Academic Editor: Brian Tomlinson
Background: In kidney transplantation (Ktx) recipients, cardiovascular
(CV) disease remains the leading cause of death. Abnormal carotid intima-media
thickness (IMT) represents a valid indicator of incipient atherosclerosis also in
this setting. Cathepsin-K (CatK) is a cysteine protease involved in vascular
remodelling, as well as in progressive atherosclerosis. In this study we
evaluated clinical predictors of CatK in Ktx recipients, with a particular focus
on its possible relationships with subclinical atherosclerosis. Methods:
Circulating CatK was measured in 40 stable Ktx recipients together with
several laboratory, clinical and echocardiography parameters. 30 healthy subjects
and 30 hemodialysis (HD) patients served as controls for CatK values. Carotid IMT
was measured in Ktx and these subjects were then categorized according to
age-gender reference cut-offs of normal IMT. Results: CatK levels were
similar in Ktx recipients and healthy subjects but significantly reduced as
compared to HD (p = 0.0001). In Ktx, at multivariate analyses CatK was
associated with the LV end-diastolic volume (LVEDVi) (
Patients with advanced chronic kidney disease (CKD) on hemodialysis (HD) treatment exhibits a remarkable risk of cardiovascular (CV) morbidity and mortality [1]. Although a successful kidney transplantation (Ktx) significantly improves such risk, the rate of CV events persists higher in Ktx recipients as compared with the general population and CV disease remains the leading cause of death, more so than infection or malignancy [2]. Increased carotid intima-media thickness (IMT) is largely considered as a footprint of incipient atherosclerosis, as well as a robust risk stratifier for CV mortality also in the Ktx setting [3]. Although Ktx often elicits a significant improvement in IMT as compared to patients remaining on maintenance dialysis [4], abnormally increased IMT persists in a large part of asymptomatic Ktx recipients and may worsen over time to frank atherosclerosis, driven by various factors such as immunosuppressive therapy, dyslipidaemia and diabetes [5, 6]. Cathepsin-K (CatK) is a cysteine protease endowed with collagenase and elastase activities, which has recently been implicated in the pathogenesis of progressive atherosclerosis [7]. High CatK mRNA/protein expression has been found in unstable atherosclerotic plaques [8] and CatK-deficiency was showed to protect atherosclerotic mice from disease progression [9]. Coronary artery disease (CAD) patients have altered circulating CatK levels; however, while some studies found direct associations between circulating CatK and the severity of CAD [10, 11], some others find opposite correlations, particularly in the presence of concomitant mineral bone disorders [12].
Recently, we reported increased CatK levels among chronic HD patients which reflected an altered bone mineral metabolism, also holding prognostic value for CV mortality [13, 14]. To the best of our knowledge, however, no study has analysed so far such protease in the context of Ktx, particularly in relationship with the presence of early, asymptomatic CV disease.
Starting from these premises, we thus aimed at conducting an exploratory study in a small cohort of Ktx recipients to evaluate clinical predictors of circulating CatK levels and its possible associations with subclinical atherosclerosis.
Fifty-eight adult kidney transplant recipients (age
Blood specimens were collected after an 8-hour overnight fast. Biochemical parameters and cardiac-specific biomarkers were measured in all patients by Cobas 8000 (Roche Diagnostics, Basel, Switerland) using the relative kits (Roche Diagnostics, Basel, Switzerland). Blood count analysis (Hb, RBC, WBC and platelet counts) was performed using ADVIA 2120i (Siemens Healthcare Diagnostics, Marburg, Germany). Fibrinogen was determined on BCS XP (Siemens, Healthcare Diagnostics, Marburg, Germany) using the Clauss method. All the above-mentioned assays were carried out according to the manufacturers’ instructions. Serum samples were centrifuged at 1227 g for 15 minutes at 4 °C and the aliquots stored at –80 °C until thawed for batch analysis. Cathepsin-K was measured in the blood using an ELISA commercially available kit (Novus Biological, Centennial, CO, US), according to the manufacturer’s instructions. The enzymatic reactions were quantified in an automatic microplate photometer. Measurements were made blind and in duplicate and levels were expressed as pg per mL. CatK was also measured in 30 healthy subjects and 30 patients undergoing chronic HD treatment who were matched with Ktx recipients for age and gender.
