Chronic kidney disease stage affects small, dense low-density lipoprotein but not glycated low-density lipoprotein in younger chronic kidney disease patients: a cross-sectional study

Chronic kidney disease stage affects small, dense low-density lipoprotein but not glycated... Background: Small, dense low-density lipoprotein (sd-LDL) and glycated LDL (g-LDL) have been associated with cardiovascular disease (CVD) in chronic kidney disease (CKD) in patients>60 years of age. Since young adult and paediatric patients have shorter exposure to Framingham-type risk factors, our study aims to determine whether younger CKD patients exhibit the same sd-LDL and g-LDL pattern. Methods: After ethics board approval, this cross-sectional study was conducted at two universities with 44 patients (mean6 standard deviation age 12.66 4.9, range 2–24 years) with CKD stage of 1–5. Laboratory parameters studied were Cystatin C (CysC), CysC estimated glomerular filtration rate (eGFR) (calculated from the Filler formula), sd-LDL, g-LDL and albumin. Lipid samples were measured for sd-LDL and g-LDL using ELISA. Non-linear correlation analysis was performed to determine the relationship between g-LDL, sd-LDL and eGFR. Clinical Trials Registration is at clinicaltrials.gov, NCT02126293, https://clinicaltrials.gov/ct2/show/NCT02126293. Results: Triglycerides, but not total cholesterol and calculated LDL, were associated with CKD stages (ANOVA P¼ 0.0091). As in adults, sd-LDL was significantly associated with CKD stages (ANOVA P¼ 0.0133), CysC eGFR (r¼0.6495, P< 0.00001), and body mass index (r¼0.3895, P¼ 0.0189), but not with age. By contrast, there was no significant correlation between g- LDL and CKD stages or CysC eGFR (P¼ 0.9678). Conclusions: Our study demonstrates that only triglycerides and sd-LDL were associated with CKD stages in this young cohort without confounding Framingham-type CVD risk factors. While larger studies are needed, this study Received: July 26, 2017. Editorial decision: August 17, 2017 V C The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 384 | G. Filler et al. suggests that lowering sd-LDL levels may be a potential target to ameliorate the long-term CVD risks in paediatric CKD patients. Key words: cardiovascular risk, chronic kidney disease, disordered lipids, Framingham-type risk factors, small dense LDL, triglycerides adults with CKD typically do not have diabetes or other more Introduction conventional cardiovascular risk factors. Lipoprotein transfer proteins are complex particles composed of 80–100 proteins per particle [organized by a single apolipopro- tein B for low-density lipoprotein (LDL) and larger particles] that Materials and methods increase the availability of fats and allow them to be taken up Methods by the cells in the body via receptor-mediated endocytosis [1, 2]. A single LDL particle is about 220–275 angstroms in diameter The study adhered to the Declaration of Helsinki. The Research and typically transports 3000 to 6000 fat molecules/particle, Ethics Boards of Western and McMaster Universities approved which vary in size depending on the number and mix of fat mol- the study as part of a cross-sectional study of biomarkers of ecules contained within [3]. The lipids that are transported by CVD in children and young adults with CKD (Western Ontario: LDL particles include all fat molecules with cholesterol, phos- REB#16962E, McMaster University: REB#12-537). The goal of the pholipids and triglycerides. The proportions of these fats vary original study was to describe FGF23 and other biomarkers in considerably. LDL particles pose a risk for cardiovascular dis- relationship to renal function in children and young adults with ease (CVD) when they invade the endothelium and become oxi- CKD and comprised 157 patients from London and Hamilton. As dized, since the oxidized forms are more easily retained by the an ancillary study, we conducted this cross-sectional sub-study proteoglycans [1, 3]. Small, dense (sd-LDL) and glycated (g-LDL) of 44 children, adolescents and young adults, who were not on LDL have been associated with CVD risk [4]. dialysis, and were recruited between 17 March 2010 and 28 It has long been known that patients with chronic kidney October 2013 with CKD from Stages 1 to 5 (CKD was diagnosed disease (CKD) usually have disordered lipid profiles and tend to according to the 2012 KDIGO clinical practice guideline [16]). succumb to cardiovascular events [5, 6]. The impaired lipid They had blood work performed as part of their regular assess- metabolism seen in patients with CKD impacts the cardiovascu- ment and had abundant serum. The original inclusion criteria lar system in several ways, namely by preventing the formation for the study period that determined the sample size were of high-density lipoprotein (HDL), impairing reverse cholesterol patients from 1.5 years to 50 years with CKD Stages 1–5 who transport, intensifying the prevailing systemic oxidative stress require regular assessments to measure their renal function and inflammation, and increasing the risk of atherosclerotic and bone status. However, for the purposes of this study, we cardiovascular disease and CKD progression [7]. Cyclically, this excluded patients>25 years of age. Additional exclusion criteria HDL deficiency and dysfunction contributes to CKD progression included patients who were younger than 1.5 years of age by promoting glomerulosclerosis and tubular damage and dys- because of the developmental changes of GFR. Clinical Trials function [8, 9]. Such patients also tend to have an increase in tri- Registration is