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Diabetic nephropathy is one the major complications seen in diabetes mellitus. It is believed that lysozyme has nephroprotective functions against renal damage which is caused by AGE (advanced glycation end products). The purpose of this present study was carried was to investigate the molecular mechanism of action of lysozyme in-vitro as previous findings have shown lysozymes ability to reduce serum AGE in diabetic mice.
Methods: An in-vitro cell model was used for the study and cell viability assays (SRB assay which measures protein density and MTT assay which measures mitochondrial activity) were performed on the cell line (HK-2) Human proximal tubular cells. RT-PCR was performed to analyse the expression of RAGE (receptor AGE acts on) and IL-6 (pro inflammatory cytokine involved in diabetic nephropathy).
Results: The data obtained from the cell viability assays showed no increase in cell viability in the presence of lysozyme compared to that seen in the AGE control. All values appear random and show no trend and show no significance. RT-PCR results also show no change in mRNA expression in RAGE and IL-6 when co-treated with lysozyme and AGE
Conclusion: No findings in this current study showed that lysozyme has an effect on AGE in diabetic nephropathy.
Advanced Glycation End products
What effects AGE has
Other Sources of AGE
Other cell mediated responses
Inflammatory cell activation and cytokine productions
Current development of study
Previous methods and findings
Ethical and Safety considerations
Thawing cell procedure
Splitting of cells
Preparations of SRB & MTT assays and dilutions
RNA isolation from cells
Preparation of PCR
SRB (optical density)
Average for all SRB hours
SRB results for new aliquot
Previous MTT assay data
MTT for all averages
MTT results for new aliquot
Possible errors for SRB
Variance and Statistical Analysis
1.1 Diabetes mellitus
Diabetes mellitus is a long term common disease which is increasing in the number of cases every year worldwide; recent figures show that the number of people who have been diagnosed with this condition is now over 2.9 million people (Diabetes.org 2012). Patients could either suffer from type I diabetes, otherwise known as juvenile diabetes, which is due to the body's incapability of producing insulin due to the destruction of beta-cells in the Islets of Langerhans. Alternatively, the second type of diabetes is known as type2, which is due to insulin resistance and poor response to the hormone.
Diabetes mellitus occasionally leads to consequent micro/macro vascular complications. Some patients may be at risk of cardiovascular disease, atherosclerosis, diabetic retinopathy, diabetic leg, foot ulcers, frequent infections and diabetic neuropathy (Patient.co.uk 2012a).
1.2 Diabetic Nephropathy (DN)
One example of a complication, which is primarily being focused on in this study, is diabetic nephropathy. It must be noted that not all diabetic patients will develop diabetic nephropathy; according to (diabetes.co.uk 2012), around 40% of type I and type II patients will suffer from diabetic nephropathy. This condition is also one of the main causes of renal failure (Navarro and Mora 2006) otherwise known as end stage renal disease (ESRD). Diabetic nephropathy is a progressive disease and is categorised into 5 main stages (diabetes.co.uk 2012); stage5 of DN is a critical period for the patient where there is a need for dialysis or even a kidney transplant in extreme cases(Vatsal Anand,onlymyhealth.com).
Diabetic nephropathy can lead to micro albuminuria and then to proteinuria when the condition becomes more advanced (stage 5). However it must be said that not all patients with micro albuminuria will develop proteinuria, but are merely more at risk. Additionally, some patients can even revert back to their normal albuminuria state from the more advanced stage. Characteristics of DN also indicate a decrease in glomerular filtration rate (GFR) and this normally occurs once proteinuria has developed (Zelmanovitz et al. 2009).
Table 1 from (Zelmanovitz et al. 2009) showcases the albumin concentration present in urine samples. Macroalbuminuria (proteinuria) shows the most albumin excretion.
A marker in diabetic nephropathy is used to measure the albumin:creatinine ratio, when the (ACR) is found to be greater than or equal to 2.5 mg/mmol (men) or 3.5 mg/mmol (women), it is indicative of the patient is suffering from Microalbuminuria(Patient.co.uk 2012b) .
Different characteristics are seen between the two types of diabetes. Type I patients primarily develop the thickening of the glomerular and tubular membrane, due to increased amount of matrix protein in the structures (Zelmanovitz et al. 2009, Abrass, Peterson and Raugi 1988, Abrass 1995). Another characteristic is also mesangial expansion (Abrass 1995) which I shall be discussing in a greater depth later. Furthermore, in type I patients, structural changes in podocytes can be involved in the pathological changes whereas in type II patients, far more complex renal lesions are seen compared to that seen in type I sufferers (Zelmanovitz et al. 2009).
Increasingly visible symptoms of diabetic nephropathy are normally seen at stage 4 (out of 5) during the diseases progression. Some patients experience nausea and vomiting, feeling fatigued, darker urine due to blood being present and fluid retention causing swelling in the legs, hands and ankles (diabetes.co.uk 2012).
1.2.2 Risk Factors
Image 1 (Tan, Forbes and Cooper 2007) showing all the possible factors and interactions in the pathogenesis of diabetic nephropathy.
From image 1, we see that multiple factors can lead to diabetic nephropathy. Metabolic factors (discussed later), genetic factors, haemodynamic factors and environmental factors (smoking and dietary intake (discussed later)) can all contribute to the activation of intracellular signalling pathways and hence cause the stimulation of inflammatory mediators. Hyperglycaemia is known to be the main risk factor contributing to diabetic nephropathy, as it can cause the thickening of the basement membrane of the kidney and up regulates the expression of VEGF (vascular endothelial growth factor) in podocytes (present in the glomerulus) thus increasing the vascular permeability (Zelmanovitz et al. 2009). Another risk factor potentially causing nephropathy is arterial hypertension (Wetzels et al. 1986, Zelmanovitz et al. 2009). Diabetic patients who have a systolic pressure less than 120 mmHg are at less risk of developing macro/micro vascular complications (Adler et al. 2000) (Zelmanovitz et al. 2009).
