Part 2: Carbamylated Sortilin Associated With Cardiovascular Calcification in Patients With Chronic Kidney Diseas

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

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Carbamylated sortilin alters ligand binding

Sortilin is a coreceptor for several ligands.8 Carbamylation of lysine significantly alters the properties of the lysine sidechain, whereby the positively charged amino group is replaced by a bulky polar group, potentially affecting ligand binding. We examined the interactions of the most frequently modified sortilin lysine sidechains found in chronic kidney disease (amino acid sequence [crystal structure sequence]), Lys95(62), Lys205(172), Lys260(227), and Lys294(261), by in silico modeling to predict possible structure-function consequences of lysine carbamylation. We used the existing sortilin ectodomain crystal structure from both the neurotensin-bound form at neutral pH (PDB Q24 file: 4PO7; Figure 3a) and the ligand-free form at acidic pH (PDB file: 6EHO; Supplementary Table S5).

At neutral pH, Lys95(62) forms a salt bridge with Glu609(576) and a hydrogen bond with the main-chain carbonyl group of Arg90(57). At acidic pH, the interaction with Glu609(576) is tighter as the distance between the amino group of Lys95(62) and the carboxyl group of Glu609(576) now allows the formation of a hydrogen bond, whereas the interaction with Arg90(57) is not present. Therefore, carbamylation at Lys95(62) may destabilize both sortilin conformations. Thus, carbamylation at Lys205 would possibly favor the acidic pH conformation of sortilin. Lys294(261) has no interactions at both neutral and acidic pH.

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Finally, at neutral pH, Lys260(227) interacts with neurotensin-Tyr11 in the ligand-binding site in the b-propeller tunnel of sortilin. As the acidic pH sortilin structure has no ligand affinity, we find no interactions of the Lys260(227) sidechain in this structure. The interaction with neurotensinTyr11 will be affected by carbamylation and might lead to a lower affinity for neurotensin. Interleukin 6 (IL-6), a known sortilin ligand,20 binds sortilin through its C-terminal tail, which contains an arginine at the equivalent position of neurotensin-Tyr11. Thus, carbamylation of Lys260(227) might probably increase the affinity for IL-6, a potential contributor to vascular calcification.

Subsequent binding studies using surface plasmon resonance spectroscopy and SortCo or SortCarb revealed more efficient binding of IL-6 to SortCarb (KD ¼ 23 nM) compared Q25 with nonmodified SortCo (KD ¼ 141 nM), suggesting carbamylated sortilin as a potential IL-6 binding partner (Figure 3b and c). Progranulin, another known sortilin ligand,22 bound to SortCo, but a binding to SortCarb could not be detected (Supplementary Figure S7A and B). Both SortCo and SortCarb are bound to human receptor-associated protein fused to a GST tag,23 but not to GST alone, supporting the Q26 structural integrity of sortilin regardless of its carbamylation status and demonstrating the specificity of our experimental setting (Supplementary Figure S7C–F).

Next, we assessed whether the altered affinity to IL-6 might affect signaling pathways involved in vascular calcification. In calcifying hSMCs, the addition of IL-6 to SortCarb promoted ALPL and RUNX2 mRNA expression (Figure 3d and e) as well as TNAP activity (Figure 3f), whereas it had no effect in combination with SortCo. These data indicate that sortilin carbamylation increases the ability of ligand binding to IL-6, which causes increased smooth muscle cell calcification.

figure 3-1

Sortilin carbamylation is associated with vascular calcification in chronic kidney disease patients On the basis of our data that carbamylated sortilin residues were found to associate with chronic kidney disease and calcification, we next studied chronic kidney disease participants of the CARVIDA study, in whom computed tomographic scans quantified CAC. Like the CARE FOR HOMe study participants, most CARVIDA participants had 2 and 3 PTMs, with lysine residues 95 and 260 frequently affected (Supplementary Figure S8A). Compared with patients without carbamylated residues, patients with 1 sortilin carbamyl-lysine residues were older (P ¼ 0.002), had lower eGFR (P ¼ 0.008), were more frequently (former) smokers (P ¼ 0.023), and had significantly higher CAC volume (P < 0.001; Figure 4a and Supplementary Table S6).

