Relationship Between Parathyroid Hormone Levels and Abdominal Aortic Calcication in Incident Hemodialysis Patients Not Treated with Calcium or Vitamin D

Vascular calcication (VC) and secondary hyperparathyroidism (SHPT) are important causes of high incidence of cardiovascular events in chronic kidney disease (CKD) patients. The relationship between parathyroid hormone (PTH) and VC is very complex. Different studies have inconsistent reports on the effect of PTH on VC. The present study investigated the correlation between PTH levels and abdominal aortic calcication (AAC) in incident hemodialysis patients who did not receive calcium, calcium-containing phosphorus binders, calcitriol or vitamin D analogs. Our data conrm that serum PTH levels is signicantly negatively correlated with AAC within a certain concentration range in incident hemodialysis patients who not treated with calcium or vitamin D.


Introduction
The incidence and mortality of cardiovascular disease (CVD) signi cantly increased in chronic kidney disease (CKD) patients. Even after strati cation by age, gender, race, and presence of diabetes, CVD mortality in end-stage renal disease (ESRD) patients treated by hemodialysis or peritoneal dialysis is 10 to 20 times higher than in the general population 1 .
Vascular calci cation (VC), de ned as the inappropriate and pathological deposition of mineral in the form of calcium phosphate salts into the vascular tissues, is a very common complication in CKD patients and associated with signi cantly increased all-cause and cardiovascular mortality [2][3][4][5] .
Secondary hyperparathyroidism (SHPT) is another common complication of CKD and has also been associated with increased cardiovascular mortality and CKD progression, especially in CKD stage 3-5 patients 6-8 .
The relationship between VC and parathyroid hormone (PTH) is very complex. A study of 1095 hemodialysis patients (aged 65-88) showed that abdominal aortic calci cation (AAC) was more severe in male patients with serum PTH levels within the upper normal range than patients with serum PTH levels within the lower normal range 9 . Another study revealed that in non-dialysis CKD stage 2-5 patients with AAC score > 6 or pelvic arterial calcifcation (PAC) score > 1 had higher serum PTH 10 . In addition, among patients receiving hemodialysis, serum PTH levels were signi cantly associated with AAC progression 11 . However, in clinical practice, it is very common that PTH levels do not match the severity of AAC. The present study examined the relationship between PTH levels and AAC in incident hemodialysis patients who did not receive calcium, calcium-containing phosphorus binders, calcitriol or vitamin D analogs in order to determine whether PTH levels are associated with the severity of AAC.

Results
Clinical characteristics and biochemistry of all patients are shown in Table 1 autosomal dominant poly-cystic kidney disease in 1. Shapiro-Wilk test showed that iPTH, AAC, diastolic blood pressure (DBP), C-reactive protein (CRP), albumin, triglycerides are skewed distribution, and age, serum calcium, serum phosphorus, systolic pressure (SBP), hemoglobin (Hb), serum total protein (TP),alkaline phosphatase (ALP), serum total cholesterol (TC), high-density lipoprotein (HDL) and lowdensity lipoprotein (LDL) are normal distribution. Because AAC is a skewd distribution which Pearson's correlation or multiple linear regression analysis is not suitable, so Spearman's rank correlation analysis was employed to analyze the relationship between AAC and other variables. Our results suggested that there was a strong negative correlations between serum PTH and AAC (Spearman's rho -0.618, P <0.001). There was a strong positive correlations between age and AAC (Spearman's rho 0.605, P <0.001). We also found that there was a moderate negative correlation between Pi and AAC (Spearman's rho -0.435 P=0.016) ( Table 2). To further analyze why there is a negative correlation between serum phosphorus and AAC, the patients were divided into two groups based on the cut-off point of 50 years old. The AAC and serum PTH of the two groups were skewed distribution, the calcium and phosphorus was normal distribution, so we used Mann-Whitney U test and T-test to compare between groups respectively.