All Ktx recipients underwent a comprehensive cardiovascular assessment including blood pressure measurement at rest by a manual sphygmomanometer, a thorough echocardiographic examination and carotid echography for IMT evaluation. Echocardiography was performed using a GE Vivid E95 (General Electric Healthcare, Illinois, USA), with electrocardiographic monitoring during the exam. Left ventricular (LV) function was assessed by computing LV ejection fraction and the fractional shortening. In addition, the LV end-diastolic volume (LVEDVi) and a body-surface indexed LV mass (LVMi) were calculated, as indicated [15]. Right ventricular function was measured as the tricuspid annular plane systolic excursion (TAPSE) and Left and Right atrial volumes (RAVi, LAVi) were also measured as recommended [16].
Measurement of carotid IMT was performed in the posterior wall of both carotid
arteries by mode B ultrasound with an ultrasonography device (LogiQ C5 premium,
GE Medical Systems, China) equipped with a linear 8 cm probe operating at 8 MHz
by an experienced operator. IMT was computed by measuring the thickness of the
innermost two layers of intima-media, 5 mm before the bifurcation of the common
carotid artery. Mean carotid IMT values were calculated as the average of
absolute dx and sx measurements. Subclinical atherosclerosis was assumed in the
presence of a mean carotid IMT value
The statistical analysis was performed using the SPSS package (version 24.0; IBM
corporation, Chicago, IL, USA), the MedCalc Statistical Software (version 14.8.1;
MedCalc Software bvba, Ostend, Belgium) and the GraphPad prism package (version
8.4.2, GraphPad Software, San Diego, CA, USA). Data were presented as mean
The final study population fulfilling the inclusion criteria consisted of 40 Ktx
recipients. Mean age was 56.6
Cathepsin-K levels in Ktx patients (n = 40) as compared with healthy matched controls (n = 30) and hemodialysis patients (n = 30). *p = 0.0001.
All | SubAth | no-SubAth | p | |
n = 40 | n = 17 | n = 23 | ||
Age (yrs) | 56.6 |
57.6 |
52.2 |
0.20 |
Gender (%Male) | 65 | 58.9 | 69.5 | 0.48 |
Dialysis vintage (mo.) | 33.5 [13–65] | 27 [14–58.5] | 33.5 [10–79.5] | 0.84 |
Ktx vintage (yrs) | 9 [3–18] | 14 [5.5–22] | 4 [2–15.7] | 0.10 |
DD Ktx (%) | 85 | 88.2 | 82.6 | 0.66 |
BMI (kg/m |
26.1 |
26.5 |
25.6 |
0.67 |
WHR (cm/cm) | 0.92 |
0.94 |
0.91 |
0.39 |
Current smokers (%) | 15 | 17.6 | 13 | 0.69 |
Diabetes (%) | 25 | 41 | 13 | 0.04 |
Coronary disease (%) | 2.5 | 1.8 | 4.3 | 0.23 |
Heart failure (%) | 5 | 5.9 | 8.7 | 0.21 |
Hypertension (%) | 65 | 70.6 | 60.9 | 0.54 |
Immunosuppressive Therapy (%): | ||||
-Corticosteroids | 82.5 | 82.3 | 82.6 | 0.86 |
-CNI | 92.5 | 94.1 | 91.3 | 0.81 |
-MMF | 72.5 | 70.6 | 82.6 | 0.61 |
-m-TORi | 10 | 11.7 | 8.6 | 0.74 |
Statin/Ezetimibe (%) | 22.5 | 29.4 | 17.4 | 0.65 |