at clinicaltrials.gov, NCT02126293, https://clinical glycerides [10]. Although cardiovascular morbidity has been trials.gov/ct2/show/NCT02126293. linked to LDL, some LDL is favourable as it serves as the main carrier for the transport of cholesterol to the cells, and LDL lev- Materials els may be normal in patients with CVD [11] and may even be reduced in CKD patients [10]. LDL consists of several subclasses The samples for the lipid study were selected at random based with distinct sizes, densities and physicochemical compositions on the abundant samples available in the repository at the [11]. sd-LDL [11] and g-LDL [4] have specifically been associated Translational Research Centre at the London Health Sciences with cardiovascular disease. More recently, a specific pattern of Centre [17] and in an effort to have an even distribution of elevated oxidized LDL [10] and sd-LDL was described with patients with CKD Stages 1–5. Other laboratory parameters that worsening CKD [12], and sd-LDL levels were associated with were necessary for our analysis were taken from our database mortality [13]. Notably, these studies were performed in typical (Microsoft Excel for Mac v.14.6.8) or from our centre’s electronic adult CKD patients over 60 years of age, where typical medical chart database. Laboratory parameters that were used Framingham-type risk factors may confound CKD-specific for this study included Cystatin C (CysC), CysC eGFR, sd-LDL, g- changes. Framingham risk factors include age, gender, total and LDL and albumin. We calculated CysC eGFR using the Filler for- HDL cholesterol, smoking, diabetes, systolic blood pressure and mula [18] and body mass index (BMI) from the patients’ meas- treatment for high blood pressure [14]. The similarities and dif- ured height and weight. Even though BMI is age-dependent in ferences between the lipid profiles of patients with metabolic children, BMI values were not converted to z-scores because syndrome and patients with CKD have been previously high- there are no z-scores available for young adults. sd-LDL and g- lighted [15]. We were interested in seeing whether children LDL were measured using commercially available ELISA kits by would also exhibit the sd-LDL elevation found with a worsening MyBioSource, Inc., San Diego, CA , USA. The sd-LDL was meas- estimated glomerular filtration rate (eGFR) where Framingham- ured with the qualitative sandwich ELISA ‘Human small dense type risk factors are typically absent. We measured sd-LDL and low density lipoprotein (sLDL) ELISA Kit (Cat.No: MBS700740)’ g-LDL in children and adolescents with CKD Stages 1–5 who using undiluted original serum samples, with a detection range were ‘not on dialysis’. We hypothesized that the concentration between 0.312 and 20 nmol/mL. The g-LDL was measured with of sd-LDL would increase with worsening kidney function while the qualitative sandwich ELISA ‘GLDL ELISA KIT (Cat.No: g-LDL would remain unaffected because children and young MBS020040)’ using undiluted original serum samples. The Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 CKD stage affects small dense LDL but not glycated LDL | 385 detection range is 0.25 –8 nmol/mL. Standards were used as per measurements in Figure 1. As seen, only very few patients had the manufacturer’s instructions. As there were some inconsis- elevated total cholesterol or LDL cholesterol levels, or decreased tencies with the expected concentrations and those of the HDL cholesterol levels. Only triglycerides were associated with standards, we report the ELISA readings as relative units (rU). the stage of CKD (ANOVA P¼ 0.0091). Figure 2 shows the rela- tionship of sd-LDL and g-LDL with CysC eGFR. Interestingly, sd- LDL cholesterol, but not g-LDL cholesterol, was associated with Statistical analysis both the CysC eGFR (Spearman r¼0.6495, P < 0.00001) and the stage of CKD (ANOVA P¼ 0.0133) (Figures 1 and 2). The patients’ Contiguous data were analysed for normal distribution using BMI also correlated with sd-LDL (Spearman r ¼0.3895, the Kolmogorov Smirnov test. For normally distributed parame- P¼ 0.0189). There was no significant correlation between CysC ters, parametric and otherwise non-parametric statistical com- eGFR and g-LDL (Figure 2, right). parator tests were applied based on the distribution of the data Median microalbumin to creatinine ratio was 19.7 g/mol using GraphPad Prism v.5.0f and Microsoft Excel version 15.34 (range 0.2–44.6, interquartile range 2.35–46.5 g/mol). Data were for Mac. Data are expressed as average6 1 standard deviation not normally distributed. Interestingly, microalbumin/creati- (SD) for normally distributed parameters, and median (25th, nine ratio correlated significantly with sd-LDL (P¼ 0.0033) and 75th percentile) otherwise. As appropriate for the distribution, with triglycerides (P¼ 0.0441). Using multivariate analysis and linear or non-linear correlation analysis was performed to adjusting for microalbumin, CysC eGFR remained significant assess the relationship between age, eGFR, sd-LDL and g-LDL. A (P¼ 0.0135). We also adjusted for BMI and for age. Both of these P-value of <0.05 was considered significant for all analyses. were not significant in the multivariate analysis. Therefore, only CysC eGFR and microalbuminuria affected sd-LDL. Results The primary diagnoses of our study cohort are given in Table 1. Discussion Patients ranged in age from 2 to 24 years, with a mean age of In an effort to unravel the cardiovascular aetiology and out- 12.66 4.9 years; 40 patients were18 years old, and 4 were comes that pose the greatest threat to patients