Association of circulating proprotein convertase subtilisin/kexin type 9 concentration with coagulation abnormalities in patients with primary membranous nephropathy

Abstract

Objectives

The aims of the study were to explore the potential associations between plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) and coagulation indexes in patients with primary membranous nephropathy (PMN).

Methods

A total of 87 patients diagnosed with PMN were enrolled in our study. 30 healthy participants were recruited to match PMN participants. Plasma PCSK9 concentrations were tested by enzyme-linked immunosorbent assay (ELISA). Correlations between PCSK9 and coagulation abnormalities in patients with PMN were analyzed using univariate and multiple linear regression analysis.

Results

Plasma PCSK9 levels in patients with PMN were significantly higher than that in healthy controls [232.0 (143.5, 359.5) ng/mL vs. 166.8 (129.7, 199.7) ng/mL; p = 0.001]. Plasma levels of PCSK9 were positively correlated with factor VIII, factor IX, factor XI, log-transformed tissue factor, protein C and protein S (r = 0.267, p = 0.013; r = 0.496, p < 0.001; r = 0.217, p = 0.045; r = 0.584, p < 0.001; r = 0.372, p = 0.001; r = 0.282, p = 0.011). In multiple linear regression analysis, PCSK9 concentration was independently and positively correlated with factor VIII, factor IX, and tissue factor (β = 0.186, p = 0.047; β = 0.325, p = 0.001; β = 0.531, p < 0.001; respectively). PCSK9 concentration was independently and negatively correlated with PT (β= −0.343, p = 0.011).

Conclusion

Plasma PCSK9 levels had good positive correlations with procoagulant clotting factors and negative correlations with PT in PMN, which might provide novel information with regard to PCSK9 and hypercoagulability in PMN.

Keywords:

• Primary membranous nephropathy
• PCSK9
• coagulation abnormalities
• hypercoagulability

Introduction

Thromboembolism is one of the major life-threatening complications of nephrotic syndrome [1–3]. The incidence of venous thromboembolic events, including deep and renal vein thrombosis as well as pulmonary embolus, has been reported to range from 3 to 48%, depending upon the site of thrombosis and the intensity of diagnostic screening [4,5].

The pathophysiology of thrombogenesis in nephrotic syndrome seems to be multifactorial and incompletely understood. The area that has garnered the most attention is disease-related coagulation abnormalities. Plasma concentrations of procoagulant proteins, such as factor V and factor VIII, are markedly elevated in nephrotic syndrome, which is predicted to amplify the coagulation cascade [6–8]. Increased protein synthesis in the liver to counterbalance the massive urinary loss of serum proteins was supposed to be the important cause of elevated levels of hemostatic proteins in nephrotic syndrome [6,9].

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged over the past decade as a regulator of cholesterol homeostasis by enabling low-density lipoprotein (LDL) receptor degradation in lysosome and then increasing circulating LDL levels [10–12]. Increased plasma concentrations of PCSK9 have been reported in nephrotic patients and mouse models and were supposed to play important roles in the development of hyperlipidemia in nephrotic syndrome [13,14]. PCSK9 is mainly secreted by the liver, however, renal PCSK9 expression increases in nephrotic patients and mouse models, and kidney-derived PCSK9 contributes significantly to the increased plasma PCSK9 concentration in nephrotic syndrome [15].

Beyond the effect on cholesterol metabolism, several studies revealed the existence of additional roles of PCSK9 in lipid-independent reactions, of which the most studied one is the effect of PCSK9 on hemostasis and thrombosis. Wang et al. demonstrated that pcsk9 deficient mice had a reduced rate of venous thrombosis and lower length of the thrombus after the inferior vena cava was partially ligated [16]. Sepsis-induced hypercoagulable state was exacerbated in PCSK9 over-expressing mice [17]. In patients with angina-like chest pain, PCSK9 concentration was negatively correlated with plasma levels of prothrombin time (PT) and cardiovascular outcomes [18]. Chiara Ricci et al. demonstrated that human recombinant PCSK9 drives an inflammatory response in macrophages, which was supposed to play a central role in the development of atherosclerotic cardiovascular disease [19]. LDL receptor and LDL receptor-related protein-1 (LRP-1) downregulates blood clotting factor VIII by mediating its endocytosis and degradation [20], PCSK9 might induce an increase in factor VIII plasma levels in theory. Basiak M et al. demonstrated that PCSK9 inhibitors reduced plasma levels of factor VII in patients with isolated hypercholesterolemia [21]. In patients with coronary artery disease and type 2 diabetes mellitus, plasma levels of tissue factors (TF), an initiator of extrinsic coagulation pathway playing a central role in atherothrombosis [22], positively correlated with plasma PCSK9 [23]. Nevertheless, there are no studies concerning the correlation between the elevated circulating PCSK9 concentration and hypercoagulability in patients with kidney disease until now.

