Cardiovascular calcification is a serious complication in patients with chronic kidney disease (CKD) [1]. In patients with end-stage renal disease, calcification occurs in the aorta and aortic valve based on bone mineral disorder, with incidences of 49–93 % and 27–100 %, respectively [2]. Aortic valve calcification induces abnormalities in cardiac function [3], and approximately 30 % of deaths in CKD patients are due to cardiovascular disease [4]. Calcification and fibrosis are the primary causes of aortic valve sclerosis [5]. Several therapeutic drugs have been developed to prevent calcification, but no drugs have been approved for treating aortic valve calcification [6].

In aortic valve calcification in patients with CKD, valvular interstitial cell-mediated processes may be associated with Ca deposition [7]; therefore, valvular interstitial cells (VIC) have been used in in vitro aortic valve calcification studies [8]. In experiments using VIC, extracted Ca is measured directly, while in vivo, calcification is mainly visualized through pathological analysis using echocardiographic or micro-computed tomography (µCT) assessment [8]. In vivo imaging techniques, such as µCT, are non- to low-invasive methods of quantifying calcification, but they are primarily only available in specialized core facilities due to the requirement of hardware equipment and personnel training [9]. Pathological analysis visualizes the location of calcification but does not present the calcifying degree of the whole tissue. Compared with pathological evaluation, the direct measurement of Ca and P is quantitative and more stable for the evaluation of calcification; furthermore, it can be conducted in a shorter time. Therefore, the direct measurement of Ca and P in animal models would help to explore the mechanism of valve calcification and discover new therapeutic drugs for aortic valve calcification, as described by Davidson et al [10]. However, in rodents, which are commonly used for animal experiments due to being easy to handle, their valves are difficult to isolate and provide less material for molecular analysis due to their small size [11]. Thus, using animal models for evaluating aortic valve calcification remains challenging in terms of cost, availability, and adaptability for quantitative determination of Ca and P using easy experimental manipulation.

Some ingenuity could resolve the problem of the aortic valve in rodents being too small to be isolated, enabling them to be used as a novel model for the study of aortic valve calcification. The sinus of Valsalva, which is located just above the aortic valve and is the origin of the coronary artery [12], contributes to valve opening/closing [13], and its calcification leads to a disorder known as valve stenosis [14]. In this study, we quantified rat aortic valve calcification by isolating the whole aortic valve, which includes three sinuses of Valsalva and their respective valve leaflets.

Calciprotein particles (CPP), which are aggregates of Ca, P, and fetuin-A proteins in the body, have been suggested as a surrogate marker and/or therapeutic targets for calcification [15]. The findings from the Phase 2b study of phytate, a known inhibitor of CPPs, together with the outcomes of CPP removal column experiments conducted in minipigs, indicate its potential utility as a surrogate marker for aortic valve calcification [16,17]. Therefore, confirming the relationship between CPP formation and calcification of the aortic valves and exploring the effects of bisphosphonates, inhibitors of CPP formation, on calcification would be useful for clarifying the characteristics of the calcification in the aortic valves. The inhibitory effect of bisphosphonates on aortic valve calcification has been confirmed in vivo using pathological staining [18,19]. We focused on two bisphosphonates with different characteristics: etidronate and FYB-931. Etidronate showed inhibitory effects on the calcification of rat and sheep valvular interstitial cells [20], while FYB-931 inhibited CPP at lower concentrations or doses than etidronate in vitro and in vivo [21,22].

In this study, the effects of etidronate and FYB-931 on in vitro calcification of human aortic valvular interstitial cells (HAVICs) [8] were tested to confirm their potential as inhibitors of aortic valve calcification. The rats with calcified aortic valves treated with the two bisphosphonates were evaluated in vivo. The Ca/P contents of the whole aortic valve were quantified to investigate whether the region collected as aortic valve has distinct calcification characteristics compared with those of the heart or aorta in the context of CPP formation as a biomarker of calcification propensity.