Hypercholesterolemia in Progressive Renal Failure Is Associated with Changes in Hepatic Heparan Sulfate - PCSK9 Interaction

Abstract

Background: Dyslipidemia is an important risk factor in CKD. The liver clears triglyceride-rich lipoproteins (TRL) via LDL receptor (LDLR), LDLR-related protein-1 (LRP-1), and heparan sulfate proteoglycans (HSPGs), mostly syndecan-1. HSPGs also facilitate LDLR degradation by proprotein convertase subtilisin/kexin type 9 (PCSK9). Progressive renal failure affects the structure and activity of hepatic lipoprotein receptors, PCSK9, and plasma cholesterol.

Methods: Uninephrectomy- and aging-induced CKD in normotensive Wistar rats and hypertensive Munich-Wistar-Frömter (MWF) rats.

Results: Compared with 22-week-old sex- and strain-matched rats, 48-week-old uninephrectomized Wistar-CKD and MWF-CKD rats showed proteinuria, increased plasma creatinine, and hypercholesterolemia (all P<0.05), which were most apparent in hypertensive MWF-CKD rats. Hepatic PCSK9 expression increased in both CKD groups (P<0.05), with unusual sinusoidal localization, which was not seen in 22-week-old rats. Heparan sulfate (HS) disaccharide analysis, staining with anti-HS mAbs, and mRNA expression of HS polymerase exostosin-1 (Ext-1), revealed elongated HS chains in both CKD groups. Solid-phase competition assays showed that the PCSK9 interaction with heparin-albumin (HS-proteoglycan analogue) was critically dependent on polysaccharide chain length. VLDL binding to HS from CKD livers was reduced (P<0.05). Proteinuria and plasma creatinine strongly associated with plasma cholesterol, PCSK9, and HS changes.

Conclusions: Progressive CKD induces hepatic HS elongation, leading to increased interaction with PCSK9. This might reduce hepatic lipoprotein uptake and thereby induce dyslipidemia in CKD. Therefore, PCSK9/HS may be a novel target to control dyslipidemia.

Keywords: PCSK9; chronic kidney disease; dyslipidemia; heparan sulfate; syndecan-1.

Graphical abstract

Figure 1.

Cholesterol profiling by FPLC in Wistar-22W (n=6), MWF-baseline (25 weeks, n=14), and MWF-CKD (48 weeks, n=14) rats. (A) FPLC profiles for plasma cholesterol for Wistar-22W, MWF-baseline, and MWF-CKD rats. The dark lines indicate the mean; the light shades indicate SEM. (B) Fold increase in VLDLc, (C) in LDLc, (D) in HDLc, and (E) in non-HDLc. Non-HDLc was calculated as the sum of VLDLc and LDLc. Data shown as mean±SEM. *P<0.05, **P<0.005, ***P<0.0001.

Figure 2.

Plasma and hepatic PCSK9 expression. (A) Measurement of plasma PCSK9 in healthy, untreated Wistar-22W (n=6), MWF-baseline (25 weeks, n=14), and MWF-CKD (48 weeks, n=14) plasma. (B) Fold change in mRNA expression of PCSK9 in livers of Wistar-22W (n=6), Wistar-CKD (48 weeks, n=7), and MWF-CKD (48 weeks, n=7) rats. (C) Measurement of PCSK9 protein by Western blot. (D) Quantification of Western blots. (E) Immunofluorescence staining of hepatic PCSK9. (F) Quantification of immunofluorescence staining of hepatic PCSK9. (G) Univariate correlation analysis of PCSK9 immunofluorescence staining with plasma creatinine. Scale bars in photomicrographs represent 150 µm. *P<0.05.

Figure 3.

Expression of HS by anti-HS mAbs 10E4 and 3G10. Expression and quantification of HS by anti-HS mAbs (A and C) 10E4 and (B and D) 3G10 in livers of Wistar-22W, Wistar-CKD, and MWF-CKD rats. Univariate correlation analysis of HS-3G10 with (E) plasma creatinine and (F) TC. Scale bar represents 150 µm. **P<0.005, ***P<0.0001.

Figure 4.

Double staining of HS with sinusoidal endothelial marker LYVE1. Confocal immunofluorescence images of hepatic HS using anti-HS mAb 3G10 with sinusoidal endothelial marker LYVE1. DAPI represents nuclear staining. Scale bars in photomicrographs represent 50 µm. *P<0.05, **P<0.01. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5.

Elongated hepatic HS in both CKD models correlates with total cholesterol, PCSK9 plasma levels and PCSK9 binding affinity. (A) Hepatic HS disaccharides are expressed as the percentage of total disaccharides. (B) Total quantity of HS in the livers of healthy Wistar-22W, Wistar-CKD, and MWF-CKD rats measured by HPLC and expressed in nanograms per milligram of tissue. (C) Fold change in mRNA expression of EXT1 in the livers of Wistar-22W (n=6), Wistar-CKD (n=7), and MWF-CKD (n=6) rats. (D and E) Univariate correlation analysis showing associations between (D) EXT1 mRNA with TC, and (E) EXT1 mRNA with PCSK9 total protein. *P<0.05. (F) Competition of heparinoids with PCSK9 binding to immobilized heparin-albumin is critically dependent on polysaccharide chain length. LMW, low molecular weight.

Figure 6.

Confocal immunofluorescence double stainings of PCSK9 (red) with anti-HS mAb 3G10 (green). PCSK9 partly colocalizes with HS at the hepatic sinusoids in Wistar-CKD and MWF-CKD rats, but this is absent in Wistar-22W rats. Scale bars represent 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 7.

Reduced VLDL binding in liver sections of CKD rats. (A) VLDL binding assay on liver sections of Wistar-22W, Wistar-CKD, and MWF-CKD rats. (B) Quantification of VLDL binding. Scale bars represent 150 µm. *P<0.05.

Figure 8.

Proposed model representing potential novel mechanism for dyslipidemia in progressive CKD. (A) HSPG presenting PCSK9 to LDLR in normal conditions. (B) Formation of HSPG-PCSK9 dimer–LDLR complex due to hyperelongated state of HS in CKD.