Collaborative Scientific Research: A Journey of Knowledge and Support

ACKNOWLEDGEMENTS

Scientific research has always been something which requires the collaboration of many reserchers.This is always to bring ideas and put them together in order to build a solid research focus which will lead to a finite solution. I can acknowlege that I am previlage to benefit from such an experience in real life.First,my suppervisor, Dr Claire Peppiatt-Wildman who has given me the opportunity to undertake such a knowlegeagle task.She did not only provided the giudance and support,she provided me with the animal model I used in carrying out the project.Secondly,I am extremely grateful for the support provided to me by Kirsti Robertson.She was there at the beginning,teaching me the techniques I used in performing this peace of work.This work was also supported by Rebecca Lilley who provided me with the data on Angiotensin-II for me to be able to compare the pericyte mediated vasoconstriction of both C5a and Angiotensin-II application.she was also there to provide me with the suport and guidance.

ABSTRACT

Ischemic acute injury of the kidney is a major cause of morbidity in kidneys. It occurs when the kidney is procured for transplantation. Reperfusion that follows a successful transplantation also increases the injury and is characterized by early inflammation. The inflammation is allo-antigen independent. Components of the innate immune system which include cytokines, chemokine and TLR’s contribute to pathogenesis of the injury. Cell mediated responses to the acute injury include macrophages, neutrophils and lymphocytes apart from the innate immune cells. In this review, we will focus on ischemic acute kidney injury and the immune mediators that participate in its pathogenesis (Jung and Rabbi, 2009).

A large number of research has been done on ischaemic acute kidney injury using different experimental methods. However, there are some major drawbacks where more research on specific area is vastly required.Looking at the morphology of the kidney, blood comes to the kidney from the heart via the afferent arteriole,goes through the glomerulus in the Bowman`s capsule and goes out via the efferent arteriole. It then forms a network of dense capillaries surrounding the renal tubule in the medulla before joining to form the vein to carry blood away from the kidney. This dense network of vessel is called the vasa recta. The vasa recta is involved in carrying blood to the kidney medulla .The endothelium of the vasa recta consist of pericyte cells. In situ in vitro studies on an isolated descending vasa recta shows that pericytes can cause constriction of the descending vasa recta in the presence of the chemoattractant anaphylatoxin complement agent, C5a.The constriction which was produced by C5a was compared to that produced by an endogenous vasoactive agent,ANG-II. Methods: Using a confocal microscope, images of a live kidney slices were monitored to identify the vasa recta .Pericyte mediated real-time changes in the vasa recta diameter was investigated using a DIC video imaging of this live kidney slices. Results: The changes in vasa recta diameter were recorded in response to bath application of C5a.It was seen that C5a causes pericyte mediated constriction at the vasa recta capillaries. This results in changes in medulla blood flow. These changes in medullary blood flow is thought to play a role in the pathogenesis of Ischaemic acute kidney injury. Conclusion: This model used in investigating how c5a mediate in situ changes in vasa recta diameter in response to pericytes is a valid complementary one. This technique may also be useful in exploring the role of tubulovascular cross talk in regulating of medullary blood flow (Crawford et al, 2012).

HYPOTHESIS

C5a causes pericyte mediated constriction at in situ vasa recta capillaries.

The magnitude of constriction evoked by C5a is greater than that of other vasoactive agents such as angiotensin II ( ANG II)

INTRODUCTION

THE GENERAL ANATOMY OF THE KIDNEY

Kidneys are vital organs to human existence. They are paired lobular organs weighing about 120-170g in adults.The kidneys are situated on the posterior abdominal wall on either side of the vertebral column with the right kidney normally lower than the left.Each of these kidneys are covered by a very thin protective uniform capsule surrounded by adipose tissue. The kidney is divided into two distinct regions,the cortex and the medulla. The medulla is made up of a number of renal pyramids which then protrude into the renal pelvis.

THE NEPHRON

The nephron is the basic functional unit of the kidney and the human kidney contains about 1 million nephrons. Each nephron consist of the bowman`s capsule which contains the glomerulus which is made up of a network of capillaries supplied by the afferent arteriole. Selective ultrafiltration occur according to size and charge. These small molecules will need to pass through the fenestration endothelium, endothelial basement membrane and epithelial podocyte.Following the ultrafilotration,the filtrate enters the tubular system and it is subsequently modified by re-absorption and secretory process.

