Uremic mouse model to study vascular calcification and “inflamm-aging”

In the current study, we present a DBA2/N mice model for induction of renal failure via adenine-enriched diet. This model is suitable for studying uremic vascular calcification and arterial “inflamm-aging” as characterized by the induction of cellular senescence, pro-inflammatory SASP, and osteogenic differentiation. Although several studies have already investigated parts of this context [13,14,15,16,17,18,19,20], our experimental setting differs in several parameter to previously published protocols and therefore combines the advantages of non-surgical disease induction by likewise minimizing the disadvantages that were so far associated with feeding of an adenine-enriched diet: (1) no surgical intervention to induce CKD in mice; (2) the modified diet with 0.2% adenine, 1% phosphate, and 6% protein content is well tolerated by the mice and the weight loss is not associated with premature dead; and (3) to our knowledge, this is the first study demonstrating the adenine-induced time-dependent uremia progression with associated MAC and SASP in DBA2/N mice.

As it is already known for DBA2 mice that they suffer from an age-dependent soft tissue calcification and are more prone to develop MAC after CKD induction than other mice strains [11], this mice strain was used for the presented adenine-based CKD model. As female mice are more prone to calcification compared to male littermates [18], only female mice were selected for this study.

CKD induction in rodents can be induced either by surgical reduction of renal mass or by nephrotoxic adenine diet [11]. Nephrectomy-based CKD models are established for rats and mice, but the spread of renal function loss and associated MAC extent is high [11]. Induction by adenine leads to a reproducible CKD condition in rodents with moderate to severe MAC progression [11]. Drawbacks of the initial protocols using 0.75% adenine are the pronounced weight loss associated with high mortality rate and biological variability in the CKD degree [11]. A reduced mortality rate and reduced body weight loss could be achieved by reduction of the adenine content (0.25% in rats, 0.2% in mice) [11]. The adenine dose of the current study with 0.2% was sufficient to induce a stable uremic condition in all mice from 6 to 12 weeks without any impact on animal survival.

By variations of the diet components, the biological variations and weight loss can be also reduced. Some studies used vitamin D-enriched diet that on the one hand increases vessel calcification, but on the other hand further promotes weight loss [11]. A study by Price et al. showed in rats that reduction of the protein content in the adenine diet to 2.5% leads to exacerbated aortic calcification compared to higher protein content of 25% [24]. However, reduction of protein content leads also to reduction in food intake [25]. To counteract the expected weight loss associated with using the adenine-based protocol for renal insufficiency induction in mice, we chose a protein content of 6%. Although the protein content is reduced compared to the standard diet, we expected less reduction in food intake [12, 25]. In our study, the diet was tolerated by the animals and the weight loss was controllable so that no premature death occurred. The body weight loss upon adenine feeding was also seen in other studies with mice receiving a diet with similar adenine content [13].

The CKD induction is reflected by histological alterations of glomerular and tubular structures corresponded with an increase of blood parameters as Bun, Crea, and Pth. These results are in line with those of previous CKD studies in C57BL/6 mice [13], ldr knockout mice [26], and rats [12]. The laboratory chemistry reference areas were found to be different in various mice strains and varies also during the stages of animal growth [27]. A direct comparison of the blood parameters between different studies is also hampered by variations in ingredients of the experimental diet used; these ingredients do not only vary in concentration [11], but also in their source, which is relevant as, e.g., the intestinal resorption of phosphorus varies depending on the source. In our study, we used a diet containing 1% phosphate which did not result in elevated phosphate plasma concentration, while other protocols often used hyperphosphatemia inducing phosphate concentration (up to 2% phosphate) [14, 15, 28]. Tani et al. recently compared 0.8% phosphate vs. high phosphate concentration (1.8%) in C57/BL6 mice. After 12 weeks of treatment, they found higher phosphate plasma concentrations in both groups and detected a stable CKD and subsequent MAC [28]. In contrast, several studies in DBA2 mice showed no hyperphosphatemia with diet phosphate concentrations of 0.5% and 0.9%, respectively [18,19,20]. These findings are in line with our results.