Discussion
The present study investigated the correlation between PTH levels and AAC in incident hemodialysis patients who did not receive calcium, calcium-containing phosphorus binders, calcitriol or vitamin D analogs. Our results suggested that lower serum PTH levels were associated with higher AAC scores in this population.
Although the prevalence of VC in CKD patients is higher, the incidence of arterial calci cation in different sites is not consistent, and the risk factors of arterial calci cation in different locations and their in uence on cardiovascular events are also different [12][13][14] .
AAC is an independent risk factor for all-cause mortality or CVD events in the non-CKD patients, peritoneal dialysis patients and hemodialysis patients [15][16][17] . The Kidney Disease Improving Global Outcomes (KDIGO) guidelines also suggest that a lateral abdominal radiograph can be used to detect the presence or absence of vascular calci cation in patients with CKD stage 3-5 to guide the management of chronic kidney disease-mineral and bone disorder (CKD-MBD) 18 .
The researches have demonstrated that PTH receptors exist in myocardial cells, vascular smooth muscle cells and endothelial cells, indicating that inappropriate (excessive or insu cient) secretion of PTH may have adverse effects on the cardiovascular system 19,20 . It has been found that PTH perfusion can lead to intense aortic medial calci cation in rats with parathyroidectomy and this effect has nothing to do with uremia or serum phosphorus levels 21 . Another study also found that cinacalcet could inhibit the calci cation of aorta and heart in 5/6 nephrectomized rats by by decreasing serum PTH levels 22 . These results suggest that PTH has a direct pro-calci cation effect, at least in the animal model of CKD.
As for the exact effects that PTH has on AAC in CKD patients, this is still a matter of debate. Studies have shown that serum PTH level is related to the severity of AAC 9, 10, 11 . However, some studies have also found that there is no association between the two or even negative correlation 12-14, 23, 24 . One possible explanation for these contradictory conclusions is that the widespread use of calcium, calciumcontaining phosphorus binder, calcitriol or vitamin D analogs affects the natural process of AAC.  27,28 . Therefore, it is di cult to draw reliable conclusions when discussing the association between PTH and AAC in the CKD population using calcium and vitamin D.
In view of this, in this study, we purposely selected incident hemodialysis patients who have not used calcium, calcium-containing phosphorus binders, calcitriol or vitamin D analogs as the research objects. Interestingly, even after eliminating the interference factors such as calcium and vitamin D, PTH and AAC are still signi cantly negatively correlated. For the negative correlation, one explanation is that lower serum PTH levels may lead to adynamic bone disease, and adynamic bone disease will impair the ability of patients handling and buffering of calcium loads and pose a higher risk of extraosseous calci cations 29,30 . It should be emphasized that although lower serum PTH levels do contribute to the risk of adynamic bone disease, there is currently no evidence that low PTH alone can represent adynamic bone disease. Another possible explanation for the negative correlation between AAC and PTH is that low serum PTH levels may only be a manifestation of malnutrition, in ammation or cachexia syndrome (MICs) and malnutrition-in ammation is associated with vascular calci cation in uremic patients 31 . However, in another study that included 97 hemodialysis patients who were followed up for one year, patients with malnutrition and chronic in ammation (de ned as serum albumin <40 g/L and hs-CRP≥28.57 nmol/L) had signi cantly higher PTH levels than the control group (241.5 pg/ml vs 161.8 pg/ml) 11 . Therefore, neither adynamic bone disease nor malnutrition can fully explain why low PTH levels can aggravate AAC. Further studies are needed to elucidate the mechanism of AAC deterioration caused by low PTH levels.
Another surprising nding of the present study is that there is also a signi cant negative correlation between AAC and serum phosphorus. Serum phosphorus plays a very important role in the occurrence and development of vascular calci cation 32 . Subgroup analysis of the MESA study indicated that each 1-mg/dl increment in serum phosphate concentration was associated with a 21%, 33%, 25% and 62% greater prevalence of coronary artery, thoracic, aortic valve, and mitral valve calci cation, respectively 33 . However, some researchers found that there was no signi cant difference in serum phosphorus levels between the AAC score >6 group and AAC score≤6 group in hemodialysis patients 10 ; Study on chinese hemodialysis population (CDCS study) also found that serum phosphorus is a risk factor for coronary artery calci cation, but not a risk factor for AAC 13 . It should be pointed out that there are de ciencies in the above studies, that is, all participants received hemodialysis or peritoneal dialysis which can effectively remove serum phosphorus and a large proportion of the participants took phosphate binders (64.6% in CDCS study). Therefore, it can not concluded that there is no correlation between serum phosphorus and AAC. In the present study, serum phosphorus was not affected by dialysis, and the patient did not use any form of phosphorus bonding agent, but there is still a signi cant negative correlation between AAC and serum phosphorus. This nding is consistent with that of Harin Rhee et al 31 , who also found that the prevalence of baseline AAC and its progression in low serum phosphorus group was signi cantly higher than that in high serum phosphorus group.
Further analysis of the patients' characteristics showed that the serum phosphate of patients under 50 years old was signi cantly higher than that of patients over 50 years old, but the AAC score was signi cantly lower than that in patients over 50 years old. We speculate that age may have a greater impact on AAC than serum phosphorus in CKD patients. Indeed, a study of young patients with ESRD who were undergoing dialysis also con rmed that there was no signi cant difference in serum phosphorus between patients with or without coronary artery calci cation 28 . Another study involving 174 Chinese patients also found that age may be the most important factor affecting coronary artery calci cation in maintenance hemodialysis patients 34 .
In our study, most of the patients were relatively young with an average age of 55.7 years, so the effect of phosphorus on AAC may not be obvious. Therefore, serum phosphorus may be statistically negatively correlated with AAC, but it does not mean that serum phosphorus has no effect on AAC from a pathophysiological perspective. Our results once again show that the mechanism of VC is very complex, and even serum phosphorus, a recognized pro-calci cation factor, has different effects on speci c arteries in speci c CKD populations.
There are several limitations to our study. First, the sample size for our study was small. More CKD stage 5D patients who did not receive calcium, calcium-containing phosphorus binder, calcitriol or vitamin D analogs would be necessary to attain adequate power in determining the correlation between serum PTH levels and AAC. Unfortunately, this was beyond our capacity. Second, We only evaluated the degree of AAC by abdominal radiographs, which is less sensitive and accurate than electron beam computed tomography (EBCT), multislice CT (MSCT). However, due to the relatively high cost and the risk of exposure to higher radiation doses, these tests cannot be performed routinely. Third, the serum PTH levels of our observation population was in the range of 31.3-594.5pg/ml. The correlation between PTH and AAC is not clear in CKD patients with serum PTH levels exceeding 600 pg/ml or higher.
In conclusion, the present study, which is the only study focused on the association between PTH levels and AAC in incident hemodialysis patients who did not receive calcium, calcium-containing phosphorus binder, calcitriol or vitamin D analogs, demonstrates that PTH levels is signi cantly negatively correlated with AAC within a certain concentration range. Inappropriate inhibition of PTH may lead to deterioration of AAC in CKD stage 5D patients.

