Similarities and Differences of Vascular Calcification in Diabetes and Chronic Kidney Disease

Introduction

The VC procedure parallels the bone morphogenesis process and could frequently be delineated as progressive atherosclerosis, culminating in diminished vascular elasticity.¹ Ultimately increasing the risk of future cardiovascular events. There are two primary forms of calcification; intimal calcification, which is present in atherosclerotic lesions of the intima, and medial calcification, also known as Mönckeberg sclerosis, which involves calcification of the smooth muscle layer of the vascular mesentery.² Cardiovascular disease is an essential terminal phase alteration in type 2 diabetes mellitus (T2DM) and CKD, and it is the most prevalent cause of mortality in the populace, wherein the high frequency of VC is a significant contributing risk factor.³

At present, an array of investigations have demonstrated that both DM and CKD are intimately interconnected with VC. Recent evidence has revealed a multitude of shared mechanisms that accelerate VC in the milieu of both diseases. These include mitochondrial oxidative stress, pro-inflammatory cytokine release, endothelial impairment, lipid metabolic aberrations, hypoxia, common signalling cascades (such as Advanced Glycation End products (AGEs)/Receptor for AGEs (RAGE)), extracellular vesicles, miRNAs, etc. Despite these shared mechanisms, the differences between the two in VC conditions are equally noticeable. VC is primarily caused by metabolic disorders such as hyperglycemia and hyperlipidemia, whereas in CKD chronology, VC is exacerbated by calcium and phosphorus imbalances, hormonal disorders, and intestinal leakage of large amounts of uremic toxins. This review will summarize recent advances in VC mechanisms in the context of CKD and diabetes. We will also highlight vascular alterations and newly detected molecular pathways as promising novel therapeutic targets. Insights into molecular mechanisms and the identification of novel targeted agents for calcification will foster the discovery of novel biomarkers and therapeutic strategies for cardiovascular disease.

Relationship Between Two Different Environments and Vascular Calcification

Although many aspects of VC pathogenesis remain elusive, it is currently recognized as a multi-factorial process that requires careful regulation and continuous suppression. Numerous pathognomonic features have been extensively documented as being correlated with VC. Prolonged hyperglycemia serves as a hallmark feature of diabetes mellitus, with its clinical association with VC firmly established. There is mounting evidence that numerous elements of metabolic syndrome (Mets) linked to type 2 diabetes may promote VC, including oxidative stress, endothelial abnormalities, dyslipidemia, disordered mineral metabolism, and elevated synthesis of pro-inflammatory cytokines. In CKD patients, VC is amplified by multiple interconnected mechanisms, including aberrant mineral metabolism, inflammatory pathways, hormonal dysregulation, and uremic toxins, all of which could potentially augment VC directly in such patients. Therefore, we will delineate recent advances in VC pathogenesis in the context of diabetes and nephropathy. The similarities and correlations of these pathways will be discussed in more detail. VC plays a critical role in the progression of both diseases.

Similarities in the Mechanisms of Vascular Calcification in Both Environments

VC is a common risk factor for both diabetes and chronic kidney disease. The mechanisms of VC in these two environments have many similarities (Table 1).

Table 1 Similarity of Vascular Calcification Mechanisms in the Two Environments

Pro-Inflammatory Factors

Chronic inflammation is a well-documented phenomenon in the development of VC and DM, in addition to Renal Disease. The present scientific consensus suggests that VC is essentially a chronic inflammatory state. There is substantial evidence to demonstrate that pro-inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6),⁴⁶ interleukin-19 (IL-19), angiopoietin-2 (Ang-2), transforming growth factor beta (TGF-β),⁴⁷ and tumor necrosis factor-alpha (TNF-α)¹² play crucial roles in the etiopathogenesis of VC.⁴⁸ Furthermore, evidence has also linked the augmented levels of TNF-α, IL-6, and interleukin-1β (IL-1β) with the initiation of VC by catalyzing the osteochondral differentiation of vascular smooth muscle cells (VSMCs).^4,5 Additionally, it is well-established that the activation of t tissue nonspecific alkaline phosphatase (TNAP) is instrumental in the formation of hydroxyapatite crystals,⁴⁹ and TNF-α promotes the expression of matrix metalloproteinase 2 (MSX2) via the nuclear factor-κB (NF-κB) signalling pathway, thereby enhancing TNAP activity and thereby promoting VC.^6,7 Furthermore, TNF-α stimulates the production of IL-6, which, in turn, induces osteochondral maturation of VSMCs through bone morphogenetic protein-2 (BMP2) mediated amplification of IL-6,^7,50 Notably, reactive ROS released by IL-6, IL-1β, and TNF-α are shown to regulate VC through activation of AKT signalling and regulation of the associated transcription factor 2 (Runx2) / core-binding factor alpha 1 (CBFA1).⁸ Reactive ROS also stimulate NF-κB and NF-κB dependent bone-induced signalling pathways⁹ and augment pro-inflammatory reactions to VSMCs.⁵¹ In addition, protein kinase A (PKA) is demonstrated to suppress the calcification of human aortic smooth muscle cells that alleviate inflammatory reactions.⁵² Research indicates that finerone (an Angiotensin-converting enzyme II receiver antagonist (MRA) mitigates the mRNA expression of M1 macrophage markers IL-1β and the pro-inflammatory factor TNF-α.⁵² Therefore, the suppression of inflammatory cytokines may serve as a promising strategy to mitigate cardiovascular risk among individuals with diabetic nephropathy.

Decreased Expression of Silent Mating Type Information Regulation 2 Homolog- 1(SIRT1)

SIRT1, a NAD-dependent deacetylase, is strongly associated with cellular metabolism, energy stress, and longevity. Acetylation of Runx2 promoter significantly controls Runx2 expression, leading to osteogenic differentiation. In an environment of heightened glucose levels, SIRT1 expression in the serum of diabetic subjects is notably decreased due to increased acetylation of Runx2 promoter in comparison with healthy individuals and under osteogenic conditions.⁵³ This implies that an increased acetylation of Runx2 promoter in cells is associated with reduced SIRT1 expression (Figure 1). Additionally, Liu et al⁵⁴ conducted an experiment demonstrating that treatment of human VSMCs with spermidine (Spd) resulted in a decrease in BMP2 and Runx2 activity on the seventh day. This, in turn, led to an up-regulation of SIRT1 in VSMCs, which ultimately reduced the expression of endoplasmic reticulum (ER) stress signal transduction molecules, contributing to CKD arterial calcification. These findings highlight the potential of SIRT1 activators in managing VC.

Figure 1 Mechanism of oxidative stress promoting vascular calcification in diabetes. In the environment of diabetes, AGEs bind to advanced glycosylation receptor (RAGE), which activates NF-kB and promotes vascular calcification by enhancing the expression of VEGF, VCAM-1 and other genes. PKC pathway increases ROS level of endothelial cells by reducing NO production.Oxidative stress promotes VC by activating p21, JNK, p38 and NF-kB. ROS excess inhibits insulin gene transcription by opening ATP-sensitive K+channels, thus inhibiting insulin production and secretion. Oxidative stress promotes VC by activating p21, JNK, p38 and NF-kB. Upregulation of SIRT1 in VSMC leads to downregulation of endoplasmic reticulum (ER) stress signal transduction components, regulating SIRT1 and ER stress signals to inhibit arterial calcification in CKD. Graphics by Figdraw.

