Introduction (491 words):
Chronic kidney disease (CKD) is a worldwide public health problem estimated to have a global prevalence of 13.4% (1). Patients with end-stage renal disease (ESRD) consume a disproportionate share of the healthcare resources (1). CKD accounts for major financial burdens to healthcare systems due to many associated comorbidities. CKD is associated with cardiovascular disease (CVD) (1). Reducing CVD events can help with reducing renal disease progression, mortality, and with improving quality of life. An identified cause for CVD events in CKD is dyslipidaemia (2). Dyslipidaemia can arise from several factors; in CKD, abnormalities in lipid metabolism and clearance seem to be major causes (2). Dyslipidaemia is associated with renal disease progression and has various negative consequences, including glomerular injury (2).
In the nephrotic patient suffering from dyslipidaemia, defective clearance of lipoprotein particles by various proteins, such as LDLR, LRP-1, and syndecan-1 (SDC-1), has been shown to lead to high levels of triglycerides (TRLs) (3). These proteins facilitate lipid uptake and subsequent lysosomal degradation, which occurs mainly in the liver, and thereby contribute to lipid homeostasis (4). PCSK9, an enzyme involved in degradation of LDLR, and enzymes involved with SDC-1 functioning, such as sulfatase, have been shown to be upregulated in CKD, leading to reduced uptake of TRLs from the circulation and further increasing the risk of CVD (5,6,7). Statins are currently the most widely used group of drugs to treat dyslipidaemia. In addition, it was found that statins reduce CVD incidence by 21-43%, which means it is not sufficient for many (8). Furthermore, statins upregulate PCSK9 levels in hepatocytes, which could limit its efficacy due to increased LDLR degradation (9). In addition, anti-PCSK9 monoclonal antibodies have been proven to be effective in treating dyslipidaemia. However, due to their high costs, they are not suitable for use in standard practice. Therefore, a cheaper alternative should be found.
In this study, we investigate the role of PCSK9 in lipoprotein receptor degradation using nephrotic disease models, which can be used to improve potential therapeutic targets. SDC-1 is a transmembrane heparan sulfate proteoglycan consisting of a core protein that provides attachment points for heparan sulfate (HS) chains. (10) In normal conditions, SDC-1 is important in selective lipoprotein uptake, contributing to the normal clearance of lipoproteins (4). In diseased conditions, however, sulfation of the side chains is observed to be decreased. (6)
Furthermore, SULF-2, an endosulfatase that removes sulfate groups, expression has been shown to be decreased. (6). Increased sulfation of the HS side chains may in turn lead to disrupted lipoprotein clearance by possible increased binding of HSPGs to PCSK9. It has been shown that HSPGs, such as syndecan-1, may present PCSK9 to LDLR in diseased conditions, thereby facilitating its degradation in lysosomes and further contributing to dyslipidaemia (11).
In this study, we investigate whether increased sulfation of syndecan-1 in CKD leads to a pathological gain of function: the ability of syndecan-1 to bind to PCSK9 and subsequently present PCSK9 to LDLR, facilitating LDLR lysosomal degradation.
Materials & Methods (1250 words)
Materials (179 words)
All animal procedures conducted previously to obtain tissue samples were in accordance with animal experimentation ethics board. Different rat models were implemented in this study to observe the levels of aforementioned proteins in healthy and CKD-model rats. Young Wistar rats with adriamycin-induced nephrotic disease (aYW) were used as experimental CKD models. Healthy young Wistar rats (hYW) were used as control. Uninephrectomised old Wistar rats (uOW) and uninephrectomised Munich-Wistar Frömter (uMWF) rats were used as experimental nephrotic disease models.
Antibodies and proteins
Various primary and secondary antibodies, as well as proteins, were used for these staining procedures. Please refer to appendix 1 for the complete list.
