Carbamylated sortilin associates with cardiovascular calcification in patients with chronic kidney disease

Abstract

Sortilin, an intracellular sorting receptor, has been identified as a cardiovascular risk factor in the general population. Patients with chronic kidney disease (CKD) are highly susceptible to develop cardiovascular complications such as calcification. However, specific CKD-induced posttranslational protein modifications of sortilin and their link to cardiovascular calcification remain unknown. To investigate this, we examined two independent CKD cohorts for carbamylation of circulating sortilin and detected increased carbamylated sortilin lysine residues in the extracellular domain of sortilin with kidney function decline using targeted mass spectrometry. Structure analysis predicted altered ligand binding by carbamylated sortilin, which was verified by binding studies using surface plasmon resonance measurement, showing an increased affinity of interleukin 6 to in vitro carbamylated sortilin. Further, carbamylated sortilin increased vascular calcification in vitro and ex vivo that was accelerated by interleukin 6. Imaging by mass spectrometry of human calcified arteries revealed in situ carbamylated sortilin. In patients with CKD, sortilin carbamylation was associated with coronary artery calcification, independent of age and kidney function. Moreover, patients with carbamylated sortilin displayed significantly faster progression of coronary artery calcification than patients without sortilin carbamylation. Thus, carbamylated sortilin may be a risk factor for cardiovascular calcification and may contribute to elevated cardiovascular complications in patients with CKD. Sortilin, an intracellular sorting receptor, has been identified as a cardiovascular risk factor in the general population. Patients with chronic kidney disease (CKD) are highly susceptible to develop cardiovascular complications such as calcification. However, specific CKD-induced posttranslational protein modifications of sortilin and their link to cardiovascular calcification remain unknown. To investigate this, we examined two independent CKD cohorts for carbamylation of circulating sortilin and detected increased carbamylated sortilin lysine residues in the extracellular domain of sortilin with kidney function decline using targeted mass spectrometry. Structure analysis predicted altered ligand binding by carbamylated sortilin, which was verified by binding studies using surface plasmon resonance measurement, showing an increased affinity of interleukin 6 to in vitro carbamylated sortilin. Further, carbamylated sortilin increased vascular calcification in vitro and ex vivo that was accelerated by interleukin 6. Imaging by mass spectrometry of human calcified arteries revealed in situ carbamylated sortilin. In patients with CKD, sortilin carbamylation was associated with coronary artery calcification, independent of age and kidney function. Moreover, patients with carbamylated sortilin displayed significantly faster progression of coronary artery calcification than patients without sortilin carbamylation. Thus, carbamylated sortilin may be a risk factor for cardiovascular calcification and may contribute to elevated cardiovascular complications in patients with CKD. see commentary on page 456Translational StatementPatients with chronic kidney disease (CKD) are highly susceptible to developing cardiovascular (CV) disease. CKD-specific CV risk factors are hitherto mainly unknown. We demonstrate that circulating sortilin is post-translationally modified by carbamylation in patients with reduced kidney function. Sortilin carbamylation leads to higher binding affinity to interleukin-6 and promotes arterial calcification ex vivo. Moreover, carbamylated sortilin is a risk factor for the presence and progression of coronary artery calcification. Our results point to carbamylated sortilin as a potential therapeutic target for hindering CV calcification in patients with CKD. see commentary on page 456 Patients with chronic kidney disease (CKD) are highly susceptible to developing cardiovascular (CV) disease. CKD-specific CV risk factors are hitherto mainly unknown. We demonstrate that circulating sortilin is post-translationally modified by carbamylation in patients with reduced kidney function. Sortilin carbamylation leads to higher binding affinity to interleukin-6 and promotes arterial calcification ex vivo. Moreover, carbamylated sortilin is a risk factor for the presence and progression of coronary artery calcification. Our results point to carbamylated sortilin as a potential therapeutic target for hindering CV calcification in patients with CKD. About 1 in 10 people worldwide experience chronic kidney disease (CKD).1Gansevoort R.T. Correa-Rotter R. Hemmelgarn B.R. Chronic kidney disease and cardiovascular risk: epidemiology, mechanisms, and prevention.Lancet. 