1.3 Advanced Glycation end products (AGE)
In the article written by (Cocchietto et al. 2008), it is stated that there is evidence of Advanced Glycation end products (AGEs) formation and accumulation in the kidney, that can ultimately lead to the development of diabetic nephropathy, with the accompaniment of reactive oxygen species. This therefore explains why persistent hyperglycaemia is a major risk of diabetic nephropathy, as AGE is continuously formed irreversibly at a faster rate when more glucose is available (Zelmanovitz et al. 2009).
Advanced Glycation end products (AGEs) are a heterogeneous group of molecules (proteins, lipids, nucleic acids) that become glycated after exposure to sugars and can accumulate over time as a result of natural aging (Tan et al. 2007). AGE can also target other organs, amongst them is the kidney, where they can accumulate and damage the organ's normal functioning. AGE formation is significantly enhanced by on-going hyperglycaemia as previously stated and oxidative stress which causes further modifications of proteins (Tan et al. 2007).
1.3.1 Biochemistry of AGE
The initial processes of non-enzymatic glycation is through the covalent binding of ketone or aldehyde groups of reducing sugars to free amino groups of protein, resulting in the formation of Amadori products and Schiff base, called the Maillard reaction; the latter being the first initial product which spontaneously rearranges itself to form Amadori products (Basta, Schmidt and De Caterina 2004).
Image 2 from (Busch et al. 2010) showing how AGE is formed by the Maillard reaction and the duration of its formation.
Image 3 from (Forbes et al. 2003) showing the overall timeline and actions of AGE.
Carbonyl intermediates which are known as Î±-dicarbonyls(3- deoxy-glucosone, methyl-glyoxal and glyoxal) are formed and accumulated (referred to as carbonyl stress) during Amadori reorganisation (Goldin et al. 2006). These Î±-dicarbonyls are able to interact with amino, sulfhydryl, and guanidine functional groups in proteins which results in denaturation and cross-linking of the targeted proteins (Lo et al. 1994) (Goldin et al. 2006).
As seen from image 4, these carbonyl intermediates can also be formed via the Polyol pathway and via intermediate lipid metabolism. The polyol pathway involves a 2 step metabolic pathway, where glucose becomes reduced to sorbitol by NAPDH and then gets metabolised into fructose by sorbitol dehydrogenase (Lorenzi 2007). In turn, the Î±-dicarbonyls are able to undergo processes such as reacting with amino groups of proteins which then ultimately results in AGE compound formation: CML (N-Îµ-carboxy-methyl-lysine), N-Îµ-carboxy-ethyl-lysine (CEL), glyoxal-lysine dimer (GOLD) and methyl-glyoxal-lysine dimer (MOLD) and Deoxyglucosone Lysine Dimer (DOLD).
Image 4 from (Tan et al. 2007) showing the possible pathways for AGE formation.
As well as glycation, CML and Pentosidine can be formed via the oxidation pathway. An interesting note to take is that AGE can fluoresce intrinsically and can often be used as a marker for AGE accumulation detection resulting in easier diagnosis. Pentosidine is a well-known used marker as it has the ability to fluoresce the tissues and plasma. Pyralline can also be formed via the non-oxidative pathway (Peppa, Uribarri and Vlassara 2012).
AGE can also become metabolised within the kidney, particularly in the proximal tubule in-vivo and in-vitro (Busch et al. 2010, Peppa et al. 2012)
1.3.2 What affects AGE has on the kidney
AGE's can cause structural changes, for example continuous cross linking and accumulation in the tissue are known to be the cause of the complications seen in diabetes (Cocchietto et al. 2008). AGE can modify structural components of the basement membrane and extracellular matrix (ECM). ECM are prone to AGE modifications; between ECM proteins that are glycated, are covalent interactions (inter/intra molecular cross links ) which can cause changes in the membrane permeability, packing density and other structural factors (Tan et al. 2007, Forbes et al. 2003). AGE therefore prevents normal functioning of tissue as it can disrupt the cell matrix due to the accumulation of tissue macromolecules that have been modified by AGE (Mitsuhashi et al. 1997a). ECM component expression levels become elevated by AGE in a dose dependent manner (Forbes et al. 2003). As mentioned before, as well as the thickening of the glomerular membrane, mesangial expansion also occurs as hyperglycaemic levels causes a rise in ECM components such as fibronectin, laminins and collagen (Forbes et al. 2003) in the mesangium which provides structural support for the glomerular capillaries. This leads to an imbalance in the synthesis of these components and its degradation, thus causing expansion and reduction of glomerular filtration surface (Abrass 1995, Zelmanovitz et al. 2009).
Collagen type IV is a major protein component in the glomerular basement membrane; the non-enzymatic productions of glycation can bind to the collagen and alter its permeability leading to urinary albumin (Zelmanovitz et al. 2009).
1.3.3 Other sources of AGE
AGE may be formed exogenously and absorbed into our circulation due to certain intake of foods for example: food containing high levels of proteins, sugar and fat and also foods that are cooked in high fat content and are processed (Tan et al. 2007).
RAGE is a multi-ligand receptor at which AGE elicits its response through their interaction; it is a member of the immunoglobulin superfamily which belongs to the MHC class III (Sparvero et al. 2009)it consists of 394 amino acids, a single hydrophobic transmembrane domain which consists of 19 amino acids and a COOH terminal cytosolic tail which consists of 43 amino acids and is highly charged (Tan et al. 2007).