Patients
This study was approved by the Ethical Committee of the Chonggang general hospital. Written informed consent was obtained from each person at recruitment. We con rm that all experiments of the study were performed in accordance with relevant guidelines and regulations. Incident hemodialysis patients who have not used calcium, calcium-containing phosphorus binders, calcitriol or vitamin D analogsage between January 2020 and june 2021 were initially screened for enrollment in the cross-sectional study.
Of the total 47 incident hemodialysis patients, patients with malignancy, decompensated liver Cirrhosi, lupus nephritis, crescentic glomerulonephritis and acute kidney injury (AKI), as well as the elderly over 75 years old were excluded. Finally, 30 patients were enrolled.

Biochemical data and vascular calcifcation
Pre-dialysis blood samples and plain radiographs were obtained at the time of enrollment. Serum intact parathormone hormone (iPTH) levels were determined by Chongqing DIAN medical laboratory ( reference range 15-65 pg/ml). AAC was determined by a lateral lumbar spine radiograph, and quantitative analysis of AAC was performed using the method previously described by kauppila et al. (total score 0-24) 35 . All Xrays were reviewed by two physicians who had the expertise to score the X-rays.

Statistical analysis
Data are presented as mean ± standard deviation (SD). The normality of the distribution was determined using the Shapiro-Wilk test. The correlation between two continuous variables was analyzed by the Pearson's correlation (normal distribution) or Spearman's correction (Skew distribution). Two continuous variables were compared using the Student's t test (normal distribution) or non-parametric test (Skew distribution). P value <0.05 is considered statistically signifcant. All computations were performed using the SPSS 20.0 software (Chicago, IL, USA).