Markedly Increased Small Dense Low-Density Lipoprotein During Acute Phase in Childhood and Adolescent Nephrotic Syndrome

Background: Hyperlipidemia is an important characteristic feature of idiopathic nephrotic syndrome (NS) in children. This study was conducted to examine the lipid proles, including small dense low-density lipoprotein (sdLDL-C), in childhood-onset NS. Methods: This retrospective study enrolled patients diagnosed with initial-onset NS in childhood and adolescence. Study parameters included lipid proles. The “alternative LDL window” comprises the number and sizes of LDL particles estimated according to non-HDL-C and TG levels. Results: A total of 39 patients were enrolled who exhibited markedly increased lipid abnormalities, including TC, TG, LDL-C, and non-HDL-C levels (TC, 409.7 TC, TG, and sizes of LDL particles estimated as non-HDL-C, 332.3). Of the 39 patients, 32 (82%) were categorized in the area of hyper-TG/-non-HDL levels, which is considered as sdLDL. A positive correlation was found between non-HDL-C and TC (r = 0.96, P < 0.001), TG (r = 0.38, P = 0.018), LDL-C (r = 0.84, P < 0.001), TC/HDL (r = 0.53, P < 0.001), and atherogenic index of plasma (r = 0.42, P = 0.008). Conclusions: Our study demonstrated markedly increased lipid proles during the acute phase of NS. Evaluation state level was calculated by subtracting the HDL-C level from the total-C level. Subjects were divided into the following four groups: normal (non-HDL-C < 170 and TG < 150 mg/dL), hyper-TG (non-HDL-C < 170 and TG ≥ 150 mg/dL), hyper-non-HDL (non-HDL-C ≥ 170 and TG < 150 mg/dL), and hyper-TG/-non-HDL (non-HDL-C ≥ 170 and TG ≥ 150 mg/dL).

Hyperlipidemia gradually tends to normalize with remission, although it remains after remission in some patients. Low-density lipoprotein (LDL) apheresis used as a treatment for patients with resistant NS supports that dyslipidemia, including high levels of LDL cholesterol (LDL-C), is associated with the development and risk factors of NS [6,7].
Hirano et al. developed an assay for the quanti cation of cholesterol and separated LDL-C particles into three or four major LDL subclasses based on density [8]. This assay is not widely used in general clinical practice due to the di culty, especially in children, to obtain su cient blood samples. Subsequently, Hayashi et al. proposed a simpler method, the "alternative LDL window," that uses conventional methods for estimating high small dense LDL-C (sdLDL-C) levels [9]. sdLDL-C level was found to be increased in patients combined with hyperlipidemia [10]. Thus, the level of sdLDL-C is a candidate for a novel risk factor, or a risk marker for atherosclerotic vascular complications such as coronary heart disease [10,11,12]. sdLDL is also currently considered as one of the lipoproteins that leads to the development of cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) [13]. Canden et al. reported that subclinical CVD and its association with risk factors were independently associated only with higher LDL-C levels in children with steroid-resistant nephrotic syndrome (SRNS) [14]. However, no evaluation of sdLDL-C levels in NS was performed. Therefore, this study was conducted to examine the cholesterol composition, including sdLDL-C level in NS referring to the "alternative LDL window," to provide a better understanding of the lipid pro les of childhood-onset NS.

Methods
This retrospective study enrolled patients diagnosed with initial-onset NS in childhood and adolescence from April 2010 to March 2020. All patients were recruited from the Department of Pediatrics at Kindai University Hospital, where they were treated with prednisolone according to the recommendations of the Japanese Society of Pediatric Nephrology. Remission was de ned as no evidence of proteinuria in earlymorning specimens for 3 consecutive days. Patients with NS caused due to other renal diseases and those with familial hyperlipidemia, receiving lipid-lowering drugs, and lack of su cient data at the time of enrollment were excluded. At the point of study entry, the height and weight [for body mass index (BMI) calculation] were measured. Study parameters were estimated before the initiation of prednisolone.
Laboratory data consisting of serum creatinine, estimated GFR, serum concentrations of total protein (TP), albumin (Alb), total cholesterol (TC), triglyceride (TG), LDL-C, HDL-cholesterol (HDL-C), uric acid, and creatinine levels were examined. Non-HDL-C level is calculated using the following formula: [non-HDL-C (mg/dL) = TC -HDL-C]. The concentrations of urinary protein and creatinine were also measured. The baseline of lipid pro les was de ned based on the National Heart, Lung, and Blood Institute (NHLBI) guidelines, which recommend universal screening of children for hyperlipidemia [15]. The average of atherogenic index of plasma (AIP) was calculated using the formula log10 (TG/HDL-C) [16].