Glycemia (mg/dL) | 96.5 |
104.6 |
90.6 |
0.06 |
Serum creatinine (mg/dL) | 1.33 [1.10–2.11] | 1.21 [1.01–1.92] | 1.38 [1.24–2.39] | 0.10 |
eGFR (CKD-Epi mL/min/1.73 m |
54.8 [32.7–66.1] | 60.9 [37.1–67.4] | 53.7 [28.4–66.2] | 0.31 |
Proteinuria (g/24 h) | 0.15 [0.10–0.40] | 0.14 [0.10–0.49] | 0.16 [0.09–0.66] | 0.82 |
Urea (mg/dL) | 55 [43.5–89.5] | 55 [45–75.5] | 54 [42–91.2] | 0.92 |
Uric Acid (mg/dL) | 6.02 |
6.07 |
5.99 |
0.86 |
Serum Phosphate (mg/dL) | 3.34 |
3.30 |
3.36 |
0.79 |
Serum Calcium (mg/dL) | 9.6 |
9.75 |
9.63 |
0.59 |
Parathormone (pg/mL) | 122.7 [68.6–167.2] | 121.8 [96.3–160.6] | 117.8 [59.5–177.7] | 0.97 |
Alkaline Phosphatase (U/L) | 80.4 |
90.2 |
74 |
0.008 |
Total Cholesterol (mg/dL) | 184.6 |
190.7 |
177.2 |
0.37 |
LDL Cholesterol (mg/dL) | 109.5 |
110.7 |
105.3 |
0.84 |
HDL Cholesterol (mg/dL) | 58.1 |
59.3 |
56.9 |
0.61 |
Triglycerides (mg/dL) | 140.5 |
169.6 |
120.6 |
0.003 |
Fibrinogen (mg/dL) | 352.3 |
363.5 |
344.9 |
0.54 |
25(OH)Vit-D (ng/mL) | 27.1 |
27.7 |
26.7 |
0.78 |
ESR (mm/h) | 15 [9–27] | 18 [9.5–26] | 14.5 [9–28.5] | 0.71 |
Albumin (g/dL) | 4.38 |
4.37 |
4.39 |
0.79 |
RBC (n × 10 |
4.68 |
4.72 |
4.87 |
0.80 |
Hb (g/dL) | 12.6 |
13.3 |
12.1 |
0.10 |
WBC (n × 10 |
7.11 |
6.08 |
8.01 |
0.11 |
PLT (n × 10 |
217 |
271.5 |
214.2 |
0.42 |
C-reactive protein (mg/L) | 3.23 [2.13–4.10] | 3.40 [2.13–4.8] | 3.23 [2.13–3.42] | 0.12 |
Ferritin (mg/dL) | 38 [16.5–97] | 39 [14.5–97] | 42 [16.5–119.7] | 0.71 |
TSAT (%) | 31.7 |
32.3 |
31.2 |
0.56 |
Serum iron (mg/dL) | 66.1 |
65.5 |
67.4 |
0.91 |
Cathepsin-K (pg/mL) | 160 [80–490] | 260 [135–490] | 140 [50–240] | 0.002 |
BMI, body mass index; CNI, calcineurin inhibitors; DD, deceased
donor; eGFR, estimated glomerular filtration rate; ESR, erythrocyte sedimentation
rate; Hb, haemoglobin; HDL, high density lipoprotein; Ktx, kidney
transplantation; LDL, low density lipoprotein; MMF, mycophenolate mofetil;
m-TORi, m-TOR inhibitors; PLT, platelets; RBC, red blood cells; TSAT, saturated
transferrin; Vit-D, vitamin-D; WBC, white blood cells; WHR, waist-hip-ratio. Data
are presented as mean ( |
All | SubAth | no-SubAth | p | |
N = 40 | n = 17 | n = 23 | ||
SBP (mmHg) | 135 |
136.5 |
133.6 |
0.66 |
DBP (mmHg) | 85 |
86.1 |
84.3 |
0.42 |
CK-MB (U/L) | 1.80 [1.15–2.20] | 1.6 [1.15–2.15] | 1.85 [1–2.32] | 0.57 |
Myoglobin (nmol/L) | 41.5 [33–78.5] | 41 [29–80.5] | 43 [33.5–85.5] | 0.37 |
hs-cTn (ng/L) | 12.4 [7.3–24.6] | 12.2 [6.7–19] | 12.1 [7.5–26.9] | 0.27 |
nt-pro-BNP (pg/mL) | 220 [67–1266] | 250 [85.5–411] | 187.5 [65–1523] | 0.92 |
Mean carotid IMT (mm) | 0.71 |
0.85 |
0.61 |
|
Right carotid IMT (mm) | 0.71 |
0.90 |
0.57 |
|
Left carotid IMT (mm) | 0.70 |
0.89 |
0.56 |
|
LAVi (mL/m |
35.1 |
29.8 |
31.2 |
0.82 |
LVMi (g/m |
162 |
161.5 |
159.9 |
0.97 |
LVEDVi (mL/m |
52.1 |
48.4 |
55.3 |
0.09 |
Ejection Fraction (%) | 59.3 |