Each receptor has a various range of ligands present (Challier et al. 2005) . RAGE protein is found in human kidney tubular epithelial cells, mesangial cells and podocytes etc. and is expressed highly in areas where there is vascular injury. Once activated by the binding of AGE, it activates various signalling transduction cascades; it can also down regulate generations of reactive oxygen species (ROS) and the activation of transcription factors such nuclear factor kappa B (NF-Ä¸B) and protein kinase C. When the transcription factor NF-Ä¸B or any other pathways become induced by AGE-RAGE interaction, pro-inflammatory cytokines such as IL-6(Navarro and Mora 2006) are released, as well as the expression of the adhesion molecules and growth factors which are heavily involved in complications of diabetes (Tan et al. 2007).
1.3.5 Other cell mediated responses AGE elicits
188.8.131.52 Inflammatory cell activation and cytokine production.
AGE can contribute to the release of pro inflammatory cytokines, growth factors and adhesion molecules (Forbes et al. 2003). Pro inflammatory cytokines such as IL-6 are known to be involved in the pathogenesis of diabetic nephropathy. It was measured in patients that suffered from an increase in the glomerular basement membrane that IL-6 levels were elevated; fibrinogen and IL-6 is the cause of the thickening of the glomerular basement membrane (Navarro and Mora 2006). A study carried out by (Navarro and Mora 2006) on diabetic mice that exhibited diabetic nephropathy, showed that there was an increase in the molecule ICAM-1 (intracellular adhesion molecule-1). ICAM-1 is a protein that is encoded by the ICAM1 gene and induced from cytokine release stimuli (IL-6 & TNF- Î±). ICAM-1 promotes the invasion of leukocytes and macrophages (inflammatory cells) to the glomeruli thus causing inflammation.
184.108.40.206 Oxidative stress
As mentioned before, oxidative stress is another key factor that plays a role in the development of diabetic nephropathy (Coughlan, Mibus and Forbes 2008). Oxidative stress is when there is an imbalance of reactive oxygen species produced by cellular metabolism and the antioxidant defences that removes the ROS. There is a build-up of excess free radicals as the antioxidant defences fails to eliminate the ROS; the free radicals become destructive as they attack nucleic acids, lipids and proteins thus causing oxidative stress (Tan et al. 2007). Oxidative stress can come from various sources such as NADH oxidase, mitochondrial pathways which become deficient and auto oxidation of glucose (Coughlan et al. 2008).The main radical which is known to be involved in the complications associated with diabetes mellitus is O2-. Studies have proven that oxidative stress and AGE modified proteins are prominent in human glomerular lesions in a diabetic, which leads to the conclusion that oxidative stress may cause cross linking to tissue and formation of AGE (Tan et al. 2007).
1.4 Development of the current study
The factors which have been discussed are all known to be involved in the pathogenesis of diabetic nephropathy. This has led to investigations of substances that have the ability to reduce AGE formation. Many interventions have been explored for targeting AGE, for example reducing dietary AGE and AGE formation inhibitors etc. (Tan et al. 2007).
(Zheng et al. 2001, Cocchietto et al. 2008, Mitsuhashi et al. 1997a) all explore the idea of whether Lysozyme is involved in reducing AGE.
In (Cocchietto et al. 2008) study, they have hypothesised that orally administered microencapsulated lysozyme can down regulate serum AGE thus reducing the severity of diabetic nephropathy and that microencapsulated lysozyme has a better efficacy to that of free lysozyme. (Zheng et al. 2001)study have hypothesised that lysozyme can increase in-vivo AGE clearance and supress macrophage recruitment in-vitro and hence improve microalbuminuria treatment. Also (Mitsuhashi et al. 1997a) have hypothesised the depletion of AGE from diabetic uremic sera can occur by using a lysozyme matrix.
Lysozyme is an antibacterial enzyme which is present in tears, egg white, saliva and other body fluids such as nasal secretions, mucus, serum etc. (Mitsuhashi et al. 1997b) (dictionary 2012).
It breaks down the bacterial cell wall by catalysing the hydrolysis of 1,4-beta-glycosidic linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan in cell walls (sigma-aldrich 2012). It contains alpha and beta folds which contains five to seven alpha helices and a three-stranded antiparallel beta sheet and has 129 single residues (College 2012).
Lysozyme contains an AGE-specific binding cysteine bounded domain otherwise known as (ABCD motif) which allows the lysozyme to sequester AGE. This hydrophilic motif can be active in-vitro as well as in-vivo, it is able to interact and shield AGE molecules thus preventing further production of ROS(Schmidt et al. 1999) (Zheng et al. 2001). Lysozyme is also able to bind to AGE at a high affinity (KD=50nM) (Mitsuhashi et al. 1997a, Zheng et al. 2001, Li, Tan and Vlassara 1995) and is able to enhance AGE uptake by macrophages (Zheng et al. 2001).
1.6 Previous methods and findings
(Zheng et al. 2001)research article investigated the effects of lysozyme on AGE removal in vivo when lysozyme was injected into the non-obese mice, db/db(+/+) (model mice which models diabetes and obesity) and last of all non-diabetic AGE infused rats. The main result of this study showed that administered lysozyme reduced endogenously-formed serum AGE and increased renal AGE excretion. Lysozyme was administered into the mice and serum levels of AGE were assessed, it showed that there was a decrease in serum AGE levels. Also urinary albumin levels were tested in both mice and lysozyme treatment declined urinary albumin excretion; clearance of AGE was significantly increased when AGE and lysozyme were administered together. The in-vitro studies also showed the increase in macrophage binding, endocytosis and degradation of AGE and this was done using cell assays, the results shows a possible sequestration of AGE. mRNA levels for inflammatory mediators TNF-Î± and IGF-1 were analysed using PCR method and a range of primers were used. AGE mediated macrophage gene induction was also suppressed in the presence of lysozyme.