2013; 382 (310–310)Google Scholar Impaired kidney function is a major independent risk factor for cardiovascular (CV) morbidity and mortality and all-cause mortality.1Gansevoort R.T. Correa-Rotter R. Hemmelgarn B.R. Chronic kidney disease and cardiovascular risk: epidemiology, mechanisms, and prevention.Lancet. 2013; 382 (310–310)Google Scholar,2Noels H. Boor P. Goettsch C. et al.The new SFB/TRR219 Research Centre.Eur Heart J. 2018; 39: 975-977Google Scholar In fact, patients with CKD are much more likely to die from CV events than to develop dialysis-requiring kidney failure.3Thompson S. James M. Wiebe N. et al.Cause of death in patients with reduced kidney function.J Am Soc Nephrol. 2015; 26: 2504-2511Google Scholar The excess calcific mineral deposition within vascular tissue observed in CKD patients contributes mainly to the increased CV risk.4McCullough P.A. Agrawal V. Danielewicz E. Abela G.S. Accelerated atherosclerotic calcification and Monckeberg's sclerosis: a continuum of advanced vascular pathology in chronic kidney disease.Clin J Am Soc Nephrol. 2008; 3: 1585-1598Google Scholar CKD facilitates post-translational modification (PTM) of proteins.5Gajjala P.R. Fliser D. Speer T. et al.Emerging role of post-translational modifications in chronic kidney disease and cardiovascular disease.Nephrol Dial Transpl. 2015; 30: 1814-1824Google Scholar PTMs, in turn, have been linked to CV calcification,6Mori D. Matsui I. Shimomura A. et al.Protein carbamylation exacerbates vascular calcification.Kidney Int. 2018; 94: 72-90Google Scholar suggesting that a better understanding of PTMs in CKD-induced calcification processes could reveal novel therapeutic targets. However, hitherto, specific CKD-associated protein modifications linked to CV calcification are unknown. Sortilin is a ubiquitously expressed member of the vacuolar protein sorting 10 protein family of intracellular sorting receptors.7Goettsch C. Kjolby M. Aikawa E. Sortilin and its multiple roles in cardiovascular and metabolic diseases.Arterioscl Throm Vas. 2018; 38: 19-25Google Scholar It is a single-pass type I transmembrane protein with various roles in protein sorting, trafficking, and cell signaling. As an endocytosis receptor, sortilin can trigger the internalization of ligands from the cell surface via endocytosis and sort ligands between intracellular compartments, such as trans-Golgi network, endosome, lysosome, and secretory pathway.8Kjolby M. Nielsen M.S. Petersen C.M. Sortilin, encoded by the cardiovascular risk gene SORT1, and its suggested functions in cardiovascular disease.Curr Atheroscler Rep. 2015; 17: 496Google Scholar Preclinical in vivo evidence suggests an important role of sortilin in the pathogenesis of vascular and metabolic disorders through contributions to arterial wall inflammation and calcification, dysregulated lipoprotein metabolism, and type 2 diabetes mellitus, all CV risk factors.7Goettsch C. Kjolby M. Aikawa E. Sortilin and its multiple roles in cardiovascular and metabolic diseases.Arterioscl Throm Vas. 2018; 38: 19-25Google Scholar In human vascular smooth muscle cells (hSMCs), intracellular sortilin regulates the loading of the procalcific protein tissue nonspecific alkaline phosphatase (TNAP) into extracellular vesicles, thereby conferring the calcification potential that contributes to microcalcification formation.9Goettsch C. Hutcheson J.D. Aikawa M. et al.Sortilin mediates vascular calcification via its recruitment into extracellular vesicles.J Clin Invest. 