Proteinuria converts hepatic heparan sulfate to an effective proprotein convertase subtilisin kexin type 9 enzyme binding partner
Authors: Pragyi Shrestha, Saleh Yazdani, Romain R. Vivès, Rana El Masri, Wendy Dam, Bart van de Sluis, Jacob van den Born
Journal: Kidney International (2021)

Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia. Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia. Translational StatementCardiovascular complications due to dyslipidemia are of concern in chronic kidney disease. Herein, we discovered that proteinuria induces hypersulfation of hepatic heparan sulfate proteoglycans, increasing their affinity to proprotein convertase subtilisin kexin type 9 (PCSK9) and causing sinusoidal accumulation of PCSK9. These changes were associated with elevated plasma triglycerides and total cholesterol, without affecting low-density lipoprotein receptor expression. These data indicate a novel mechanism behind dyslipidemia in chronic kidney disease. At present, where current cholesterol-lowering strategies to treat dyslipidemia in chronic kidney disease are still insufficient and novel PCSK9 inhibitors are being introduced, our study opens new venues for future development of cost-effective heparin mimetics as PCSK9 inhibitors. Cardiovascular complications due to dyslipidemia are of concern in chronic kidney disease. Herein, we discovered that proteinuria induces hypersulfation of hepatic heparan sulfate proteoglycans, increasing their affinity to proprotein convertase subtilisin kexin type 9 (PCSK9) and causing sinusoidal accumulation of PCSK9. These changes were associated with elevated plasma triglycerides and total cholesterol, without affecting low-density lipoprotein receptor expression. These data indicate a novel mechanism behind dyslipidemia in chronic kidney disease. At present, where current cholesterol-lowering strategies to treat dyslipidemia in chronic kidney disease are still insufficient and novel PCSK9 inhibitors are being introduced, our study opens new venues for future development of cost-effective heparin mimetics as PCSK9 inhibitors. Chronic kidney disease (CKD) is a global health problem affecting >10% of the people worldwide.1Bikbov B. Purcell C.A. 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In the current study, we have studied the effects of proteinuria on hepatic HSPGs, PCSK9, and their effects on lipoprotein metabolism using Adriamycin (doxorubicin)–induced proteinuria model. This model is a pure proteinuria model (due to the damaging effect of Adriamycin on glomerular podocytes) without loss of renal function (glomerular filtration rate).34Jeansson M. Björck K. Tenstad O. Haraldsson B. Adriamycin alters glomerular endothelium to induce proteinuria.J Am Soc Nephrol. 2009; 20: 114-122Crossref PubMed Scopus (118) Google Scholar We observed that proteinuria induces dyslipidemia and hypersulfation of hepatic HSPGs, ultimately affecting HSPG-PCSK9 interaction and the localization of PCSK9 in the livers. Based on these data, we propose a novel mechanism and treatment strategy in proteinuria-induced dyslipidemia. Proteinuria was induced in 3-month-old male Wistar rats (weighing 180–200 g) by single injection of Adriamycin (1.8 mg/kg body weight), as described by Yazdani et al. (n = 8).35Yazdani S. Hijmans R.S. Poosti F. et al.Targeting tubulointerstitial remodeling in proteinuric nephropathy in rats.DMM Dis Model Mech. 2015; 8: 919-930Crossref PubMed Scopus (13) Google Scholar Healthy, age- and sex-matched rats with saline injection served as controls (n = 6). At week 12, organs were harvested after saline perfusion and preserved in liquid nitrogen. Creatinine in plasma and urine was measured by an enzymatic UV assay (Roche Modular P, Mannheim, Germany), and total urinary protein was measured by turbidimetric assay (Roche Modular P). Experimental procedures were performed according to the national guidelines for the care and use of laboratory animals,and approved by the local Animal Ethics Committee of the University of Groningen. Rat plasma samples were fractionated by fast protein liquid chromatography, as previously described by Bartuzi et al.22Bartuzi P. Billadeau D.D. Favier R. et al.CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL.Nat Commun. 2016; 7: 1-11Crossref Scopus (82) Google Scholar Hepatic lipoprotein receptors, PSCK9 and HS, were visualized by (confocal) immunofluorescence. Details on tissues, antibodies, and quantification of the stainings are given in Supplementary Table S2. Liver homogenates from control rats and Adriamycin-treated animals were used for immunoblotting, as described by Fedoseienko et al.21Fedoseienko A. Wijers M. Wolters J.C. et al.The COMMD family regulates plasma LDL levels and attenuates atherosclerosis through stabilizing the CCC complex in endosomal LDLR trafficking.Circ Res. 2018; 122: 1648-1660Crossref PubMed Scopus (42) Google Scholar Antibodies used were as follows: rabbit polyclonal anti-LDLR (1:300 in phosphate-buffered saline/1% bovine serum albumin; Pab8804; Abnova, Taipei City, Taiwan); rabbit monoclonal antibody (mAb) anti–LRP-1 (1:7500 in phosphate-buffered saline/1% bovine serum albumin; Abcam 92544, Cambridge, UK); and rabbit anti-mouse PCSK9 (552C; 1:300 in phosphate-buffered saline/1% bovine serum albumin), which was kindly provided by Jayson d'Horton (University of Texas Southwestern Medical Center, Dallas, TX).36Rashid S. Curtis D.E. Garuti R. et al.Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9.Proc Natl Acad Sci U S A. 2005; 102: 5374-5379Crossref PubMed Scopus (520) Google Scholar,37Hyock J.K. Lagace T.A. McNutt M.C. et al.Molecular basis for LDL receptor recognition by PCSK9.Proc Natl Acad Sci U S A. 2008; 105: 1820-1825Crossref PubMed Scopus (277) Google Scholar Goat anti-rabbit IgG horseradish peroxidase (DAKO, Heverlee, Belgium) was used as a secondary antibody. Proteins were isolated from liver cryosections of control rats and Adriamycin-treated rats and measured as described by Fedoseienko et al.21Fedoseienko A. Wijers M. Wolters J.C. et al.The COMMD family regulates plasma LDL levels and attenuates atherosclerosis through stabilizing the CCC complex in endosomal LDLR trafficking.Circ Res. 2018; 122: 1648-1660Crossref PubMed Scopus (42) Google Scholar A total of 100 μl of samples at concentration 0.625 μg/100 μL of total protein was blotted on a polyvinylidene difluoride membrane with the aid of a BIODOT (BioRad, Hercules, CA) device, and dot blot was done as previously described by Adepu et al.28Adepu S. Katta K. Tietge U.L.F. et al.Hepatic syndecan-1 changes associate with dyslipidemia after renal transplantation.Am J Transplant. 2014; 14: 2328-2338Crossref PubMed Scopus (10) Google Scholar As a positive control, recombinant syndecan-1 was spotted on the membrane. Wells without any spotted sample, but incubated with primary and secondary antibodies, served as negative controls. Total mRNA was isolated from liver tissues of control rats and Adriamycin-treated group using RNeasy mini kit (Favorgen Biotech Corp., Ping-Tung, Taiwan), as described by Yazdani et al.35Yazdani S. Hijmans R.S. Poosti F. et al.Targeting tubulointerstitial remodeling in proteinuric nephropathy in rats.DMM Dis Model Mech. 2015; 8: 919-930Crossref PubMed Scopus (13) Google Scholar Housekeeping gene was endogenous Gapdh. Transcripts for Ldlr, Pcsk9, Lrp-1, Syndecan-1, Sulf 2, enzymes involved in HS synthesis and degradation, and enzymes involved in cholesterol biosynthesis were measured (primers in Supplementary Table S1). All primers were purchased from Sigma Aldrich (St. Louis, MO), except Syndecan-1, Ldlr, and Sulf2, which were from Qiagen (Hilden, Germany). The cycle procedure was as described previously by Yazdani et al.35Yazdani S. Hijmans R.S. Poosti F. et al.Targeting tubulointerstitial remodeling in proteinuric nephropathy in rats.DMM Dis Model Mech. 2015; 8: 919-930Crossref PubMed Scopus (13) Google Scholar Negative controls are included using the samples without cDNA (polymerase chain reaction–grade water and mRNA sample mix where no reverse transcriptase was added while making cDNA). Measurements were done in triplicate. From each rat, ∼30 mg frozen liver tissue was collected. Five liver samples per group were pooled, resulting in 2 pooled samples (control rats and Adriamycin-treated rats). Extraction and purification of HS was performed as described previously in Ledin et al.38Ledin J. Staatz W. Li J.-P. et al.Heparan sulfate structure in mice with genetically modified heparan sulfate production.J Biol Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Disaccharide analysis of HS was performed by reverse-phase ion-pair high-performance liquid chromatography, as described by Hijmans et al.27Hijmans R.S. Shrestha P. Sarpong K.A. et al.High sodium diet converts renal proteoglycans into pro-inflammatory mediators in rats.PLoS One. 2017; 12e0178940Crossref PubMed Scopus (18) Google Scholar Disaccharide isolation and analyses of each pool were performed twice in triplicate. PCSK9 plasma levels in control rats and Adriamycin-treated rats were determined using rat PCSK9 enzyme-linked immunosorbent assay kit (SEK80005; Sino Biological Inc., Beijing, China). The ability of heparin/nonanticoagulant heparin derivatives to compete with heparin-albumin for binding to PCSK9 was evaluated by enzyme-linked immunosorbent assay. Details can be found in Supplementary Methods S1. Analyses were performed using GraphPad version 5 (La Jolla, CA). Mann-Whitney U-test was used for statistical significance between the groups. Data are given as means ± SEM. Associations were analyzed by nonparametric Spearman correlation. P < 0.05 was taken as statistically significant. Clinical parameters in control rats and Adriamycin-treated rats were assessed 12 weeks after saline or Adriamycin injection (Table 1). Adriamycin-treated rats developed