Again with the studies by (Cocchietto et al. 2008) a similar result in AGE clearance was seen in-vivo. It also showed in this case that RAGE was down regulated in the presence of lysozyme meaning less AGE is able to elicit its action through this receptor. The study showed the effects of orally administered lysozyme microencapsulated as chitosan-coated alginate microspheres in vivo has better efficacy of that of non-encapsulated lysozyme. The idea of encapsulated lysozyme significantly reducing serum AGE levels in vivo is better than that of free lysozyme is currently being further studied by the researchers in labs.
In-vitro studies have been performed by (Mitsuhashi et al. 1997a) where they have hypothesised the depletion of reactive AGE from diabetic uremic sera using a lysozyme-linked matrix, they also used AGE derived from in-vitro practices and identified the AGE proteins that are susceptible to AGE modifications. As they have found lysozymes ability to deplete AGE, it gives us a good foundation and background for our current in-vitro study.
1.7 Current Study
It is from the previous studies and findings that we now want to develop our study to investigate and comprehend a possible mechanism of action of lysozyme. Previous studies have been performed in-vivo where AGE serum levels were tested and analysed; we now want to perform in-vitro studies by using an in-vitro model. An in-vitro study (Mitsuhashi et al. 1997a) was previously done however was only performed on diabetic sera and not on renal cells and a mechanism of action was not established. An in-vitro model is specifically used to investigate a mechanism of action of a substance and provides a cheaper and faster alternative to in-vivo testing. However it doesn't provide homeostatic mechanisms and pathways that are found in animals, in-vivo studies represent more similar responses to that in humans, however it is not sure that exactly the same response will be seen (GALLO 2012). Due to ethical issues involved in-vivo testing, we want to investigate whether lysozyme has the ability to improve cell viability in the presence of AGE using cell viability assays which will be performed in-vitro. We want to observe whether lysozyme co-treated with AGE can affect mRNA expression of RAGE and IL-6, this is a similar to the previous studies in literature by using real time PCR method. Lastly we want to observe macrophage invasion in renal cells when co- treated with lysozyme and AGE, again similar to previous work (Zheng et al. 2001), which showed lysozymes ability to supress macrophage. Preliminary studies were performed to select an appropriate cell line in-vitro model for the studies; the reasons for selection will be discussed later. All results obtained from the cell viability assays and invasion assays will be analysed in a spectrometer at a wavelength of 570nm. A Corbett Rotor-6000 is the instrument used for the RT-PCR method.
The aims that are being explored in this project are to understand the molecular mechanisms involved in the protective action of lysozyme in diabetic nephropathy and to analyse their scavenger like activities of AGE (advanced glycation end products) in vitro.
3 Ethical and Safety considerations
As all experiments have to be carried out in sterility, many precautions had to be carried out to maintain the sterility of the experiments and equipment and the safety of the people carrying out the experiments.
As most of the experiments are carried out in the hood the following precautions should be taken:
Gloves should be worn at all times to avoid skin contact with substances.
Disinfectant should be used on gloves each before opening the incubator and before working under the hood.
Lab coat must be worn at all times to avoid toxic substances and dyes going on clothes and on skin.
Hair should be tied up.
Sterile components should be only opened and closed in the hood.
Before opening and closing bottles under the hood, the rim of the bottle and the lid should be flamed under the Bunsen burner.
Lids should be closed tightly and wrapped around with Para film only under the hood.
Substances such as DMSO need to be disposed correctly as DMSO is toxic.
When using substances such as DMSO, it should be used under the hood with a fan extractor as it could be inhaled.
Vials and plates under the hood should not be left open when taking it out of the hood as it could cause contamination and destroy the experiment.
The incubator should not be left open as it could contaminate all cells in flasks in the incubator.
Bleach should be used when flasks are contaminated and then should be disposed of.
All waste should be disposed in toxic waste bin and not in normal waste bins.
While carrying out experiments under the hood, a slight distance should be kept between the chair and the hood in case any substances spill on you.
Chairs should be tucked in after each experiment under the hood to avoid tripping over.
Every week the lab should be cleaned with disinfectant: the microscopes, surfaces, tables, hood, centrifuge to avoid contamination.
After every experiment under the hood, the area must be cleaned with disinfectant through ugly to avoid contaminations into other experiments.
Before the opening of the hood, the fan should be turned on and U.V light should be turned off for the hood to remain sterile.
The gas tap should be turned on first for the Bunsen and then should be lit.
If gas is smelt then gas should be immediately turned off via the main tap.
When closing the lid of the hood, gas tap and Bunsen tap must be switched off and any waste must be removed.
Pipettes must be cleaned with disinfectant before using under the hood to avoid contamination.
Vials in the fridge containing components should be kept in a rack to avoid it falling and spilling on the floor.
Pipette tips must be disposed in its designated disposal container.
4.1 Thawing cells process
When this process was performed, a new aliquot of cells were needed for the experiments. The aliquots of cells were stored in the liquid nitrogen and then thawed and split so this cell line will be ready for the experiments.
20mL flask was labelled with the cell line, the date of the aliquot of frozen cells that we will be split and the date of the split
20mL of the HK-2 control medium was put in the flask. The components of the HK-2 are seen below in the splitting method. The medium dilutes the DMSO (Dimethyl sulfoxide) when freezing a new aliquot.
Water in a tray was placed in an incubator to allow the water to become warmer for the defrosting of the frozen cells that are already in the liquid nitrogen.
The vial containing the HK-2 cells in the liquid nitrogen tank is taken out and was put in the tray containing water to allow the cells to defrost.
Once it became defrosted, a small amount of cells was pipetted and put into the medium in the flask.
The pipette then drew up the medium with the cells remaining in the pipette, and was pipetted back and forth into the flask and all most of the content in the vial was fully suspended in the medium.
To make sure the cells are present are in the flask, the flask was checked under the microscope.
The flask was rubbed with alcohol on the bottom and placed in the incubator. It remained in the incubator until a significant number of cells have grown and they have become confluent. Once they are, it is ready to split.