Primary membranous nephropathy (PMN) became the most common primary glomerular disease in China nowadays [24]. Compared with other primary nephrotic diseases, PMN is associated with the highest risk for developing thrombosis, even adjusted for gender, proteinuria, and serum albumin by multivariable analysis [25]. The aim of this present study was to explore the potential associations between PCSK9 and coagulation indexes including PT, activated partial thromboplastin time (APTT), and blood clotting factors in patients with PMN.

Materials and methods

Study design and population

The present study protocol complied with the Declaration of Helsinki and was approved by the Ethics Committee of Shandong Provincial Hospital affiliated with Shandong First Medical University. Every patient signed the informed written consent before enrolling in this study.

We consecutively recruited patients diagnosed with PMN who completed the determination of PCSK9 and coagulation factor tests from March 2021 to September 2022. The inclusion criteria for PMN were as follows: biopsy-diagnosed MN or patients with positive anti-PLA2R antibody test [26]. Major exclusion criteria included: (1) patients with autoimmune diseases, diabetes mellitus, hepatitis, liver cirrhosis or abnormal liver function, (2) tumor history, (3) drug-induced secondary MN, (4) patients at high risk of hypercoagulable state, such as the acute phase of infection, recent trauma, surgery, pregnancy and cancer, (5) patients receiving hormones, (6) lipid-lowering medications could increase circulating PCSK9 concentration, patients taking any lipid-lowering agents within 3 months prior to entering the study were excluded, (7) anti-coagulation drugs could prolong clotting time, patients taking any anti-coagulation agents within 1 month prior to entering the study were excluded. (8) glucocorticoid use might contribute to a hypercoagulable state [27], patients receiving immunosuppressive drugs prior to the study enrollment were excluded. During the study period, 117 primary MN patients were initially screened at our hospital, 30 were further excluded, of whom, 7 had hepatitis B infection, 2 had colorectal cancer, 4 used oral contraceptives, 9 patients had prophylactic anticoagulation treatment, and 8 patients received glucocorticoid therapy before enrollment. Thus, a total of 87 patients were recruited for this study.

Clinical evaluation

Clinical data were obtained for all patients at admission, including age, sex, blood pressure measurements, history of smoking, hemoglobin, platelet count, serum albumin (ALB), serum lipids profile, urinalysis, quantification of proteinuria, blood urea nitrogen, serum creatinine (Scr), estimated glomerular filtration rate (eGFR), anti-PLA2R antibody, and pathological results. eGFR was calculated using an Scr-based equation adjusted for coefficients for age and gender by modified abbreviated MDRD equation based on data from Chinese CKD patients: eGFR (ml/min per 1.73 m²) =186 × [Scr (mg/dl)]^−1.154×age^−0.203× (0.742 if female).

Conventional coagulation tests

Coagulation tests including PT, APTT, and factor assays were performed on an automated coagulation analyzer (ACL TOP 700, Beckman Coulter, USA). PT, APTT, and fibrinogen were measured with the clotting method by using HemosIL RecombiPlasTin, SynthASil, and HemosIL Fibrinogen-C XL reagents, respectively (Instrumentation Laboratory SpA). Coagulation factors were assayed by an APTT-based clotting assay using SynthASil reagent for factor VIII, factor IX, and factor XI. Antithrombin III (AT III) and protein C levels were determined by chromogenic assays (HemosIL liquid antithrombin and HemosIL Protein C, respectively; Instrumentation Laboratory SpA), and protein S activity was measured by clotting assay (HemosIL Free Protein S, Instrumentation Laboratory SpA).