Similarities in Oxidative Stress

Oxidative stress has been demonstrated to be strongly associated with vascular endothelial dysfunction. The primary mechanistic pathways involved in vascular endothelial cell dysfunction include PKC, AGE/RAGE, and additional pathways. Specifically, PKC reduces the production of nitric oxide (NO),¹⁴ which is the key regulatory factor for the normalization of vascular function, which is a critical regulatory factor for the proper function of the vascular system. Furthermore, the AGEs/RAGE pathway promotes vascular endothelial cell inflammation and thrombosis by increasing the content of reactive oxygen species (ROS) within endothelial cells (ECs), stimulating the activation of NF-κB, and enhancing the expression of vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), and other genes.^13,14 (Figures 1) Antioxidant stress is also implicated in VC by stimulating the expression of p21, c-Jun N-terminal kinase (JNK), p38, and NF-κB. Furthermore, other animal experiments have confirmed the role of NADPH oxidase family protein 1 (Nox1) in oxidative stress associated with diabetes-induced atherosclerosis,¹⁵ (Figures 1) while a similar role is observed in CKD. NADPH oxidase inhibitors, such as Dextromethorphan (DXM), effectively decrease ROS production and impede calcifying mediators-induced VSMCs calcification.^55,56

Oxidative Stress - Downstream Signaling Pathway

Adequate scientific research suggests that an increase in oxidative stress is often associated with an increase in the expression of RUNX2 and alkaline phosphatase (ALP), and the calcification of VSMCs.⁵⁷ Furthermore, the generation of hydrogen peroxide from a variety of sources in blood vessel cells is considered a significant contributor to oxidative stress in atherosclerotic lesions.¹⁹ As such, inhibiting the activation of the H2O2-activated AKT signalling pathway has the potential to suppress the increased expression of RUNX2 and the calcification of VSMCs.⁸ Concurrently, it has been established that the accumulation of advanced oxidation protein products (AOPPs) can promote the osteoblast differentiation of human VSMCs through the activation of the ERK signalling pathway, thereby stimulating VC.¹⁶ Other studies have revealed that the oxidative stress triggered by the ERK1/2 MAP kinase^17,58 and p38 MAPK pathway can promote bone induction¹⁷ of VSMCs. Furthermore, oxidative stress can trigger the apoptosis of VSMCs and the progression of VC.^59,60 It is increasingly being recognized that the upregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) can lead to a decrease in antioxidant enzyme expression (including superoxide dismutase (SOD)) and an increase in mitochondrial reactive oxygen species (mtROS) production, which are associated with VC.^61, In addition, recent studies have identified an increase in posttranslational protein modification via oxidative stress and O-linked β-n-acetylglucosamine modification (o-glcnac acylation), which is linked to calcium flux^62,63 and the regulation of calcium-dependent signalling.^61,64 The calcium-sensing protein STIM1 is known to regulate intracellular calcium flux and intracellular calcium influx through store-operated calcium portals.⁶⁵ Additional research has demonstrated that an increase in calcium flux, calcium signalling activation and ER stress in STIM1δ / ΔVSMCs can lead to STIM1-deficient O-GlcNAcylation and VSMC calcification.¹⁸ Sirtuin 3 (SIRT3) also plays a crucial role in mediating the protective effects of PGC-1α on mitochondrial oxidative stress and VC.⁶⁶ It is worth noting that a recent study has highlighted the essential role of antioxidants in defending against ROS attacks.⁶⁷ Therefore, antioxidants should be included as an integral component of modern treatment strategies. Antioxidant therapy has shown promising results in preventing and slowing the development of VC in patients with CKD and DM.

Similarities in Endothelial Dysfunction

The role of endothelial dysfunction in the development of cardiovascular disease has been extensively investigated. Endothelial cells serve as vital regulators of vascular tone through the release of vasodilators such as NO and vasoconstrictors like endothelin-1 (ET-1). In response to physical strain and autonomic molecules, ECs control the behaviour of VSMCs and contribute to the maintenance of vascular health.^10,68 The potential regulatory roles of NO and ET-1 in VC have been the focus of recent studies. Evidence suggests that NO can inhibit TGF-induced Smad2/3 phosphorylation, thereby preventing SMC calcification and osteochondrocyte transformation.¹⁰ Conversely, ET-1 has been shown to contribute to VC by suppressing calcification inhibitors like matrix Gla protein (MGP) and osteoprotegerin (OPG). Moreover, ET-1 also promotes the production of pro-calcification mediators like BMP2 and osteopontin (OPN).¹¹ In an in vitro study conducted by Sanchez-Duffhues et al.¹² IL-1β and TNF-α were found to suppress the expression of bone morphogenetic protein receptor type II (BMPR2) in endothelial cells. This suppression was associated with activation of the BMPR-c-Jun N-terminal kinase (JNK) signaling axis, a pathway that plays a crucial role in inflammation-induced endochondral metaplasia and promotes endothelial osteogenic transformation. Individuals with CKD and hyperglycemia are at a relatively high risk of developing VC. High levels of glucose can induce mineral metabolic imbalance in ECs by increasing calcium and potassium uptake, leading to an irregular phosphorylation of endothelial nitric oxide synthase (eNOS) and overexpression of osteopontin through the p38 pathway.⁶⁹ These alterations ultimately undermine endothelial function and lead to the development of VC.

The beneficial effects of metformin, an anti-diabetic medication, on VC have been demonstrated in animal models. Metformin has been shown to mitigate VC through activation of the adenosine monophosphate kinase (AMPK)/eNOS/TGF-β1 signaling pathway.⁷⁰ In summary, the regulation of vascular tone by ECs in response to autonomic molecules and physical strain is critical for maintaining vascular health. The identification of key pathways such as NO and ET-1 as potential targets for the prevention and treatment of VC provides important insights into the development of novel therapeutic strategies for this prevalent disease.

Oxygen Deficit and Vascular Calcification

Hypoxia significantly enhanced the protein expression of HIF-1α (Hypoxia-Inducible Factor-1α), GLUT1 (Glucose Transporter 1), and VEGFA (Vascular Endothelial Growth Factor A) mRNA expression. The activity of RUNX2, SOX9 (Sex-determining region Y (SRY)-box 9), OCN (Osteocalcin), and ALP (Alkaline phosphatase) proteins was also increased. A time-dependent accumulation of calcium was detected within the extracellular matrix (ECM) of VSMCs in response to hypoxia.³⁶ Hypoxia-induced osteochondrogenesis and differentiation of VSMCs is triggered by HIF-1-dependent and mitochondria-derived reactive oxygen species, subsequently promoting VC. The expression of OCN and ALP genes, regulated by osteochondrogenic transcription factors, also increased in VSMCs under hypoxia.²⁰ Several studies have indicated that hypoxia-induced ROS generation is mediated by HIF-1 activation in VSMCs.⁷¹ In addition, hypoxia enhances osteochondral differentiation and extracellular matrix calcification through ROS. It is believed that excess ROS generated during hypoxic conditions elevates the expression of HIF-1α.¹⁹ Furthermore, Zhao et al⁹ reported that mitigation of mitochondrial ROS generation can hinder phosphate-induced VSMCs osteogenic differentiation.

Lipid Metabolism Disorders

VC is often associated with disruptions in lipid metabolism. A well-researched correlation between diabetes and atherosclerosis is the concentration of small, dense, low-density lipoprotein (sdLDL-C).⁷² Notably, the prevalence of type B (with small-sized LDL particles) is higher in patients with diabetes,⁷³ which contributes to the development of VC.⁷⁴ Furthermore, the formation of sdLDL is strongly associated with insulin resistance and elevated triglycerides.⁷³ Although the concentration of LDLC in patients with moderate to advanced CKD does not appreciably increase, oxidized low-density lipoprotein cholesterol (oxLDLC) and sdLDL-C tend to rise.^22,23 In experimental conditions of high phosphate exposure, fat cells have been found to induce vascular smooth muscle cell calcification by secreting adipokines such as VEGF.⁷⁵ PGC-1α, as a metabolic master regulator, controls downstream target genes in the nucleus and mitochondria through the recruitment of coactivators. It plays a pivotal role in mitochondrial biogenesis, fatty acid oxidation, and adipogenesis.^76,77 In a pioneering study, Feng et al²¹ demonstrated that the activation of the PGC-1α/SIRT3 pathway can inhibit VC. This suggests that the PGC-1α/SIRT3 pathway may be a promising therapeutic target for managing VC. Additionally, statins have been shown to reduce cholesterol levels and lesion areas in mice fed a high-fat “Western” diet. Therefore, it is hypothesized that the adjustment of triglycerides and the use of statins may assist in delaying the onset of VC in patients.

