Elevated red blood cell count levels are associated with increased vascular calcification in hemodialysis patients

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Elevated red blood cell count levels are associated with increased vascular calcification in hemodialysis patients

https://doi.org/10.21203/rs.3.rs-4725104/v1

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Introduction:

Coronary artery calcification (CAC) is a predictor of poor survival and cardiovascular outcome in end-stage renal disease patients.We conducted this study to assess the risk factors for vascular calcification in maintenance hemodialysis patients.

Methods:

A total of 71 cases of HD patients who underwent hemodialysis in Hunan Provincial People's hospital from January 2022 to September 2022 were enrolled. The general clinical data and Laboratory results of the patients were collected.Chest CT examination was performed,and the CAC score was calculated.

Results: CAC occurred in 41 ( 57.75%) of the 71 HD patients in the study.In the logistic regression analysis, CAC in these patients was significantly predicted by an diabetes (OR [95% CI]: 6.20 [1.37–28.11], P = 0.02) and red blood cell count (OR [95% CI]: 2.29 [1.05–5.02], P = 0.04).The ROC curve was applied to analyzing the red blood cell count; the area under the curve (AUC) was 0.66 (P = 0.02) and clinical diagnostic cutoff value = 2.99 (sensitivity = 75.6% and specificity = 56.7%) .

Conclusion: The incidence rate of CAC in HD patients was high.Diabetes and red blood cell count were independent risk factors for CAC in HD patients.

Vascular calcification

Hemodialysis

Diabetes

Cardiovascular disease

Risk factors

Chronic kidney disease has become a global public health problem. It has been estimated that the prevalence of CKD in Chinese adults has exceeded 10% (1–2). The number of ESRD patients is increasing yearly. Patients with end-stage renal disease on chronic hemodialysis often develop various cardiovascular complications, including vascular calcification, cardiovascular calcification, and coronary artery disease (3). Song et al have verified the effects of intravenous sodium thiosulfate on vascular calcification in dialysis patients with end-stage renal disease (4). There are two types of vascular calcification, including intimal and medial calcification. Vascular calcification causes arterial stiffness through medial calcification and myocardial ischemia through intimal calcification. Vascular calcification is a common complication associated with chronic kidney disease and a major cause of morbidity and mortality in patients with end-stage renal disease (5). Previous studies have identified traditional factors, including advanced age, male sex, diabetes, and smoking, as major risk factors for vascular calcification in HD patients (6–7). However, the pathogenesis of vascular calcification is complex and unclear, and it is particularly important to evaluate the effect of hemodialysis on hematological parameters in HD patients.

Previous studies have suggested that a variety of factors are associated with coronary artery calcification in hemodialysis patients. Xiong et al report suggested that coronary artery calcification in maintenance hemodialysis patients was closely related to age, hemodialysis time, infection, lipid metabolism disorder, and urea toxin level, but not to osteoporosis (8). Besides, it was reported that the levels of hemoglobin, erythrocyte sedimentation rate, C-reactive protein, and hepcidin were different between hemodialysis patients and healthy volunteers (9). These abnormally regulation of markers may be related to the poor survival of HD patients. However, there are few studies on the risk factors causing CAC in maintenance hemodialysis patients.

Therefore, in our research, we expect to find the markers that have the greatest influence on CAC in HD patients, so as to promote the prognosis of HD patients, and provide effective interventions.

Patients

The primary AVF patency rate of ESRD patients who had an AVF created between January 2022 and September 2022 at the Nephrology Department of Hunan Provincial People's Hospital was retrospectively analyzed. The study population comprised 100 adult patients (aged > 18 years) on maintenance hemodialysis for at least six months and had an arteriovenous fistula for vascular access. After excluding patients with a history of thrombosis, surgical interventions, infection at the arteriovenous fistula site or patients with incomplete data,finally 71 patients were included.Patients were divided into non-calcification group( CAC score ≤ 4 points) and calcification group( 5 points ≤ CAC score ≤ 15 points) .