Ln-(CAC+1) at baseline correlated with the presence and increase of post-translationally carbamylated sortilin (Supplementary Table S7). Furthermore, Ln-(CAC þ 1) was also associated with male gender, diabetes mellitus, antidiabetic and lipid-lowering medication, higher age, serum IL-6 and hemoglobin A1c, and lower eGFR and serum high-density lipoprotein (Supplementary Table S7). Nevertheless, the association between carbamylated sortilin residues and baseline Ln-(CAC þ 1) remained significant after adjustment for all variables mentioned above (P ¼ 0.029; Table 3).

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Next, we assessed whether the presence of carbamylated residues has an impact on CAC progression. The presence of carbamylated sortilin (vs. no carbamylation) increased the progression of CAC (P ¼ 0.047). In detail, chronic kidney disease patients with carbamylated sortilin (n ¼ 33) exhibited a significantly increased CAC progression over a median follow-up time of 4.4 years (Figure 4b), with an annual increase in median CAC volume of 30.32 Q27 (interquartile range ¼ 84.0) (Figure 4C). In contrast, chronic kidney disease patients without sortilin carbamylation (n ¼ 8) revealed an annual change in median CAC volume of 1.63 (interquartile range ¼ 8.54) and no significant CAC progression over the follow-up period (Figure 4b and c). After adjusting for covariates associated with CAC progression (baseline CAC volume, age, gender, body mass index, eGFR, diabetes mellitus, serum IL-6, serum high-density lipoprotein, and lipid-lowering medication), carbamylation status was no longer associated with CAC progression (P ¼ 0.286).

Patients with carbamyl-lysine residue 260 exhibited the highest increase in CAC volume than patients with no carbamylated residue or carbamyl-lysine residues other than 260 (Supplementary Figure S8B).

Overall, the data indicate that post-translational carbamylated sortilin is associated with CAC volume and its progression in patients with chronic kidney disease.

DISCUSSION

In various experimental in vitro and in vivo studies and 2 independent prospective cohorts, we demonstrate that patients with chronic kidney disease exhibit a specific pattern of post-translational carbamylated sortilin lysine residues in the circulation, which can also be detected in the vascular wall. Furthermore, sortilin carbamylation was associated with CAC in chronic kidney disease patients, independent of age, kidney function, and other risk factors for calcification. Mechanistically, we could show that sortilin carbamylation increases its ability to bind IL-6 and acts directly on vascular cells to promote vascular calcification (Figure 4d). Taken together, this study revealed a novel nontraditional risk factor for calcification, which potentially contributes to the high burden of cardiovascular diseases in chronic kidney disease.

Protein carbamylation can be mediated by cyanate, which is developed during the spontaneous decomposition of urea or is generated by myeloperoxidase-catalyzed thiocyanate oxidation at sites of inflammation.24 We showed that chronic kidney disease patients with higher urea levels due to reduced kidney function exhibited more sortilin carbamylation residues than healthy controls. However, we found no association between total levels and activity of myeloperoxidase and sortilin carbamylation, suggesting uremia as a primary driver for sortilin carbamylation in chronic kidney disease. In addition, our data demonstrate that age, NT-proBNP, and a lower intake of aldosterone antagonists are associated with more carbamylated residues. Our data are consistent with previous reports that identified aging as a significant mediator of protein carbamylation.25 It has been shown that the antihypertensive drugs hydrochlorothiazide and amlodipine affect homocitrulline levels, a urea cycle-related amino acid, and a carbamylation-derived product.26 Whether intake of aldosterone antagonists lowers homocitrulline, and thus reduces carbamylated residues in chronic kidney disease patients, is unknown. Drechsler et al. demonstrated a correlation between serum carbamylated albumin and NTproBNP, which was associated with heart failure in dialysis-dependent chronic kidney disease patients.27 Whether carbamylation of sortilin may also serve as a risk factor for heart failure is not known, but increased coronary calcification with potentially subsequent harmful effects on heart function could explain the association between carbamylated sortilin residues and NTproBNP levels in our study.