Microelement

The principal emphasis of this research pertains to the significance of magnesium and zinc in the prevention of VC. Numerous randomized studies have demonstrated that magnesium, via the antagonism of calcification-promoting protein (CPP) maturation, can inhibit the progression of VC.²⁴ Magnesium has been observed to negatively regulate osteogenic transdifferentiation and VC²⁵ by enhancing the activity of its transporter, transient receptor potential melastatin 7 (TRPM7), and its invasion into cells, resulting in the expression of anti-calcification proteins such as bone morphogenetic protein 7 (BMP-7), OPN, and MGP. Furthermore, magnesium has been shown to inhibit the WNT /β-catenin signalling pathway and repress Pit-1 expression^26,27 within VSMCs. The anti-calcification influence of magnesium is mediated by its ability to impede the WNT /β-catenin pathway, thereby preventing calcium from penetrating VSMCs and osteogenic transdifferentiation.⁷⁸ In addition, magnesium has the potential to prevent the formation of extracellular hydroxyapatite, thereby preventing VSMC calcification.⁷⁹ Magnesium has also been shown to influence bone formation (Runx2, Smad1, and Osterix) by down-regulating microRNA expression (miR-133a, miR-30b, and miR-143a).²⁸ Emerging evidence has highlighted the role of zinc in oxidative stress resistance and its potential in mitigating the progression of VC.²⁹ Specifically, zinc may deter phosphate-induced arterial calcification by inhibiting the activation of NF-κB light chain enhancer. In individuals suffering from type 2 diabetes and CKD, serum zinc levels have been found to correlate positively with T50 (a shorter T50 indicates a heightened propensity to calcify).²⁹ Thus, the judicious supplementation of magnesium, zinc, and various other trace elements may provide a promising approach to delaying the progression of VC.

AGE/RAGE Signal Cascade

AGEs refer to a class of proteins that are indirectly modified by the automatic oxidation of carbohydrates and reactive carbonyl compounds that are formed through the AGE process. The circulating AGEs in patients on dialysis, including pentanoside, are often found to be elevated.⁸⁰ This class of molecules has been shown to act through a feedforward cycle, which results in the upregulation of RAGE expression and the exacerbation of oxidative stress.^32,33 Upon interaction between AGEs and RAGE, RAGE initiates a downstream signaling cascade through PKC-ζ, which is mediated by p38 mitogen-activated protein kinase (MAPK), TGF-β and NF-κB, and PKC.^30,31 Studies have shown that RAGE activation by AGE/RAGE signaling can stimulate the generation of reactive ROS through specific pathways such as TGF-β, NF-κB, and Nox-1.³² Evidence suggests that members of the AGE/RAGE signaling cascade, including p38 MAPK and ERK1/2, are phosphorylated following RAGE activation.³⁴ Moreover, TGF-β signaling can lead to the phosphorylation of Smads, a family of transcriptional regulators.⁸¹ This implies that the AGE/RAGE cascade is also associated with Smad activation and TGF-β signaling.^33–35 RAGE signaling has also been implicated in various mitotic pathways that regulate VSMC calcification. The p38 MAPK pathway is an essential component of AGE/RAGE-induced VSMC differentiation. The role of AGE/RAGE signaling in diabetes-induced VC is attributed to the calcification caused by oxidative stress and the AGE-induced transformation of VSMC phenotype.^36,37 In addition, the AGE/RAGE signal has been proven by activating and lower SOD Nox-1 expression to increase the oxidative stress and promotes diabetes mediated VC.^32,82

Furthermore, studies have demonstrated that AGE/RAGE signaling can directly affect ALP activity, where p38 MAPK plays a key role.^83,84 Janda et al⁸⁵ have found that elevated serum levels of fetal protein A (fetuin-A) are a positive indicator of increased AGE deposition in arteries, suggesting that fetuin-A may indirectly influence the AGE/RAGE pathway, particularly in the presence of inflammatory molecules. Studies have also linked AGEs to matrix alterations, specifically to the formation of irreversible crosslinks of stromal collagen and elastin, resulting in an increase in ECM stiffness and the development of arteriosclerosis. Non-enzymatic glycosylation of collagen renders it resistant to enzymatic degradation and contributes to its accumulation in the arterial wall.^38,39 RAGE mediates PI-induced VSMC calcification⁸⁶ in vitro and VC⁸⁷ in enpp1 − / − mice. An example of AGE-induced matrix alterations is seen in enpp1 − / − mice, where AGEs alter the glomerular basement membrane, leading to increased albumin permeability, endothelial cell pathology, and the activation of pro-atherogenic and prothrombotic genes. Ultimately, this may lead to an impediment in the selective absorption of high-density lipoprotein (HDL) ester and HDL cholesterol from peripheral cells, thereby impeding the reverse transport of cholesterol.⁸⁸

Extracellular Vesicles

Apart from the release of secretory vesicles by specialized cells, which carry, for example, hormones or neuro- transmitters, all cells are capable of secreting various types of membrane vesicles, known as extracellular vesicles, and this process is conserved throughout evo- solution from bacteria to humans and plants.^89,90 Notably, these vesicles carry microRNAs (miRNAs, miRs), mRNAs, and proteins that undergo profound changes in protein composition as a result of elevated phosphate levels.⁹¹ From VSMCs, the apoptotic bodies of cells undergoing apoptosis (ABS) can also serve as a form of calcium crystal in the nuclear architecture, similar to matrix vesicles (MV).⁹² In physiological conditions, these vesicles remain free of crystals as they are equipped with potent mineralization inhibitors such as fetuin-A and MGP.^93,94 However, an increase in phosphatemia and hypercalcemia can stimulate the secretion of matrix vesicles by human VSMCs, potentially triggering the mineralization of the extracellular matrix in bone.⁴⁰ In addition, it has been found that apoptosis induced by high phosphate may be related to extracellular phosphate-induced VSMC calcification.⁴⁰ Conversely, other research has concluded that phosphate-induced VSMC calcification is independent of apoptosis or extracellular vesicles.⁹⁵ Therefore, whether extracellular vesicles can be novel targets for VC remains to be investigated.