A small amount of cells were then frozen in another vial in DMSO and frozen in the liquid nitrogen.
4.2 Splitting of cells on human kidney proximal tubular epithelial cells (HK-2)
This process was performed every time before an experiment was done; as cells continue to grow and use up vital nutrients in the culture medium, they will begin to die if there no available nutrients for the growing cells. Cells needed to be split when they become confluent; this normally takes 2-3 days after it being split.
All steps in this method were performed under the sterile hood. When opening and placing flasks in the incubator, disinfectant must be used on the gloves and on the bottom of the flask. When opening and closing of components, the lid and vial must be flamed and shut with Para film to keep it sterile.
Empty flask was seeded with 5mL of medium and labelled with the cell line used how many cells you wish to later seed in the flask and the date of the splitting procedure.
If medium is not made then prepare new 50 mL of medium with the following components:
500µL ([ ]i=200nM)
25µL ([ ]i=10mg/mL)
5µL ([ ]i=50mg/mL)
T3 5 pg/mL
2.5µL ([ ]i=1mg/mL, diluted 1:10000)
Sodium selenite 5ng/mL
1µL ([ ]i=250µg/mL)
1µL ([ ]i=250µg/mL)
1µL ([ ]i=500µg/mL)
2mL of medium was added into the vial and label.
The previous cells that were split before and were already in the incubator were taken out of the incubator and its medium that was in the flask was discarded in a waste beaker.
2mL of Phosphate Buffer Saline (PBS) was added into the empty flask and washed around the surface of the flask.
The PBS was discarded in the waste beaker.
2mL of Trypsin (diluted from 10x to 1x) was added to the flask with a 5 mL pipette and the flask was in the incubator for 1 minute.
The same pipette was used to pipette up and down to make sure the cells became detached on the surface of the flask, when the cells began to detach, the trypsin liquid became an opaque colour.
With the vial containing the medium, 2mL of trypsin was placed with the cells into this vial.
With another 5mL pipette, 2mL of PBS was measured and put in the empty flask to wash the flask and any remaining cells that were left were collected. Again the pipetting up and down technique was done on the surface to make sure all the cells became detached and washings were put into the vial.
To ensure that all the cells became attached, the empty flask was checked under the microscope.
On the weighing scales, the vial was balanced with another vial containing water and both vials were faced opposite each other in the centrifuge which was precooled to 4°c beforehand, it was centrifuged at 1290 rotations per minute for 7 minutes.
After the 7 minutes was completed the Bürker chamber was prepared with the clear slide on top of the chamber.
The medium that was present in the vial was discarded, ensuring that the pellet of the cells that were present at the bottom of the vial was not discarded with the medium.
The vial was flicked a few times to re-suspend the cells in the little medium that was left and then some medium was added into the vial (approximately 400µL or more).
The total amount of medium present in the vial was measured and the cells were re -suspended in the medium using a P200 pipette ensuring that the cells were fully re-suspended in the medium.
Then with the P20, 10µL of this was taken and put on each side of the Bürker chamber at a 90° angle ensuring that the clear slide on the top did not move.
The cells were counted on the chamber and 4 regions were counted in each chamber. It doesn't necessarily mean only the 4 corners need to be counted, if not a lot of cells are present then you can select another 4 square regions but the cells in same 4 square regions will have be counted in the other chamber.
The volume needed to seed a certain number of cells in a new flask was calculated.
The required quantity of cells was taken from the vial and pipetted into the new flask in a slanted angle into the medium.
This flask was placed in the incubator making sure the bottom of flask was rubbed with disinfectant beforehand.
4.3 Preparation of SRB assay and MTT assay and dilutions
Plates were seeded with HK-2 cells in wells of 96 with medium for HK-2 which includes 0.1% BSA but with no FBS, the BSA doesn't induce the growth of the cells so this was used for medium in the wells.
We wanted 5000 cells in 100µL (so this is each well)
There are 64 wells in 1 plate and there were 3 plates all together so there is a total of 192 wells.
19200µL was the overall volume of medium but was rounded to 20mL for excess amounts.
5000 cells x 192 wells= 96 0000 cells in 19.2mL
However we wanted this for 20mL so the number of cells we require is 1000000 cells.
Plates were left for 48 hours then the 72 hours plate is treated with the AGE and LZ dilutions calculated below. 48 hour plate is then treated a day later and the same for the 24 hour plate.
Dilutions for treatment on cells for 3 plates:
As 4 wells were used for each condition, 100µL was put in each well so a total of 400µL was used.
These are the following conditions the cells were treated with:
AGE 1µM, 10µM, 20µM
LZ 1µM, 10µM, 20µM
AGE 1µM+LZ 1 µM, AGE 1 µM+LZ 10 µM, AGE 1 µM+ LZ 20 µM
AGE 10 µM+LZ1 µM, AGE 10 µM +LZ 10µM, AGE 10 µM + LZ 20 µM
AGE 20 µM+LZ1 µM, AGE 20 µM+ LZ 10 µM, AGE 20 µM+ LZ 20 µM
These dilutions were prepared from stock solutions of AGE and Lysozyme. The AGE is at its initial concentration of 151. 52µM and the Lysozyme of 357 µM. First the volume for each condition was calculated before the stock solution is made.
The control for HK-2 without FBS and plus BSA was used as the control. This was the scheme of the plates that we have used for the assays:
AGE 1 µM+LZ 10 µM
AGE 1 µM
AGE 1 µM+ LZ 20 µM
AGE 10 µM
AGE 10 µM+LZ1 µM
AGE 20 µM
AGE 10 µM +LZ 10µM
LZ 1 µM
AGE 10 µM + LZ 20 µM
LZ 10 µM
AGE 20 µM+LZ1 µM
LZ 20 µM
AGE 20 µM+ LZ 10 µM
AGE 1µM+LZ 1 µM
AGE 20 µM+ LZ 20 µM
As 400µLwas amount used for each condition, it was multiplied by 3 as 3 plates are being used and an additional 200µL was being added for excess. This equals to a total of 1400µL for the treatment of AGE and LZ controls (not the co-treatments).
