Carbamylation versus Carboxylation—A Clash Culminating in Vascular Calcification?

Abstract

In their recent work in Acta Physiologica, Kaesler et al. identify a novel mechanistic link between the uremic environment in chronic kidney disease (CKD) and vascular calcification [1]. Medial vascular calcification (VC) is an inappropriate deposition of calcium-phosphate, mostly as hydroxyapatite, in the medial layer of the arteries [2]. This VC increases with aging and is strongly accelerated by CKD [2]. The intricate and multifaceted pathogenesis of VC is tightly linked to calcium–phosphate imbalance. When calcium and phosphate concentrations exceed their solubilities in the plasma, spontaneous complexation and formation of extraosseous minerals could occur that is physiologically balanced by a mineral buffering system [3]. In CKD patients, bone demineralization and hyperphosphatemia strain the physiological mineral buffering system [2]. Thereby, an increased formation of calcium–phosphate particles can occur, which in turn can induce pro-inflammatory cascades. The stimulation of this pro-inflammatory effect is further exacerbated by the accumulation of uremic toxins in the plasma of CKD patients [4]. Vascular smooth muscle cells (VSMC) are particularly susceptible to calcium–phosphate particle stress and respond with phenotypic changes, including activation of inflammatory pathways, release of pro-calcific transmitters and extracellular vesicles as well as remodeling of the extracellular matrix. All these changes favor a local pro-calcific microenvironment [2]. From this perspective, rectifying a deranged mineral buffering system in CKD holds great potential to prevent VC and reduce cardiovascular mortality.

Several factors of the mineral buffering system, such as pyrophosphate and fetuin-A, have been linked to an anticalcific function [2, 3]. Additionally, a decisive role has been attributed to vitamin-K-dependent GLA proteins [5]. Contrary to osteocalcin (bone GLA protein), matrix GLA protein (MGP) is a potent extraosseous calcification inhibitor. MGP is a ~12-kDa protein that was originally identified from bone matrix but is also highly expressed in soft tissues. Its critical role was identified in MPG-deficient mice that die from rupture of their calcified arteries before they reach an age of 2 months. Interestingly, the anticalcific effects of MGP might involve several mechanisms [5]. MGP directly adsorbs hydroxyapatite crystals and is associated with inhibition of crystal growth but may also inhibit bone morphogenic protein 2, an important activator of pro-calcific effects in VSMCs [5].

The function of MGP is regulated by posttranslational modifications, such as phosphorylation and carboxylation. Besides serine phosphorylation, the gamma-carboxylation of glutamate residues by gamma-glutamyl carboxylase (GGCX) and vitamin K as co-factor is important for the anti-calcific function of MGP [5]. In pseudoxanthoma elasticum, ectopic calcification occurs due to pyrophosphate deficiency, but the phenotype can be replicated by GGCX deficiency, independent of pyrophosphate levels [6]. Therefore, the functional status of MGP is decisive for its biological function.

The powerful protective effect of MGP has sparked various investigations on its role in vascular homeostasis [5]. The circulating inactive form (dephosphorylated as well as un- or under-carboxylated) of MGP is increased in CKD and is associated with the severity of VC. Some studies indicate a link between inactive MGP and VC even beyond CKD [5]. As MGP activation by carboxylation requires vitamin K, both vitamin K deficiency and the vitamin K antagonist warfarin have been linked to VC. Vitamin K deficiency was also described as a feature of CKD [5]. However, clinical studies with vitamin K supplementation in CKD remain inconsistent with unclear cardiovascular benefits [5, 7]. Disturbances in lipoprotein-mediated vitamin K transport may impair the effects of vitamin K supplementation in dialysis patients [8]. Therefore, further understanding of vitamin K homeostasis in health and disease is of critical importance to harvest a putative therapeutically potential.

In their current work, Kaesler et al. provide fascinating new insight into the complex regulation of vitamin K homeostasis in CKD (Figure 1) [1]. Earlier, they observed reduced activity of GGCX in rats with adenine-induced kidney disease, together with increased levels of undercarboxylated MGP [9]. Interestingly, vitamin K supplementation in these animals had a beneficial effect on GGCX activity [9], but the underlying mechanisms were unclear. Now, Kaesler et al. identify posttranslational modification of GGCX by carbamylation as one underlying mechanism for its reduced activity in CKD [1]. The unique uremic environment in CKD fosters a reactive milieu with some typical posttranslational protein modifications [4]. The high urea levels in CKD can form isocyanic acid, which then induces carbamylation as a chemical modification of proteins, altering their function [4]. Carbamylation has been shown for multiple proteins, such as LDL, uromodulin, or albumin [4]. Now, the authors show that GGCX activity is directly reduced by its carbamylation, while vitamin K2 addition could prevent this effect [1]. Furthermore, a screening approach identified binding partners of GGCX, where chrysin was shown to increase GGCX activity in rat liver microsomes. However, this effect was only present in microsomes from healthy animals and not in microsomes from rats with adenine-induced CKD. Nonetheless, chrysin prevented calcification of VSMC, which was paralleled by apparent restoration of MGP status in VSMCs. Chrysin is a natural flavonoid and has been associated with multiple protective mechanisms in the cardiovascular system [1]. It is also one more in an ever-growing list of flavonoids associated with protective effects during VC, alongside for example, quercetin and fisetin. Multiple mechanistic effects may contribute to the protective effects of chrysin on VSMC calcification beyond the effect on GGCX.

Although the current exploratory experiments have some limitations, Kaesler et al. highlight the importance of carbamylation as a functionally relevant modification in uremia. Therefore, the interaction of vitamin K and carbamylation warrants further study to answer remaining questions. Can vitamin K ameliorate the carbamylation of other targets? Since carbamylation affects a wide range of proteins and is linked to inflammatory processes [4], this protective effect of vitamin K may reach beyond MGP and VC. What concentrations should be targeted to translate these findings to clinical studies with CKD patients receiving vitamin K substitution? On the other hand, could inhibition of carbamylation improve vitamin K-dependent activation of MGP? This seems especially intriguing, since free amino acids could compete with proteins as carbamylation targets, and dietary l-lysine protects against VC in rats with adenine-induced CKD [10]. Thus, while the current findings advance our understanding of mechanisms underlying disturbed calcific homeostasis in CKD, many new questions arise, and the promising perspective of therapeutic modification of vitamin K homeostasis to the benefit of patients remains on the horizon.

The authors declare no conflicts of interest.