Blood collection and measurement of plasma PCSK9 and TF

Fasting blood samples were obtained for all patients at admission and collected into EDTA-containing tubes. Plasma samples were prepared by centrifugation at 3500 × g twice for 10 min each and were stored in aliquots at −80 °C until analysis.

Plasma PCSK9 levels were determined using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). An anti-human PCSK9 monoclonal antibody (Sino Biological, Beijing, China) was used to coat the ELISA plate. PCSK9 in standard (Sino Biological, Beijing, China) and test samples became bound to the monoclonal antibody. Unbound reagents were washed away, and a horseradish peroxidase-conjugated mouse anti-Human PCSK9 monoclonal antibody (Sino Biological, Beijing, China) was added, forming an immune complex adherent to the plate. If PCSK9 was present in the reaction well, the chromogenic substrate exhibited a blue colour and then became yellow after adding a stop solution. The optical absorbance was measured at 450 nm. Plasma TF levels were measured in PMN patients and control individuals by using a commercially available ELISA kit (Elabscience, Wuhan, China).

Statistical analysis

SPSS 23.0 software was used for data analysis. Clinical and laboratory data were expressed as mean ± standard deviation (SD) for normally distributed continuous data or median with quartile range for skewed distributed continuous data. The normal distribution of the variables was tested by the Shapiro–Wilk test. Categorical variables were expressed as frequencies and percentages. Comparisons between categorical data were performed with Chi-Squared tests. Continuous variables were compared by Student’s t tests assuming equality of variances or not depending on the results of the Levene’s test, or non-parametric Mann–Whitney test. To evaluate the association between log-transformed PCSK9 and other parameters, Pearson correlation analysis was used. The linear regression model was applied to assess the factors influencing coagulation factors in PMN patients. Only variables with p < 0.05 in the univariate linear regression analysis were used in the multiple linear regression. Stepwise multivariable linear regression analysis was performed to determine the relationship between PCSK9 and PT. A 2-tailed p < 0.05 was considered statistically significant.

Results

General characteristics of the study participants at diagnosis

In the present study, we consecutively enrolled 87 patients with PMN. A total of 30 healthy participants were recruited to match PMN participants according to age and sex. The general characteristics of all patients and healthy controls are shown in Table 1. Of the 87 PMN patients, 39 (44.8%) were females and 48 (55.2%) were males. The mean age of the patients was 49.2 ± 13.4 years. 65.5% (57/87) of the patients had nephrotic-level proteinuria. Plasma levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) were significantly higher and ALB levels were significantly lower in the PMN patients compared with those in the healthy controls (p < 0.05). There were no significant differences in age or sex between the two groups.

Table 1. General characteristics of the study participants at diagnosis.

Characteristics	Healthy controls	Patients with PMN	p Value
Number	30	87	–
Age (mean ± SD) (years)	45.2 ± 12.8	49.2 ± 13.4	0.153
Sex, Male (%)	15 (50.0%)	48 (55.2%)	0.624
Albumin (mean ± SD) (g/L)	45.9 ± 2.2	27.0 ± 5.5	<0.001
Urinary protein (mean ± SD) (g/24h)	–	5.4 ± 4.4	–
Nephrotic proteinuria, n (%)	–	57 (65.5%)	–
Scr (mean ± SD) (μmol/L)	68.9 ± 12.7	65.5 ± 35.1	0.604
eGFR (mean ± SD) (ml/min/1.73 m^2)	107.7 ± 21.2	118.8 ± 31.5	0.076
PLA2R Abs (median, quartile) (RU/mL)	–	24.42 (2.47,83.98)	–
TC (mean ± SD) (mmol/L)	4.4 ± 0.8	7.2 ± 2.3	<0.001
TG (mean ± SD) (mmol/L)	0.9 ± 0.2	2.3 ± 1.5	<0.001
LDL-C (mean ± SD) (mmol/L)	2.5 ± 0.6	4.6 ± 1.7	<0.001
Leukocytes (mean ± SD) (×10^9/L)	2.0 ± 0.5	6.4 ± 2.3	0.701
Neutrophils (mean ± SD) (×10^9/L)	3.2 ± 0.8	3.7 ± 1.7	0.130
Platelets (mean ± SD) (×10^9/L)	218.0 ± 52.1	257.7 ± 59.4	0.002

PMN: primary membranous nephropathy; SD: standard deviation; Scr: serum creatinine; eGFR: estimated glomerular filtration rate; PLA2R: phospholipase A2 receptor; Abs: antibodies; TC: total cholesterol; TG: Triglycerides; LDL-C: low density lipoprotein cholesterol.