Similarity of microRNA in Vascular Calcification

Recently, a study has found a number of miRNAs in diabetes, CKD, and coronary heart disease (CAD), which have a high prevalence of calcification: miR-21, and miR-146a are common in these diseases and have a regulatory role in the transdifferentiation of VCm and SMCs into osteoblast-like cells. Current evidence suggests that miRNAs are implicated in modulating various intracellular pathways that regulate osteogenic / chondrogenic phenotypic transitions in VSMCs, Including WNT/β-catenin pathway,⁹⁶ PI3K signaling pathway,⁴¹ STAT3 signaling pathway⁴² or TGF-β1 /SMAD signaling pathway.⁴³ These miRNAs exert a critical role in osteogenic/chondrogenic transdifferentiation of VSMCs by controlling various cellular operations under conditions of hyperphosphatemia, such as gene expression,^{44,45,97–99} inflammasome activation,¹⁰⁰ apoptosis,^98,101 senescence,^42,98 or endoplasmic reticulum stress.¹⁰¹

A study by Zhou et al¹⁰² confirmed that the expression of miR-21 is stimulated by hemodynamic forces (such as oscillatory shear stress (OSS)) and facilitates the pro-inflammatory response in human umbilical vein endothelial cells (HUVECs) by augmenting the binding of c-Jun to the miR-21 promoter region. In endothelial cells, miRNAs suppress the expression of genes associated with NF-κB signalling, potentially playing a role in the progression of endothelial disease. Additionally, miR-146a-5p has been identified as a suppressor of NF-κB¹⁰³ Diminished expression of miR-146a might result in diminished efficacy of suppression of target genes involved in NF-κB and other cytokine production and signalling pathways. Furthermore, interleukin-1 receptor-associated kinase (IRAK)1, TNF receptor-associated factor 6 (TRAF6), and IRAK2 have been identified as self-targets of miR-146a,¹⁰⁴ further highlighting its role in mitigating inflammatory cytokine production and pro-inflammatory conditions. The role of epigenetics⁴⁴ and the currently recognized microRNAs related to VC.^45,105 This research sheds light on the potential role of miRNAs in VC and the complex regulatory network involving various miRNAs and their targets in the progression of cardiovascular disease.

Differences in Vascular Mechanisms Between the Two Environments

The development of VC is a complex and multifaceted process involving a number of mechanisms, such as chronic inflammation, oxidative stress, and dysregulation of bone-mineral metabolism. While both DM and CKD share certain similarities in their aetiologies, they also exhibit several significant differences in their respective mechanisms of development (Table 2). In the context of DM, hyperglycemia and hyperlipidemia serve as primary metabolic ecosystems that drive the progression of VC. In contrast, CKD is associated with a variety of metabolic disorders that contribute to the exacerbation of VC, such as disturbances in calcium and phosphorus metabolism, hormonal imbalances, and increased release of uremic toxins from the gastrointestinal tract. These disparities further underscore the distinct pathologies underlying VC in the two conditions.

Table 2 Differences in Vascular Calcification in the Two Environments

Differences in Oxidative Stress

The investigation of elevated blood sugar as a potent stimulus of oxidative stress has shown that it amplifies the circulatory flux of citric acid, stimulating NF-κB activation.¹³³ On the converse, an excessive production of reactive ROS can repress insulin gene transcription, mediated by the activation of ATP-sensitive K+ channels, thereby inhibiting the generation and release of insulin. Alternatively, the augmented blood glucose level in diabetic patients facilitates the accumulation of advanced glycation end products (AGEs), which bind to the receptor for advanced glycation (RAGE), initiating inflammatory signalling and escalating oxidative stress.^107,108 (Figure 1) In addition, within the context of CKD, phosphorus instigates oxidative stress in VSMCs by inciting an imbalance between antioxidant and reactive ROS production mechanisms,^9,109,134 and phosphate absorption by PIT1 may lead to intracellular alkalization.¹³⁵ Zhao et al⁹ have established for the first time that mitochondrial ROS-driven p65 nuclear translocation is implicated in the hyperphosphate-induced inflammation in VSMCs. This finding further supports the notion that phosphate-induced ROS production and oxidative stress are both significant contributors to this phenomenon.^109,135 The precise mechanisms by which elevated blood sugar and phosphorus contribute to oxidative stress in CKD remain to be fully elucidated. Future research should focus on elucidating the complex interplay between these factors and their potential impact on the progression of CKD.

Loss of Klotho Expression

It is well known that dysregulated mineral metabolism and exceptionally high levels of phosphorus and calcium are the most essential factors for the development and occurrence of calcification in CKD patients.¹⁰⁶ The critical importance of minerals to human anatomy was recently underscored by an observational cohort study.¹³⁶ Calcium homeostasis is primarily regulated by two major hormones, parathyroid hormone (PTH) and 1.25-dihydroxyvitamin D3 (1,25D3). In contrast, phosphorus homeostasis is predominantly controlled by the fibroblast growth factor-23 (FGF-23)/klotho axis (Klotho is a soluble anti-aging protein predominantly expressed in the kidney as a co-receptor for FGF23). Additionally, PTH and 1.25D3 mediate calcium homeostasis. In the context of CKD, the loss of Klotho expression can instigate calcification and osteoblast differentiation of VSMCs¹³⁷ (Figure 2B). An investigation revealed that pretreating Klotho protein in human umbilical vein endothelial cells co-treated with inorganic sulfate (IS) decreased the expression of superoxide radicals and monocyte chemoattractant protein-1 (MCP-1)¹¹⁰ (Figure 2A). Additional research has also highlighted the role of the FGF-Klotho axis in VC in CKD patients, exacerbating calcium and phosphorus dysregulation¹³⁸ (Figure 2C). Evidence suggests that calcimimetic agents can decrease calcium and phosphorus levels in the aorta¹³⁹ and prevent the progression of VC in uremic mice.¹⁴⁰

Figure 2 Mechanism of calcium and phosphorus imbalance in chronic kidney disease. (A) In the environment of chronic kidney disease, the decline of renal function leads to the dysfunction of P excretion and the increase of serum Pi level, and the decrease of 1.25(OH)2D3 secretion leads to the decrease of serum Ca level. At the same time, renal injury changes the intestinal microenvironment and reduces the absorption of Ca. In addition, in the environment of chronic kidney disease, hyperparathyroidism and increased PTH levels can not only promote the activity of osteoblasts and osteoclasts, thereby promoting calcification, but also increase the serum Pi level and promote changes in the intestinal microenvironment. (B) PTH, FGF-23, Klotho, and 1,25D have feedback loops to maintain calcium and phosphorus homeostasis. (C) In the environment of chronic kidney disease, the hormone levels of PTH, 1,25D and FGF-23 are dysregulated, thereby increasing the osteogenic activity of osteoclasts and promoting vascular calcification. Graphics by Figdraw.

The dependence of the sodium phosphate transporter on solute carrier membrane protein (SLC) group is crucial. Type II transporters (SLC34 family) consist of renal API-IIa (SLC34A1), intestinal API-IIb (SLC34A2),¹⁴¹ and NaPi-IIc (SLC34A3).¹⁴² The renal NaPi-IIa (SLC34A1) incorporates NaPi-IIaelectrogenic NaPi-IIa/b and electroneutral NaPi-IIc. These molecules are regulated by numerous factors, including parathyroid hormone (PTH) and FGF23, which are instrumental in mineral metabolism.^143,144 The SLC20 family encompasses two proteins: Pit 1 (SLC20A1) and PiT-2 (SLC20A2). The phosphate signaling in osteoblasts and VSMCs may be orchestrated by the SLC20 transporter family. Pit 1 and PiT-2 are considered indispensable for Pi influx in systemic cells.¹⁴⁵ A study indicated that phosphate uptake in VSMCs occurs through type III sodium-dependent P-cotransporters PiT-1 and PiT-2. These PiT transporters exhibit contrasting functions on VSMCs under hyperphosphatemic conditions.¹⁴⁶ PiT-1 exerts a pathological effect by stimulating osteogenic differentiation of VSMC and the discharge of calcium phosphate-laden vesicles.^111,112,146 Downstream effects of Pit-1 encompass Runx2/ core-binding factor alpha 1 (CBFA1) and osteopontin transcription.¹⁴⁶ In contrast, PiT-2 deficient VSMC exhibited increased stromal calcification, which may be mediated by suppression of osteoclast inhibitor osteopontin.¹¹²