with CKD, pre- between the ages of 19 and 24. Of these patients, 23 were male vious studies in adults have examined the use of lipid and 21 were female. CKD stage distribution was as follows: biomarkers to predict and monitor patient CVD outcomes, since Stage 1¼ 4, Stage 2¼ 7, Stage 3¼ 13, Stage 4¼ 8, Stage 5¼ 12. traditional risk factors do not fully explain the high incidence of Average age (6 1 SD) was 14.16 2.5 years for CKD Stage 1, CVD in CKD, and traditional lipid measures do not sufficiently 11.16 3.6 years for CKD Stage 2, 12.36 4.3 years for CKD Stage 3, predict these outcomes in patients with CKD [19–22]. This effort, 20.46 6.5 for CKD Stage 4 and 13.86 5.9 years for CKD Stage 5. however, may be confounded by the Framingham factors seen Median BMI and laboratory values are given in Table 2. Neither in adults that are absent in children. sd-LDL nor eGFR correlated with age. Total cholesterol, LDL As predicted, the lack of correlation between CysC eGFR and cholesterol, HDL cholesterol and triglycerides are summarized g-LDL seen in our study suggests that traditional Framingham- in Figure 1. We also provide the sd-LDL and g-LDL like cardiovascular risk factors such as glycosylation may not be important factors in the cardiovascular morbidity and mortality Table 1. Patients breakdown by CKD aetiology of non-diabetic CKD patients. The sd-LDL, however, was associ- ated with CysC eGFR, stage of CKD and BMI, thereby confirming CKD aetiology adult data [23] and solidifying the notion that a CKD patient’s sd-LDL level may be a very important atherogenic risk factor, Hereditary Acquired and can be used as a predictor for CVD morbidity and mortality Renal dysplasia 5 Renal transplant 13 [12, 19, 24–26]. The results of this study also conformed to the Genetic conditions 12 Haemolytic uraemic 4 significantly higher levels of sd-LDL compared with LDL seen in syndrome patients with coronary artery disease, and this relationship’s Obstructive uropathy 3 Spina bifida and 2 association with the incidence of cardiovascular events inde- neurogenic bladder pendently of LDL itself [27, 28]. Similar to past studies [29], our Idiopathic Fanconi 1 Glomerulonephritis, 3 study also suggests that sd-LDL may be a more suitable marker syndrome FSGS than LDL for measuring CVD, which is particularly important Ischaemic acute 1 considering the most recent paediatric/adolescent guidelines kidney injury that only use LDL as a marker for dyslipidaemia and atheroscle- rosis [30, 31]. Such as autosomal recessive polycystic kidney disease, nephronopthisis and syndromes associated with renal dysplasia. Our study has several limitations, including its retrospective FSGS, focal segmental glomerulosclerosis. design, the wide age range of our patients and the modest sam- ple size. Absolute BMI was used, and it is possible that the corre- Table 2. Median laboratory values lation between BMI and sd-LDL was therefore confounded by age, although age was not correlated with sd-LDL. A strength of Blood parameter Median (Q1, Q3) Unit our study is the use of CysC eGFR rather than Schwartz eGFR; the former is a more accurate biomarker for kidney function, BMI 19.0264.35 kg/m and by the virtual elimination of Framingham factor confound- CysC 2.28 (1.25–4.61) mg/L 2 ers, a trait that cannot be replicated in adult studies. The for- CysC eGFR 36.5 (16.75–71.25) mL/min/1.73 m mula used for calculating the eGFR is the only formula that has Albumin 43.5 (40.8–46.0) g/L been validated in both children and adults [32]. g-LDL 0.23 (0.14–0.59) rU Without the aforementioned confounding Framingham risk sd-LDL 2.47 (1.74–3.98) rU factors seen in adults, these results bring us closer to Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 386 | G. Filler et al. Fig. 1. Lipid parameters by CKD stage. Small Dense LDL Glycated LDL 25 4 n.s., p=0.9678 Spearman r=-0.6495, p<0.00001 0 0 0 306090 120 030 60 90 120 CysC eGFR CysC eGFR Fig. 2. sd- and g-LDL versus CysC eGFR using the Filler formula [18]. Average age (6 1 SD) was 14.16 2.5 years for CKD Stage 1; 11.16 3.6 years for CKD Stage 2; 12.36 4.3 years for CKD Stage 3; 20.46 6.5 for CKD Stage 4; and 13.86 5.9 years for CKD Stage 5. determining a mechanism for the elevated sd-LDL levels seen grouped cholesterol is either ‘good’ or ‘bad’; its use as a bio- in patients with CKD and to addressing their role in CVD as the marker may in fact be measuring more fundamental variables leading cause of death in patients with CKD. It also contributes [12], and our focus may have to narrow to a greater understand- to scepticism regarding our historically clear-cut view that ing of sub-fractions of lipoprotein transfer lipids. Furthermore, Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Small Dense LDL [rU] glycated LDL [rU] CKD stage affects small dense LDL but not glycated LDL | 387 5. Filler G. Challenges in pediatric transplantation: the impact our findings in children, echoed by studies in adults, also strongly indicate that there is a need for a faster, less expensive, of chronic kidney disease and cardiovascular risk factors on less laborious and more efficient way of measuring the different long-term outcomes and recommended management strat- classes of LDL, particularly sd-LDL. Comparable to strategies egies. Pediatr Transplant 2011; 15: 25–31 employed within the SHARP trial [33], our results suggest that 6. Kaysen GA. Lipid and lipoprotein metabolism in chronic kid- targeting sd-LDL may be a viable option for reducing long-term ney disease. J Ren Nutr 2009; 19: 73–77 7. Vaziri ND. HDL abnormalities in nephrotic syndrome and cardiovascular