Study design

The data were collected from their medical records at the time of AVF were created.These data included demographic and clinical information such as age, sex, duration of hemodialysis, comorbidities, and medication history. In addition, clinical and biochemical parameters were measured, including predialysis complete blood count, albumin, uric acid, creatinine, cholesterol, triglycerides, calcium, phosphorus, intact parathyroid hormone, and ferritin. Agatston calcium scores was evaluated by means of non-contrast cardiac CT.

This study protocol was reviewed and approved by the Ethics Committee of Hunan Provincial People’s Hospital.The study involving human participants adhered to ethical standards set by the institution and the national research committee. It was in accordance with the principles outlined in the 1964 Helsinki Declaration and its later amendments or similar ethical standards. All patients provided written informed consent.

Statistical analysis

All data were analyzed using SPSS 19.0. Continuous data are expressed as mean ± standard deviation and compared using the independent t-test (normal distribution) or Mann-Whitney U test (non-normal distribution). Categorical variables are reported in frequency and percentage, and compared using the chi-square test. Risk factors for calcification were identified by univariate and multivariate logistic regression analyses. A P < 0.05 was considered statistically significant. Multivariate logistic regression analysis was used to identify the independent risk factors related to outcome.The receiver operating characteristic (ROC) analysis with the area under the curve (AUC) was used to evaluate the predictive performance of developing calcification in HD patients.

Baseline demographic and clinical characteristics of HD patients with and without calcification

The baseline demographic and clinical characteristics of the patients with and without calcification are summarized in Table 1 and Table 2. There were 30 patients in the non-calcification group and 41 patients in the calcification group. Among patients in the non-calcification group, 14 (46.7%) were male, and the mean age was 60.23 ± 13.21. In the calcification group, 25 (61.0%) were male and the mean age was 60.93 ± 12.55. The baseline characteristics including height,weight,blood pressure,HD time, smoking history and the portion of comorbid hypertension were generally not significantly different. Higher portion of patients in the calcification group had comorbid diabetes compared with non-calcification group(Table 1).

Table 1

Characteristics of HD patients with and without calcification
Characteristics	n	calcification group(n = 30)	non-calcification group(n = 41)	t/\(\:{\varvec{Z}/\varvec{\chi\:}}^{2}\)
Gender
Male	39	14(46.7)	25(61.0)	1.43	0.23
Female	32	16(53.3)	16(39.0)
Age(year)	—	60.23 ± 13.21	60.93 ± 12.55	-0.23	0.82
Height(m)	—	160.97 ± 7.85	160.85 ± 9.08	0.06	0.96
Weight(kg)	—	56.30 ± 12.23	59.86 ± 12.65	-1.19	0.24
BMI	—	21.82 ± 4.10	22.95 ± 3.16	-1.31	0.19
DBP (mmHg)	—	80.13 ± 10.21	80.54 ± 10.42	-0.16	0.87
SBP (mmHg)	—	146.40 ± 24.72	150.78 ± 22.95	-0.77	0.45
HD time(months)	—	22.0(9.5,62.0)	30.0(13.5,51.0)	-0.71	0.48
Smoking history	41	16(53.3)	25(61.0)	0.42	0.52
Comorbidities
Hypertension	59	23(76.7)	36(87.8)	1.53	0.22
Diabetes	22	4(13.3)	18(43.9)	7.57	< 0.01

DBP, diastolic blood pressure; SBP, systolic blood pressure;HD,hemodialysis.

Compared with the calcification group, patients in the non-calcification group had significantly higher fibrinogen(4.2(3.7 ~ 5.3)vs. 3.6༈3.1 ~ 4.5༉; P=0.01), lower RBC (3.0 ± 0.8 vs. 3.4 ± 0.7; P < 0.01),higher ferritin (194.0༈85.2 ~ 288.0༉ vs. 44.9༈18.0 ~ 170.5༉; P < 0.01) higher red blood cells count (3.0 ± 0.7 vs. 3.5 ± 0.7; P < 0.01) and lower calcium (2.2༈2.0 ~ 2.3༉vs. 2.3༈2.2 ~ 2.4༉; P < 0.01). There were no significant differences in other parameters between the two groups(Table 2).

