the enzymatic activity of APRT is crucial for the normal recycling of adenine. [97–99].

Figure 1. Adenine metabolism pathways. APRT: adenine phosphoribosyl transferase; PRPP: 5-phosphoribosyl-1-pyrophosphate; AMP: adenosine monophosphate; IMP: inosine monophosphate; AMPD1: adenosine monophosphate deaminase 1; ADA: adenosine deaminase; PNP: purine nucleoside phosphorylase; XO: xanthine oxidase; PRPS1: phosphoribosyl pyrophosphate synthetase 1; PRPPs: phosphate diphosphokinase; ADSL: adenylosuccinate lyase; ATIC: 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase; ADSS: Adenylosuccinate synthase 2; ADP: adenosine diphosphate; ATP: adenosine triphosphate; AdeR: adenine receptors.

There is growing body of evidence that adenine metabolism is important for both renal function and disease [100–102]. Acting beyond its traditional intracellular roles, adenine can also function as an extracellular signaling molecule, engaging with receptors widely expressed across various organs, the kidney included [103,104]. Intriguingly, an orphan G protein-coupled membrane receptor originally cloned from dorsal root ganglia has been subsequently shown to be a receptor selective for adenine [92,105,106]. Although the highest expression of this adenine receptor (AdeR) has been found in the dorsal root ganglion, significant mRNA levels of the receptor have also been found in other organs such as the kidney [105]. The AdeR is expressed in the vasculature in the cortical region, including glomerular arterioles, and throughout the collecting duct system [107–109]. AdeR binds adenine with high affinity and potentially reduces cellular cAMP levels [41]. Adenine itself can act as an extracellular signaling molecule, but dietary adenine is metabolized in the intestines before it contributes to various biological processes (Figure 1) [110,111]. Currently, the only known pathway for endogenous adenine production in mammals so far is the intracellular polyamine synthesis pathway, where adenine is produced as a byproduct [104]. Numerous concentrative nucleoside transporters (CNTs) and equilibrative nucleoside transporters (ENTs) possess the ability to facilitate the transportation of nucleobases, specifically adenine [112]. While ENTs facilitate diffusion, CNTs function as active transporters that rely solely on Na⁺ ions [113]. Both ENT1 and ENT2 have been observed to participate in nucleobase transport within cells derived from healthy human kidney (renal proximal tubule cells) as well as renal cancers [114, 115]. Nevertheless, the ENT1 transporter is unlikely to play a significant role in renal adenine transportation due to its low affinity for adenine [116,117]. The two nucleoside transporters exhibiting the highest expression levels in the kidney are CNT1 and ENT3, therefore potentially being involved in adenine transport [118–120]. Initially described as a sodium-independent lysosomal transporter with an optimal pH range of 5.5 to 6.5, ENT3 has recently been detected on the mitochondrial membranes and the cell surface in select human cell lines derived from various tissues. These findings indicate that ENT3 is localized differently within subcellular compartments across various cell types [118,119,121]. Given its abundance in the kidney, this transporter may indeed play a vital role in renal function. In summary, endogenous adenine is transported from cytosol to the extracellular space via transporter proteins, before it functions as a signaling molecule through the activation of the G-protein-coupled AdeR [108,122].