risks in paediatric CKD patients, particularly for those who are in the later stages of CKD. The mechanism is not chronic kidney disease. Nat Rev Nephrol 2016; 12: 37–47 well understood. Upregulation of GPIHBP1 in a model of 5/6 8. Zhao YY, Wang HL, Cheng XL et al. Metabolomics analysis nephrectomy in rats and subsequent lipoprotein lipase defi- reveals the association between lipid abnormalities and oxi- dative stress, inflammation, fibrosis, and Nrf2 dysfunction ciency which interferes with normal metabolism of VLDL and chylomicrons may play a role [7]; however, a recent in-depth in aristolochic acid-induced nephropathy. Sci Rep 2015; 5: review did not offer a clear explanation [34]. The question is 12936. how to lower sd-LDL levels? Possible options are fibrates and 9. Zhao YY, Vaziri ND, Lin RC. Lipidomics: new insight into kid- ney disease. Adv Clin Chem 2015; 68: 153–175 LDL apheresis, however, fibrates are often not well tolerated and LDL apheresis is invasive and access to this treatment 10. Kaysen GA. New insights into lipid metabolism in chronic kidney disease. J Ren Nutr 2011; 21: 120–123 modality is limited [35]. Newer fibrates such as fenofibrate in a simvastatin combination have recently demonstrated good 11. Hirayama S, Miida T. Small dense LDL: An emerging risk factor safety profiles in adults and even reduced proteinuria [36]. for cardiovascular disease. Clin Chim Acta 2012; 414: 215–224 In summary, this study confirms the specific pattern of sd- 12. Chu M, Wang AY, Chan IH et al. Serum small-dense LDL abnormalities in chronic renal disease patients. Br J Biomed LDL being associated with worsening eGFR in the absence of any association with g-LDL. The results strongly suggest that Sci 2012; 69: 99–102 this pattern is CKD specific and not confounded by 13. Shen H, Xu Y, Lu J et al. Small dense low-density lipoprotein Framingham-type risk factors. cholesterol was associated with future cardiovascular events in chronic kidney disease patients. BMC Nephrol 2016; 17: 143 Acknowledgements 14. Kannel WB, Dawber TR, Kagan A et al. Factors of risk in the development of coronary heart disease–six year follow-up We would like to thank the Translational Research Centre experience. The Framingham Study. Ann Intern Med 1961; 55: under the leadership of Dr Douglas Fraser and the expert 33–50 sample handling by Carolina Gillio-Meina for handling and 15. Kaysen GA. Metabolic syndrome and renal failure: similar- processing our samples in the repository. We also thank Ms ities and differences. Panminerva Med 2006; 48: 151–164 Marta Kobrzynski for her expert editing. Finally, we thank 16. Chapter 1: Definition and classification of CKD. Kidney Int Mr Connor Smith, chemistry student at Western University, Suppl (2011) 2013; 3: 19–62 for his assistance with the ELISA measurements. 17. Gillio-Meina C, Zielke HR, Fraser DD. Translational research in pediatrics IV: solid tissue collection and processing. Pediatrics 2016; 137 Authors’ contributions 18. Filler G, Lepage N. Should the Schwartz formula for estima- tion of GFR be replaced by cystatin C formula? Pediatr Nephrol Conception or design: G.Filler, C.F., C.M., S.T.; providing 2003; 18: 981–985 intellectual content of critical importance to work described: 19. Stenvinkel P. Chronic kidney disease: a public health priority G.Filler, C.F., C.M., S.T.; performed the experiments: G.Filler, and harbinger of premature cardiovascular disease. J Intern C.S.; analysis and interpretation of data, or both: G.Filler, Med 2010; 268: 456–467 G.Fusch; drafting the article or revising it: G.Filler, L.S.; final 20. Rubin C, Nolin TD, Himmelfarb J. Are biomarkers useful for approval of version to be published: G.Filler, S.T., C.M., C.S., assessing cardiovascular risk in patients with chronic kid- L.S., G.Fusch, C.F. ney disease? Curr Opin Nephrol Hypertens 2007; 16: 506–511 21. Kanbay M, Siriopol D, Saglam M et al. Serum sclerostin and adverse outcomes in nondialyzed chronic kidney disease Conflicts of interest statement patients. J Clin Endocrinol Metab 2014; 99: E1854–E1861 22. Tonelli M, Muntner P, Lloyd A et al. Association between None declared. LDL-C and risk of myocardial infarction in CKD. J Am Soc Nephrol 2013; 24: 979–986 References 23. Savic J, Zeljkovic A, Bogavac-Stanojevic N et al. Association 1. Dashti M, Kulik W, Hoek F et al. A phospholipidomic analysis of small, dense low-density lipoprotein cholesterol and galectin-3 in patients with chronic kidney disease. Scand J of all defined human plasma lipoproteins. Sci Rep 2011; 1: 139 2. Dashty M, Motazacker MM, Levels J et al. 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Curr Opin Lipidol 2011; 22: 254–261 1687–1697 Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 388 | G. Filler et al. dyslipidemia: Evidence Report and Systematic Review for 26. Austin MA, Breslow JL, Hennekens CH et al. Low-density lipo- protein subclass patterns and risk of myocardial infarction. the US Preventive Services Task Force. JAMA 2016; 316: JAMA 1988; 260: 1917–1921 634–644 27. Ai M, Otokozawa S, Asztalos BF et al. Small dense LDL choles- 32. White CA, Akbari A, Doucette S et al. Effect of clinical varia- terol and coronary heart disease: results from the bles and immunosuppression on serum cystatin C and beta- Framingham Offspring Study. Clin Chem 2010; 56: 967–976 trace protein in kidney transplant recipients. Am J Kidney Dis 28. Arai H, Kokubo Y, Watanabe M et al. Small dense low- 2009; 54: 922–930 density lipoproteins cholesterol can predict incident cardio- 33. Baigent C, Landray MJ, Reith C et al. The effects of lowering vascular