Table 2

Laboratory results of HD patients with and without calcification
Characteristics	non-calcification group(n = 30)	Calcification group(n = 41)	Z/t	P
HDL (mmol/L)	1.0(0.8 ~ 1.2)	1.0(0.8 ~ 1.2)	-1.03	0.31
LDL (mmol/L)	1.6(1.4 ~ 2.1)	1.6(1.1 ~ 1.9)	-0.93	0.36
TC (mmol/L)	3.7(3.3 ~ 4.3)	3.5(2.9 ~ 4.1)	-0.73	0.47
TG (mmol/L)	0.5(0.3 ~ 1.1)	0.9(0.4 ~ 1.3)	-1.63	0.10
APTT	27.4(26.1 ~ 29.1)	27.0(25.1 ~ 28.2)	-1.43	0.15
FDP	4.8(2.2 ~ 5.8)	3.4(2.1 ~ 6.0)	-1.00	0.32
Fibrinogen	4.2(3.7 ~ 5.3)	3.6(3.1 ~ 4.5)	-2.53	0.01
Antithrombin III activity	93.58 ± 11.32	89.09 ± 12.54	1.55	0.13
PTA	101.7(86.1 ~ 115.2)	104.1(92.1 ~ 117.8)	-0.49	0.63
PT (sec)	10.7(10.3 ~ 11.3)	10.5(9.9 ~ 11.3)	-0.75	0.45
PLT (×10⁹/L)	173.0 ± 61.3	162.7 ± 64.2	0.68	0.50
MPV (fl)	10.1(9.1 ~ 10.7)	10.1(9.8 ~ 10.7)	-0.77	0.44
PDW (fl)	16.0(12.0 ~ 39.7)	15.8(10.7 ~ 18.6)	-0.82	0.41
MONO (×10⁹/L)	0.4(0.3 ~ 0.6)	0.5(0.3 ~ 0.7)	-0.41	0.68
NEU (×10⁹/L)	4.6(3.4 ~ 5.7)	3.9(2.9 ~ 4.7)	-1.93	0.05
LYMPH (×10⁹/L)	0.8(0.6 ~ 1.2)	1.0(0.7 ~ 1.2)	-0.59	0.55
PLR	190.1(147.2 ~ 273.3)	162.5(133.5 ~ 217.0)	-1.29	0.20
MLR	0.5(0.4 ~ 0.7)	0.5(0.3 ~ 0.6)	-0.34	0.73
NLR	4.8(3.08.1)	3.8(2.7 ~ 5.6)	-1.37	0.17
WBC (×10⁹/L)	6.1(5.2 ~ 7.8)	5.5(4.5 ~ 6.4)	-1.76	0.08
RBC (×10⁹/L)	3.0 ± 0.8	3.4 ± 0.7	-2.17	0.03
RDW (fl)	51.0 ± 7.2	50.9 ± 6.4	0.09	0.93
HB(g/L)	83.0(67.3 ~ 101.3)	93.0(79.0 ~ 107.0)	-1.65	0.10
Fer (ng/ml)	194.0(85.2 ~ 288.0)	44.9(18.0 ~ 170.5)	-2.51	0.01
ALB (g/L)	37.3(35.2 ~ 41.7)	36.9(33.6 ~ 39.3)	-1.08	0.28
β2MG (mg/L)	32.8 ± 15.2	32.9 ± 13.7	-0.02	0.98
Glucose	4.8(4.1 ~ 6.3)	4.6(4.0 ~ 6.1)	-0.65	0.52
Ca (mmol/L)	2.2(2.0 ~ 2.3)	2.3(2.2 ~ 2.4)	-2.82	< 0.01
P(mmol/L)	1.9 ± 0.6	1.8 ± 0.6	0.67	0.51
K (mmol/L)	4.6 ± 1.0	4.4 ± 0.7	0.87	0.39
CRP	17.8(3.1 ~ 20.2)	17.8(8.7 ~ 20.2)	-0.02	0.99
PCT (ng/ml)	0.6(0.2 ~ 0.9)	0.7(0.3 ~ 1.0)	-0.11	0.91
D-dimer (ug/ml)	1.6(0.6 ~ 1.7)	0.9(0.5 ~ 1.6)	-1.30	0.19
EGFR	5.2(3.3 ~ 8.8)	5.6(4.1 ~ 7.8)	-0.29	0.77
Creatinine (umol/L)	822.5(530.5 ~ 1160.0)	808.7(599.5 ~ 973.0)	-0.06	0.95
Uric Acid (umol/L)	411.5(319.0,526.8)	388.0(327.1,478.0)	-1.18	0.24
Urea nitrogen (mmol/L)	21.0 ± 8.5	19.9 ± 6.3	0.66	0.51