About Phosphate Dependency Bone Induction Signal Cascade

NF-kB Signaling Pathway

Within VSMCs, an increase in extracellular phosphate levels initiates the activation of NF-κB.^113,147,148 The NF-κB signalling pathway partially stimulates the calcification of VSMCs by inducing the expression of MSX2 and elevating the levels of CBFA1, which subsequently augments the expression of ALP.^148,149 TNF-α strengthens the expression of MSX2 in VSMCs via the NF-κB pathway, leading to the initiation of osteogenic/chondrogenic transdifferentiation of VSMCs.¹⁴⁹ The receptor activator of NF-κB ligand (RANKL)/RANK system significantly contributes to VC through NF-κB activation.¹⁵⁰

SGK1/NF-kB Signaling Pathway

Phosphate has been established as a cardiovascular toxin, particularly affecting VSMCs.¹⁵¹ It has been observed that phosphate can induce the expression and activation of serum and glucocorticoid-induced kinase (SGK1) in VSMCs.¹⁵² By interfering with the SGK1/NF-kB signaling pathway, phosphate could potentially maintain the anti-calcification environment of VSMCs, thereby improving VC during hyperphosphatemia.¹⁵² In a similar vein, IL-18 may activate the SGK1/NF-kB osteoinductive pathway,¹⁵³ potentially contributing to the exacerbation of phosphate-induced calcification in VSMCs.¹⁵³

WNT/β-Catenin Signaling Pathway

The WNT/β-catenin signaling pathway serves a critical role within the osteoinductive signaling cascade and also functions as a mediator of VC.^26,154,155 Phosphate stimulation results in activation of the WNT/β-catenin pathway, leading to increased downstream effects of MSX2 and subsequent promotion of osteogenic transdifferentiation and calcification of VSMCs via direct upregulation of CBFA1¹⁵⁶ and PIT1 gene expression.¹⁵⁵ Moreover, the WNT/β-catenin pathway may also play a role in VC by inducing the expression of matrix metalloproteinase (MMP)2 and MMP9 in VSMCs.¹⁵⁷ Similarly, RNA interference studies confirmed the role of the MSX gene in TNAP andexpression. Additionally, paracrine WNT signaling is integral for the refinement of osteogenic TNAP expression downstream of BMP2 and Sonic hedgehog (Shh).¹⁵⁸

mTOR Signaling Pathway

In VSMC, klotho expression is down-regulated by phosphate,¹⁵⁹ an effect associated with activated mTOR signalling that enhances VC.^159,160

The protein kinase referred to as the mammalian target of rapamycin (mTOR) belongs to the family of PI3K-associated kinase (PIKK), a class of serine/threonine kinases. mTOR forms two distinct protein complexes, referred to as mTOR Complex 1 (mTORC1) and 2 (mTORC2), which play vital roles in various cellular processes, including protein synthesis, cell growth, and proliferation. Excessive activation of mTOR has been observed in various types of human cancers, as well as in age-related diseases such as atherosclerosis, obesity, diabetes, and Alzheimer’s disease. In a nutritionally adequate state, mTOR signalling is activated, and conversely, mTOR signalling is blocked, with it being reported that mTORC1 is specifically regulated by nutrient.¹⁶¹ The upregulation of mTOR by phosphate is particularly intriguing, as phosphates are essential nutrients involved in energy metabolism. Protein digestion leads to the production of amino acids and phosphates. It is likely that mTORC1 is involved in the signalling cascade activated by phosphate, which in turn activates the mTOR cascade in VSMCs, leading to a downregulation of Klotho expression. This downregulation can enhance the calcification of VSMCs.¹⁶² This upregulation of mTOR in a uremic environment is yet to be fully understood. It has been reported that mTOR inhibitors can be considered as an upregulation of Klotho. As CKD is associated with severe kidney and systemic Klotho deficiency, these drugs that upregulate Klotho have been shown to protect the kidney and vascular system. Additionally, there is substantial evidence that supports the broad-spectrum anti-atherosclerotic effects of mTOR inhibitors, and experimental studies have demonstrated that rapamycin can act as a stabilizer of atherosclerotic plaques and even promote plaque regression.¹⁶³ In animal models (such as apolipoprotein E-deficient mice), rapamycin has been shown to effectively reduce inflammation, inhibit progression, and enhance the stability of atherosclerotic plaques,¹⁶⁴ Thus, it can be anticipated that mTOR inhibitors could be used to prevent or delay the onset of atherosclerosis.

BMP-2 Signaling Pathway

TNF-α-induced VSMC calcification is associated with increased BMP2 signalling.¹⁶⁵ Phosphate has been observed to stimulate BMP-2 expression in VSMCs Phosphate induces BMP-2 expression in VSMCS.^166,167 Additionally, BMP-2 promotes VSMC calcification by stimulating the expression of PIT-1, which is upregulated and the activation of SMAD signalling pathways.¹⁶⁵ Notably, phosphate has also been observed to promote TGFβ1 expression in VSMCs.^168,169 Moreover, downstream osteoinductive signals of TGFβ1 include the transcription factor NFAT5 (also known as TonEBP), which mediates the upregulation of CBFA1 in VSMCs through SOX9. Interestingly.^10,168,169 TGFβ1 may also contribute to VC by inducing cellular senescence, including the upregulation of plasminogen activator inhibitor PAI-1, which plays a pro-calcification role.^10,168

Differences in Endothelial Dysfunction

Endothelial impairment has been established as a primary concern in individuals with CKD and has been found to persistently elevate throughout the course of the disease. Studies have consistently demonstrated that parathyroid hormone (PTH) and hyperphosphatemia significantly contribute to the progression of endothelial impairment.^170,171 Specifically, PTH has been found to trigger osteoblast transdifferentiation in endothelial cells through the activation of the extracellular signal-regulated kinase (ERK)1/2 and NF-κB signaling cascades.¹²⁷ Conversely, an experimental study has demonstrated that uremia, a significant complication of CKD, can lead to the amplification of fibroblast growth factor (FGF) signaling within endothelial cells, which exacerbates atherosclerosis.¹¹⁴ Additionally, recent studies have found that fluctuations in the functional properties of low-density lipoprotein-cholesterol (LDL-cholesterol) can further instigate endothelial impairment in CKD patients.¹¹⁵ Similarly, another in vitro experiment revealed that triggered by urinary toxin IS, injured endothelial cells generate excessive microvesicles, which modulate calcium accumulation, inflammation, and osteogenic transdifferentiation of SMCs.¹⁷² The same time, uremic toxins perturb endothelial function in CKD patients by competitively inhibiting eNOS.¹⁷⁰ Recent studies have indicated that modifications in the functional properties of low-density lipoprotein-cholesterol may contribute to the development of endothelial impairment in patients with CKD. However, in the context of diabetes, hyperglycemia exacerbates hemodynamic damage, specifically shear stress, which can suppress NO production and induce alterations in endothelial phenotype. These changes can lead to alterations in vascular permeability and leukocyte recruitment during inflammation.¹⁷³ It has been suggested that in the diabetic milieu, a decrease in the abundance of proteoglycans may provide protection against the development of atherosclerosis.¹¹⁶

Vitamin D and Vitamin K Deficiency

CKD is strongly associated with VC through Vitamin D deficiency. In fact, a vitamin D deficit often presents in individuals affected by CKD¹⁷⁴ (Figure 2A). The potential mechanisms that link CKD and VC may involve the capacity of Vitamin D to inactivate RUNX2 regulation, as demonstrated in recent studies.^175,176 Furthermore, individuals with CKD often experience elevated FGF23 concentrations due to a compromised Glomerular Filtration Rate (GFR). This elevation in FGF23 consequently impairs vitamin D levels.¹⁷⁷ Moreover, 1.25(OH)2D3 suppresses NF-kB and TNFα, key signalling molecules involved in bone-induced signalling cascades.^117,178 In a similar study involving patients with Type 1 Diabetes, vitamin D deficiency was associated with an increased risk of Coronary Artery Calcification (CAC).¹¹⁸ Therefore, vitamin D supplements have shown beneficial effects on VC in CKD.