3. Adenine-induced renal dysfunction

Although physiological level doses of adenine play an important role, high doses of adenine can be used to induce kidney disease in animals [85]. A search in the Google Scholar database indicates that the first report of a renal problem caused by adenine was published in 1902, and described 2,8-DHA crystals were deposited in the kidneys following adenine administration [123]. This focused subsequent interest of toxicologists on the kidney and its function [124]. The rat model of adenine-induced CKD was first proposed by Yokozawa in 1986, in which adenine feeding or gavage caused tubular obstruction and massive loss of renal units, leading to azotemia, toxin accumulation, and disorders of electrolyte and amino acid metabolism, resulting in renal failure [34,90]. Now, the procedures of adenine-induced CKD in mice and rats are well established and widely used. As shown in Figure 2, feeding mice and rats with diets containing 0.075–0.75% adenine or intraperitoneally injecting them with an adenine solution for several weeks resulted in the development of CKD in both species. In general, the recommended induction dosage for rats is 0.75% w/w, while for mice, it is 0.20% w/w. Rats are more tolerant to adenine, so the dosage can typically be set at 0.75%. Conversely, mice are more sensitive or less tolerant to adenine intake, so the dosage for mice is generally controlled at 0.20%. Additionally, taste enhancers such as casein may need to be added to the adenine solution for mice [44]. It is, however, crucial to note that elevating the dosage beyond these established benchmarks can culminate in renal toxicity or acute kidney failure. Furthermore, higher doses may also have detrimental effects on other organs, thereby deviating from the intended modeling of CKD and potentially leading to the construction of different disease models such as acute kidney failure or reproductive dysfunction. It is also important to consider the ethical implications and scientific validity of using very high doses, as it can result in a significant number of animal deaths within a short period. On the other hand, reducing the induction dosage below the recommended levels may lead to a more gradual and persistent decline in kidney function. However, this could result in excessively long experimental periods or the absence of the phenotypic expression of CKD.

Figure 2. General experimental scheme of adenine-induced CKD. Adenine is usually given at a dose of 0.75% w/w for rats or 0.20% w/w for mice, and is mixed into normal diets for ad libitum consumption (note: it is advisable to add a certain level of casein to the diets for mice). The progression of CKD may be studied for up to, or beyond, 4 weeks following the start of adenine feed administration.

Symptoms of adenine-induced CKD via the different modes of administration are very similar but each has its own advantages and disadvantages [125]. Gavage, for example, allows precise control of the dose of adenine given to the animal; however, due to the invasive nature of this method, it may cause stress to the animal, which can have confounding effects on the development of CKD. In contrast, the addition of adenine to food provides a less invasive method of continuous administration, thereby reducing the stress to which the animal is subjected. Of course, differences in food intake between individual animals can lead to dose inconsistencies, but standardized measures can be used to improve the reliability and reproducibility of study results. Intraperitoneal (IP) administration, although not common in adenine-induced CKD studies, can be applied in studies about CKD-related gastrointestinal dysfunction [126]. However, IP administration may not accurately mimic the natural pathway of human exposure to adenine, and the injection process itself may introduce confounding factors, such as inflammation or local tissue damage, that can affect disease progression. Therefore, considering all factors, CKD is now frequently induced in rodents via feeding administration of adenine to model the disease in preclinical experimental studies.

Renal disease occurs at non-physiologically high concentrations of adenine due to the fact that exogenously administered adenine is not only metabolized to AMP by APRT, but also becomes a substrate for XO, which is converted to 8-hydroxyadenine, and subsequently to the highly insoluble 2,8-DHA, and then the 2,8-DHA forms intratubular precipitates that lead to chronic tubulointerstitial injury, tissue remodeling of crystalline interstitial inclusions, inflammatory and fibrotic processes, renal insufficiency, and metabolic abnormalities similar to the symptoms of CKD in humans [16,34,95,127]. Certainly, the etiology of adenine-induced chronic renal failure is different from that of human chronic renal failure, especially because of the low incidence of crystalline nephropathy as a causative factor in human disease[128]. Despite the different etiology, the adenine model has been exemplary in elucidating the mechanisms of extrarenal comorbidities associated with CKD. Therefore, this method of animal model establishment is commonly used to study the pathological mechanisms of CKD [11,44,79,129,130]. Moreover, it is possible to study the progression of different disease stages in CKD by controlling for the progression after induction periods [131–133]. Additionally, as adenine is administered by gavage or mixed in feed, the process of kidney disease induction is simple and straightforward and as it circumvents the need for surgical intervention. Importantly, adenine-induced renal disease can recapitulate the clinical symptoms of human kidney disease and demonstrates a high degree of reproducibility in experimental settings [39].