proteinuria, without loss of renal function. They developed significant increase in plasma TG and TC (very-low-density lipoprotein cholesterol [VLDLc], LDLc, and HDLc) levels and in non-HDLc (VLDLc + LDLc) levels (Figure 1). Proteinuria was positively associated with plasma TG (r = 0.86; P = 0.0003) and TC levels (r = 0.96; P < 0.0001). Overall, herein, we show that proteinuria induced by Adriamycin causes hyperlipidemia in rats.Table 1Clinical characteristics of healthy controls and Adriamycin rats at 12 weeksParametersControls (n = 6)Adriamycin (n = 8)P valueBody weight, g472 ± 12446 ± 9NSBlood pressure, mm Hg Systolic blood pressure144 ± 7137 ± 5NS Diastolic blood pressure87 ± 789 ± 3NS Mean blood pressure108 ± 8106 ± 4NSRenal function Plasma creatinine, μmol/l26 ± 126 ± 1NS Creatinine clearance, ml/min4.8 ± 0.24.8 ± 0.2NS Proteinuria, mg/24 h50 ± 16343 ± 63aP < 0.005.Plasma lipid parameters, mmol/l Triglycerides1.49 ± 0.204.74 ± 1.29aP < 0.005. Total cholesterol1.58 ± 0.094.00 ± 0.76aP < 0.005.NS, no significant difference.Data represent mean and SEM.a P < 0.005. Open table in a new tab NS, no significant difference. Data represent mean and SEM. Because LDLR, LRP-1, and syndecan-1 are the major hepatic receptors for VLDL and LDL, we studied their expression by immunofluorescence staining, quantitative reverse transcriptase–polymerase chain reaction, and Western blot or dot blotting (Figure 2). We observed sinusoidal staining pattern, because all 3 receptors are present at the basolateral membranes of hepatocytes. No differences were observed in staining intensity or distribution (Figure 2a–f) and mRNA expression (Figure 2g–i) of all the receptors between the groups. Western blot and dot blot analysis confirmed no changes in total protein expression of all receptors (Figure 2j and k) between the groups. These data indicate that proteinuria does not affect expression of hepatic lipoprotein receptors. Despite no changes in hepatic lipoprotein receptors, Adriamycin-treated rats showed significant increase in serum TG and TC (VLDLc, LDLc, and HDLc) levels and in non-HDLc (VLDLc + LDLc) levels. Therefore, next, we investigated the expression of genes involved in endogenous cholesterol synthesis. We observed no significant differences in mRNA expression of Acat1, Acat2, Mvd, Hmgcr, Hmgcs1, Mvk, and Sqle (Supplementary Table S1) in between the groups. This excludes increased endogenous cholesterol production as a cause behind nephrotic dyslipidemia. Because PCSK9 hampers hepatic lipoprotein uptake by LDLR degradation, we measured plasma PCSK9 levels. No significant difference was observed (Figure 3a). Interestingly, however, plasma PCSK9 positively associated with plasma TG (r = 0.59; P = 0.03), TC (r = 0.71; P = 0.0061), and proteinuria (r = 0.63; P = 0.022), suggesting PCSK9 might be involved in proteinuric dyslipidemia (Figure 3b–d). Proteinuria did not affect the mRNA and protein levels of hepatic PCSK9 (Figure 3e and f). We also evaluated hepatic localization of PCSK9 in both groups by confocal immunofluorescence microscopy (Figure 3g). In proteinuric rats, PCSK9 was clustered and condensed in sinusoids compared with the finer granular cytoplasmic staining in control rats, indicating that proteinuria induces changes in the distribution of hepatic PCSK9. Because both PCSK9 and lipoproteins can bind to HS, next we investigated the effects of proteinuria on the structure of hepatic HS. First, we performed immunofluorescence staining using anti-HS mAbs 10E4 and 3G10. As reported before, anti-HS mAb 10E4 binds to HS domains having mixed N-acetylated and N-sulfated disaccharide units.39van den Born J. Salmivirta K. Henttinen T. et al.Novel heparan sulfate structures revealed by monoclonal antibodies.J Biol Chem. 2005; 280: 20516-20523Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar The mAb 3G10 reacts with HS neoepitopes containing unsaturated uronic acid residues, generated by digesting HS with heparitinase I.40David G. Bai X.M. Van Der Schueren B. et al.Developmental changes in heparan sulfate expression: in situ detection with mAbs.J Cell Biol. 1992; 119: 961-975Crossref PubMed Scopus (393) Google Scholar Both mAbs 10E4 and 3G10 marked the sinusoids of the liver. No significant changes in intensity and localization of 10E4 and 3G10 epitopes were observed between the groups, although 10E4 tended to be reduced and 3G10 tended to be increased in proteinuric rats (Figure 4a–d). Because reduced intensity of 10E4 HS might be the result of either lower expression levels of HSPGs, such as syndecan-1, or a lower total amount of HS (3G10 stains all HS chains), we calculated the expression of 10E4 relative to both syndecan-1 core protein and to 3G10. The relative expression of 10E4 to both syndecan-1 core protein and to 3G10 was significantly reduced in proteinuric rats (Figure 4e and f), indicating that the lower 10E4 staining is not due to lower syndecan-1 or lower HS