disease in an urban Japanese cohort: the Suita LDL cholesterol with simvastatin plus ezetimibe in patients study. J Atheroscler Thromb 2013; 20: 195–203 with chronic kidney disease (Study of Heart and Renal 29. Koba S, Yokota Y, Hirano T et al. Small LDL-cholesterol Protection): a randomised placebo-controlled trial. Lancet is superior to LDL-cholesterol for determining severe 2011; 377: 2181–2192 coronary atherosclerosis. J Atheroscler Thromb 2008; 15: 34. Mikolasevic I, Zutelija M, Mavrinac V et al. Dyslipidemia in 250–260 patients with chronic kidney disease: etiology and manage- ment. Int J Nephrol Renovasc Dis 2017; 10: 35–45 30. Gooding HC, Rodday AM, Wong JB et al. Application of Pediatric and Adult Guidelines for treatment of lipid levels 35. Filler G, Lee M, Hegele RA. Barriers to the implementation of among US adolescents transitioning to young adulthood. lipoprotein apheresis in Canada. Can J Cardiol 2017; 33: 409–411 JAMA Pediatr 2015; 169: 569–574 36. Filippatos TD, Elisaf MS. Safety considerations with fenofi- 31. Lozano P, Henrikson NB, Morrison CC et al. Lipid screening in brate/simvastatin combination. Expert Opin Drug Saf 2015; 14: childhood and adolescence for detection of multifactorial 1481–1493 Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Kidney Journal Oxford University Press

Chronic kidney disease stage affects small, dense low-density lipoprotein but not glycated low-density lipoprotein in younger chronic kidney disease patients: a cross-sectional study

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Abstract

Background: Small, dense low-density lipoprotein (sd-LDL) and glycated LDL (g-LDL) have been associated with cardiovascular disease (CVD) in chronic kidney disease (CKD) in patients>60 years of age. Since young adult and paediatric patients have shorter exposure to Framingham-type risk factors, our study aims to determine whether younger CKD patients exhibit the same sd-LDL and g-LDL pattern. Methods: After ethics board approval, this cross-sectional study was conducted at two universities with 44 patients (mean6 standard deviation age 12.66 4.9, range 2–24 years) with CKD stage of 1–5. Laboratory parameters studied were Cystatin C (CysC), CysC estimated glomerular filtration rate (eGFR) (calculated from the Filler formula), sd-LDL, g-LDL and albumin. Lipid samples were measured for sd-LDL and g-LDL using ELISA. Non-linear correlation analysis was performed to determine the relationship between g-LDL, sd-LDL and eGFR. Clinical Trials Registration is at clinicaltrials.gov, NCT02126293, https://clinicaltrials.gov/ct2/show/NCT02126293. Results: Triglycerides, but not total cholesterol and calculated LDL, were associated with CKD stages (ANOVA P¼ 0.0091). As in adults, sd-LDL was significantly associated with CKD stages (ANOVA P¼ 0.0133), CysC eGFR (r¼0.6495, P< 0.00001), and body mass index (r¼0.3895, P¼ 0.0189), but not with age. By contrast, there was no significant correlation between g- LDL and CKD stages or CysC eGFR (P¼ 0.9678). Conclusions: Our study demonstrates that only triglycerides and sd-LDL were associated with CKD stages in this young cohort without confounding Framingham-type CVD risk factors. While larger studies are needed, this study Received: July 26, 2017. Editorial decision: August 17, 2017 V C The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 384 | G. Filler et al. suggests that lowering sd-LDL levels may be a potential target to ameliorate the long-term CVD risks in paediatric CKD patients. Key words: cardiovascular risk, chronic kidney disease, disordered lipids, Framingham-type risk factors, small dense LDL, triglycerides adults with CKD typically do not have diabetes or other more Introduction conventional cardiovascular risk factors. Lipoprotein transfer proteins are complex particles composed of 80–100 proteins per particle [organized by a single apolipopro- tein B for low-density lipoprotein (LDL) and larger particles] that Materials and methods increase the availability of fats and allow them to be taken up Methods by the cells in the body via receptor-mediated endocytosis [1, 2]. A single LDL particle is about 220–275 angstroms in diameter The study adhered to the Declaration of Helsinki. The Research and typically transports 3000 to 6000 fat molecules/particle, Ethics Boards of Western and McMaster Universities approved which vary in size depending on the number and mix of fat mol- the study as part of a cross-sectional study of biomarkers of ecules contained within [3]. The lipids that are transported by CVD in children and young adults with CKD (Western Ontario: LDL particles include all fat molecules with cholesterol, phos- REB#16962E, McMaster University: REB#12-537). The goal of the pholipids and triglycerides. The proportions of these fats vary original study was to describe FGF23 and other biomarkers in considerably. LDL particles pose a risk for cardiovascular dis- relationship to renal function in children and young adults with ease (CVD) when they invade the endothelium and become oxi- CKD and comprised 157 patients from London and Hamilton. As dized, since the oxidized forms are more easily retained by the an ancillary study, we conducted this cross-sectional sub-study proteoglycans [1, 3]. Small, dense (sd-LDL) and glycated (g-LDL) of 44 children, adolescents and young adults, who were not on LDL have been associated with CVD risk [4]. dialysis, and were recruited between 17 March 2010 and 28 It has long been known that patients with chronic kidney October 2013 with CKD from Stages 1 to 5 (CKD was diagnosed disease (CKD) usually have