A 1-4= 100µl x 4= 400µl. For 3 plates x 3= 1200µL
B 1-4= the volume was calculated using initial concentration x initial volume = final concentration x final volume formula. 1400µL x 1µM/151.52µM= 9.24µL and 1.4mL of medium was required for the dilution.
C 1-4= 1400µL x 10µM/151.52µM= 92.4 µL. 1.3mL of medium.
D 1-4= 1400µL x 20µM/151.52µM=184.8 µL. 1215.2µL of medium.
E 1-4= 1400µL x 1µM/357µM=3.92µL. 1396.08µL of medium.
F 1-4= 1400µL x 10µM/357µM=39.2µL. 1360.8µL of medium.
G 1-4= 1400µL x 20µM/357µM=78.4µL. 1321.6µL of medium.
For the co-treatments, the calculation was different as each concentration is needed 3 times for each plate. As we used AGE and lysozyme combined in one well, we used 50µL of each to make it up to 100µL in each well; therefore the concentration had to be doubled when calculating the volumes.
50µL of each x4= 200µL x by 3 for treatments in one plate =600µL. This was multiplied by 3 for 3 plates which gives a total of 1800µL and an additional 200µL is added for excess which gives a total of 2000µL of each concentration that needs to be made. (note: concentrations are doubled)
H 1-4 = 2000µL x 2µM/151.52µM= 26.4µL. 1973.6µL of medium.
A 5-8= 2000µL x 20µM/151.52µM=264µL. 1736µL of medium.
B 5-8= 2000µL x 40µM/151.52µM=528µL. 1472µL of medium.
C 5-8= 2000 µL x 2µM/357µM= 11.2µL. 1988.8µL of medium.
D 5-8= 2000 µL x 20µM/357µM= 112.04µL. 1888µL of medium.
E 5-8= 2000 µL x 40µM/357µM= 224µL. 1776µL of medium.
TOTAL of AGE required: 1104.84µL
TOTAL of LZ required: 468.76µL
Starting concentration of AGE= 10mg/mL
Starting concentration of LZ= 5mg/mL
Required concentration of AGE= 15mg/1500µL
Required concentration of LZ= 6mg/1200µL
To prepare the stock solution for AGE, 15mg was measured out and dissolved in sterile 1.5 ml of PBS in a 2 mL vial. To prepare the stock solution for LZ, 5mg was measured out and dissolved in sterile 1 ml PBS in a 2 mL vial. Both vials were put on the vortex until all the substance is dissolved in the PBS. From this stock solution, the calculated volume of AGE and LZ were mixed with the calculated medium to make up the various dilutions. These were then kept in the fridge and taken out just before the treatment. Each time this was used, it should be vortexed to ensure no substance settled to the bottom of the vial and that it was all evenly dispersed.
4.4 SRB Assay
This assay involved the use of Sulforhodamine B which is a protein dye that can electrostatically bind to protein basic amino acid residues of TCA fixed living cells. This assay determines the protein density within the cell (Voigt 2005).
The medium was discarded in the wells with a multichannel pipette and the wells were washed with 100µL of non-sterile PBS.
50µL of TCA is put in every well. TCA was used to fix the cells at the bottom of the cell.
The cells were left in the TCA solution for 1 hour in the fridge at 4°c, this allows the cells to sufficiently fixed at the bottom of the well.
After an hour, the TCA was discarded using the multichannel pipette.
The plates were then washed with 200µL of distilled water, the water was chucked away in to the sink and then plate was patted dry on the tissue paper. This was done 5 times.
We then waited until the plates were completely dry and sometimes were put under sunlight to allow the drying process to be quicker.
When the plates were completely dry, the cells were coloured with SRB solution (Sulforhodamine B which is a protein dye that can electrostatically bind to protein basic amino acid residues of TCA fixed living cells)
To make SRB solution, 400mg of SRB should be dissolved in 100mL of 0.4% acetic acid, rotate on agitator to dissolve all of SRB in solution.
The cells were left with SRB for 30 minutes at room temperature.
After 30 minutes, the SRB was discarded.
The plates were then washed 3 times with 1% acetic acid. 400µL was used in the first wash then the second and third 200µL was used. After each wash, the acetic acid was discarded in the sick and patted dry on tissue paper before the next wash.
A cotton bud was used to clean the walls of the wells for a better wash.
The plates then had to become completely dry.
Once dried, we used 100µL of 10mM of Trisbase at pH 10.5 in every well.
Using a multichannel pipette, the solution was pipetted up and down in each well so the substance in the well were all dissolved in Trisbase solution.
To prepare Tribase solution, 157.6mg was dissolved in 100ml of water and small drops of NaOH was added to adjust the pH to 10.5, the rest of the solution was made up with water. The pH was tested using a pH tester.
The plates were read on the spectrometer at the wavelength of 570nm.
4.5 MTT assay
The MTT assay is a method whereby viable cells are determined. It involves measuring the reduction of yellow MTT by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. Reduction occurs in living cells so cell viability can be measured (Sylvester 2011).
The same dilutions were prepared as earlier however this time we wanted to investigate whether AGE and lysozyme pre-incubated before treatment will have any effect on the cells as previous MTT, AGE and LZ were not pre incubated.
3 plates were used, 1 hour, 2 hour and 4 hours. The co treatments were pre incubated (with AGE and LZ together) for 1 hour and then after cells were treated with this in one plate, the dilutions were then pre-incubated for another hour then cells in another plate were treated with this. We then waited another 2 hours and the cells were treated in the 4 hour plate. All plates were left for 24 hour incubation. The plate scheme used in SRB was used in this assay also.