Despite a correlation between vitamin K deficiency and enhanced VC severity in CKD patients, this correlation did not consistently lead to increased cardiovascular disease or mortality.Vitamin K deficiency was associated with increased severity of VC in patients with CKD, but, paradoxically, vitamin K deficiency was not consistently associated with cardiovascular disease and mortality.¹²⁰ Potential mechanisms for vitamin K’s ability to reduce calcification propensity include an increase in the activity of vitamin K-dependent calcification inhibitors or a reduction in inflammation by inhibiting innate immune system components.¹⁷⁹

MicroRNAs and Epigenetic Modifications at Different Points in Vascular Calcification

There were different epigenetic modifications to VC in the two environments. During hyperphosphatemia, different epigenetic modifications, including DNA methylation, histone modifications, and dysregulation of microRNAs, contribute to osteoinduced cell signaling.^119,180 It has been confirmed that methylation in the SM22α promoter region can induce VC at higher pi levels. It has been reported that the downregulation of miR-29b and activation of WNT/β-catenin signalling may be involved in VC in IS-induced CKD.¹⁸¹ A potential lncRNA H19/miR-138/TLR3 axis¹²¹ was predicted, and its downstream microRNAs were enriched in the NF-κB signalling pathway, which may be related to VC in CKD. Interestingly, microRNAs play a role in the diabetic environment by affecting vascular dysfunction and inflammatory status. As endothelial miR-126 affects vascular dysfunction and inflammatory status,¹²² it is thought that activation of miR-21 may be a key event associated with the development of diabetes and atherosclerosis. It may trigger endothelial activation, affecting foam cell formation, circulating monocyte adhesion, macrophage apoptosis, and phagocyte clearance.¹²³ In the diabetic environment, miR-21 functions again appear in inhibitory mechanisms of antioxidant response, regulating ROS homeostasis through Krev interaction trapping protein (KRIT)-1 and silencing the major mitochondrial antioxidant enzyme SOD2.¹⁸² Therefore, miRNAs could serve as potential therapeutic targets for the treatment of VC.

Intestinal Microbiota and Uremic Toxins

As a source of uremic toxins, the gut microbiome represents a significant contributor to cardiovascular disease in CKD. It executes a pivotal role in human health and the onset of kidney disease.^124,183 It is hypothesized that the altered gut microbiome and its metabolic byproducts in CKD may be implicated in the VC process.¹⁸⁴ A number of studies have shown that uremic toxins such as indoxyl sulfate (IS), p-cresol (PC), p-cresol sulfate (PCS), uric acid, etc., released by the gut microbiome, stimulate oxidative stress and augment the inflammatory state by upregulating proinflammatory cytokines such as TNF-α and IL-6.¹⁸⁵ In addition, endotoxin (LPS) and short-chain fatty acids (SCFA) are specific categories of gut microbiome-derived metabolites that are capable of causing systemic inflammation if the intestinal barrier is compromised. (Figure 3). Uric acid may be synthesized in endothelial cells, leading to local vascular smooth muscle cell proliferation and migration through the release of platelet derived growth factor, thereby promoting inflammation through the release of monocyte chemoattractant protein-1, and intensifying oxidized low-density lipoprotein.¹²⁵ Furthermore, urate deposits serve as a trigger for local intravascular inflammation, which can stimulate the progression of atherosclerosis and the formation of adjacent inflammatory plaques.^126,186,187 As a result, maintaining the balance of the gut flora is crucial for the therapeutic management of CKD patients, as well as patients with end-stage renal disease (ESRD).

Figure 3 Uremic toxin and intestinal flora disorder. Lipopolysaccharide and short chain fatty acid imbalance, promote intestinal flora disorder, promote intestinal microorganisms and uremic toxins to destroy intestinal barrier. Uremic toxins such as IS increase TNF-α and IL-6, leading to an increased inflammatory state by promoting oxidative stress. Graphics by Figdraw.

Hormone Levels

The hormone release rates in the respective environments are not consistent, which in turn results in variations in the mechanisms associated with VC. The experimental findings from both in vivo and in vitro models propose that the regulation of calcification is mediated by a combination of hormones: angiotensin II,¹⁸⁸ aldosterone,^189,190 and glucocorticoid,¹⁹¹ such as estrogen, thyroid, adiponectin, parathyroid, FGF23, calcitriol, testosterone, insulin, etc., exert protective effects against VC. Recent animal investigations have suggested that the increase of PTH triggers the transformation of aortic endothelial cells into chondrocytes¹⁷¹ and the PTH receptor 1 (PTHR1) signaling of endothelial cells plays a pivotal role in PTH-induced osteogenesis and is correlated to angiogenesis.¹⁹² It is acknowledged that insulin resistance is closely associated with diabetes, and insulin stimulates the release of endothelial NO, which in turn oxidizes lipoproteins, decelerates the rate of intimal calcification, and suppresses vasa vasorum formation.¹²⁸ Upon the occurrence of insulin resistance (IR), the higher concentration of free fatty acids in the liver prompts the body to amplify the absorption and very low-density lipoprotein (VLDL) triglyceride production and secretion to compensate; therefore, the susceptibility to blood vessel calcification escalates. Iguchi et al have demonstrated that individuals with higher IR levels are predisposed to develop thin cap fibroatheroma (TCFA) and exhibit notably thinner caps.¹⁹³ Elevated insulinemia reduces OPG and MGP levels, activates BMP2, and enhances carboxy-terminal propeptide of type I collagen (CBFPa-1) and ALP levels, culminating in the bone-like transformation of stromal cells.¹²⁹ As such, hormone levels are equally indispensable for the progression of VC.

Persistent Activation of the Renin-Angiotensin-Aldosterone System (RAAS)

The role of RAAS in the pathophysiology of hypertension, cardiovascular and renal diseases is well known.CKD complications such as RAAS activation can induce endothelial dysfunction and atherosclerosis. These pathologies coexist during the progression of CKD and exacerbate VC. VSMCs express the mineralocorticoid receptor, and the activation of mineralocorticoid promotes VC.¹³⁰ As renal function declines, the standard defence mechanism, phosphorus (Pi) and calcium (Ca) endocrine axis, becomes overwhelmed and disrupted. Pi can directly induce the expression of aldosterone synthase in the adrenal gland and vascular tissues, which may be related to FGF-23-mediated activation of RAAS.¹³¹ Increased levels of angiotensin II increase the production of aldosterone that activates sodium-phosphate cotransporter (Pit-1), leading Pi to enter the VSMC. In addition, aldosterone promotes inflammatory processes by inducing TNF-α.¹⁹⁴ Furthermore, vascular injury caused by excessive activation of mineral corticosteroid receptor (MR) can be manifested as inappropriate vascular remodelling, endothelial dysfunction and increased vascular stiffness.¹³²

Therapeutic Strategies for Vascular Calcification in Two Environments

Research on the treatment of VC targets still has important clinical significance, the next (Table 3) summed up in two kinds of environments drugs for the treatment of VC.