Equipment and software
The Cryotome Leica CM1950 machine was used for cryosectioning of the rat kidneys and livers. The Leica Microscope DM4000B equipped with a Leica DFC345FX camera and set at magnification 200x was used to visualise the immunofluorescent-stained sections under the fluorescent microscope. The Leica Suite Application version 4.8.0 software was used to capture digital images of the stained sections. The Leica Microscope DMIL LED was used to visualise the live HepG2 cells.
ImageJ version 2.2.0 software was used to quantify the level of immunofluorescence in the sections. GraphPad Prism version 7.0d software was used to carry out the statistical analyses of our results.
Methods (941 words)
Cell culture
To study the presence and interaction of various proteins in individual cells we used HepG2 cells as a model for human hepatocytes. The cells were cultured in cell flasks using Dulbecco’s modified eagle medium (DMEM) and were passaged at varying dilutions to maintain desirable confluence, allowing to perform further assays. Trypsin was used to promote cell detachment during passaging into different flasks. These cells were cultured in a humidified atmosphere at 37℃.
Cell immunofluorescence staining
Syndecan-1 expressed by the HepG2 cells was detected through immunofluorescence staining. To this end, HepG2 cells were cultured on coverslips and fixed with 4% paraformaldehyde in preparation for staining. Triton 0.1% was used to permeabilise the cell membrane. Cells were incubated for 1 hour with the following primary antibody: monoclonal mouse anti-human syndecan-1. Different dilutions of primary antibody for syndecan-1 were made with 10% normal rat serum (NRS) in PBS. The dilutions were done to optimise the signal sensitivity of the staining. Subsequently, cells were washed with PBS. All cells received a secondary antibody after addition of primary detecting antibody for the aforementioned protein targets. Cells additionally received a third antibody conjugate attaching to the secondary antibody. This additional second and third antibody allows for fluorescence signal amplification and improvement in signal detection sensitivity. In addition, a negative control lacking the primary detecting antibody was included for comparing staining. A PBS wash was performed after each antibody step. Finally, tyramide TRITC (1:50) is added to obtain fluorescence signal from the bound antibodies. DAPI was used for cell nuclear staining.
Immunofluorescence stainings on cryosections
Immunofluorescence stainings were performed to detect the cellular localisation of the proteins of interest (PCSK9, LDL-R, LRP-1, and Syn-1) on hYW (n=6), aYW (n=8), uOW (n=13), and uMWF (n=12) kidney cryosections. These cryosections were first air-dried on slides, fixed in acetone, and then rehydrated in phosphate buffer solution (PBS). Hydrogen peroxide and bovine serum albumin were used to block endogenous peroxidase and non-target proteins respectively to reduce non-specific binding. Next, the primary antibody was applied for 1 hour on all sections except the negative controls. The sections were washed in PBS and then the secondary antibody that contains the immunofluorescent tag was added to all the sections for 30 minutes. After washing, the fluorescent dye (TRITC) was applied as well as DAPI. The slides were illuminated under a fluorescent microscope and digital images were taken. Quantifying the mean immunofluorescence of the tissue stainings allows for measuring and comparing the protein expression levels in different conditions.
bFGF binding assays
Basic fibroblast growth factor (bFGF) ligand binding assays were performed to determine the extent of bFGF receptors present in the rat livers. bFGF is a known heparan sulfate proteoglycan. The higher the immunofluorescence detected on the sections, the more bFGF receptors present, which could indicate more ligand binding between bFGF and its receptors. This increased binding suggests increased sulfation of the GAG side chains. The hypothesis would be supported if more sulfation was observed in diseased conditions of the rat livers. This could be due to more bFGF receptors present or increased binding capacity in diseased conditions. (7)
The cryosections were air-dried, fixed in 4% formalin, and washed with TSM buffer. Hydrogen peroxide and bovine serum albumin were used to reduce nonspecific binding. The sections were incubated with bFGF for 2 hours, excluding the negative control. The sections were washed with TSM, and then all were incubated with the primary antibody for 1 hour, followed by the second and then third antibody for 30 minutes each. TRITC and DAPI were applied after washing. The slides were illuminated under the microscope, and digital images were taken.