Coagulation abnormalities in patients with PMN

As shown in Figure 1(a), in this PMN cohort, the average fibrinogen levels in patients with PMN were significantly higher than that in healthy controls (3.9 ± 0.9 g/L vs. 3.1 ± 0.4 g/L; p < 0.001). Elevated factor VIII activity (higher than the upper normal limit of 150%) was found in 79.3% (69/87) of the patients (Figure 1(b)). Factor IX activity was found to be increased with values exceeding 150% in 35 (40.2%) patients, the median level of factor IX was 141.8%, which was close to the upper normal limit (Figure 1(c)). The median factor XI activity was 113.6% with 10 out of 87 patients (11.5%) exhibiting values above 150% (Figure 1(d)). Except for one patient exhibiting decreased factor XI activity, none of the tested patients had subnormal plasma factor VIII, IX, and XI activity. Besides these intrinsic coagulation factors, plasma levels of TF, the initiator of the extrinsic coagulation pathway, were also detected. Plasma TF levels in patients with PMN were significantly higher than that in healthy controls [20.2 (9.5, 44.6) vs.; 4.0 (2.4, 6.8); p < 0.001] (Figure 1(e)).

Figure 1. Scatter plot shows the distributions of coaugulation-related parameters. (a) Fibrinogen levels in PMN and health controls. (b–d) Plasma factor VIII, factor IX and factor XI activity in PMN. (e) Tissue factor levels in PMN and health controls. (f–h) Activity of protein C, protein S and AT III in PMN. (i,j) PT and APTT levels in PMN and health controls. PMN, Primary membranous nephropathy; AT III, antithrombin-III; PT, Prothrombin time; APTT, Activated partial thromboplastin time.

For natural inhibitors of coagulation, protein C, protein S, and AT III were determined in this study (Figure 1(f–h)). The median level of protein C was 130.0% with values exceeding 140% in 26 (29.9%) patients. The median level of protein S was 122.1% with values exceeding 149.0% in 17 (19.5%) patients. None of the patients had protein C and protein S deficiency in this cohort. The median level of AT III was 89.5%, which was close to the lower limit of normal, with 29.9% of patients (26/87) exhibiting values below 83% and 2 (2.3%) patients above 128%.

Although most of the patients in this cohort had PT levels in the normal range, the average PT levels in patients with PMN were significantly lower than that in healthy controls [10.4 ± 0.8 s vs. 10.7 ± 0.5 s; p = 0.005] (Figure 1(i)). APTT levels were similar between PMN patients and healthy controls (Figure 1(j)).

Since patients with NS were more likely to have thromboembolic events, coagulation factor levels in patients with and without NS were then compared. PMN patients with NS had significantly higher levels of fibrinogen, factor VIII, factor IX, factor XI, protein S, and lower levels of PT than patients without NS. However, elevated levels of fibrinogen, factor VIII, factor IX, and tissue factor could still be found in PMN patients without NS (Supplementary Figure 1).

Plasma PCSK9 levels and associations with coagulation-related parameters in patients with PMN

ELISA results showed that plasma PCSK9 levels in patients with PMN were significantly higher than that in healthy controls [232.0 (143.5, 359.5) ng/mL vs. 166.8 (129.7, 199.7) ng/mL; p = 0.001] (Figure 2). Plasma PCSK9 levels were also detected in 7 patients with focal and segmental glomerulosclerosis (FSGS), 10 patients with IgA nephropathy (IgAN), and 6 patients with minimal change disease (MCD). There was no significant difference in the levels of PCSK9 between patients with PMN and other pathological types (Supplementary Figure 2).