disordered lipid profiles and tend to according to the 2012 KDIGO clinical practice guideline [16]). succumb to cardiovascular events [5, 6]. The impaired lipid They had blood work performed as part of their regular assess- metabolism seen in patients with CKD impacts the cardiovascu- ment and had abundant serum. The original inclusion criteria lar system in several ways, namely by preventing the formation for the study period that determined the sample size were of high-density lipoprotein (HDL), impairing reverse cholesterol patients from 1.5 years to 50 years with CKD Stages 1–5 who transport, intensifying the prevailing systemic oxidative stress require regular assessments to measure their renal function and inflammation, and increasing the risk of atherosclerotic and bone status. However, for the purposes of this study, we cardiovascular disease and CKD progression [7]. Cyclically, this excluded patients>25 years of age. Additional exclusion criteria HDL deficiency and dysfunction contributes to CKD progression included patients who were younger than 1.5 years of age by promoting glomerulosclerosis and tubular damage and dys- because of the developmental changes of GFR. Clinical Trials function [8, 9]. Such patients also tend to have an increase in tri- Registration is at clinicaltrials.gov, NCT02126293, https://clinical glycerides [10]. Although cardiovascular morbidity has been trials.gov/ct2/show/NCT02126293. linked to LDL, some LDL is favourable as it serves as the main carrier for the transport of cholesterol to the cells, and LDL lev- Materials els may be normal in patients with CVD [11] and may even be reduced in CKD patients [10]. LDL consists of several subclasses The samples for the lipid study were selected at random based with distinct sizes, densities and physicochemical compositions on the abundant samples available in the repository at the [11]. sd-LDL [11] and g-LDL [4] have specifically been associated Translational Research Centre at the London Health Sciences with cardiovascular disease. More recently, a specific pattern of Centre [17] and in an effort to have an even distribution of elevated oxidized LDL [10] and sd-LDL was described with patients with CKD Stages 1–5. Other laboratory parameters that worsening CKD [12], and sd-LDL levels were associated with were necessary for our analysis were taken from our database mortality [13]. Notably, these studies were performed in typical (Microsoft Excel for Mac v.14.6.8) or from our centre’s electronic adult CKD patients over 60 years of age, where typical medical chart database. Laboratory parameters that were used Framingham-type risk factors may confound CKD-specific for this study included Cystatin C (CysC), CysC eGFR, sd-LDL, g- changes. Framingham risk factors include age, gender, total and LDL and albumin. We calculated CysC eGFR using the Filler for- HDL cholesterol, smoking, diabetes, systolic blood pressure and mula [18] and body mass index (BMI) from the patients’ meas- treatment for high blood pressure [14]. The similarities and dif- ured height and weight. Even though BMI is age-dependent in ferences between the lipid profiles of patients with metabolic children, BMI values were not converted to z-scores because syndrome and patients with CKD have been previously high- there are no z-scores available for young adults. sd-LDL and g- lighted [15]. We were interested in seeing whether children LDL were measured using commercially available ELISA kits by would also exhibit the sd-LDL elevation found with a worsening MyBioSource, Inc., San Diego, CA , USA. The sd-LDL was meas- estimated glomerular filtration rate (eGFR) where Framingham- ured with the qualitative sandwich ELISA ‘Human small dense type risk factors are typically absent. We measured sd-LDL and low density lipoprotein (sLDL) ELISA Kit (Cat.No: MBS700740)’ g-LDL in children and adolescents with CKD Stages 1–5 who using undiluted original serum samples, with a detection range were ‘not on dialysis’. We hypothesized that the concentration between 0.312 and 20 nmol/mL. The g-LDL was measured with of sd-LDL would increase with worsening kidney function while the qualitative sandwich ELISA ‘GLDL ELISA KIT (Cat.No: g-LDL would remain unaffected because children and young MBS020040)’ using undiluted original serum samples. The Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 CKD stage affects small dense LDL but not glycated LDL | 385 detection range is 0.25 –8 nmol/mL. Standards were used as per measurements in Figure 1. As seen, only very few patients had the manufacturer’s instructions. As there were some inconsis- elevated total cholesterol or LDL cholesterol levels, or decreased tencies with the expected concentrations and those of the HDL cholesterol levels. Only triglycerides were associated with standards, we report the ELISA readings as relative units (rU). the stage of CKD (ANOVA P¼ 0.0091). Figure 2 shows the rela- tionship of sd-LDL and g-LDL with CysC eGFR. Interestingly, sd- LDL cholesterol, but not g-LDL cholesterol, was associated with Statistical analysis both the CysC eGFR (Spearman r¼0.6495, P < 0.00001) and the stage of CKD (ANOVA P¼ 0.0133) (Figures 1 and 2). The patients’ Contiguous data were analysed for normal distribution using BMI also correlated with sd-LDL (Spearman r ¼0.3895, the Kolmogorov Smirnov test. For normally distributed parame- P¼ 0.0189). There was no significant correlation between CysC ters, parametric and otherwise non-parametric statistical com- eGFR and g-LDL (Figure 2, right). parator tests were applied based on the distribution of the data