Previously published results by Shanahan´s group showed the importance of premature vascular aging for MAC in children with advanced CKD in vivo [4]. Compared to controls, human vessels and VSMC showed properties of premature vascular aging and increased mineralization, which was partly due to activation of the pro-inflammatory SASP [4]. The SASP is established in different cell types in response to DNA damage and includes potent osteo-inductive and pro-inflammatory mediators like Bmp-2, Il-1β, and Il-6 [4, 29,30,31,32]. These data suggest that the paracrine secretion of pro-inflammatory and/or osteo-inductive factors by senescent VSMC may be an important driver of human uremic MAC. However, animal models with a comparable pathophysiology are required to investigate signal transduction pathways and treatment options. As already shown by Santana et al., adenine-induced CKD is associated with chronic inflammation in C57/BL6 mice [13]. However, the causal link to MAC remains unclear, as this study aimed to investigate the inflammatory-based renal insufficiency [13]. The investigated model presented here might bridge this gap. Thoracic aortas from uremic DBA2/N mice show properties of premature aging, osteogenic differentiation, and enhanced mineralization. Comparable to the situation in CKD children, the mouse aortas have enhanced expression of p21, Bmp-2, Sox-9, Saa1, Il-6, and Il-1β. In CKD children, the Il-6 plasma concentration was significantly elevated and showed a high correlation to the extent of coronary artery calcification [4]. Therefore, we investigated a pro-inflammatory multiplex panel in our mouse model. Interestingly, out of the 34 investigated cytokines, only the plasma concentration of Saa, Il-6, and Mcp-1 were increased. Possibly, a vicious cycle of inflammation during uremia leads to the predictive effect of cytokine release and cardiovascular death as shown for Saa and Il-6 [33,34,35]. In addition, studies have shown that the pathways of Il-1β and Il-6 are interconnected [36]. Recently, we could show that Il-6 is induced during calcification progression, but not directly induces mineralization, while Il-1β induces an osteogenic driven auto-loop in smooth muscle cells [37]. Il-1β and Il-6 are known middle-sized uremic toxins influencing osteogenic differentiation [9].

Limitations of the current protocol are (a) only female DBA2/N mice were investigated so that a transfer to other sex and strains is pending, (b) no blood and pulse pressure data were obtained, and (c) this study was designed as proof-of-experimental protocol study so that an interventional approach in the current model is also pending.

As some studies already exists for C57/BL6 mouse strains and CKD induction upon adenine diet [15, 16, 28], the current protocol should be transferable to other strains. A correlation between blood and pulse pressure changes with the extent of MAC was successfully shown in an adenine rat model [12]. Some animal treatment studies with therapeutic treatment are already available. The protective effect of tissue non-specific Alp inhibition in an adenine-based CKD mouse model was recently shown [28]. A further study has shown the potential of a treatment strategy by induction of an endogenous regulator against MAC, peroxisome proliferator-activated receptor-gamma coactivator-1 alpha in a CKD rat model [38]. Also, a magnesium-based phosphate binder as therapeutic option was tested in an adenine-based CKD rat model [39].

As up to now, specific treatment options in the clinical situation for reduction or prevention of disease progression are still missing, and of current research interest [1, 3], the described study protocol here might be beneficial in further studies.

We used an adenine-based diet to induce CKD in DBA2/N mice with subsequent chronic uremia resulting in MAC and associated SASP situation in the vessel wall of the mice. The described situation is comparable to the human CKD situation recently published [4] in several points: (1) premature aortic aging (e.g., p21), (2) osteogenic trans-differentiation (e.g., Bmp-2), and (3) and vessel mineralization.

Because CVD is a strong prognostic factor in CKD, understanding its pathogenesis as well as evaluating and testing of novel therapeutic drugs are urgently needed to provide new therapeutic concepts that help to reduce the high cardiovascular mortality of these patients, and therefore, suitable animal models are necessary.

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