With respect to the adenine-induced CKD, the adenine-induced model may have some advantages over other CKD models, including the UUO model, the 5/6 nephrectomy model, the folic acid toxicity model, and the cisplatin model. Combined with those described in the historical literature and practiced in the authors’ laboratories, the major advantages of the adenine-induced model are that it does not require surgical construction, it can be administered by diet to reduce animal suffering and human labor, it can be controlled for CKD disease stages [79,134,135]. Furthermore, the optimization of the dietary ratios can reduce the animal mortality rate during the study, while taking into account the disease development in the animal model, thereby ensuring reproducibility issues [44]. In addition, this model is particularly suitable for the study of CKD with metabolic disorders [136–139]. Given that the mechanisms underlying CKD remain elusive, exploring and contrasting various CKD models could yield comprehensive insights into the pathophysiology. Nevertheless, in the adenine-induced CKD model, it is clear that inflammatory abnormalities, oxidative stress, fibrosis, renal metabolic disorders and ferroptosis are involved in CKD progression [11,140–143].

Tubular cells are highly susceptible to nephrotoxic injuries, in particular, the metabolically active and energy-intensive proximal tubular epithelial cells (PTECs) [144]. Notably, crystal deposition and tubular damage in the animal model of adenine-induced CKD are mostly located in the proximal tubular compartment (Figure 3) [135,145]. The accumulation of crystal deposits within renal tubular epithelium leads to mechanical obstruction, interstitial inflammation, and programmed cell death [146]. Notably, a mouse model with CKD induced by an adenine-rich diet exhibits the distinctive characteristics of human CKD patients with renal filtration failure and cardiovascular symptoms [79,147,148]. In addition, the formation of 2,8-DHA crystals in the kidney was found in both the adenine-induced CKD mouse model and human patients with APRT deficiency [149–151]. Thus, the adenine-induced CKD model, which is a laboratory animal model, basically summarizes many of the clinical manifestations of patients with clinical CKD [144]. As is inherent in all animal models of human disease, any model of kidney disease in animals can only serve as a substitute and will never be identical to human kidney disease.

Figure 3. Process of 2,8-DHA crystal granuloma formation. Supersaturation of 2,8-DHA in the urine leads to the formation of 2,8-DHA crystal nuclei. These nuclei adhere to renal tubular cells, creating a nidus for further crystal growth and leading to the formation of crystal plugs in the tubular lumen, causing obstruction of the tubules. Very small crystal plugs were internalized and likely degraded by renal tubular cells, while medium-sized crystal plugs trigger dedifferentiation and proliferative growth of renal tubular cells, eventually resulting in the translocation of plugs into the interstitium. Within the interstitial compartment, renal tubular cells cooperate with macrophages and other cells in the formation of interstitial crystal granulomas that isolate the crystal plug from the renal parenchyma and initiate crystal degradation.

4. Major mechanisms of adenine-induced CKD

When given non-physiological doses of adenine via the diet, adenine is metabolized to form 2,8-DHA, which rapidly accumulates crystal deposits in the renal tubules and leads to recurrent episodes of renal injury that leads to CKD transformation, characterized by a loss of renal function (increased proteinuria and blood urea nitrogen, plasma uric acid, and plasma creatinine) and tubular injury (increased number of dilated tubules, increased cellular debris in the tubular lumen and increased fibrosis) [84,145]. This sequence of events is similar to other animal models of induced CKD, and little is known about the in vivo pathogenesis of the CKD by excessive adenine and 2,8-DHA crystal accumulation. With the increase in the number of days of adenine dietary intake, the model animals showed gradual wasting, chills and cold limbs, significantly enlarged kidneys, grayish-white kidneys with granular surfaces, and elevated serum creatinine (SCr) and blood urea nitrogen (BUN), and other biochemical indexes of CKD [34, 79]. In this article, the main mechanisms involved in adenine-induced CKD are summarized.