LDL particle size and LDL window determination
The "alternative LDL window" comprises the number and sizes of LDL particles estimated according to the plasma non-HDL-C and TG levels. Non-HDL-C is a surrogate marker for apoB [17]; therefore, it was used instead of apoB. Non-HDL-C level was calculated by subtracting the HDL-C level from the total-C level. Subjects were divided into the following four groups: normal (non-HDL-C < 170 and TG < 150 mg/dL), hyper-TG (non-HDL-C < 170 and TG ≥ 150 mg/dL), hyper-non-HDL (non-HDL-C ≥ 170 and TG < 150 mg/dL), and hyper-TG/-non-HDL (non-HDL-C ≥ 170 and TG ≥ 150 mg/dL).

Statistical analysis
Quantitative data are presented as mean ± standard deviation (SD). Pearson's correlation coe cients were used to analyze the correlations between variables. A P value of < 0.05 was considered as statistically signi cant.

Discussion
It is well known that TC, TG, and LDL-C levels are markedly increased during the nephrotic state. Especially in the initial onset, dyslipidemia appears frequently compared to that during the relapse phase. The magnitude of dyslipidemia in NS is considered to be directly associated with the severity of proteinuria [18]. In our study, signi cant dyslipidemia with not only high levels of TC, TG and LDL-C, but also non-HDL levels were noted. Non-HDL-C exhibits several bene ts beyond LDL-C and can be used for lipid management when a patient's samples show a TG level of > 400 mg/dL or non-fasting state [19]. As unifying the fasting samples at the onset is di cult, evaluating non-HDL-C level is considered to be useful, especially in children. Several studies reported high non-HDL-C levels in patients with metabolic syndrome. In our study, the non-HDL-C level of patients with NS was signi cantly higher than previously reported data [20,21]. Furthermore, we focused on the LDL particle size to evaluate non-HDL-C level. TG is a powerful inverse determinant of LDL particle size [9,17]. In other words, the higher the TG value, the smaller the particle size of LDL, which is recognized as sdLDL. Using the "alternative LDL window," understanding the condition of dyslipidemia at a glance in the acute phase of NS is easy. When patients belong to the area of hyper-TG/-non-HDL, it implies that among those patients with sdLDL, 82% with hyper-TG/-non-HDL levels could be exposed to the risk of endothelial cell damage.
Endothelial cell damage contributes to the enhancement of vascular permeability, and it is considered to be one of the important factors in the development of NS. Sharma et al. reported that patients with SRNS exhibited high levels of endothelial dysfunction markers [22]. sdLDL is a lipoprotein that strongly induces arteriosclerosis and is closely associated with cardiovascular events. Using these criteria, the majority of our patients were found exhibit a considerable risk of developing premature vascular disease. Hyperlipidemia is also involved in the pathogenesis of various acute complications in NS [23,24].
Interestingly, Ambrosch et al. provided novel clues on the atherogenic mechanisms of sdLDL, which sensitize vascular cells to in ammatory signals more effectively than normal-sized LDL particles [25].
The oxidative modi cation of LDL plays a vital role in the pathogenesis of several vascular diseases [26,27]. In addition, endothelial dysfunction and in ammation occur in even an asymptomatic proteinuria state [28]. AIP is used to predict the angiographic complexity of peripheral artery disease, and it has been con rmed to be signi cantly correlated with other important atherosclerosis indexes such as LDL-C particle size and sdLDL-C [16,29]. It was classi ed into 3 risk levels for CVD according to the following values: −0.3 to 0.1 for low risk, 0.1-0.24 for medium risk, and > 0.24 for high risk [30]. In our study, 69% of the patients exhibited high levels of AIP, which implies they may exhibit a high-risk condition during the acute phase, although it may be a short period. Clinically, no obvious complications were observed in both acute and remission phases in our study. This is not surprising as children basically do not present with an underlying illness and may demonstrate fewer complications than adults. In our study, we found that AIP positively and signi cantly correlated with lipid pro les such as TG, LDL-C, and TC/HDL negatively correlated with TP and Alb. The latter ndings generally normalize, and hyperlipidemia also tends to disappear with the resolution of proteinuria. However, some studies reported persisting lipid anomalies during remission [31,32]. In one study of 30 patients with childhood-onset steroid-sensitive NS (SSNS), dysregulation of lipids, including increased TC levels, and LDL-C were persistent during the follow-up at 4-15 years after the completion of steroid therapy [33]. The hyperlipidemic pro les remained despite the remission, especially in frequently relapsing NS cases [31]. Furthermore, subclinical cardiovascular disease and its association with risk factors are detected in children with SRNS [14]. In addition, lowering cholesterol levels using HMG-CoA reductase inhibitors during childhood may reduce the risk for atherosclerotic changes [34]. Hence, lipid-lowering agents may be of bene t in certain patients with NS such as those with SRNS.
Currently, the number of young patients improving from pediatric to adult renal care progressively increased due to improved management worldwide. It appears possible that even SSNS, which was previously believed to be entirely benign, may engender an increased risk for hypertension, atherosclerosis, and CKD in adulthood [35]. In our study, no signi cant correlation existed between the data of lipid pro les and eGFR. However, the CKD in Children Study reported that the presence of dyslipidemia was associated with the reduction of eGFR in patients with nonglomerular disease [36].
The limitations of this study include the small number of patients. Further studies are required to examine the changes in the levels of lipid pro les in individuals at the remission phase, which could help predict the severity and prognosis. In addition to increasing the number of patients, considering the measurement of aortic pulse wave velocity, carotid intima media thickness, and left ventricular mass is necessary to evaluate the risk factors of cardiovascular disease.