Table 3 Comparison of the Treatment of Vascular Calcification in Two Environments

Metformin

Currently, metformin is the first-line medication to treat T2DM in most guidelines and is used daily by >200 million patients. Surprisingly, the mechanisms underlying its therapeutic action are complex and are still not fully understood. Metformin activates protein kinase (AMPK) through phosphorylated AMP, and by activating AMPK signalling in VSMC, the RANKL gene is an essential inducer of VC. Metformin can down-regulate the overexpression of the receptor activator of RANKL gene.²²⁴ Metformin has been shown to alleviate VC through the AMPK-eNOS-NO pathway.¹⁹⁶ In the rat model of adenine-induced CKD, Metformin has shown that the expression of inflammatory cytokines TNF-a and IL-1β and to decrease serum PTH and FGF-23 hormone levels significantly are reduced in the kidneys of rats treated with Metformin.¹⁹⁵ Metformin can improve macrophage dysfunction, reduce NLRP3 inflammatory vesicles, and inhibits ROS production.²²⁵ Therefore, Therefore, metformin is currently one of the most promising therapies to reduce VC. However, Interestingly, More insights into the benefits of metformin on healthspan and lifespan in humans will hopefully be provided by the MILES (Metformin in Longevity Study) and TAME (Targeting Aging with Metformin) clinical trials.¹⁹⁷ The ability of metformin to promote health or increase lifespan in healthy populations remains to be determined.

Thiazolidinediones (TZD)

TZDs exhibit their therapeutic effects by stimulating the activity of nuclear peroxisome proliferator-activated receptor gamma (PPARγ). This stimulation of PPARγ signalling has been implicated in the regression of VC. Previous research indicates that PPARγ is capable of regulating the proliferation of VSMCs induced by platelet-derived growth factor-induced protein (PI)²²⁶ Further studies have shown that the administration of a PPAR-γ inducer was able to suppress NF-kB-dependent secretion of inflammatory cytokines in a rodent model.¹⁹⁸ Similarly, pioglitazone, a thiazolidinedione-like drug, has been shown to mitigate TNF-α-induced endothelial dysfunction in patients with diabetes.¹⁹⁹ A retrospective study involving 156 patients with T2DM demonstrated that rosiglitazone significantly reduced TNF-α, interleukin-6, high-sensitivity C-reactive protein (hs-CRP), and malondialdehyde (MDA) levels.²⁰⁰ Based on these findings, thiazolidinedione (TZD) drugs can be utilized as the primary treatment for VC. TZDs are commonly utilized as second-line oral hypoglycemic drugs. However, there is emerging evidence suggesting that long-term use of TZDs may exacerbate cardiac dysfunction.²²⁷ Therefore, it is crucial for healthcare professionals to consider multiple factors when administering TZD drugs.

Sulfonylurea

Sulfonylureas, such as glimepiride, elicit insulin release by virtue of their ability to inhibit ATP-sensitive K+ channel receptors located in pancreatic β cells.²²⁸ This phenomenon is underscored by a study demonstrating that treatment with glimepiride decreased renal NLRP-3 expression in a rodent model of adenine-induced CKD.^201,204 In addition, in diabetic mice treated with glibenclamide, it was demonstrated that VC development was delayed by down-regulating IL-10, IL-18, TGF-β, and mRNA levels.²⁰² A study on whether diabetes treatment has a beneficial effect on calcification markers showed that anti-diabetic treatment with glibenclamide for 18 weeks resulted in a significant reduction in circulating OPG, suggesting that this may slow the progression of VC.²⁰³ This finding suggests that sulfonylureas may exert anti-calcifying effects through the manipulation of inflammatory and oxidative pathways.

Dipeptidyl Peptidase-4 (DPP-4) Inhibitor

The dipeptidyl peptidase-4 (DPP4) is a crucial enzyme in the regulation of the incretin system. This system, which is responsible for regulating blood glucose levels, functions by suppressing the hormone glucagon-like peptide 1 (GLP-1). By blocking the activity of DPP4, DPP4-inhibitors (DPP4-i) can enhance GLP-1 concentration, thereby controlling blood glucose levels.²²⁹ A study conducted on high-fat diet-induced obesity-related glomerulopathy (ORG) rat model demonstrated the ability of ghrelin to suppress local inflammation by reducing the presence of mast cells and macrophages, and by decreasing the expression of proinflammatory cytokines such as TNF-a and IL-6.²³⁰ Interestingly, a random clinical study on T2D patients suggested that linagliptin treatment for 4 weeks prevented impairment of renal endothelial functions²⁰⁵ However, DPP4-I is currently controversial, and recent studies have shown that DPP-4 inhibition can induce cancer metastasis.²⁰⁶ However, this controversy in the carcinogenic mechanism of diabetes has been shown to be complex, including excessive ROS formation, destruction of essential biomolecules, chronic inflammation and impaired healing phenomena, which together lead to carcinogenic effects in diabetic patients. A recent study by Zendehdel et al²³¹ found that patients with type 1 diabetes had a 19% increased risk of developing cancer, particularly gastric, cervical, and endometrial cancers. This risk is higher compared to the risk observed in patients with CKD. Compared to CKD, although it can promote kidney cancer itself, some other anti-tumour drugs (such as cisbo) can themselves have a harmful effect on kidney function, leading to renal cell carcinoma. The combination of cancer with impaired renal function worsens patients’ outcomes and complicates their management and treatment.

Selective Sodium-Glucose Transporter-2 Inhibitors (SSGT −2)

SGLT-2 inhibitors, such as empagliflozin, function by inhibiting glucose reuptake in the proximal tubules of the nephron.²³² In an ApoE−/− mouse model, studies have indicated that chronic empagliflozin administration for eight weeks resulted in a decrease in atherosclerotic lesions and a reduction in TNF-α and IL-6 levels.²³³ Furthermore, clinical trials have provided support for the anti-inflammatory effects of empagliflozin in reducing atherosclerosis in individuals with T2DM.²⁰⁷ A subsequent analysis of the therapeutic efficacy of empagliflozin on the Kidney Disease Improving Global Outcomes (KDIGO) classification of CKD suggested that SGLT-2 inhibitors may exhibit cardioprotective and nephroprotective effects.²³⁴ A recent comprehensive meta-analysis of 11 cardiovascular outcome trials (CVOTs) examining the effects of SGLT-2 inhibitors on cardiorenal outcomes in individuals with type 2 diabetes agreed with these findings.²³⁵

GLP-1 Agonists

GLP-1 primarily modulates postprandial glucose surge through augmenting insulin production and suppressing glucagon release.²³⁶ Several recent studies have further elucidated the effects of GLP-1 and its receptor agonists on cardiac and vascular physiology, inflammatory responses, and platelet function.²³⁷ For instance, Liraglutide, a GLP-1 agonist, has been shown to significantly reduce pro-inflammatory cytokines, such as TNF-α and IL-6, thus attenuating local inflammation.²³⁰ Similarly, Exenatide, another GLP-1 receptor agonist, has demonstrated the ability to mitigate VC by downregulating RANKL expression.²⁰⁸ Thus, these data suggest that GLP-1 agonists may have therapeutic potential in the management of VC. Furthermore, it has been observed that co-administration of liraglutide with GLP-1 agonists has reduced the risk of cardiovascular events in patients with diabetes.²³⁸ The cardiovascular safety profile of liraglutide in patients with type 2 diabetes appears to be relatively reassuring.

Alpha-Glucosidase Inhibitors (AGI)

AGI exhibits a promising role in the treatment of VC by virtue of its ability to suppress postprandial hyperglycemia through the restraint of α-glucosidase in the brush border of the small intestinal mucosa. Studies have demonstrated the efficacy of acarbose, an α-glucosidase inhibitor, in the management of type 2 diabetes (DM) in rodent models. Acarbose treatment was observed to diminish oxidative stress and reduce reactive ROS levels.²³⁹ Additionally, it was shown to have an anti-inflammatory effect, thereby dampening pro-inflammatory factors.^209,210 It is noteworthy that AGI displays the same therapeutic efficacy as other anti-diabetic medications in controlling glycosylated haemoglobin and blood glucose levels, and its effect is comparable to metformin. Despite its promising potential, AGI is currently underutilized in clinical practice, particularly in patients with advanced CKD, due to its limited safety profile in these patients. However, further research on AGI’s safety in severe renal impairment is warranted to determine its therapeutic potential in this patient population.