4.1. Inflammations

Renal inflammation is a protective response that is induced following kidney injury, which seeks to eliminate the cause of injury and establish tissue repair [152]. Studies have shown that 2,8-DHA crystals can induce renal tubular cell injury and promote an inflammatory phenotype. This involves the activation of toll-like receptors (TLRs) on renal mononuclear phagocytes, the expression of high-mobility group protein B1 (HMGB1), and the activation of nuclear factor-kappa B (NF-κB) in B cells [135]. If left unresolved, persistent generation of 2,8-DHA crystals can lead to a series of biochemical pathways involving immune system cells and intrinsic renal cells. In the context of chronic inflammation, DHA crystal-induced renal damage is characterized by sustained inflammation with increased expression of the chemokine Ccl5 and monocyte infiltration, mainly composed of F4/80⁺ macrophages and resident renal dendritic cells, as well as ErHr3⁺ monocytes and monocyte-derived macrophages representing a subset of F4/80⁺ cells [153–155]. Macrophages accumulate around damaged cortical and medullary tubules and often form interstitial granulomas around the crystals [156]. In this process, tubular cells in cooperation with macrophages overgrow and transfer crystals into the interstitium, restoring the patency of the tubular lumen; this was followed by degradation of interstitial crystals as a result of granulomatous inflammation [135, 157]. If renal tubular injury and persistent inflammation are not resolved, the intrinsic cells of the kidney exhibit increased reactivity to the inflammatory and fibrotic processes in the environment of chronic inflammation. Changes in their gene expression profile enable them to produce and release more pro-fibrotic cytokines, driving fibroblast differentiation and progressive transformation toward myofibroblasts. This process is associated with oxidative stress and cell death (Figure 4) [152, 158].

Figure 4. Processes of the primary mechanism of adenine-induced renal inflammation.

4.2. Oxidative stress

Oxidative stress is already found in early stages of renal disease and increases with declining kidney function [159–162]. Numerous studies demonstrate renal oxidative stress in the adenine-induced CKD model [163–167]. The occlusion of renal tubules by 2,8-DHA crystals not only affects the tubules themselves but also imposes a pressure load on the peritubular capillaries, resulting in subsequent renal hypoxia [131, 134, 168]. Hypoxia accelerates degradation of cellular ATP and increases the production of hypoxanthine, which activates renal XO, subsequently leading to the production of oxidative stress [169]. For instance, in adenine-induced mouse model, Keiichi Ohata et al. found that the levels of HIF-1α mRNA and XO activity in the kidneys of the adenine diet group were significantly higher than those of the normal diet group, but the levels of catalase mRNA were significantly lower than those of the normal diet group; thus, higher levels of oxidative stress promote the progression of renal tubulointerstitial injury in the adenine model [165]. Furthermore, in their research on an adenine-induced CKD model, Ali, Badreldin H. et al. found that the concentrations of glutathione and superoxide dismutase, markers of oxidative stress, were significantly increased in an adenine-induced CKD model. Meanwhile, reactive oxygen species directly impaired mitochondrial function, DNA synthesis, triggered genomic instability and induced cell death [164]. These findings suggest that adenine is closely related to oxidative stress (Figure 5).

Figure 5. Processes of the major mechanism of adenine-induced renal oxidative stress.

4.3. Programmed cell death

The pathophysiology of renal diseases is significantly connected with programmed renal cell death, including apoptosis, necroptosis, ferroptosis, pyroptosis, and autophagy, as is now recognized [170–176]. In rodent models of adenine-induced nephropathy, where crystal deposition and tubular injury are predominantly observed in the proximal tubular compartment, Muhammad Ali Khan et al. confirmed the pathobiological process of ferroptosis within these structures. Their research employed an experimental rat model of adenine-induced nephropathy and extended to adenine-exposed human primary proximal tubular epithelial cells (PTEC). Notably, in rats subjected to an adenine-rich diet, substantial elevations in iron accumulation and lipid peroxidation, specifically 4-hydroxynonenal (4-HNE), were discovered within the tubules—hallmarks signaling ferroptosis. Furthermore, in human primary PTEC cultures challenged with adenine, substantial rise in mitochondrial superoxide production, mitochondrial depolarization, DNA damage, and incidence of necrotic cell death were observed, when compared to untreated PTEC. Detailed molecular studies into adenine-challenged PTEC confirmed these findings, highlighting a significant reduction in the lipid-repairing enzyme, glutathione peroxidase 4 (GPX4), and a corresponding increase in 4-HNE. Such changes further validate the occurrence of ferroptotic cell death in the context of excessive adenine. Furthermore, Ferrostatin-1 (a potent inhibitor of ferroptosis) treatment attenuated PTEC death in the adenine-induced CKD model by targeting the aforementioned ferroptosis pathway through the inhibition of lipid peroxidation, which further demonstrated that ferroptosis plays a more important role in the adenine-induced CKD model [143, 177]. Notably, other types of programmed cell death, such as cellular apoptosis and autophagy, have also been reported in adenine-induced CKD (Figure 6) [38, 178–183].