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Recent studies identified the role of sortilin in the pathogenesis of vascular and metabolic disorders but mainly focused on tissue expression of sortilin rather than exploring the function of the soluble form.7 Circulating sortilin may originate from cellular shedding of their luminal domain, as demonstrated in vitro, 28–30, and from secreted sortilin-packed extracellular vesicles.9,31 Carbamylation leads to alterations in charge, structural, and functional properties of proteins, mediating loss of function and potentially pathophysiological cellular and molecular responses.5 We established for the first time a specific lysine residue carbamylation pattern of circulating sortilin in chronic kidney disease, using a targeted MS approach, and predicted functional and structural alterations by in silico modeling. We propose that lysine carbamylation will favor the acidic pH conformation of sortilin, affecting the pH-dependent ligand affinity of sortilin. Previous crystal structure studies demonstrated that at pH 5.5, which represents an environment similar to that of late endosomes, sortilin undergoes conformational changes and dimer formation, making known binding sites unavailable for ligand binding.32,33

Lysine 260 forms a hydrogen bond to a tyrosine of neurotensin inside the b-propeller tunnel, intimating that other ligands that bind similarly to neurotensin would also be affected. Our investigation found increased affinity of IL-6 to carbamylated sortilin, suggesting participation in IL-6 signaling pathways. Several studies suggested that IL-6 may contribute to vascular calcification in chronic kidney disease.22,34,35 functional role in calcific vascular pathology. Soluble carbamylated sortilin promoted vascular calcification in vitro and ex vivo by increasing osteogenic target genes, TNAP activity, and matrix mineralization. Moreover, the addition of IL-6 to carbamylated sortilin further increased osteogenic target genes and TNAP activity.

Carbamylation has been associated with extracellular matrix alteration,36 oxidative stress,25 endothelial dysfunctions,37 and atherosclerosis,38 all involved in calcification initiation and progression. Although Mori et al. reported that protein carbamylation promotes vascular calcification through carbamylation of mitochondrial proteins,6 no specific target proteins were identified. A recent study demonstrated that carbamylation of uromodulin resulted in a loss of its anti-calcific properties in vitro. 39 Both studies used Western blot to detect carbamylation in general. We demonstrated that in vitro carbamylated sortilin mimics the specific in vivo lysine modification pattern found in chronic kidney disease patients using MS. Thus, our study provides clues to specific mechanisms connecting chronic kidney disease, protein carbamylation, IL-6, and calcification.

Previously, high serum sortilin levels were associated with both abdominal aortic calcification and cardiovascular events independent of traditional Framingham risk factors in a community-dwelling cohort of men aged >50 years.10 In low- to intermediate-risk chest pain patients, sortilin levels did not associate with the severity of coronary artery disease.40 In this study, we could show that patients with chronic kidney disease have higher sortilin levels than controls with normal kidney function. Sortilin levels did not increase with decreased kidney function once a patient had a chronic kidney disease with eGFR <60 ml/min per 1.73 m2. However, we observed an increase of carbamylated sortilin residues with decreased kidney function. Similarly, Kalim et al. reported no association between total and carbamylated albumin levels in chronic kidney disease patients.41 Sortilin carbamylation, resulting from impaired kidney function, might further augment vascular calcification related to chronic kidney disease-associated mineral disturbances. Our study has several strengths and limitations. Strengths include analyzing data obtained in vitro, ex vivo, and in patients using different experimental approaches. The careful characterization of patients with chronic kidney disease, including standardized questionnaires to assess participants’ characteristics, in-person study visits conducted by trained study nurses, and measurements of many laboratory values and calcification, is a particular strength of the study. The small cohort may appear to limit our study; however, we used independent chronic kidney disease cohorts with follow-up data for calcification to validate the carbamylation status of sortilin by laborious targeted MS. The clinical findings were made in German chronic kidney disease patients and may thus restrict the generalizability of the findings to other countries or ethnicities. Finally, although the study design allowed adjustment for many important confounders, residual confounding, as in any observational study, cannot entirely be ruled out.

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Conclusion

In conclusion, this is the first study demonstrating specific amino acids that are post-translationally modified in circulating sortilin. Our data suggest that the presence of chronic kidney disease promotes the carbamylation of circulating sortilin and that carbamylated sortilin exhibits procalcific properties, as demonstrated in vitro, ex vivo, and by the presence in calcified tissue. Sortilin carbamylation was associated with CAC, even after adjustment for age, kidney function, and several other risk factors, suggesting that prevention of sortilin carbamylation might reduce the risk for cardiovascular calcification and thus cardiovascular complications in patients with chronic kidney disease. Thus, our results point to carbamylated sortilin as a contributor to the cardiovascular disease burden in chronic kidney disease patients.

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