2016; 126: 1323-1336Google Scholar The ectodomain of plasma membrane-bound sortilin can be shed and secreted into the circulation.7Goettsch C. Kjolby M. Aikawa E. Sortilin and its multiple roles in cardiovascular and metabolic diseases.Arterioscl Throm Vas. 2018; 38: 19-25Google Scholar In a community-dwelling cohort of men aged >50 years, we reported an association of high sortilin serum levels with aortic calcification and CV events, suggesting sortilin as a CV risk factor in the general population.10Goettsch C. Iwata H. Hutcheson J.D. et al.Serum sortilin associates with aortic calcification and cardiovascular risk in men.Arterioscl Throm Vas. 2017; 37: 1005-1011Google Scholar A role of sortilin in CKD, a patient population with a marked increase in CV calcification, continues to defy elucidation. Most studies have focused on the function of cellular sortilin rather than exploring the biological function of the circulating soluble form.7Goettsch C. Kjolby M. Aikawa E. Sortilin and its multiple roles in cardiovascular and metabolic diseases.Arterioscl Throm Vas. 2018; 38: 19-25Google Scholar Therefore, achieving a better understanding of the mechanistic relationship between circulating sortilin and the regulatory impact of PTM in CKD will broaden the knowledge of sortilin in CV calcification. This study interrogates the hypothesis that PTM of circulating sortilin is involved in the development of CV calcification in patients with CKD. The CARE FOR HOMe study has been previously described in detail.11Zewinger S. Rauen T. Rudnicki M. et al.Dickkopf-3 (DKK3) in urine identifies patients with short-term risk of eGFR loss.J Am Soc Nephrol. 2018; 29: 2722-2733Google Scholar A subset of 97 patients was used for the analysis. The study design of the Dan-NICAD 1 trial has been described previously.12Nissen L. Winther S. Isaksen C. et al.Danish study of Non-Invasive testing in Coronary Artery Disease (Dan-NICAD): study protocol for a randomised controlled trial.Trials. 2016; 17: 262Google Scholar For the analysis, we identified a subset of 97 enrolled patients with estimated glomerular filtration rate (eGFR) >60 ml/min and performed frequency matching based on age, sex, body mass index, smoking status, diabetes mellitus, and CV disease. Identification was performed blinded for sortilin levels. The CARVIDA is a substudy of the German Chronic Kidney Disease study.13Eckardt K.U. Barthlein B. Baid-Agrawal S. et al.The German Chronic Kidney Disease (GCKD) study: design and methods.Nephrol Dial Transplant. 2012; 27: 1454-1460Google Scholar Only samples of CARVIDA patients included in the trial in Aachen, Germany, were used (n = 78). Computed tomographic imaging was performed on a Dual Source CT scanner (SOMATOM Definition Flash or Force; Siemens), as previously described.14Saritas T. Reinartz S.D. Nadal J. et al.Epicardial fat, cardiovascular risk factors and calcifications in patients with chronic kidney disease.Clin Kidney J. 2019; 13: 571-579Google Scholar With a median follow-up time of 4.4 years, 41 of 78 patients agreed or were available for a second computed tomographic scan during the second CARVIDA visit in 2019 and 2020. Femoral arteries were obtained during autopsies from patients with and without CKD from RWTH Aachen University, Germany. The study was approved by the ethical committee of the RWTH Aachen University (ethical votes EK180/14 and EK239/11) and performed according to the Declaration of Helsinki. Recombinant proteins were carbamylated in vitro by O-methylisourea bisulfate solution (pH 11.0) at 25 °C for 3 hours, as previously described.15Kimmel J. Guanidation of proteins.Meth Enzymology. 1967; 11: 584-589Google Scholar PTM was identified using MALDI–time-of-flight (TOF) mass spectrometry (MS; Ultraflex III; Bruker-Daltonic), as previously described.16Jankowski V. Schulz A. Kretschmer A. et al.The enzymatic activity of the VEGFR2 receptor for the biosynthesis of dinucleoside polyphosphates.J Mol Med (Berl). 2013; 91: Scholar were with in in a to mass and a of study are as the of independent or of was using the and and were using the A or with or was performed to 2 For or analysis of by was performed for with and with were by the by with were by analysis of by In the are as or as median and for with are as was used to study the association between and or for between 3 were using analysis of by of carbamylated sortilin residues were using as described S. Rauen T. Rudnicki M. et al.Dickkopf-3 (DKK3) in urine identifies patients with short-term risk of eGFR loss.J Am Soc Nephrol. 2018; 29: 2722-2733Google S. D. et is a of in and in chronic kidney S. L. et and cardiovascular Heart J. 2017; 38: Scholar Coronary artery calcification was to was using and 1 or were in was as follow-up follow-up time in of with in as the and a for carbamylation was used for the analysis of In was by the of analysis of for the at and the in was were performed using or are in we sortilin serum levels in patients with CKD from the CARE FOR HOMe S. Rauen T. Rudnicki M. et al.Dickkopf-3 (DKK3) in urine identifies patients with short-term risk of eGFR loss.J Am Soc Nephrol. 