Antioxidants

Oxidative stress is considered to play a pivotal role in the progression of VC, which has been established as a standard mechanism for the development of VC in conditions such as DM and CKD. Research indicates that various nutrients and dietary plant metabolites exhibit antioxidant activities, such as α- and γ-tocopherols, retinol, β-cryptophanes, ascorbic acid, α- and β-carotene, lutein and zeaxanthin, and lycopene.^211,212 For example, Vitamin E has been shown to act as an antioxidant and has been identified as a compound that can decelerate VC progression.²¹² Furthermore, Vitamin C, possessing vital antioxidant functions, is capable of reducing reactive ROS levels and maintaining vascular and endothelial functionality.²¹³ Other dietary plant metabolites, such as quercetin,²⁴⁰ curcumin,¹⁸⁴ resveratrol,²⁴¹ ursolic acid and others, can also function as antioxidants and exert protective effects against VC.In a systematic review and meta-analysis of the effects of age and dose on the glycemic impact of resveratrol in managing type 2 diabetes, it was demonstrated that resveratrol significantly reduced blood glucose, insulin, and Glycosylated Hemoglobin, Type A1C (HbA1c) levels in patients aged 60 years.²⁴² Therefore, the positive and significant role of antioxidant compounds in CKD is undeniable, highlighting the significant role they play in disease prevention and intervention through the regulation of free radicals.The consumption of these antioxidant compounds through diet, including fruits, vegetables, and grains, plays a crucial role in disease prevention and intervention.

Angiotensin-Converting Enzyme II Receptor Antagonist (MRA)

An experimental study was performed, in which angiotensin di-receptor (AT2) knockout rats were utilized. Results indicated that these rats demonstrated diminished aortic ring calcification in comparison to controls, indicating that AT2 stimulation can inhibit phosphate-induced VC.²⁴³ Finenone, an MR antagonist, can inhibit reactive ROS production, decrease oxidative stress, and prevent endothelial dysfunction, thereby providing control over VC.^214,244 It is worth noting that the expression of pro-inflammatory molecules and macrophages plays a crucial role in the development and manifestation of VC. A study conducted on rats with CKD demonstrated that finenone therapy reduced the activity of matrix metalloproteinase-2,9 (MMP-2,9) in plasma, thereby curtailing VC and oxidative stress.²¹⁵ In addition, MR antagonists can also block the pro-inflammatory effects of IL-6.²¹⁶ Further, MRAs are considered a promising therapeutic strategy in diabetic kidney disease. Additionally, other non-steroidal MRAs have shown anti-albuminuric effects in diabetic kidney disease. Whether the concurrent administration of MRAs with other nephroprotective drugs, such as sodium-glucose cotransporter-2 (SGLT2) inhibitors, can provide synergistic protective effects requires further investigation.

Combating Mineral Disorders

It has been documented that phosphate-binding agents augment P excretion and diminish VC by reducing FGF-23 in clinical trials pertaining to phosphate disturbances.²⁴⁵

In a rat model, Mg alleviated inflammatory damage associated with oxidative stress.²⁴⁶ Zinc has been shown to inhibit ROS-induced LDL-C oxidation.⁴⁸ Similarly, inadequate Zn intake was associated with increased ROS production,²⁹ which is essential in reducing the burden of VC.²¹⁹ Cinacalcet is a second-generation calcifying agent, and a clinical meta-analysis of Cinacalcet showed that Cinacalcet reduced PTH synthesis and secretion, decreased serum calcium, and was often accompanied by a decrease in phosphate, thereby reducing VC.²⁴⁷ Klotho is a critical protein that protects the kidney. Its deficiency is implicated in the pathogenesis and progression of CKD. Conversely, an increase in Klotho levels results in improved kidney function and delays CKD progression. Therefore, mineral supplementation may become a therapeutic agent for VC in the future.

Vitamin Supplement and New Therapies

Vitamin K supplementation is essential for VC.²²⁰ I In previously publicized studies, vitamin K-dependent carboxylation of glutamate residues activates MGP in hemodialysis patients, and VC is regulated and, to a certain extent, inhibited by MGP.²⁴⁸ Moreover, preliminary results from clinical trials imply that vitamin K supplementation reduces VC.²⁴⁹ Additionally, paricalcitol and lower clinical doses of paricalcitol may thwart VC.²¹⁸ However, a clinical randomized controlled trial revealed no significant distinction in the progression of coronary artery calcification (CAC) or valvular calcification between treatment with osteopontin and paricalcitol in patients with CKD over 48 weeks.²⁵⁰ A more advanced drug now available in clinical studies for the treatment of VC is an inositol hexaphosphate (IP6) formulation called Sanifit’s SNF472.²⁵¹ Recent studies have identified an OEG2-derived inositol phosphate (OEG2-IP4) that may have clinical implications in the prevention and treatment of VC.²⁵² Novel therapies may help prevent VC progression in CKD and DM. Further clinical studies are needed to determine clinical efficacy.

Statins

Statins promote low-density lipoprotein metabolism and increase high-density lipoprotein concentration by inhibiting hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the body’s cholesterol synthesis process.²¹⁷ This has been evaluated in several randomized controlled clinical trials, each showing that statins have a significant LDL-lowering effect.^221,253 However, the effect of statins on VC is still controversial. A prospective, multi-country study using continuously computed tomography angiography (CT) scans at 2-year intervals to detect atherosclerotic plaque progression showed that patients taking statins had a faster rate of lesion calcification, a lower rate of atherosclerosis progression and a lower occurrence of high-risk plaque features compared to controls.²²² This means that statins do not affect progression in the percentage of coronary artery disease stenosis severity but induce plaque phenotypic transformation. Another prospective study suggests a possible mechanism by which statins promote calcium accumulation in arterial walls by inhibiting vitamin K-dependent proteins and participating in vascular protection.²⁵⁴ In contrast, Doran et al²²³ recently suggested that statin therapy promotes the reversal of significant microcalcification in mice. Another study found that treating coronary heart disease patients treated with statin with PCSK9 inhibitor evolocumab can significantly reduce LDL cholesterol and plaque regression. Therefore, whether for patients with CKD or DM, long-term statin use can significantly prolong the survival of patients by reducing apolipoprotein a, but there is also an impact on VC. Generally speaking, the benefits outweigh the disadvantages.

Conclusions and Perspectives

Due to meticulous investigation into their mechanisms, significant breakthroughs have been achieved in the pharmacotherapy of two diseases. Studies have established a link between diabetes and CKD with VC. Recent data suggest the existence of multiple, convergent mechanisms accelerating VC within the framework of these diseases. These mechanisms involve mitochondrial oxidative stress, pro-inflammatory cytokine release, endothelial dysfunction, lipid metabolic disorders, hypoxia, and common signalling cascades. Additionally, extracellular vesicles and miRnas are also implicated in VC. Although these factors share some similarities, there are also distinct differences. For example, metabolic disorders are the primary factors for VC in diabetes, while excessive calcification in CKD is intensified by calcium and phosphorus imbalances, hormone dysregulation, and the release of massive amounts of uremic toxins from the intestine. Despite significant advances in the research of VC within diabetic and CKD milieus, including the elucidation of mechanistic aspects and the initiation of clinical pharmacological trials, the complex and heterogeneous pathophysiological mechanisms underlying VC within these contexts have yet to be fully understood. This complexity has posed significant challenges for the identification of optimal therapeutic targets and the advancement of drug development. Furthermore, it is important to emphasize the need for increased research on the pathophysiological mechanisms and preventative measures of VC. By addressing these aspects of the disease, we can develop more effective strategies to mitigate the negative consequences of VC and ultimately improve patient outcomes.