Figure 2. Scatter plot shows the levels of PCSK9 in PMN patients and health controls. PCSK9 was highly skewed, so natural log transformation was used for the analysis. PMN, Primary membranous nephropathy; PCSK9, proprotein convertase subtilisin/Kexin type 9.

Correlation analysis was performed to clarify the association between log-transformed PCSK9 levels with parameters in all patients (Figure 3 and Table S2). In our cohort, circulating PCSK9 concentration was not significantly associated with age, sex, ALB, TC, TG, LDL-C, Scr, anti-PLA2R antibody, or 24h proteinuria levels (r= −0.013, p = 0.907; r= −0.095, p = 0.379; r= −0.029, p = 0.789; r = 0.170, p = 0.115; r = 0.104, p = 0.339; r = 0.138, p = 0.203; r = 0.173, p = 0.109; r = 0.095, p = 0.401 r = 0.168, p = 0.123). Interestingly, good correlations were found between PCSK9 and coagulation-related parameters. As shown in Figure 3, plasma levels of PCSK9 were positively correlated with factor VIII, factor IX, factor XI, log-transformed tissue factor, protein C and protein S (r = 0.267, p = 0.013; r = 0.496, p < 0.001; r = 0.217, p = 0.045; r = 0.584, p < 0.001; r = 0.372, p = 0.001; r = 0.282, p = 0.011). No significant correlations were observed with D-dimer, fibrinogen, or AT III. More importantly, we observed significant negative correlations of PT and APTT with plasma levels of PCSK9 in the present study (r= −0.420, p < 0.001; r= −0.269, p = 0.012, respectively).

Figure 3. Association of PCSK9 and coagulation-related indicators in PMN patients. PMN, Primary membranous nephropathy; PCSK9, proprotein convertase subtilisin/Kexin type 9; AT III, antithrombin-III.

Increased protein synthesis in the liver to counterbalance the massive urinary loss of serum proteins was supposed to be the important cause of elevated levels of hemostatic proteins in nephrotic syndrome. In order to eliminate the impact of potential associations between circulating PCSK9 concentration and coagulation indexes with other confounders, univariate and multiple linear regression analyses were further conducted (Table 2). In univariate analysis, factor VIII, factor IX and factor XI were indeed positively correlated with 24h proteinuria and/or negatively correlated with serum ALB levels factor, while TF showed marginal correlations with ALB and proteinuria. In the further multivariate analysis, LnPCSK9 was independently associated with plasma levels of factor VIII, factor IX, and TF.

Table 2. Independent determinants of clotting factors to log-transformed PCSK9.

Parameters	Factor VIII				Factor IX				Factor XI				Log-transformed TF
	Univariate analysis		Multivariate analysis		Univariate analysis		Multivariate analysis		Univariate analysis		Multivariate analysis		Univariate analysis		Multivariate analysis
	Beta	p Value	Beta	p Value	Beta	p Value	Beta	p Value	Beta	p Value	Beta	p Value	Beta	p Value	Beta	p Value
Age	0.206	0.057			−0.070	0.521			−0.039	0.725			0.066	0.556
Sex	0.054	0.622			−0.082	0.452			−0.019	0.861			0.013	0.906
Albumin	−0.502	<0.001	−0.439	<0.001	−0.310	0.004			−0.217	0.045			−0.206	0.065
LDL-C	0.308	0.004			0.305	0.004			0.328	0.002			0.167	0.136
Scr	0.387	<0.001	0.284	0.003	0.377	<0.001	0.265	0.004	0.141	0.195			0.261	0.019
Proteinuria	0.419	<0.001			0.416	<0.001	0.254	0.007	0.314	0.003	0.202	0.046	0.198	0.079
Protein C	0.257	0.021			0.412	<0.001	0.212	0.028	0.472	<0.001	0.338	0.001	0.279	0.015
Protein S	0.189	0.090			0.379	<0.001			0.385	<0.001	0.241	0.019	0.266	0.021
AT III	−0.149	0.177			0.042	0.706			0.093	0.400			−0.059	0.608
LnPCSK9	0.267	0.013	0.186	0.047	0.496	<0.001	0.325	0.001	0.217	0.045			0.584	<0.001	0.531	<0.001

LDL-C: low density lipoprotein cholesterol; Scr: serum creatinine; TF: tissue factor; AT-III: antithrombin-III.