Figure 6. Processes of the major mechanism of adenine-induced renal programmed cell death.

4.4. Metabolic disorders

Multiple metabolic pathways, including those involving adenine, tryptophan, bile acids, phospholipids, creatinine, and fatty acid metabolism, have also been reported to be dysregulated in the adenine-induced CKD model [137–139, 163, 184–187]. In a study exploring biomarkers of early stages of CKD, Toshihiro Kobayashi et al. applied metabolomics to blood samples collected from adenine-induced CKD rats for 28 days, and analyzed them to find that indolyl sulfate, phenyl sulfate, p-cresol sulfate, and phenylacetylglycine were increased with the progression of renal hypoplasia, suggesting that tryptophan and glycocholic acid may reflect the development of CKD [138]. Ying-Yong Zhao et al. identified 12 endogenous metabolites in the urine of CKD rats, and the changes in these metabolites suggest that disturbances of amino acid metabolism, phospholipid metabolism, and energy metabolism are associated with adenine-induced CKD rats [185]. It was also found that indoxyl sulfate and p-cresyl sulfate were significantly increased in adenine-induced CKD rats and stimulate progressive tubulointerstitial fibrosis by increasing the expression of transforming growth factor-β1 (TGF-β1) [187]. Similarly, in an animal model of adenine induction and in patients with early-stage CKD, it was found that reduced levels of tryptophan metabolite 5-MTP and its regulatory enzyme, TPH-1, causes renal injury by activating NF-κB and inhibiting Nrf2 pathways, and this dysfunction leads to accumulation of renal extracellular matrix and progression of renal fibrosis [188, 189]. Tomoko Kimura et al. showed that the main cause of anemia in CKD is a lack of adequate erythropoietin (EPO), but other factors may also be involved, including dysregulation of iron metabolism, using an adenine-induced mouse model of CKD [40]. Overall, these studies suggest that dysregulation of various metabolic pathways, including those involving adenine, tryptophan, bile acids, phospholipid, creatinine, and fatty acid metabolism, is associated with the development and progression of adenine-induced CKD (Figure 7). Moreover, the findings regarding the role of specific metabolites in promoting renal injury and fibrosis through activation of pro-fibrotic pathways highlight the potential therapeutic targets for the treatment of CKD.

Figure 7. Processes of the major mechanism of adenine-induced renal metabolic disorders.

4.5. Fibroblast/myofibroblast activation

When renal tubular epithelial cells (RTECs) are persistently injured, such as with sustained 2,8-DHA accumulation, RTECs produce a large number of growth factors and cytokines, including TGF-β, PDGF, hedgehog, and Wnt ligands [190–193]. These cytokines secreted by injured RTECs can accelerate the regeneration of injured RTECs and promote the differentiation of fibroblasts into myofibroblasts [194]. Activated myofibroblasts undergo rapid proliferation and produce a large amounts of ECM components, such as fibronectin and collagen, which contribute to the progression of renal tubulointerstitial fibrosis [195]. For instance, numerous studies have demonstrated that adenine significantly upregulated the expression of type IV collagen, fibronectin, and overall extracellular matrix in the kidneys of mice with adenine-induced kidney injury, resulting in disruption of TGF-β/Smad signaling and epithelial mesenchymal transition (EMT) in RTECs [166, 196–200]. During this process, the activation of TGF-β1 stimulates ERK, p38 MAPK, and JNK pathways, mediating the progression of renal fibrosis [166, 196, 201]. Additionally, fibroblast-derived αvβ1 integrin, a critical regulator of renal fibrosis, is the only αv integrin on renal fibroblasts capable of binding to and activating TGF-β. Inhibiting αvβ1 integrin in an adenine-induced model has been shown to the activation of the TGF-β complex and ameliorate renal failure and fibrosis [202]. Furthermore adenine was found to upregulate Wnt1 protein expression in the kidneys of CKD rats, along with renal β-catenin protein expression. This was accompanied by an upregulation of downstream proteins, including Snail1, Twist, MMP-7, PAI-1, and FSP1, suggesting that the overactivity of the Wnt/β-catenin signaling pathway promotes myofibroblast differentiation and renal injury through RAS activation [203]. Therefore, adenine-induced fibroblast/myofibroblast activation communicates is associated with multiple signaling pathways, forming a feedback loop that exacerbates the progression of renal fibrosis by leading to exacerbation of EMT (Figure 8). Overall and mechanistically, it should be noted that adenine-diet animal model primarily damages the kidneys, with the main damage occurring in the proximal tubules. Importantly, this model can develop CKD-related complication that mimic those seen in humans, such as vascular calcification, mineral and bone disorders, muscle loss, and others [79, 204–208]. As outlined in the literature above, in experimental animals with adenine-induced CKD, inflammation and oxidative stress contribute to the repair of renal tubular damage while simultaneously increasing the expression of pro-fibrotic cytokines and collagen deposition to aid in the recovery of kidney damage. However, the persistent production of 2,8-DHA crystals can lead to renal tubule epithelial cell death via multiple pathways, including apoptosis, necroptosis, ferroptosis, and others. This process involves multifactorial upstream signal transduction abnormalities, including TGF-β/Smad and Wnt/β-catenin signaling disruptions, among a series of interrelated factors. Therefore, the precise mechanism underlying adenine-induced CKD still require thorough investigation.

