**2. Animal Models of CKD**

Animal models invariably provide valuable insights into the molecular mechanisms of diseases and their underlying pathology. However, none of the prevailing models reproduces the complexity of CVD in CKD [22]. While few non-transgenic rodent models are employed to study CV calcification in CKD [23], one such variant method is the reduction of renal mass via nephrectomy (Figure 3). Five-sixths nephrectomy is limited by the variability in CV calcification, the high mortality rate in patients with advanced CKD, and the necessity for surgery as an irreversible method [24]. Administration of dietary adenine is another strategy to initiate CKD in animal models (Figure 3); adenine is transformed to 2,8-hydroxyadenine, which precipitates in the urinary tract due to its low water solubility [25]. This causes nephrotoxicity, which is similar to clinical CKD [24]. The main disadvantage of this model is the weight loss of the animals due to reduced food intake. The adenine model is a reversible CKD model, because there is no need for surgery, which eases its implementation and handling. As neither five-sixths nephrectomy nor adenine diet alone initiate CV calcification, either high-phosphate or high-fat diet are used as a second trigger (Figure 3) [26]. Both models show similarities—hyperphosphatemia, increased plasma creatinine, and enhanced blood urea nitrogen, but the CV calcification outcome is not consistent. The reasons for this are di fferences in trial times and the high variability in diet phosphate and calcium concentrations [24]. The sensitivity to CV calcification also depends on the genetic background, age, and gender of the animals [27]. Female mice show higher susceptibility to CV calcification than males [28], which is the opposite to what observed in humans, wherein men tend to have higher average coronary artery calcium scores than women [29]. This might sugges<sup>t</sup> that the hormone status affects vascular calcification and should be considered when planning experiments. CV calcification variabilities in CKD are mostly seen in mice but tend to be strain-dependent [22]. The most commonly used mouse strain is C57Bl/6, which is resistant to the development of hypertension, glomerulosclerosis, and proteinuria. In addition, it shows decreased activity in the renin–angiotensin–aldosterone system, which is important for fibrosis development after five-sixths nephrectomy [27,30]. In summary, compromises have to be made in choosing the right CKD animal model. Therefore, it is essential to agree on standards using rodent models within the CVD–CKD research field.

**Figure 3.** Schematic presentation of rodent non-transgenic animal models of cardiovascular calcification in CKD. HFD: high-phosphate diet; CV: cardiovascular; P: phosphate; Ca: calcium. The figure was partially created using Servier Medical Art, licensed under a Creative Commons Attribution 3.0 Unported License. Arrows indicate CV calcification induced by 5/6 nephrectomy and 0.25 % adenine diet. Fork indicates kidney areas, which are removed during 5/6 nephrectomy.

### **3. Therapeutic Concepts of CV Calcification in CKD**