The tubular system of the kidney is divided into functional distinct regions known as the proximal convoluted tubule, the loop of Henle, the distal convoluted tubule and the collecting duct. Within the tubule re-absorption is enhanced by increased surface area due to the presence of cellular microvilli forming a characteristic apical brush border. The urine becomes concentrated as it passes through the loop of Henle and the collecting duct before reaching the pelvis.

RENAL FUNCTION

The kidney is involve in several physiological role. This includes the maintenance of electrolyte balance and the balance of extracellular fluid. These is achieved via the process of ultrafiltration of plasma blood via the glomerulus, followed by active and passive reabsorption of solutes as the filtrate passes through the various segments of the nephron. The key function is to excrete waste products ( e.g creatinine),drugs and excess acid.The kidney is also involve in the metabolism of small proteins and vitamin D as well as the production of erythropoietin and prostaglandins.

Acute renal injury is characterized by renal function deteriorating within days. This deterioration results in the inability of the kidney to dispose toxic products, nitrogenous in nature, and maintain the balance of fluid and electrolyte. In the last fifty years, several important studies of Ischemic acute injury and renal failure of the kidney; and the mechanisms that cause dysfunction in the kidney have been done.

1 Ischaemic injury

According to Anrather and Iadecola (2016) ischemic injury or ischemia is a condition in which the blood supply to the surrounding tissues is highly reduced. The major cause of ischemic injury is the restriction of the blood vessels which leads to a reduction of oxygen and nutrients required followed by insufficient removal of metabolites. Ischemia can rapidly affect different organs of the body that primarily includes heart, brain, kidney, bowel, and limb. Due to lack of oxygen permanent damage takes place within 3-4 minutes at body temperature after the first initiation (Russo et al. 2014).

The duration and the magnitude of ischemia are influenced by the level of cell injury. Research suggests that all organs are equally susceptible to ischemic injury (Schweickert et al. 2016). In the case of cardiac ischemia, the myocardium receives an insufficient blood flow which is characterized by chest pain known as angina pectoris that leads to long-term accretion of cholesterol in the arteries (Nicotera and Orrenius 2013). The chronic form of brain ischemia results in the formation of vascular dementia. Transient ischemic attack (TIA), commonly termed as mini stroke is a major outcome of brain ischemia. Kidneys are highly affected by ischemia that leads to decrease in the rate of glomerular filtration mainly caused by renal artery stenosis (Schweickert et al. 2016). Limb ischemia is abridged as the six P’s that include pain, paralysis, pallor, paresthesia, poikilothermia and pulseless. Zhai et al. (2015) state that due to hypoxia, the nerve endings of the limb tissues get damaged which results in foot drop. Ischemic colitis and mesenteric ischemia refer to the inflammatory process of the large and the small intestine that forms the bowel system.

1.1 Ischemic Acute Kidney Injury

Acute Kidney Injury, induced by ischemia (reperfusion) contributes to a high rate of mortality and morbidity (Kashani et al. 2013). During the process of successive re-oxygenation at the reperfusion stage, a large number of reactive oxygen species (ROS) are generated. The ROS triggers a series of harmful cellular responses that leads to inflammation, cell death and acute kidney failure (Pohlmann et al. 2013). According to Chawla and Kimmel reperfusion of kidney is a dynamic process that includes inflammation and involvement of specific mediators (adhesion molecules, cytokines) in a multifaceted interaction. “Lipid peroxidation” and “oxidative stress” are the primary factors that contribute to the inflammation process during ischemia. The renal blood flow decreases by 45%-50%, and hence the rate of glomerular filtration decreases (Lameire et al. 2013). Vascular alterations in ischemic kidney damage the actin skeleton of the endothelial layer that leads to separation of cells from single endothelial layers. Thus, an alteration takes place in the vascular reactivity and endothelial barrier function. The state of hypoxia also reduces the process of matrix breakdown and leads to fibrosis.

Ischemic acute kidney injury is generally characterized by different kinds of symptoms that primarily includes reduction in the amount of urine, swelling of legs due to fluid retention as the kidneys are not being able to eliminate wastes, difficulty in breathing, fatigue, drowsiness, seizures, prolonged chest pain, continual nausea (Chawla and Kimmel 2012). Kashani et al. (2013) broadly classified the injury into three main phases as follows:

• Initiation Phase: Characterized by gradual decrease in glomerular filtration rate and sudden increase in blood creatinine and nitrogen. It also includes microvascular hemodynamic changes
• Maintenance Phase: Characterized by rapid decrease in glomerular filtration rate with an effective increase in blood creatinine and nitrogen level
• Recovery Phase: Characterized by restoration of tubular function with decrease in levels of blood nitrogen and creatinine