Figure 8. Processes of the major mechanism of adenine-induced renal fibroblast/myofibroblast activation.

5. Research applications of the adenine-induced CKD model

Beyond shedding light on the pathophysiological underpinnings of renal diseases, the adenine-induced animal model of kidney disease serves a pivotal role in evaluating the efficacy of various therapeutic interventions. These interventions include not only pharmacological agents but also chemical and natural compounds. As illustrated in Table 2, a curated selection of studies employing this model emphasizes the diverse array of experimental therapies tested against CKD triggered by adenine. It should be noted that all compounds listed are in the preclinical stage, as their beneficial effects on CKD trials have involved experimental animals.

Table 2. Adenine-induced animal models for testing therapeutic interventions in CKD.

Chemicals/natural products	Dosage	Mechanisms	Molecular targets	References
Sitagliptin	2.5, 10 mg/kg/d, 35 d	↓Inflammation ↓Oxidative stress	↓TNF-α,↓TGF-β,↓IL-1β ↑TAC,↑GR,↑SOD,↑Catalase,↓8-OHdG	[209]
Sildenafil	0.1, 0.5, 2.5 mg/kg/d, 5 w	↓Inflammation ↓Oxidative stress ↓Apoptosis	↓IL-1β,↓IL-10 ↑CAT,↑Glutathione reductase,↑SOD,↑TAC ↓NF-kB,↑p-MAPK,↓Caspase 3	[210]
Neoxanthin	5 mg/kg/d, 7 d	↓Renal aging ↓Oxidative stress ↓Endoplasmic reticulum stress ↓Inflammation ↓Apoptosis	↓P16,↓p21,↓γH2AX,↑Ki67 ↓MDA,↑SOD,↑CAT,↓8-OHDG ↓p-PEAK,↓p-EIF2a,↓ATF4,↓CHOP ↓TNFα,↓IL-1β,↓NF-κB ↓Caspase-3,↓Bax,↑BCL-2	[211]
Pemafibrate	0.3 mg/kg/d, 4 w	↓Inflammation ↓Oxidative stress ↓Renal fibrosis	↓MCP-1,↓IL-1β,↓TNF-α,↓IL-6 ↑Catalase ↓Collagen-I,↓Collagen-III,↓Fibronectin,↓PAI-1,↓α-SMA,↓TGF-β	[212]
Sodium thiosulfate	2 g/kg/d, 3 w 400 mg/kg/d, 2 w	↓CKD-induced hypertension ↓CKD-induced vascular calcification	↑H2S,↑Thiosulfate ↑CAT,↑GPx,↓TBARS,↓SOD	[213, 214]
Melatonin	10 mg/kg/d, 3 w	↓CKD-induced hypertension ↓Inflammation	↓TMAO/TMA ↓TGF-β1 signaling pathways,↑AMPK,	[196, 215]
Topiroxostat	1 or 3 mg/kg, 2 w	↓Macrophage infiltration ↓Renal injury	↓HO-1, ↓HIF-1α, ↓MCP-1, ↓XO, ↓L-FABP	[168]
Veverimer	0.8, 1.5, 2.4 or 0.7, 1.4, 2.3 g/kg, 9 w	↓CKD-induced metabolic acidosis	↓Intestinal acid,↑Serum bicarbonate	[216]