Median microalbumin to creatinine ratio was 19.7 g/mol using GraphPad Prism v.5.0f and Microsoft Excel version 15.34 (range 0.2–44.6, interquartile range 2.35–46.5 g/mol). Data were for Mac. Data are expressed as average6 1 standard deviation not normally distributed. Interestingly, microalbumin/creati- (SD) for normally distributed parameters, and median (25th, nine ratio correlated significantly with sd-LDL (P¼ 0.0033) and 75th percentile) otherwise. As appropriate for the distribution, with triglycerides (P¼ 0.0441). Using multivariate analysis and linear or non-linear correlation analysis was performed to adjusting for microalbumin, CysC eGFR remained significant assess the relationship between age, eGFR, sd-LDL and g-LDL. A (P¼ 0.0135). We also adjusted for BMI and for age. Both of these P-value of <0.05 was considered significant for all analyses. were not significant in the multivariate analysis. Therefore, only CysC eGFR and microalbuminuria affected sd-LDL. Results The primary diagnoses of our study cohort are given in Table 1. Discussion Patients ranged in age from 2 to 24 years, with a mean age of In an effort to unravel the cardiovascular aetiology and out- 12.66 4.9 years; 40 patients were18 years old, and 4 were comes that pose the greatest threat to patients with CKD, pre- between the ages of 19 and 24. Of these patients, 23 were male vious studies in adults have examined the use of lipid and 21 were female. CKD stage distribution was as follows: biomarkers to predict and monitor patient CVD outcomes, since Stage 1¼ 4, Stage 2¼ 7, Stage 3¼ 13, Stage 4¼ 8, Stage 5¼ 12. traditional risk factors do not fully explain the high incidence of Average age (6 1 SD) was 14.16 2.5 years for CKD Stage 1, CVD in CKD, and traditional lipid measures do not sufficiently 11.16 3.6 years for CKD Stage 2, 12.36 4.3 years for CKD Stage 3, predict these outcomes in patients with CKD [19–22]. This effort, 20.46 6.5 for CKD Stage 4 and 13.86 5.9 years for CKD Stage 5. however, may be confounded by the Framingham factors seen Median BMI and laboratory values are given in Table 2. Neither in adults that are absent in children. sd-LDL nor eGFR correlated with age. Total cholesterol, LDL As predicted, the lack of correlation between CysC eGFR and cholesterol, HDL cholesterol and triglycerides are summarized g-LDL seen in our study suggests that traditional Framingham- in Figure 1. We also provide the sd-LDL and g-LDL like cardiovascular risk factors such as glycosylation may not be important factors in the cardiovascular morbidity and mortality Table 1. Patients breakdown by CKD aetiology of non-diabetic CKD patients. The sd-LDL, however, was associ- ated with CysC eGFR, stage of CKD and BMI, thereby confirming CKD aetiology adult data [23] and solidifying the notion that a CKD patient’s sd-LDL level may be a very important atherogenic risk factor, Hereditary Acquired and can be used as a predictor for CVD morbidity and mortality Renal dysplasia 5 Renal transplant 13 [12, 19, 24–26]. The results of this study also conformed to the Genetic conditions 12 Haemolytic uraemic 4 significantly higher levels of sd-LDL compared with LDL seen in syndrome patients with coronary artery disease, and this relationship’s Obstructive uropathy 3 Spina bifida and 2 association with the incidence of cardiovascular events inde- neurogenic bladder pendently of LDL itself [27, 28]. Similar to past studies [29], our Idiopathic Fanconi 1 Glomerulonephritis, 3 study also suggests that sd-LDL may be a more suitable marker syndrome FSGS than LDL for measuring CVD, which is particularly important Ischaemic acute 1 considering the most recent paediatric/adolescent guidelines kidney injury that only use LDL as a marker for dyslipidaemia and atheroscle- rosis [30, 31]. Such as autosomal recessive polycystic kidney disease, nephronopthisis and syndromes associated with renal dysplasia. Our study has several limitations, including its retrospective FSGS, focal segmental glomerulosclerosis. design, the wide age range of our patients and the modest sam- ple size. Absolute BMI was used, and it is possible that the corre- Table 2. Median laboratory values lation between BMI and sd-LDL was therefore confounded by age, although age was not correlated with sd-LDL. A strength of Blood parameter Median (Q1, Q3) Unit our study is the use of CysC eGFR rather than Schwartz eGFR; the former is a more accurate biomarker for kidney function, BMI 19.0264.35 kg/m and by the virtual elimination of Framingham factor confound- CysC 2.28 (1.25–4.61) mg/L 2 ers, a trait that cannot be replicated in adult studies. The for- CysC eGFR 36.5 (16.75–71.25) mL/min/1.73 m mula used for calculating the eGFR is the only formula that has Albumin 43.5 (40.8–46.0) g/L been validated in both children and adults [32]. g-LDL 0.23 (0.14–0.59) rU Without the aforementioned confounding Framingham risk sd-LDL 2.47 (1.74–3.98) rU factors seen in adults, these results bring us closer to Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 386 | G. Filler et al. Fig. 1. Lipid parameters by CKD stage. Small Dense LDL Glycated LDL 25 4 n.s., p=0.9678 Spearman r=-0.6495, p<0.00001 0 0 0 306090 120 030 60 90 120 CysC eGFR CysC eGFR Fig. 2. sd- and g-LDL versus CysC eGFR using the Filler formula [18]. Average age (6 1 SD) was 14.16 2.5 years for CKD Stage 1; 11.16 3.6 years for CKD Stage 2; 12.36 4.3 years for CKD Stage 3; 20.46 6.5 for CKD Stage 4; and 13.86 5.9 years for CKD Stage 5. determining a mechanism for the elevated sd-LDL levels seen grouped cholesterol is either ‘good’ or ‘bad’; its use as a bio- in patients