Part 1: Carbamylated Sortilin Associated With Cardiovascular Calcification in Patients With Chronic Kidney Disease

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

Part 1: Carbamylated sortilin associated with cardiovascular calcification in patients with chronic kidney disease

Keywords: chronic kidney disease, cardiovascular, kidney

Translational Statement

Patients with chronic kidney disease are highly susceptible to developing cardiovascular disease. Chronic kidney disease-specific cardiovascular 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 cardiovascular calcification in patients with chronic kidney disease.

Introduction

Sortilin, an intracellular sorting receptor, has been identified as a cardiovascular risk factor in the general population. Patients with chronic kidney disease are highly susceptible to developing cardiovascular complications such as calcification. However, specific chronic kidney disease-induced posttranslational protein modifications of sortilin and their link to cardiovascular calcification remain unknown. To investigate this, we examined two independent chronic kidney disease 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 chronic kidney disease, sortilin carbamylation was associated with coronary artery calcification, independent of age and kidney function. Moreover, patients with carbamylated sortilin displayed the 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 chronic kidney disease.

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About 1 in 10 people worldwide experience chronic kidney disease.1 Impaired kidney function is a major independent risk factor for cardiovascular morbidity and mortality and all-cause mortality.1,2 In fact, patients with chronic kidney disease are much more likely to die from cardiovascular events than to develop dialysis-requiring end-stage renal Q14 disease.3 The excess calcific mineral deposition within vascular tissue observed in chronic kidney disease patients contributes mainly to the increased cardiovascular risk.4 chronic kidney disease facilitates post-translational modification (PTM) of proteins.5 PTMs, in turn, have been linked to cardiovascular calcification,6 suggesting that a better understanding of PTMs in chronic kidney disease-induced calcification processes could reveal novel therapeutic targets. However, hitherto specific chronic kidney disease-associated protein modifications linked to cardiovascular calcification is unknown.

Sortilin is a ubiquitously expressed member of the vacuolar protein sorting 10 protein family of intracellular sorting receptors.7 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.8 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 cardiovascular risk factors.7 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.9 The ectodomain of plasma membrane-bound sortilin can be shed and secreted into the circulation.7 In a community-dwelling cohort of men aged >50 years, we reported an association of high sortilin serum levels with aortic calcification and cardiovascular events, suggesting sortilin as a cardiovascular risk factor in the general population.10 A role of sortilin in chronic kidney disease, a patient population with a marked increase in cardiovascular 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.7 Therefore, achieving a better understanding of the mechanistic relationship between circulating sortilin and the regulatory impact of PTM in chronic kidney disease will broaden the knowledge of sortilin in cardiovascular calcification.

This study interrogates the hypothesis that the PTM of circulating sortilin is involved in the development of cardiovascular calcification in patients with chronic kidney disease.

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METHODS

Cardiovascular and Renal Outcome in chronic kidney disease 2–4 Patients–The Fourth Homburg evaluation (CARE FOR HOMe) cohort

The CARE FOR HOMe study has been previously described in detail.11 A subset of 97 patients was used for the analysis.

Dan-NICAD 1 cohort

The study design of the Danish study of noninvasive testing in coronary artery disease (Dan-NICAD 1) trial has been described previously.12 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 cardiovascular disease. Identification was performed blinded for sortilin levels. Cardiovascular In-Depth Assessment (CARVIDA) cohort The CARVIDA is a substudy of the German Chronic Kidney Disease study.13 Only samples of CARVIDA patients included in the trial in Aachen, Germany, were used (n ¼ 78). Computed tomographic imaging was performed on a Dual Source CT scanner (SOMATOM Definition Flash or Force; Siemens), as previously described.14 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/ Q15 2020.

Statistical analysis

Experimental study data are presented as mean SD; n indicates the number of independent experiments or number of patients. Normality was tested using the Shapiro-Wilk test, and quantile plot and variance heterogeneity were tested using the Brown-Forsythe test. A paired or unpaired 2-tailed Student t-test with equal or unequal variances was performed to compare 2 groups. For comparison among 3 treatment groups, 1- or 2-way analysis of variance followed by Tukey post hoc was performed for data with normal distribution and equal variance. Data with skewed distribution were assessed by the Kruskal-Wallis test followed by the Dunn post hoc test. Data with unequal variances were tested by Welch analysis of variance followed by Dunnett T3 post hoc test.

In clinical studies, continuous data are presented as mean SD when normally distributed or as the median and interquartile range for variables with skewed distribution. Categorical data are presented as percentages. Pearson/Fisher c2 test was used to study the association between categorical variables and unpaired Student t-test or Mann-Whitney U test for continuous variables. Differences between the 3 groups were compared using a 1-way analysis of variance followed by the Sidak post hoc test. Least-square means multivariate-adjusted numbers of carbamylated sortilin residues were calculated using generalized linear models, as described previously.