HDL, high-density lipoprotein; LDL, low-density lipoprotein;TC, total cholesterol; TG, triglyceride;APTT,activated partial thromboplastin time;FDP,Fibrin degradation products;PTA,prothrombin activity;PT,prothrombin time;PLT, platelets; MPV, mean platelet volume; PDW, platelet distribution width; MONO,monocyte;NEU, neutrophil;LYMPH,lymphocyte;WBC, white blood cells; RBC, red blood cells;RDW,Red cell distribution width; HB, hemoglobin;Fer, ferritin; ALB, albumin; β2MG, β2 microglobulin;Ca, calcium; P, phosphorus;K,potassium;CRP,C-reactive protein;PCT, procalcitonin.

Logistic regression analysis

Logistic regression analysis was applied to show the influencing factors of calcification, and the results showed: Diabetes[1.82(1.37-1.00), P = 0.02] and RBC(red blood cells)[0.83 (1.05–5.02), P = 0.04]were influencing factors. Patients with diabetes and who had higher RBC had the greater risk of calcification. The details are shown in Table 3 and Fig. 1 below.

Table 3

Results of multivariate logistic regression analysis of calcification in HD patients
Characteristics	B	Wald	P	OR	95%CI
Characteristics	B	Wald	P	OR	Lower	Upper
Constant	1.63	0.56	0.45
Sex (Female)	-0.38	0.41	0.52	0.68	0.21	2.18
Age (year)	-0.01	0.16	0.69	0.99	0.95	1.04
Diabetes	1.82	5.59	0.02	6.20	1.37	28.11
TC (mmol/L)	0.00	0.00	1.00	1.00	0.44	2.28
Fibrinogen	-0.46	2.97	0.09	0.63	0.38	1.06
WBC (×10⁹/L)	-0.31	3.63	0.06	0.73	0.53	1.01
RBC (×10⁹/L)	0.83	4.29	0.04	2.29	1.05	5.02

TC, total cholesterol; WBC, white blood cells; RBC, red blood cells.

The ROC curve was applied to analyzing the red blood cell count; the area under the curve (AUC) was 0.66 (P=0.02) and clinical diagnostic cutoff value = 2.99 (sensitivity = 75.6% and specificity = 56.7%).In other words, a red blood cell count exceeding 2.99 preliminary indicates vascular calcification, with a diagnostic accuracy of 66% (Fig. 2).

To the best of our knowledge, there are few studies on the correlation between CAC and HD patients. Previous studies have believed that vascular calcification is a passive process of calcium salt deposition on the vascular wall caused by the imbalance of calcium and phosphorus metabolism (10–11). Recently, a large number of reports have shown that vascular calcification is an active and highly regulated complex pathological process similar to bone development (12). Vascular calcification is mainly characterized by increased stiffness and decreased compliance of the vascular wall, accompanied by hypertension, atherosclerosis, diabetes and chronic kidney disease, which is one of the important reasons for the high morbidity and mortality of cardiovascular and cerebrovascular diseases (13–14).

The risk factors of VC include traditional cardiovascular risk factors, including age, smoking, diabetes, dyslipidemia, and CKD related risk factors, such as blood calcium, blood phosphorus, hyperparathyroidism, dyslipidemia, hypertension.In our study,the incidence of CAC in HD patients was assessed by coronary CT scans in a well-defined cohort of HD patients, and the association between CAC occurrence and clinical measures in HD patients was analyzed (15). We found that CAC occurred in 41 (57.75%) of the 71 HD patients.Patients with diabetes and who had higher RBC had a higher risk of CAC.