MTT was stored in a sterile condition because if contaminated, the organisms can metabolise the MTT.
An automatic tip was used and 10µL of MTT was put in every well. The 1 hour plate was treated with MTT first, then an hour later the 2 hour plate was treated, 2 hours later was the 4 hour plate.
The plates were then placed in the incubator and we waited for 4 hours from time of treatment of each plate.
After incubation, we checked the places under the microscope to see purple crystals were present in the well.
The medium present in the wells were aspirated using the multichannel pipette.
If there was any residue remaining on the walls of the wells, we used cotton bud to clean the walls.
200µL of DMSO (Dimethyl sulfoxide) was added into each well and was pipetted up and down to dissolve the crystals.
If there was a drop that remained in the tips when pipetting a new condition, then the tips had to be changed to avoid contamination.
2 wells H11 and H12 contained 200µL of DMSO and these wells were set as the blanks.
The plates were read in spectrometer at 570nm.
After the plates were read, the waste was thrown into a waste beaker and not the bin as the DMSO is toxic.
4.6 Real- time PCR for ADMEC cells
RT-PCR is a technique where it is possible analyse mRNA production. RT-PCR was performed to analyse RAGE and IL-6 mRNA. RAGE is the receptor that AGE works on. IL-6 is a pro & anti-inflammatory cytokine which is secreted by macrophages and T cells to stimulate an immune response. Inflammation plays a critical role in diabetic neuropathy and it is known that IL-6 is a cytokine involved in this. RT-PCR consists in two phases: the isolation of mRNA and the following amplification of a gene of interest.
4.6.1 RNA isolation from cells
The medium was aspirated from the wells and cells were washed with 400µL of PBS.
400µL of RNA lysis buffer was added in each well. It was pipetted up and down in each well to ensure complete lysis had occurred.
The lysates were put in a green vial which is able to block DNA.
The green vials were then put in the centrifuge for 12,000g speed for 1 minute at room temperature. The flow through with RNA is collected.
Equal volume of 70% of ethanol was added (400µL) and was pipetted up and down for it to mix.
This lysate was added in the new 2mL tube of the orange columns which is the perfect bind RNA column.
This was centrifuged at 10,000g for 1 minute at room temperature.
The flow through in the orange columns was discarded.
500µL of RNA wash Buffer I was added and was centrifuged at 10,000g for 15 seconds at room temperature and the flow through was discarded.
600µL of RNA wash buffer II was added and centrifuged at 10,000g for 15 seconds and discarded.
The flow through was discarded but the collecting tube was kept. This was centrifuged again at 10,000g for 1 minute at room temperature for drying.
The collecting tube was discarded but the orange column was placed in a 1.5mL sterile tube.
50µL of sterile RNAse free dH20 was added in the orange column.
This was centrifuged again at 5000g for 1 minute at room temperature for elution.
RNA was collected in the sterile tube and the column was discarded.
1µL of RNAsi- inhibitor was added and kept at -80°c.
4.6.2 Preparing the PCR
4µL buffer 5x concentration(total is 20 µL so diluted 5x to 1x), 1µL Reverse Transcriptase and 11µL water DEPC (Diethyl pyrocarbonate) were needed for the procedure. The total of components equal to 16µL which was put in a 0.5mL sterile vial.
4µL of mRNA was put into each vial.
The vials were put in a thermo-cycle regulator for an hour where the temperature would fluctuate from high to low temperature; this was done to enable the reverse transcriptase to work.
We obtain cDNA from the mRNA however we would only obtain 0.5µL which is a very low amount so we then amplify to obtain more cDNA.
A primer is used for this procedure however we want to dilute the primer 1:10. So 2µL of primer and 18µL if water DEPC was used to dilute the primer.
We wanted to analyse RAGE, IL-6 and 18-S (housekeeping gene) expression so there are individual forward and reverse primers for each primers. Only 0.5 µl of cDNA were required in a total volume of 10 µl.
Solution for the analysis: 5µL of master mix buffer
0.5µL of primer forward
0.5µL of primer reverse
3.5µL of H20 DEPC
0.5µL of cDNA.
8) The vials were put in the Corbett Rotor-6000 to performed the RT-PCR.
5 Statistical Analysis
Experimental data were analysed through computer assisted ANOVA statistical analysis software using Graph pad software. Anova + Dunnet Post Test was used. Differences of P<0.05 were considered to be significant.
6.1 SRB assay optical densities measurement
Table 1: This table shows the optical density for all incubation hours measured in the spectrometer at 570nm. The table also shows the average and standard deviation of the optical densities for each hour. 24 hours was performed 4 times and 48 and 72 hours were performed 3 times. P value <0.9999 i.e. differences are not significant.To determine the cell count, we performed an SRB assay on the HK-2 cells after treated with lysozyme and AGE. The optical densities were measured using a spectrometer for 24 hours 48 hours and 72 hours incubation time. Table 1 shows the individual experiment optical densities and their mean and standard deviations, the results read that the densities are of similar values and show no differences between the control condition and the treatments. Evidently, the variances between all experiments performed for each incubation hour are relatively high as the standard deviations (SD) are large compared to the mean; therefore we can conclude an unreliable set of data was maybe obtained. The P values obtained show no significant difference in the optical densities measurements between any of these test conditions.
Table continues overleaf
Treatments for SRB assay (concentrations in µM)
AGE 10+ LZ 1
AGE 10+ LZ 10
AGE 10+ LZ 20
AGE 20+ LZ 1
AGE 20+ LZ 10
AGE 20+ LZ 20
Optical densities for Incubation time: 24 Hours
Incubation time: 48 Hours
Incubation time: 72 Hours
6.1.2 Pooled data for 24, 48 and 72 hours SRB assays showing the test of control expressed as a percentage of control.
Table 2 shows the pooled data for the incubation hours expressed as percentage difference from the control. The control represents 100% viability; however AGE controls show an increase in viability compared to that of the control, the co-treatments with lysozyme with AGE show no significant improvement in cell viability compared to AGE control percentage. The random large standard deviations again show the high variance between experiment percentage values. ANOVA statistical test also shows values are not significant.