Coronary artery calcification (CAC) volume was log-transformed (i.e., natural logarithm, ln; ln[CAC + 1]) to reduce skewness. Bivariate correlation was assessed using Eta (1 nominal variable and 1 metric variable) or Pearson correlation coefficients (if both variables were metric). Change in CAC volume per year was calculated as follows: [(CAC follow-up – CAC baseline) / follow-up time in months] * 12. Analysis of variance with change in CAC volume as the dependent variable and a fixed-effect term for carbamylation (yes vs. no) was used for the analysis of CAC progression. In addition, this model was adjusted by the use of analysis of covariance for the covariates CAC at Q18 baseline and the variables. P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism (Prism Software Inc., version 9) or SPSS (version 26.0).

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RESULTS

Carbamylated sortilin increases with kidney function decline

Initially, we assessed sortilin serum levels in patients with chronic kidney disease from the CARE FOR HOMe study11 and found increased sortilin levels compared with a matched control group with normal kidney function from the Dan-NICAD 1 trial (Table 1). Circulating proteins are prone to PTM in patients with chronic kidney disease Q20 .5 Therefore, we performed a detailed mapping of PTM residues of circulating sortilin in participants of the CARE FOR HOMe study and healthy control subjects (Supplementary Table S1) using MALDI-TOF/TOFMS. Compared with control subjects, chronic kidney disease patients had 8 of the 30 lysine residues that were predominately carbamylated in the extracellular domain of sortilin (Supplementary Table S3 Q21 and Supplementary Figure S1). Representative MS spectra from a control subject and a patient with chronic kidney disease are illustrated in Figures 1a and b. The specificity of the signal was supported by MALDI-TOF/TOF-MS/MS spectra (Supplementary Figure S2A). Quantification revealed a chronic kidney disease stage-dependent increase of carbamylated residue number (Figure 1c) and intensity of carbamylated sortilin peptides (Figure 1d). Lysine residue 205 was equally modified in all chronic kidney disease stages, whereas residues 95, 260, and 294 were more often modified in advanced chronic kidney disease stages (Figure 1e).

Next, we assessed associations between sortilin carbamylation and baseline characteristics (Supplementary Table S2) in the CARE FOR HOMe study. We observed an age-dependent increase of sortilin carbamylation residues (Table 2). Lower kidney function, based on eGFR based on serum cystatin c and creatinine (eGFRcys-area), and higher urea and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were associated with higher carbamylated sortilin residues (Table 2). The associations remained significant after adjustment for age and gender (Table 2). chronic kidney disease patients under Q22 aldosterone antagonist medication displayed reduced sortilin carbamylation (Table 2), whereas there was no difference in eGFRcys-area (no, 50.6 23.2 ml/min per 1.73 m2; yes, 43.5 22.9 ml/min per 1.73 m2; P ¼ 0.236) and urea (no, 68.6 40.1 mg/dl; yes, 72.3 40.9 mg/dl; P ¼ 0.729) between patients with or without aldosterone antagonists. Besides urea, myeloperoxidase may mediate protein carbamylation in cardiovascular disease.19 However, we found no association between total levels and activity of myeloperoxidase and sortilin carbamylation (Supplementary Figures S3A and B).

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Furthermore, we assessed the presence of carbamylated sortilin in human femoral arteries using MS imaging. Carbamylated peptide SEDYGK*NFK* (m/z 1172) was highly present in calcified femoral arteries from chronic kidney disease patients and absent in noncalcified femoral arteries (Figure 1f). MS/MS spectra supported the identification of SEDYGK*NFK* (Supplementary Figure S2B). SEDYGK*NFK* is located close to calcified areas in the tunica media (Figure 1g). In contrast, the mass-signal intensity of non–post-translationally modified peptide SEDYGKNFK (m/z 1086) was higher in control arteries (Figure 1f).

Taken together, compared with controls with normal kidney function, patients with chronic kidney disease have higher sortilin serum levels and exhibit post-translational carbamylated sortilin in the circulation, which can also be detected in the vasculature.

Carbamylated sortilin promotes smooth muscle cell calcification Given our finding that carbamylated sortilin is localized to calcified areas, we next assessed the effect of sortilin carbamylation on vascular calcification in vitro and ex vivo. To determine the functional relevance of sortilin carbamylation, we induced in vitro carbamylation of recombinant sortilin (SortCarb) by urea and detected a similar carbamyl-lysine residue pattern as detected in humans in vivo (Supplementary Figure S4A and Supplementary Table S4). In vitro, carbamylation did not alter the protein integrity of SortCarb compared with control mock-modification (SortCo), as assessed by gel electrophoresis and Western blot (Supplementary Figure S4B and C). Neither SortCo nor SortCarb exhibited cytotoxic effects on hSMCs (Supplementary Figure S5A). SortCo and SortCarb were equally taken up by hSMCs (Supplementary Figure S5B). In calcifying hSMCs, SortCarb induced the proosteogenic transcripts ALPL (þ56%; P ¼ 0.039) and RUNX2 Q23 (þ25%; P ¼ 0.014; Figure 2a and b), as well as tissue non-specific alkaline phosphatase activity (þ46%; P ¼ 0.007; Figure 2c), compared with SortCo. On a functional level, SortCarb significantly augmented matrix calcification (Figure 2d and e). Finally, in an ex vivo organ culture model, SortCarb increased calcification in rat aortic rings (Figure 2f and g).

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To be continued, click here for Part 2.