58.5 |
57 |
0.58 |
Vmax (m/s) | 2.06 |
1.92 |
2.45 |
0.22 |
TAPSE (mm) | 21.8 |
23.5 |
20 |
0.43 |
E/e’ | 9.4 |
7.1 |
11.5 |
0.04 |
Fractional Shortening (%) | 3.20 |
3.35 |
3.65 |
0.75 |
RAVi (mL/m |
18.3 |
14 |
23 |
0.40 |
SBP, systolic blood pressure; CK-MB, creatine-kinase MB; DBP,
diastolic blood pressure; E/e’, early diastolic peak left ventricular inflow
velocity (E)/early diastolic peak lateral mitral annular velocity (e’) ratio;
hs-cTn, highly-sensitive c-troponin; IMT, carotid intima-media thickness; LAVi,
left atrial volume index; LVEDVi, left-ventricular end diastolic volume index;
LVMi, left ventricular mass index; nt-pro-BNP, n-terminal pro Brain Natriuretic
Peptide; RAVi, right atrial volume index. TAPSE, tricuspid annular plane
excursion; Vmax, peak aortic valve velocity. Data are presented as mean
( |
At univariate analyses, circulating CatK levels were inversely correlated with
mean carotid IMT (R = –0.350; p = 0.02), Ktx vintage (R = –0.365;
p = 0.02), total cholesterol (R = –0.325; p = 0.04) and
alkaline phosphatase (R = –0.374; p = 0.01) while a direct association
was found with left-ventricular end diastolic volume index (R = 0.851; p
= 0.001). In a multivariate model including all univariate significant
correlates, IMT (
Univariate correlation coefficient | p | |
mean IMT | –0.350 | 0.02 |
(log)Ktx vintage | –0.365 | 0.02 |
Total cholesterol | –0.325 | 0.04 |
Alkaline phosphatase | –0.374 | 0.01 |
LVEDVi | 0.851 | 0.001 |
Multivariate standardized correlation coefficient ( |
p | |
mean IMT | –0.545 | 0.05 |
LVEDVi | 0.514 | 0.05 |
(log)Ktx vintage | –0.333 | 0.05 |
Total cholesterol | –0.278 | 0.13 |
Alkaline phosphatase | 0.278 | 0.26 |
Multiple R = 0.95, R |
Univariate correlates of (log transformed) Cathepsin-K levels in Ktx patients. Black and white dots refer to individuals with or without subclinical atherosclerosis, respectively.
Carotid IMT values were directly correlated to age (R = 0.484; p =
0.002), glycemia (R = 0.334; p = 0.03), triglycerides (R = 0.450;
p = 0.004) and alkaline phosphatase (R = 0.372; p = 0.01)
while, as reported above, an inverse correlation was found with CatK levels (R =
–0.350; p = 0.02). In a multivariate model with IMT as the dependent
variable, age (
Univariate correlation coefficient | p | |
(log)Cathepsin-K | –0.350 | 0.02 |
Age | 0.484 | 0.002 |
Glycemia | 0.334 | 0.03 |
Triglycerides | 0.450 | 0.004 |
Alkaline phosphatase | 0.372 | 0.01 |
Multivariate standardized correlation coefficient ( |
p | |
Age | 0.433 | 0.001 |
Triglycerides | 0.294 | 0.03 |
(log)Cathepsin-K | –0.202 | 0.05 |
Glycemia | –0.017 | 0.87 |
Alkaline phosphatase | 0.221 | 0.16 |
Multiple R = 0.69, R |
Univariate correlates of mean carotid IMT in Ktx patients. Black and white dots refer to individuals with or without subclinical atherosclerosis, respectively.
