Vitamin K preserves gamma-glutamyl carboxylase activity against carbamylations in uremia: Implications for vascular calcification and adjunct therapies

Abstract

Aim: Vascular calcification contributes to morbidity and mortality in aging and is accelerated in diabetes and in chronic kidney disease. Matrix Gla Protein is a potent inhibitor of vascular calcification, which is activated by the vitamin K-dependent gamma-glutamyl carboxylase (GGCX). However, through a currently unidentified mechanism, the activity of GGCX is reduced in experimental uremia, thereby contributing to the promotion of vascular calcifications. In this study, we aim to identify the cause of these functional alterations and to stimulate the enzyme activity by potential GGCX binding compounds as a new avenue of therapy.

Methods: Two rodent models of experimental uremia and human carotid plaques were assessed for GGCX activity and modifications, as well as calcification. In silico compound screening via BindScope identified potential binding partners of GGCX which were further validated in functional assays for enzymatic activity changes and for in vitro calcification. Mass spectrometry was applied to monitor molecular mass changes of the GGCX.

Results: Mass spectrometry analysis revealed post-translational modifications of the GGCX in uremic rats and mice, as well as in calcified human carotid plaques. Functional assays showed that the post-translational carbamylation of GGCX reduced the enzyme activity, which was prevented by vitamin K2. Chrysin, identified by compound screening, stimulated GGCX activity, reduced calcium deposition in VSMCs, and oxidized GGCX at lysine 517.

Conclusion: In conclusion, this study clearly demonstrates that the vitamin K-dependent enzyme GGCX plays a significant role in uremic calcification and may be modulated to help prevent pathological changes.

Keywords: carbamylation; chronic kidney disease; vascular calcification; vitamin K.

The source of the GGCX protein structure is uniprot, (image in the upper left of the graphical): this is the weblink: https://www.uniprot.org/uniprotkb/P38435/entry

FIGURE 1

Samples were obtained from two established animal models of chronic kidney disease. These include Wistar rats with adenine‐induced nephropathy and C57BL/6 mice subjected to 5/6 nephrectomy in. (A) Serum urea in sham rats (n = 6), compared to serum urea in adenine nephropathy (AD) (n = 9), and serum urea in adenine nephropathy plus vitamin K2 (AD+K2) (n = 7; *p < 0.05; **p < 0.001). (B) Serum urea in sham‐operated mice (n = 6) compared to 5/6 nephrectomized mice (5/6Nx) (n = 5) (**p < 0.001). (C) Characteristic matrix‐assisted laser desorption/ionization (MALDI) MS spectrum of SD‐Gel pieces of aortas from adenine‐fed rats; the arrows indicate the modifications by carbamylation (shift of the molecular mass from 443 to 487) exemplary spectrum (n = 3). (D) Characteristic matrix‐assisted laser desorption/ionization (MALDI) MS‐spectrum of SDS‐Gel of aortas from adenine plus vitamin K2 fed rats with a distinct mass peak at position 443; exemplary spectrum (n = 3).

FIGURE 2

Overexpression and functional activity of human GGCX in HEK293T cells. Human GGCX was overexpressed in HEK239T cells, isolated by FACS sorting and purified membrane fractionation. The functionally active enzyme was used to assess the effects of carbamylation and vitamin K on GGCX function. (A) Human GGCX expression vector co‐delivered with Hyperactive PiggyBac transposase expression vector to obtain mOrange fluorescent GGCX overexpressing HEK293T cells; line = 90 µm. (B) Relative expression of GGCX mRNA in HEK293T cells compared to GGCX‐overexpressing HEK293T cells (**p<0.001). (C) In vitro carbamylation of purified GGCX: GGCX enzyme was purified by membrane fractionation from GGCX‐overexpressing HEK293T cells and subjected to in vitro carbamylation by potassium cyanate (carb), and in the presence of potassium cyanate plus vitamin K (carb +vitamin K), respectively (*p<0.05, **p<0.001). (D) GGCX activity in carotid arteries versus serum urea of carotid endarterectomy patients; linear regression not significant; dashed line to separate normal and increased serum urea levels. (E) GGCX activity versus total calcium content of renal arteries (r ² = 0.50) (p = 0.01).

FIGURE 3

Effects of chrysin on GGCX activity and calcium content. Effects of chrysin were tested on GGCX containing microsomes isolated from livers from healthy and adenine‐fed rats (A, B, E) and on human VSMCs (C, D). (A) GGCX activity of liver microsomes, isolated from healthy and adenine‐fed rats, treated with chrysin (1, 5, 10 μM), each to vehicle control (0.001% DMSO) (n = 3). (B) Total calcium content in human immortalized VSMC, after 14 days of osteogenic treatment in the presence of chrysin (5, 10 μM), each to vehicle control (0.001% DMSO) and normal control medium (ctrl) (n = 8). (C) Real‐time PCR measurement, relative expression of RUNX2 to relative expression of GAPDH; ctrl: Normal control medium, OG: Osteogenic medium, OG + Chr: Osteogenic medium plus chrysin (10 μM); after 5 days of treatment; C1‐3 Exemplary images of alizarin red staining on VSMCs treated with (1) normal control medium, (2) osteogenic differentiation medium, (3) osteogenic differentiation medium plus chrysin (10 μM); scale bar =10 μM. (D) Flow cytometry analysis of ucMGP positive cells (% of single cells), 14 days after treatment with normal medium (ctrl), osteogenic medium (OG) and osteogenic medium plus chrysin (10 μM) (OG + Chr), respectively. (E) Characteristic mass spectrum of vehicle‐treated GGCX (intensity vs. mass to charge ratio) of fragments 516–520 (n = 3). (F) Characteristic mass spectrum of chrysin‐treated GGCX (intensity vs. mass to charge ratio) of fragments 516–520; shift in mass peak indicated by red arrow (n = 3).

FIGURE 4

Redox status of VSMCs after 14 days of ostegenic differentiation, compared to normal medium, baseline and after stimulation with H₂O₂, all measured in measurement buffer. (A) Cytosolic baseline redox status of VSMCs treated with 5 μM chrysin plus osteogenic medium to the vehicle to standard medium as control condition; n = 3. (B) Cytosolic redox status after challenge with 10 μM H₂O₂, VSMCs treated with 5 μM chrysin plus osteogenic medium to the vehicle to standard medium as control condition (n = 3); (C) Mitochondrial baseline redox status of VSMCs treated with 5 μM chrysin plus osteogenic medium to the vehicle to standard medium as control condition (n = 4). (D) Mitochondrial redox status after challenge with 10 μM H₂O₂, VSMCs treated with 5 μM chrysin plus osteogenic medium to the vehicle to standard medium as control condition (n = 3).