L-Lysine	20 mmol/kg/d, 5 w	↓CKD-induced vascular calcification	↓iPTH, ↑Plasma arginine and homoarginin	[217, 218]
Topiroxostat	1, 3 mg/kg/d, 4 w	↓Oxidative stress	↓XO	[165]
Citrate	216, 746 mg/kg/d, 6, 8, 12, 16 w	↓Inflammation ↓Endoplasmic reticulum stress	↑Th17/Treg balance ↓CHOP	[219, 220]
Roflumilast	0.5, 1, 1.5 mg/kg/d, 6, 8, 12, 16 w	↓Inflammation	↓TGF-β,↓TNF-α,↓IL-1β,↓ICAM-1,↓MCP-1,↓Fibronectin	[221]
Sunitinib	25 mg/kg/d, 4 w	↓Oxidative stress ↓Apoptosis	↑Catalase,↑GSH,↓MDA ↓p-ERK1/2,↓Caspase-3,↓p53,↓BCL-2	[166]
Diacerein	25, 50 mg/kg/d, 28 d	↓Inflammation ↓Oxidative stress	↓TLR4/MYD88/TRAF6/NF-κB	[222]
Upacicalcet	0.2, 1 mg/kg/d, three times a week for 3 weeks	↓CKD-induced secondary hyperparathyroidism	↓iPTH	[223]
LDN-193189	8 mg/kg/d, 5 w	↓CKD-induced anemia	↓Hepcidin	[224]
PBI-4050	50, 100, 200 mg/kg/d, 3 w	↓Renal injury	↑GPR40	[225]
EOS789	8.0 g/d, 14 d	↓CKD-induced mineral metabolism imbalance and dysregulation	↓Serum phosphorus,↓FGF-23	[226]
Cinacalcet	10 mg/kg, 12 w	↓CKD-induced secondary hyperparathyroidism ↓EndMT	↓PTH ↑CD31,↓FSP1,↓α-SMA	[227]
Dapagliflozin	1 mg/kg/d, 21 d	↓Mitochondrial damage	↓TGF-β1/MAPK	[228]
Isoliquiritigenin	20, 40 mg/kg/d, 8 w	↓CKD-induced vascular calcification	↓ALP,↓ET-1	[229]
Rutin	100 mg/kg/d, 8 w	↓Inflammation	↓HO-1,↓PLA-2	[230]
Perilla peptides	0.2, 0.3, 0.6 g/kg/d, 4 w	↓Inflammation ↓Apoptotic ↓Oxidative stress	↓IL-1β,↓IL-6 ↑BCL-2,↓BAX,↓Caspase3 ↑CAT,↑GSH-Px,↑GSH,↓MDA	[231]
Chinese chive polysaccharides	50, 100, 200 mg/kg/d, 5 w	↓Oxidative stress ↓Inflammation	↑SOD,↑GSH,↓MDA ↓TNF-α,↓IL-1β,↓IL-6,↑IL-10,↓TGF-β	[140]
Honokiol	5 mg/kg/d, 4 w	↓Renal fatty acid oxidation	↑CPT1A,↑ACOX1,↑ACADL,↑Mitochondrial respiratory complex III, IV	[232]
Laminaria japonica Polysaccharide	50, 100, 200 mg/kg/d, 5 w	↓CKD-induced electrolyte disturbance	↑Calcium, phosphorus and magnesium in urine ↓Potassium, magnesium and phosphorus in serum ↑Calcium in serum	[233]
Betulinic acid	30 mg/kg/d, 28 d	↓ECM deposition	↓TGF-β,↓CTGF,↓Fibronectin,↓Collagen I,↓Hydroxyproline	[234]
Morin hydrate	20, 40 mg/kg/d, 15 d	↓Inflammation ↓ECM deposition	↓MCP-1,↓COX-2 ↓CATD	[235]
Ameliorative	30 mg/kg/d, 28 d	↓ECM deposition	↓TGF-β,↓CTGF,↓Fibronectin,↓Collagen I	[197]
Dihydroartemisinin	30 mg/kg/d, 21 d	↓Renal injury	↓DNA-methyltransferase 1,↑Klotho	[236]