1.2 The role of the immune system during renal injury

It has been realised that excessive immune activation has shown to play an important role in the development tubuloinerstitial injury.The pathophysiological events which follows obstructive nephropathy include the infiltration of macrophages, monocytes and T-lymphocytes into the instertitium surrounding the renal tubules. It has also been suggested that the following ischaemic acute kidney injury, renal epithelial cells release inflammatory mediators and chemokines that turn to stimulate the infiltration and proliferation of lymphocytes and macrophages in the affected area(lange-Sperandio et al,2002).T helper 2 CD4+ lymphocytes has been linked to the progression of Disease most especially to the development of a profibrotic phenotype(Wynn,2004).In addition T helper one CD4+ T-cells induce a potent inflammatory response by producing interferon-Y (IFN—Y).CD8+ T-lymphocyte also play an important role as it directly targets the renal cells and induce apoptosis(Robertson et al..,2004; Wynn,2004).The mechanism of activation of invading lymphocytes is not well understood, however, the area of interest now is the interaction between the innate and adaptive immune systems. A prominent component of the innate immune response which maybe important in this role is the complement system.This is due to the fact that the complement system directly influence the adaptive immune response.

2. Innate immunity

The innate immune response is a non-specific response to pathogen. It recognises threats to homeostasis by invaders using non-memory immunological mechanisms. Microbes usually expresses surface markers that are not expressed by the host. These pathogen associated molecular patterns (PAMPs) are usually recognised by components of the innate immunity such as the C- reactive protern,Toll-like receptors and mannose binding lectins, and initiate an immune response depending on molecular recognition.

2.1 The complement system

This is a complex biochemical cascade which is made up of approximately 28 serum and membrane-bound proteins which constitute almost 10% of all serum proterns.This system makes up part of the innate immune response. This heat sensitive component observed by Ehrlich was called the “complement” due to the fact that it complement other elements of the immune system. As part of the innate immune response, it is involve in the rapid defend of the host against invading pathogens (Morgan and Walport,1991).This system can also take part in some aspects of the acquired immune response.Forexample,during the inductive phase it can contribute to the recognition and presentation of non-self-antigen by triggering the activation, maturation and proliferation of B cells (Carrooll,2000; Nweilson et al..,2000).In addition to this,it also play a role in the solubilisation of immune reactions( Frank and Fies,Atkinson,1988).The components of the complement system often appears to exist as inactive pro-enzymes, as such, they require cleavage by a proteolytic enzyme in order to become active.

2.2 Complement system in renal injury

Ischaemic reperfusion injury ( IRI) occurs when tissues are temporarily deprived of blood supply and which perfusion is restored eliciting an intense inflammatory. It is a very common scenario after tissue transplantation and as such, it determines the survival of the graft. It is a common cause of acute renal failure in kidneys and allografts and it is associated with a high mortality rate. IRI causes activation and migration of immune system cells such as neutrophils to the site of injury. This will result in the release of cytokines, forming reactive oxygen species; acute tubular necrosis and activation of the complement system. There has always been debates for some time now about the role the complement system plays during the progression of renal ischaemic reperfusion injury (IRI).Studies using C3,C4 and C6 deficient mice have demonstrated that C3 and C6 mice are significantly protected from IRI (Leien et al,2003,Thurnman et al,2003).In addition, the role produced by C3 during IRI has been demonstrated in vivo (Zhen et al..,2006).Further evidence for the enrolment of the alternative complement pathway in IRI has also been provided in studies using FB deficient mice.

2.3 Complement activation

Similar to the coagulation system , the complement effects are generated via an enzymatic cascade amplification process. This cascade is initiated via three possible pathways ( Walport, 2001).

• The classical pathway which is initiated following the complement fixation with immunoglobulins of the IgG or IgM isotypes in complex with their antigen( Roitt et al..,2001 );
• Alternative pathway which is initiated following contact with cellular surfaces including those of various pathogenic bacteria,parasites,viruses,virally infected cells and fungi (Pangburn et al..,1984),as well as some host tissues;
• Lectin pathway which becomes activated when it uses it`s mannose binding lectin to recognise pathogens expressing terminal mannose or N-acetyl glucosamine surface (Turner,1996;Matsushita et al, 1992).