Seventeen patients (42.5%) showed evidence of subclinical atherosclerosis (mean
IMT
Cathepsin-K levels in Ktx patients with (n = 17) or without (n = 23) subclinical atherosclerosis and in healthy controls (n = 30). *p = 0.01; **p = 0.002.
At logistic regression analyses, alkaline phosphatase (OR 1.873; 95% CI 1.001–3.515; p = 0.05), triglycerides (OR 1.200; 95% CI 1.001–1.454; p = 0.05) and, above all, CatK (OR 0.761; 95% CI 0.569–0.918, p = 0.04) were confirmed as significantly associated with the presence of subclinical atherosclerosis while diabetes and E/e’ values were apparently not (Table 5). In such regard, CatK also displayed a remarkable diagnostic capacity in identifying Ktx patients with subclinical atherosclerosis, showing an area under the curve of 0.763 (95% CI 0.601–0.926; p = 0.001), with an optimal cut-off of 140 pg/mL holding a sensitivity of 76.47 (95% CI 50.1–93.2) and a specificity of 78.26 (95% CI 56.3–92.5).
Unit of increase | OR | 95% CI | p | |
Cathepsin-K | 100 pg/mL | 0.761 | 0.569–0.918 | 0.04 |
Alkaline Phosphatase | 10 U/L | 1.873 | 1.001–3.515 | 0.05 |
Triglycerides | 10 mg/dL | 1.200 | 1.001–1.454 | 0.05 |
Diabetes | Presence | 8.145 | 0.449–147.879 | 0.15 |
E/e’ | 1 unit | 1.107 | 0.968–1.265 | 0.21 |
Findings from our study raises two main points for discussion. First, Cathepsin-K levels in Ktx recipients were lower as compared with individuals on maintenance hemodialysis (HD). No less important, such levels were almost comparable to those measured in healthy controls. Hence, Ktx seems to normalize the altered balance in CatK which characterizes HD patients [13, 14]. As previously observed, increased CatK in HD patients are largely influenced by the severity of bone mineral alterations and inflammation [14]. Ktx is known to ameliorate most of the complications related to advanced CKD. In particular, tangible benefits on systemic inflammation, oxidative stress and, above all, fluid and mineral metabolism become already evident few weeks after transplantation and persist over time as long as the implanted kidney continues working [21]. Indeed, our Ktx recipients displayed on average normal or nearly normal parathormone and inflammatory indexes and no independent correlations were found between CatK levels and such parameters. Conversely, a robust, inverse association was found between CatK levels and Ktx vintage; this would reinforce the hypothesis that a longer lasting recovery in renal function may play a crucial role also in normalizing systemic CatK release and activity.
CatK usually abounds in lysosome of osteoclasts and macrophages, which is in line with its well-established function of extracellular matrix remodelling [22].
Nevertheless, CatK also promotes leukocyte recruitment and elicits pro-inflammatory processes through cross-talk with the coagulation cascade [23, 24]. As CatK is upregulated in various systemic inflammatory and autoimmune diseases [25], a direct inhibitory effect of immunosuppressive therapy on circulating CatK levels in our Ktx cohort cannot be in principle excluded and would deserve appropriate investigation by targeted mechanistic studies. No less important, the strong independent correlation found with the left ventricle end-diastolic volume merits further examination as it suggests a biological involvement of CatK also in the cardiac morpho-functional adaptations which characterize renal patients. Such a hypothesis would pair well with recent studies demonstrating a direct role of cysteine proteases in pathological cardiac remodelling, particularly in chronic heart failure [26, 27].