It is worth noting that animal models of kidney disease, regardless of the inducing agents or triggers used, commonly exhibit exacerbated oxidative damage, which is a shared mechanism leading to renal inflammation and fibrosis [237]. Given these findings, the natural products with antioxidant properties outlined in Table 2 may offer therapeutic potential for managing adenine-induced kidney injury. However, it must be acknowledged that determining the most effective natural products may be challenging due to differences in testing conditions and experimental designs among different laboratories. For the prevention and treatment of CKD, the use of adenine-induced animal models to evaluate drug effectiveness is well-established, which will contribute to the development of novel medications and drive advancements in the field of nephrology.

6. Miscellaneous

Adenine-induced animal models are valuable experimental tools for elucidating the mechanisms underlying CKD and for studying its common complications. For instance, vascular calcification and bone pathology are frequent complications in CKD progression, scenarios often observed in clinical CKD patients [238–241]. Most of the CKD animal models do not report cardiovascular-related complications. However, 0.75% adenine diet for 6 weeks showed most of the vascular calcification changes expected in CKD animal models [242–244]. Additionally, Neven et al. demonstrated bone and mineral disorders for the first time in the adenine-model of induced CKD [245], and subsequently, several studies have utilized the adenine-induced CKD model to assess CKD-related osteoporosis and mineral bone disease [42, 43, 246, 247]. Indeed, abnormal mineral metabolism in patients with clinical nephropathy, which leads to secondary hyperparathyroidism, has been replicated in the adenine-induced CKD model [44, 223, 246]. It’s also worth noting that male CKD patients may develop gonadal abnormalities leading to impotence, decreased libido, reduced testicular size, impaired spermatogenesis, and gynecomastia [248–250]. Yuichi Adachi et al. first reported that adenine-induced CKD reduces testicular function by inhibiting 17β-hydroxysteroid oxidoreductase, which is crucial for converting androstenedione to testosterone [251], and they reported that the hormonal status in male rats with adenine-induced CKD changes over time post-induction [252]. In conclusion, adenine diet animal models not only present a stable progression of CKD but also model multiple comorbidities, effectively mimicking the complexities of human CKD progression. Consequently, they are indispensable for the assessment of disease mechanisms and therapeutic strategies in preclinical CKD studies.

7. Conclusion and future perspectives

The adenine-induced CKD animal model holds great promise for future CKD research. This model can facilitate drug screening and efficacy evaluation, paving the way to the discovery of innovative therapeutic targets and medications. Furthermore, it serves as a valuable tool for investigating the underlying pathological mechanisms of CKD, facilitating a comprehensive understanding of molecular, cellular, and tissue-level changes. Future research may involve integrating omics technologies, such as transcriptomics, proteomics and metabolomics to harness this preclinical animal model for revealing the complex pathogenesis and pathophysiological alterations of CKD. Additionally, the model offers a prime opportunity to be leveraged for the development of innovative therapeutic strategies (e.g. for addressing APRT deficiency), including cellular therapies and gene editing technologies, aiming to ultimately reverse or cure the disease. Finally, interdisciplinary collaborations can further enhance research efforts, exploring the interconnections between common complications of CKD and other fields such as cardiovascular, reproductive and orthopedic medicine. These perspectives demonstrate the vast potential and broad application prospects of adenine-induced CKD animal models in advancing our understanding and treatment of CKD.

Authors’ contributions

Yang Q.: conceptualization, project administration, and writing; Su S.Y., and Luo N.: data curation; Cao G.: Funding acquisition.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The authors acknowledge financial support from the Zhejiang Province Traditional Chinese Medicine Science and Technology Project (No. 2021ZQ023); the Natural Science Foundation of Zhejiang Province, China (No. LQ23H280004); and the Zhejiang Chinese Medical University University-level research projects (No. 2021JKJNTZ008B).