In order to eliminate the impact of potential association with circulating PCSK9 concentration and PT with age, sex, ALB, LDL-C, Scr, proteinuria, a stepwise multivariate linear regression analysis was used to determine the relationship between PCSK9 levels and PT after adjusting for these confounders (Table 3). The results indicated that a negative association between PCSK9 levels and PT remained significant after adjustment for age, gender, ALB, LDL-C, Scr, proteinuria (β = −0.392, p = 0.001, Table 3). Even if adding protein C, protein S, log-transformed TF, and AT III to the multivariate linear regression model, the negative correlation of PCSK9 levels and PT remained statistically significant (β = −0.343, p = 0.011). However, the significant difference between PCSK9 and APTT disappeared in the multivariable regression stepwise model (p = 0.160, Table S2).

Table 3. Independent determinants of PT to log-transformed PCSK9.

	PT
	Beta	p Value
Model 1^a	−0.426	<0.001
Model 2^b	−0.392	0.001
Model 3^c	−0.343	0.011

The multivariable regression stepwise models are shown. The log-transformed PCSK9 is the dependent variable.

^aUnivariable regression analysis of log-transformed PCSK9.

^bAdjusted for age and sex, albumin, LDL- C, Scr and proteinuria.

^cAdjusted for age and sex, albumin, LDL-C, Scr, proteinuria, protein C, protein S, log-transformed TF and AT III.

PT: Prothrombin time; LDL-C: low-density lipoprotein cholesterol; Scr: serum creatinine; TF: tissue factor; AT III: antithrombin-III.

Among the 87 patients, 5 patients (5.7%) had venous thrombosis, including 4 patients with deep vein thrombosis and 1 patient with both renal vein thrombosis and pulmonary embolus. As shown in Table S3, patients with venous thrombosis had significantly higher levels of D-dimer, insignificantly higher levels of fibrinogen, TF, and factor VIII, and insignificantly lower levels of protein C and AT III compared with patients without venous thrombosis. Plasma PCSK9 levels were comparable between patients with and without venous thrombosis in this cohort.

Discussion

In the present study, we were the first to evaluate the association of circulating PCSK9 concentration with coagulation indexes in patients with PMN. The major findings of our study were that plasma PCSK9 levels had good positive correlations with procoagulant clotting factors and negative correlations with PT in PMN, even adjusted for proteinuria, ALB, and other confounders. Our results provide novel information with regard to PCSK9 and hypercoagulability in PMN.

Coagulation abnormalities have been extensively studied in nephrotic patients. Although there were conflicting results, the most frequent alterations were: increased plasma levels of procoagulant proteins factor I, V, VII, VIII, X, and decreased levels of anticoagulant protein AT III [6,28,29]. Protein C and protein S have been reported to be decreased, preserved or upregulated [6,28,29]. In our cohort, markedly elevated plasma levels of fibrinogen, factor VIII, factor IX, and tissue factor have been observed in patients with PMN. Among the anticoagulant proteins tested in this study, AT III showed a tendency to decrease, while plasma concentrations of protein C and protein S seemed to be elevated in our PMN patients. Urinary loss of hemostatic proteins of similar sizes with albumin and increased hepatic compensatory synthesis of procoagulant proteins with high molecular weights were the two major explanations for hemostasis disorders in nephrotic syndrome at present. However, these couldn’t explain the fact that factor IX (56 KD) and protein C (62 KD) elevated markedly while plasma concentrations of factor XI (160 kD) seemed not to be that high in previous reports and our study. There might be other mechanisms for clotting abnormalities.

The published results of PT and APTT in nephrotic syndrome were also conflicting. PT and APTT decreased markedly in puromycin aminonucleoside-induced nephrotic rats [30]. Huang et al. showed insignificantly decreased PT and significantly decreased APTT in patients with MN [31]. Chioma L. Odimegwu et al. demonstrated lower PT and higher APTT in nephrotic children [32]. In our cohort, APTT levels of PMN patients were comparable with that of healthy controls, while PT levels in patients with PMN were significantly lower than that in healthy controls, despite most of the patients in this cohort had PT levels in the normal range. Our results supported the suggestion that there was a net shift in the hemostatic balance toward a prothrombotic milieu in nephrotic patients [9,33].