Treatments (concentrations in µM)
Percentage difference from control values
Table 2: This table shows the average percentage difference from the control for SRB assay for 24, 48 and 72 hours. The co treatments with AGE + LZ show random positive and negative viability values. The standard deviations are fairly high for some treatments showing there were big variations between each experiment. All values are P>0.9999 i.e. differences are not significant.
The graph shows that lysozyme does not affect the viability of cells as results show that the co treatments with AGE and lysozyme appear very random. The AGE controls in fact show an increase in protein density, this was not expected as AGE is known to be damaging to cells so the number of cells should be lower than that of the control. What is unusual is that the highest concentration of AGE 20µM and the lowest concentration of lysozyme seemed to have increase cell viability and LZ 20µM
Figure 1: This graph shows the percentage difference from control for SRB assay for 24, 48 and 72 hours. The 24 hours was done 4 times whereas 48 and 72 hours were performed 3 times. The results appear very random as there is no trend seen between the incubation hours. The AGE controls and LZ controls show no trend; nor do the co-treatments.
6.1.3 SRB results for new aliquot of HK-2 cells
Table 3: This table shows the average percentage viability vs the control for SRB assay for 24, 48 and 72 hours. However the SRB assay was done on new aliquot HK-2 cells as the previous results did not show a trend. These results again show no trend between incubation hours and the AGE controls and LZ controls show no trend nor do the co-treatments. The experiment was not repeated. The graph is below. All values are P>0.9999.
Figure 5: This graph shows the average percentage viability vs the control for SRB assay for 24, 48 and 72 hours. However the SRB assay was done on new aliquot HK-2 cells as the previous results did not show a trend. These results again show no trend between incubation hours. The AGE controls and LZ controls show no trend nor do the co-treatments.
6.2 Previous MTT assay data for 72 H
Percentage viability vs cells in medium
Figure 6: This table and graph shows the average percentage viability vs the control for MTT assay for different concentrations of AGE. (Collected before). There is a clear trend between the concentrations, as AGE concentration increases, the percentage viability decreases meaning more proximal cells are damaged when AGE is at a higher concentration. Similar results were seen in 24 and 48 H. Anova + Dunnet Post Test p<0.001 showing significance.
Figure 7: This table shows the average percentage viability vs the control for MTT assay for 1, 2 and 4 hours. 4 hour was performed once. The data shows no clear trend between incubation times. All values are P>0.99220.127.116.11 MTT for 1 hour, 2 hours and 4 hours averages
Figure 8: This graph shows the average percentage viability vs the control for MTT assay for1, 2 and 4 hours. The graph shows no clear trend between incubation times. However with the longest incubation time shows more detrimental effect on the cells but again they have no significance.
6.2.2 MTT averages for new aliquot of HK-2 cells
1 HOUR C
2 HOUR C
4 HOUR B
Figure 9: This table shows the average percentage viability vs the control for MTT assay for1, 2 and 4 hours but on a new aliquot of cells. The table shows no clear trend between incubation times. All values are P>0.9999.
Figure 10: This table shows the average percentage viability vs the control for MTT assay for1, 2 and 4 hours but on a new aliquot of cells. The graph shows no clear trend between incubation times. All values are P>0.9999
6.3 RT PCR results for ADMEC
Figure 12: This graph shows the mRNA expression for IL-6 in the presence of AGE, lysozyme and the co treatment. In the presence of AGE and lysozyme co treatment, there is no reduction in expression.
Figure 11: This graph shows the mRNA expression for RAGE in the presence of AGE, lysozyme and the co treatment. In the presence of AGE and lysozyme co treatment, there is no reduction in expression.
7.1 Previous findings
As mentioned in the introduction, a group of researchers in 2001 (Zheng et al. 2001) have discovered the lysozyme from egg white has scavenger like activity of AGE as it was seen that when lysozyme was administered into mice intravenously, it was able to increase the renal clearance of AGE, improve microalbuminuria and to reduce macrophage recruitment. The work then followed up by (Cocchietto et al. 2008) showed that when microencapsulated lysozyme is orally administered to diabetic rats, it is able to reduce the concentration of serum AGE in the circulation and their deposition in the kidneys and again prevented the development of microalbuminuria, glomerular and renal hypertrophy as well as overexpression of AGE receptors. Literature and experimental studies have led to the development of further study of the molecular mechanism of lysozymes protective function in diabetic nephropathy.
Due to ethical issues of using animals for testing in studies, the idea was to study the molecular mechanism of lysozyme in vitro; therefore the first phase of the study was to find an appropriate cell model that we can use for our study. All results from this study have been performed previously by PhD researcher Davide Gallo. The possibilities of cell lines that were available were: LLC-PK1 (Porcine kidney proximal tubular epithelial cells), ADMEC (Human Adult Dermal Microvascular Endothelial Cells)(primary culture) and HK-2 (Human kidney proximal tubular epithelial cells). From these cell lines, the in vitro stability and the ease of use were the parameters that were required for this study. Prior to my arrival, viability assays were performed on the three cell lines; firstly using ADMEC cells, an SRB assay was performed when treated prior for 72 hours with AGE at several concentrations: 15nM, 150nM, 750nM, 1.5µM. The results in turn did not show any significant variations in the parameter that was being measured as all the concentrations of AGE showed similar optical density compared to that of the control. In theory, what should have been observed is a decrease in densities of AGE compared to that of the control. A similar result was seen with the MTT assay where the treatment was at 24, 48 and 72 hours at