Increasing evidences have revealed the relationship between diabetes and vascular calcification (16). It has been reported that the prevalence of VC in diabetic patients is higher than that in non-diabetic patients, which may be due to the presence of more risk factors in such a population. In a study of 101 non-dialysis stage 5 CKD patients, the undialyzed stage 5 CKD patients with diabetes have higher prevalence rate and more severity of vascular calcification than those without diabetes. Age and diabetes were independent risk factors for vascular calcification in non-dialysis stage 5 CKD patients,which is consistent with our findings (17). The pathogenesis of VC is a highly complex and cell-regulated process. Mechanistic studies of VC have focused on calcium and phosphorus homeostasis, release of calcifying vesicles, changes in extracellular matrix, loss of inhibition, and cell differentiation. Zhang et al (18) have revealed that impaired intracellular calcium homeostasis enhances protein O-GlcNAcylation and promotes vascular calcification and stiffness in diabetes. Moreover, Kay et al (19) have found the role of AGE/RAGE signaling in diabetes-mediated VC. Thus, diabetes can be used as an independent risk factor for VC in patients with CKD, which may provides clinical evidence for the influencing factors of CAC in HD patients.

Red blood cells are the main particulate components of circulating blood, which play an important role in physiological hemodynamics and participate in the lesions of arterial wall. Currently, there is rare study focused on the relationship between RBC and vascular calcification (20–21). Meanwhile, vascular calcification in patients with CKD is common, and EPO is widely applied for renal anemia in patients with CKD treatment. Previous researches also speculated that RBCs collide with the arterial wall and release heme-Fe + + with a high toxicity for arterial cells, including endothelial and smooth muscle cells (SMCs) cardiomyocytes, as well as neuron(20). Therefore, based on these findings, we concluded that there may be a vicious circle between calcifications and RBC/iron, which promotes exponential development of both valvular and vascular calcifications in association with CV risk factors, including aging, tobacco, and dyslipidemia. Report from Tziakas, D.N.et.al (22) showed that, isolated RBC membranes are able to promote vascular calcifications, independently of hemoglobin and depending on NO release by membranes. Membranes from RBCs deficient in NO synthase have limited procalcifying effects.

Our clinical data supported this point in HD patient. To our knowledge, this is the first study to investigate the association between RBC and calcification in HD patients. Our research indicated that the level of red blood cell count was positively correlated with the risk of CAC in HD patients.

In conclusion, we firstly found a correlation between serum red blood cell levels and vascular calcification in maintenance hemodialysis patients. Besides, diabetes plays a vital role in CAC in HD patients. Nevertheless,this study was a single-center study with a relatively small sample size, and future larger studies are needed to verify our findings.

This study provides clinical evidence for the influencing factors of CAC in HD patients. Diabetes and red blood cell count were independent risk factors for CAC.The underlying mechanisms deserve further exploration. Our results may provide a new theoretical basis for the treatment of CAC in the future.

Acknowledgement

None.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This research was supported by Natural Science Foundation of Hunan Province of China (Grant No.2021JJ40297) and 2024 Haidian Hospital Research Project-Youth Project（(Grant No.KYQ2024007)）for study design and execution.

Author Contributions

F.Z. and J.Y. wrote the draft of paper; GL.L. and Y.J.collected the data and follow-up,SS.F. and YY.Y. performed the data analysis;YM.L.,YY.C., and X.L. revised the paper.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author XL upon reasonable request.

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No competing interests reported.

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Elevated red blood cell count levels are associated with increased vascular calcification in hemodialysis patients

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Patients

Study design

Statistical analysis

Results

Baseline demographic and clinical characteristics of HD patients with and without calcification

Logistic regression analysis

Discussion

Conclusion

Declarations

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This research was supported by Natural Science Foundation of Hunan Province of China (Grant No.2021JJ40297) and 2024 Haidian Hospital Research Project-Youth Project（(Grant No.KYQ2024007)）for study design and execution.

References

Additional Declarations

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