PCSK9 has emerged as an important modulator of circulating cholesterol levels and a therapeutic target. Increased plasma concentrations of PCSK9 have been reported in nephrotic patients and mouse models, and knockout of Pcsk9 ameliorates dyslipidemia in nephrotic mouse models indicating the contribution of PCSK9 to the dyslipidemia of nephrotic syndrome [34]. In human studies, some reported that plasma PCSK9 was correlated with either TG or TC in patients with nephrotic syndrome [35,36], and some reported the change in PCSK9 from the acute phase to the remission phase was correlated with the change in TC in the nephrotic patients [34]. As mentioned in the introduction section, beyond the effect on cholesterol metabolism, the role of PCSK9 in hemostasis and thrombosis become a research hotspot in recent years. Correlations between PCSK9 and coagulation indexes, including factor VIII, factor VII, TF, and PT, have been observed in cardiovascular diseases. In our cohort, PCSK9 didn’t show significant correlations with lipid profile levels, probably due to the small sample size and that only 65.5% of the patients enrolled in this study had nephrotic-level proteinuria. Surprisingly, plasma PCSK9 showed strong correlations with factor VIII, factor IX, TF, and PT, even in multiple linear regression analyses in PMN patients. Decreased ALB levels or increased proteinuria levels were indeed independently correlated with elevated coagulation factors either. Our results indicated that besides increased hepatic synthesis and urinary loss, elevated PCSK9 might be another factor responsible for the elevated procoagulant proteins. Molina-Jijon et al. found renal PCSK9 expression was upregulated in the collecting duct of nephrotic patients and animals, suggesting that the kidney might be a major source of plasma PCSK9 in nephrotic syndrome [15]. Whether kidney-derived PCSK9 is one possible explanation for kidney disease-related hypercoagulation deserves to be further investigated.

There were some limitations in this study. First, the possible causal association of PCSK9 and coagulation indexes was not been confirmed because this was an observational study. PCSK9 knock-out animals might be used to demonstrate its role in hypercoagulation in glomerulonephritis in the future. The effects of PCSK9 inhibitors on hypercoagulation in glomerulonephritis are also worth expecting. Second, only 5 thromboembolism events were found in this study. Probably due to the small sample size, patients with venous thrombosis didn’t show significantly higher levels of coagulant factors and lower levels of anticoagulant factors. The current study only observed the association between PCSK9 and hypercoagulability, but couldn’t demonstrate the relationship between PCSK9 and hypercoagulability with thrombosis. Third, PCSK9 levels were detected in a few patients with pathological types other than PMN in the current study, the association of PCSK9 with coagulation abnormalities in patients with other pathological types should be determined in larger cohorts.

Conclusions

The present study for the first time showed positive correlations of PCSK9 with clotting factors and a negative relation of PCSK9 with PT in PMN, suggesting that PCSK9 may induce and/or promote hypercoagulability in PMN. This novel finding must be confirmed in larger cohorts, and its clinical implications are worthy of further investigation.

Ethics approval and consent to participate

This study was approved by the ethics committee of Shandong Provincial Hospital affiliated to Shandong First Medical University (the approval number is SZRJJ:NO.2022-111).

Author contributions

Huizi Zhu drafted the manuscript, did the ELISA work, and collected the clinical data, and analysis. Qian Meng drafted the manuscript, collected the clinical data, and analysis. Xiang Liu participated in collecting the related clinical and pathological data. Chunjuan Zhai did the statistical analysis. Jing Sun and Rong Wang did the whole revision process. Liang Xu acted as the co-corresponding author and did the study design and manuscript revision. Xiaowei Yang acted as the corresponding author, and did the whole study design and the manuscript revision.

Acknowledgements

The authors thank the nurses of the Department of Nephrology of Shandong Provincial Hospital Affiliated with Shandong First Medical University for their support in collecting data from this work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets used during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

This work was supported by grants of Taishan Scholars Program of Shandong Province [No. ts201712090], Academic promotion programme of Shandong First Medical University [No. 2019QL022], and China international medical foundation [No. Z-2017-24-2037].