Conclusion
Clinically, lipid pro les such as TC and TG are routinely evaluated in patients with NS. De Silva et al recommended that the baseline lipid pro le, including TC, TG, HDL-C, and LDL-C, should be evaluated in children with SRNS [37]. Our results also propose the evaluation of lipid pro les and the need for careful characterization of patients. The "alternative LDL window" and AIP are relatively easy to obtain using routine biochemical parameters and help understand the state of hyperlipidemia in patients with NS. Abbreviations AIP; atherogenic index of plasma, Alb; albumin, BMI; body mass index, CKD; chronic kidney disease, CVD; cardiovascular disease, HDL-cholesterol (HDL-C)LDL; Low-density lipoprotein, LDL-C; low-density lipoprotein choresterol, NHLBI; National Heart, Lung, and Blood Institute, NS; nephrotic syndrome, sdLDL-C; Small dense low-density lipoprotein, SRNS; steroid-resistant nephrotic syndrome, TC; Total cholesterol, TG; Triglyceride, Declarations Ethics approval and consent to participate The Institutional Ethics Committee of the Faculty of Medicine of Kindai University approved this study (2020-065). Consent was obtained from a parent or guardian on behalf of any participants under the age of 16. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.

Consent to publish
All authors, patient and her parent consented for this publication.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interest
The authors declare that they have no competing interest.

Funding
This study was not received any funding.
Author's Contributions KS and MO conceived and designed the study. KM, TE, TM and RO carried out participant enrollment and managed data collection. TT led data analysis and specimen management and wrote the manuscript. TT provided guidance and expertise on data analysis and interpretation. YM assisted in data analysis and manuscript preparation. All authors contributed to the nal manuscript.