2018; 29: 2722-2733Google Scholar and increased sortilin levels with a with kidney function from the Dan-NICAD 1 trial proteins are to PTM in patients with P.R. Fliser D. Speer T. et al.Emerging role of post-translational modifications in chronic kidney disease and cardiovascular disease.Nephrol Dial Transpl. 2015; 30: 1814-1824Google Scholar Therefore, we performed a of PTM residues of circulating sortilin in of the CARE FOR HOMe study and and using with CKD patients of the lysine residues that were carbamylated in the extracellular domain of sortilin and from a and a patient with CKD are in and The of the was by revealed a CKD increase of carbamylated and of carbamylated sortilin was modified in all CKD residues and were more modified in advanced CKD levels and of with and kidney is the with kidney function 1 is the with kidney function FOR HOMe ml/min body mass CARE FOR and in CKD CKD, chronic kidney cardiovascular Dan-NICAD study of testing in coronary artery estimated glomerular filtration rate based on serum and are as and as is the with kidney function 1 CKD is the with kidney function FOR HOMe in a new body mass CARE FOR and in CKD CKD, chronic kidney cardiovascular Dan-NICAD study of testing in coronary artery estimated glomerular filtration rate based on serum and are as and as we between sortilin carbamylation and in the CARE FOR HOMe We observed an increase of sortilin carbamylation residues kidney based on eGFR on serum and and higher and levels were associated with higher carbamylated sortilin residues The for age and CKD patients displayed reduced sortilin carbamylation was in ml/min ml/min = and = between patients with or without may protein carbamylation in CV S. lipoprotein carbamylation as a mechanistic for atherosclerotic vascular disease.Clin Scholar However, we association between levels and activity of and sortilin carbamylation and of the of sortilin residues with in the CARE FOR HOMe study (n = and aged aged ml/min ml/min ml/min ml/min ml/min FOR and in CKD estimated glomerular filtration rate based on serum and Kidney was into eGFR was on the of the chronic kidney disease according to the The were by median from were Only were and aged ml/min in a new CARE FOR and in CKD estimated glomerular filtration rate based on serum and Kidney was into eGFR was on the of the chronic kidney disease according to the The were by median from were Only were we the presence of carbamylated sortilin in human arteries using was highly in calcified arteries from CKD patients and in arteries the of is to calcified in the In the of modified was higher in arteries with with kidney patients with CKD have higher sortilin serum levels and post-translational carbamylated sortilin in the which can be detected in the that carbamylated sortilin to calcified we the of sortilin carbamylation on vascular calcification in vitro and ex vivo. To the of sortilin we in vitro carbamylation of sortilin by and detected a as detected in in vivo and In carbamylation the protein of with as by and and on and were by In the alkaline phosphatase = and factor 2 = and as as tissue nonspecific alkaline phosphatase activity = with a significantly calcification and in an ex vivo increased calcification in aortic and and carbamylated type I and as as activity with Our that carbamylated soluble sortilin vascular cells and promotes vascular calcification in vitro and ex vivo. Sortilin is a for M. Nielsen M.S. Petersen C.M. Sortilin, encoded by the cardiovascular risk gene SORT1, and its suggested functions in cardiovascular disease.Curr Atheroscler Rep. 2015; 17: 496Google Scholar of lysine significantly the of the lysine the is by a ligand We examined the of the modified sortilin lysine in CKD and by in to of lysine carbamylation. We used the sortilin ectodomain from the at and the at a with and a with the of the with is as the between the of and the of the of a the with is Therefore, carbamylation at may sortilin is a with at are at Thus, carbamylation at the of sortilin. has at and at with in the in the of sortilin. As the sortilin has ligand we of the in The with will be by carbamylation and to a affinity for 6 a sortilin Kjolby M. S. et sortilin in cells and Clin Invest. Scholar sortilin through its which an at the of Thus, carbamylation of increase the affinity for a potential to vascular J. M. et 6 is linked to coronary artery calcification in patients with chronic kidney 2019; Scholar binding studies using surface plasmon resonance and or revealed more binding of to = with = suggesting carbamylated sortilin as a potential binding and sortilin T. et endocytosis levels of the Scholar to a binding to could be detected