Another important aspect of our study pertains the strong interplay found in Ktx
recipients between CatK levels and subclinical atherosclerosis, as assessed by
carotid IMT evaluation. Such observation is corroborated by various findings.
First, we found a remarkable, inverse relationship between circulating CatK
levels and mean IMT values. Of note, such an association remained independent
from potential confounders in two different multivariate models employing, in
turn, CatK and IMT as the dependent variable. Second, Ktx patients with evidence
of incipient atherosclerosis—that is displaying a mean IMT
We cannot clarify the exact biological meaning of the reduced circulating CatK levels found in the presence of incipient atherosclerosis. One possible explanation relies on a systemic down-regulation of this protein to compensate the early vascular damage and prevent disease progression towards plaque formation. It is widely acknowledged that CatK activity is essential for normal vascular tissue remodelling [28]. However, as elsewhere demonstrated [8], an enhanced CatK activity promotes instability and rupture of atherosclerotic plaques, while disrupting the CatK gene reduces plaque progression and induces fibrotic transition [29]. Accordingly, CatK levels in patients with overt coronary heart disease are positively correlated with plaque volume but inversely associated with the fibrotic content [11].
Unfortunately, to the best of our knowledge, no clinical or mechanistic studies have so far evaluated CatK in early atherosclerosis. Hence, this “first glance hypothesis” would need confirmation by focused studies modelled on such particular conditions. Immunosuppressant agents, particularly m-TOR inhibitors, are known to limit atherosclerosis progression in Ktx recipients by exerting a direct effect on immune cells at the vascular wall level [30]; by the same token, a similar inhibitory effect on CatK expression cannot in principle be excluded. Nevertheless, all study participants were under chronic immunosuppressive therapy and no differences in the rate of different medications prescribed were noticed between the two study subgroups.
Our study has some limitations that deserve mentioning. First, the sample size was relatively small, although enough powered to avoid overfitting of the statistical models. Larger studies in more heterogeneous cohorts with respect to type of Ktx, immunosuppressive regimen, residual renal function, severity of atherosclerosis and CV comorbidities are necessary to generalize our findings as selection bias cannot be fully excluded. Second, the lack of a longitudinal observation prevents to evaluate whether fluctuations in CatK over time pair with structural changes in IMT. This information would be crucial for refining the biological interpretation of the interplay between CatK and IMT, as well as for explaining whether a causal relationship exists between this factor and early vascular damage. In this view, we also acknowledge that the lack of additional instrumental information on vascular function and status (e.g., pulse wave velocity, carotid wall shear stress, plaque composition…) may limit findings interpretation: future studies encompassing a larger pattern of cardiovascular functional examinations in relationship with circulating CatK measurement would therefore be advocated.
In this study, we found decreased CatK levels in Ktx as compared to chronic HD patients. Future studies are needed to confirm the usefulness of CatK as a biomarker for early CV risk stratification and to clarify the exact pathophysiological mechanisms underlying the close relationships with the atherosclerotic process in the Ktx setting.
Conceptualization—DB, GC; Methodology—DB, GC, VA; Formal Analysis—DB; Investigation—PP, VA, AC, ER, NC; Data Curation—DB, MG, OT; Writing - Original Draft Preparation—DB, GC; Writing - Review & Editing—DB, GC; Supervision—MA, DPF; All authors read and approved the final manuscript.
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Local Ethics Committee (Comitato Etico Regione Calabria-Area Centro-397/2019).
The Authors wish to thank Valeria Spanu for the precious contribution in data collection. The Authors also wish to thank the peer reviewers for their opinions and suggestions.
This research received no external funding.
The authors declare no conflict of interest. Davide Bolignano is serving as one of the Editorial Board members of this journal. Giuseppe Coppolino is serving as one of the Editorial Board members and Guest editors of this journal. We declare that Davide Bolignano and Giuseppe Coppolino had no involvement in the peer review of this article and have no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Brian Tomlinson.
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