with CKD and to addressing their role in CVD as the marker may in fact be measuring more fundamental variables leading cause of death in patients with CKD. It also contributes [12], and our focus may have to narrow to a greater understand- to scepticism regarding our historically clear-cut view that ing of sub-fractions of lipoprotein transfer lipids. Furthermore, Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Small Dense LDL [rU] glycated LDL [rU] CKD stage affects small dense LDL but not glycated LDL | 387 5. Filler G. Challenges in pediatric transplantation: the impact our findings in children, echoed by studies in adults, also strongly indicate that there is a need for a faster, less expensive, of chronic kidney disease and cardiovascular risk factors on less laborious and more efficient way of measuring the different long-term outcomes and recommended management strat- classes of LDL, particularly sd-LDL. Comparable to strategies egies. Pediatr Transplant 2011; 15: 25–31 employed within the SHARP trial [33], our results suggest that 6. Kaysen GA. Lipid and lipoprotein metabolism in chronic kid- targeting sd-LDL may be a viable option for reducing long-term ney disease. J Ren Nutr 2009; 19: 73–77 7. Vaziri ND. HDL abnormalities in nephrotic syndrome and cardiovascular risks in paediatric CKD patients, particularly for those who are in the later stages of CKD. The mechanism is not chronic kidney disease. Nat Rev Nephrol 2016; 12: 37–47 well understood. Upregulation of GPIHBP1 in a model of 5/6 8. Zhao YY, Wang HL, Cheng XL et al. Metabolomics analysis nephrectomy in rats and subsequent lipoprotein lipase defi- reveals the association between lipid abnormalities and oxi- dative stress, inflammation, fibrosis, and Nrf2 dysfunction ciency which interferes with normal metabolism of VLDL and chylomicrons may play a role [7]; however, a recent in-depth in aristolochic acid-induced nephropathy. Sci Rep 2015; 5: review did not offer a clear explanation [34]. The question is 12936. how to lower sd-LDL levels? Possible options are fibrates and 9. Zhao YY, Vaziri ND, Lin RC. Lipidomics: new insight into kid- ney disease. Adv Clin Chem 2015; 68: 153–175 LDL apheresis, however, fibrates are often not well tolerated and LDL apheresis is invasive and access to this treatment 10. Kaysen GA. New insights into lipid metabolism in chronic kidney disease. J Ren Nutr 2011; 21: 120–123 modality is limited [35]. Newer fibrates such as fenofibrate in a simvastatin combination have recently demonstrated good 11. Hirayama S, Miida T. Small dense LDL: An emerging risk factor safety profiles in adults and even reduced proteinuria [36]. for cardiovascular disease. Clin Chim Acta 2012; 414: 215–224 In summary, this study confirms the specific pattern of sd- 12. Chu M, Wang AY, Chan IH et al. Serum small-dense LDL abnormalities in chronic renal disease patients. Br J Biomed LDL being associated with worsening eGFR in the absence of any association with g-LDL. The results strongly suggest that Sci 2012; 69: 99–102 this pattern is CKD specific and not confounded by 13. Shen H, Xu Y, Lu J et al. Small dense low-density lipoprotein Framingham-type risk factors. cholesterol was associated with future cardiovascular events in chronic kidney disease patients. BMC Nephrol 2016; 17: 143 Acknowledgements 14. Kannel WB, Dawber TR, Kagan A et al. Factors of risk in the development of coronary heart disease–six year follow-up We would like to thank the Translational Research Centre experience. The Framingham Study. Ann Intern Med 1961; 55: under the leadership of Dr Douglas Fraser and the expert 33–50 sample handling by Carolina Gillio-Meina for handling and 15. Kaysen GA. Metabolic syndrome and renal failure: similar- processing our samples in the repository. We also thank Ms ities and differences. Panminerva Med 2006; 48: 151–164 Marta Kobrzynski for her expert editing. Finally, we thank 16. Chapter 1: Definition and classification of CKD. Kidney Int Mr Connor Smith, chemistry student at Western University, Suppl (2011) 2013; 3: 19–62 for his assistance with the ELISA measurements. 17. Gillio-Meina C, Zielke HR, Fraser DD. Translational research in pediatrics IV: solid tissue collection and processing. Pediatrics 2016; 137 Authors’ contributions 18. Filler G, Lepage N. Should the Schwartz formula for estima- tion of GFR be replaced by cystatin C formula? Pediatr Nephrol Conception or design: G.Filler, C.F., C.M., S.T.; providing 2003; 18: 981–985 intellectual content of critical importance to work described: 19. Stenvinkel P. Chronic kidney disease: a public health priority G.Filler, C.F., C.M., S.T.; performed the experiments: G.Filler, and harbinger of premature cardiovascular disease. J Intern C.S.; analysis and interpretation of data, or both: G.Filler, Med 2010; 268: 456–467 G.Fusch; drafting the article or revising it: G.Filler, L.S.; final 20. Rubin C, Nolin TD, Himmelfarb J. 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Can J Cardiol 2017; 33: 409–411 JAMA Pediatr 2015; 169: 569–574 36. Filippatos TD, Elisaf MS. Safety considerations with fenofi- 31. Lozano P, Henrikson NB, Morrison CC et al. Lipid screening in brate/simvastatin combination. Expert Opin Drug Saf 2015; 14: childhood and adolescence for detection of multifactorial 1481–1493 Downloaded from https://academic.oup.com/ckj/article-abstract/11/3/383/4469410 by Ed 'DeepDyve' Gillespie user on 20 June 2018

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Clinical Kidney JournalOxford University Press

Published: Oct 12, 2017

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