and and are to human protein to a Petersen C. Nielsen M.S. C. et for ligand J. Scholar to the of sortilin of its carbamylation and the of we the altered affinity to involved in vascular calcification. In the of to and and as as activity in with that sortilin carbamylation the of ligand binding to which increased smooth muscle cell calcification. the of that carbamylated sortilin residues were to with CKD and calcification, we CKD of the CARVIDA in computed tomographic the CARE FOR HOMe study CARVIDA 2 and 3 PTMs, with lysine residues and with patients without carbamylated patients with sortilin residues were = eGFR = were more = and significantly higher and at with the presence and increase of post-translationally carbamylated sortilin was associated with sex, diabetes mellitus, and higher age, serum and and eGFR and serum lipoprotein the association between carbamylated sortilin residues and for all = associated with as obtained by multiple sortilin ml/min mellitus, body mass coronary artery estimated glomerular filtration rate based on serum and interleukin has as the of the is The for the was = for a high of were to = in a new body mass coronary artery estimated glomerular filtration rate based on serum and interleukin 6. The has as the of the is The for the was = for a high of were to = we the presence of carbamylated residues has an impact on The presence of carbamylated sortilin increased the progression of = In CKD patients with carbamylated sortilin (n = a significantly increased progression a median follow-up time of 4.4 with an increase in median of = In CKD patients without sortilin carbamylation (n = revealed an in median of = and progression the follow-up and for associated with progression age, sex, body mass index, diabetes mellitus, serum serum and carbamylation was associated with progression = Patients with the increase in than patients with carbamylated or residues than the that post-translational carbamylated sortilin associates with and its progression in patients with CKD. In various in vitro and in vivo studies and 2 independent we demonstrate that patients with CKD a specific of post-translational carbamylated sortilin lysine residues in the which can be detected in the vascular sortilin carbamylation was associated with in CKD independent of age, kidney and risk factors for calcification. we could that sortilin carbamylation its to and on vascular cells to vascular calcification study revealed a novel risk factor for calcification, which contributes to the high of CV in CKD. carbamylation can be by which is during the of or is by at of C. et of to a potential carbamylation involved in the of atherosclerotic 2018; Scholar We that CKD patients with higher levels to reduced kidney function more sortilin carbamylation residues than However, we association between levels and activity of and sortilin suggesting as a for sortilin carbamylation in CKD. In demonstrate that age, and a of are associated with more carbamylated Our are with that identified as a of protein L. C. V. et al.Protein carbamylation is a of A. 2016; Scholar It has been that the and a and et of on plasma in patients with 2017; Scholar of and carbamylated residues in CKD is unknown. et a between serum carbamylated and which was associated with in CKD C. S. et al.Protein carbamylation is associated with and mortality in patients with Int. 2015; Scholar carbamylation of sortilin may as a risk factor for is increased coronary calcification with on function could the association between carbamylated sortilin residues and levels in studies identified the role of sortilin in the pathogenesis of vascular and metabolic disorders mainly focused on tissue of sortilin rather than exploring the function of the soluble form.7Goettsch C. Kjolby M. Aikawa E. Sortilin and its multiple roles in cardiovascular and metabolic diseases.Arterioscl Throm Vas. 2018; 38: 19-25Google Scholar sortilin may from cellular of their as in et factor is in with levels by and V. J. of the domain of the in the cell T. T. et sortilin is by and its circulating levels are associated with cardiovascular risk 2016; Scholar and from secreted extracellular C. Hutcheson J.D. Aikawa M. et al.Sortilin mediates vascular calcification via its recruitment into extracellular vesicles.J Clin Invest. 2016; 126: 1323-1336Google C.M. T. et al.Sortilin mediates the and of in with two Scholar leads to in and of of function and cellular and P.R. Fliser D. Speer T. et al.Emerging role of post-translational modifications in chronic kidney disease and cardiovascular disease.Nephrol Dial Transpl. 2015; 30: 1814-1824Google Scholar We for the time a specific lysine carbamylation of circulating sortilin in CKD, using a targeted and predicted and by in We that lysine carbamylation will the of sortilin, the ligand affinity of sortilin. studies that at which an to that of sortilin and binding for ligand D. Nielsen et and ligand binding in the domain of 2017; N. into factor 2018; Scholar a to a of the that ligands that to be Our increased affinity of to carbamylated sortilin, suggesting in studies suggested that may contribute to vascular calcification in T. et endocytosis levels of the R. chronic inflammation and vascular 2019; L. into the role of interleukin 6 in vascular calcification of chronic kidney disease.Nephrol Dial Transpl. 2018; Scholar a study on cellular sortilin that of the intracellular domain of sortilin vascular C. Hutcheson J.D. Aikawa M. et al.Sortilin mediates vascular calcification via its recruitment into extracellular vesicles.J Clin Invest. 2016; 126: 1323-1336Google Scholar we that post-translational modification of soluble sortilin a role in calcific vascular carbamylated sortilin vascular calcification in vitro and ex vivo by target and Moreover, the of to carbamylated sortilin increased target and has been associated with extracellular S. S. S. et of carbamylation on type I and its to human 13: Scholar L. C. V. et al.Protein carbamylation is a of A. 2016; Scholar T. et lipoprotein Heart J. Scholar and D. et carbamylation and Am Soc Nephrol. Scholar all involved in calcification and et reported that protein carbamylation promotes vascular calcification through carbamylation of D. Matsui I. Shimomura A. et al.Protein carbamylation exacerbates vascular calcification.Kidney Int. 2018; 94: 72-90Google Scholar specific target proteins were A study that carbamylation of in a of its in I. N. et vascular calcification by with Scholar studies used to carbamylation in We that in vitro carbamylated sortilin the specific in vivo lysine modification in CKD patients using Thus, study to specific CKD, protein and calcification. high serum sortilin levels were associated with aortic calcification and CV events independent of risk factors in a community-dwelling cohort of men aged >50 C. Iwata H. Hutcheson J.D. et al.Serum sortilin associates with aortic calcification and cardiovascular risk in men.Arterioscl Throm Vas. 2017; 37: 1005-1011Google Scholar In to sortilin levels with the of coronary artery Winther S. et al.Sortilin as a for cardiovascular disease Scholar In we could that patients with CKD have higher sortilin levels than with kidney function. Sortilin levels increase with kidney function a patient a CKD with eGFR ml/min However, we observed an increase of carbamylated sortilin residues with kidney function. et reported association between and carbamylated levels in CKD et carbamylation and chronic kidney disease progression in the Chronic Dial Transplant. Scholar Sortilin from kidney vascular calcification to CKD-associated mineral Our study has and obtained in ex and in patients using The of patients with CKD, to study by study and of and calcification, is a of the The cohort may to we used independent CKD cohorts with follow-up for calcification to the carbamylation of sortilin by targeted The were in German CKD patients and and may the of the to or the study design for important as in be In is the study specific that are post-translationally modified in circulating sortilin. Our that the presence of CKD promotes the carbamylation of circulating sortilin and that carbamylated sortilin procalcific as in ex and by the presence in calcified Sortilin carbamylation was associated with for age, kidney and risk suggesting that of sortilin carbamylation the risk for CV calcification and CV complications in patients with CKD. Thus, results point to carbamylated sortilin as a to CV disease in CKD the We and for their We and for the and as of the CARE for HOMe This was by the Research to and Research to and by the of the of RWTH Aachen to to the of the German of and the and the with Sortilin, and cardiovascular calcification in chronic kidney to important roles in the pathogenesis of vascular and metabolic such as cardiovascular calcification. modifications of sortilin or are the of its the role of carbamylated sortilin to the pathogenesis of cardiovascular calcifications in chronic kidney disease. therapeutic available to the carbamylation in chronic kidney disease patients cardiovascular calcifications to be

Topics & Concepts