Following the activation of the complement system all the pathways converge into the terminal pathway of the complement , which begins with the production of c3-concertase.This enzyme act by cleaving the third complement (C3) to C3a and C3b.C3b when it covalently binds to nucleophilic activator surface(-OH,cabohydrates and amine groups of proteins),it acts as an Opsonin.C3b is unstable and last in the activated form for only a few milliseconds.This is especially in the absence of an appropriate surface to which it bind, results in its inactivation by reaction with water(Roitt et al..,2001).The short half-life allows it to bind only to its immediate vicinity of activation. When C3b binds to non-host surfaces it activates the complement via the alternative pathway. This facilitates the binding of phagocytes( neutrophils ,macrophages, and monocytes) that possess receptors for C3b(Roitt et al..,2001).C3b also covalently interact with C3-convertase to produce c5-convertase(Barnum,1995). The C5-convertase cleaves c5 to C5a and C5b.C5b associates in a non-covalent interaction with C6, C7, C8 and C9 to form the membrane attack complex (MAC).MAC forms Pores on the membrane and inserts itself into the cell membrane.

The complement factors have a range of fuctions.This include mediation of inflammatory responses, increasing vascular permeability, chemotaxis, opsonisation of pathogens for phagocytosis and activation of B-cells (Volanakis et al..,1992).These functions are expressed when C3a and C5a bind to their receptors, C3aR and C5aR respectively, which are found on various cell types (Barnum ,1995). As compared to C3a,C5a is a more potent activator of cells originating from myeloid progenitor lineage, such as neutrophils and mononuclear phagocytes,as well as glial cells ( Nataf et al..,1999).C5a is also a chemotactic factor of many cells and also induces the degranulation of mast cells and basophils. It induces the expression of adhesion molecules on the endothelial cells ( Foreman et al..,1994;Mulligan et al,1997),thus causes the recruitment and infiltration of macrophages and lymphocytes to the sites of inflammation.C5a also induces macrophages to produce a range of pro-inflammatory cytokines ( including TNF-alpha and IL-1) and other number of chemokines including CXC and CC (Czermak et al..,1999; Mccoy et al..,1995).C5a has shown to act like an acute phase cytokine by itself.It modulates the expression of acute phase proteins, some complement proteins and C-reactive proteins(Mccoy et al..,1995 ; Szalai et al ,2000).in contrast to C5a,C3a is much less potent than C5a.Despite the above reason C3a still activates phagocytes and mast cells though its chemotactic activity is more restricted (Roitt et al..,2001).Many of these C3a and C5a inducible cytokines have been implicated in ischaemic acute kidney injury.

2.4 : Consequences of complement activation

The complement system is often activated in an event to remove invading pathogenic organisms such as bacteria. This maybe archived directly through the formation of MAC or indirectly though the activation of phagocytosis and removal of the immune complexes.

2.4.1: Generation of anaphylatoxins

The activation of the complement system leads to the cleavage of complement proteins to form small, biologically active molecules c3a and c5a.These two complement components exercise a variety of functions, including cellular chemotaxis during inflammation and the ability to stimulate the release of histamine from mast cells.Thier inflammatory effects are mediated by their action on specific receptor, C3aR and C5aR ( Wetsel,1995).

2.4.2: Augmentation of the acquired immune response

Complement receptor 2 ( CR2) provides one link between innate and the acquired immunity. This receptor is located on the surface of B-cells and it is involve in promoting the uptake of opsonised antigen for processing and presentation of T-lymphocytes (Jacquiersarlin et al..,1995).I can also be seen on the surface of dendritic cells where it is able to interact with B-cell cells ,allowing the complement system to play a major role in B-cell maturation ( Fisher et al..,1998)

Amongst the above, in humans, the complement system consist of proteins which regulate it`s activity at various stages. These regulatory proteins help to prevent uncontrolled activation of the complement system, maintaining homeostasis and prevent host damage by restricting the effects produced by the complement to target antigen. This system also take part in opsonisation and cell lysis.

3. Inflammatory Cells in Ischemic Kidney Injury

Ischemic injury produces both innate and adaptive immune response. The innate response is triggered by causing injury in a non-antigen explicit fashion that includes inflammation of different cells that specifically includes the neutrophils, natural killer cells (NK), macrophages/monocytes, natural killer T cells (NKT). On the other hand, maturation of DC and antigen presentation initiates the adaptive response (Gomez et al. 2014). According to Linkermann et al. (2014) some tubular cells possesses Toll-like receptors (TLR) that initiate activation of the inflammatory cells apart from producing pro-inflammatory cytokines such as TNF-alpha, RANTES etc.

Neutrophils attach itself to the endothelium within 25-30 minutes after reperfusion and accumulate in the kidney (peritubular capillary of the outer medulla). The attachment produces a series of proteases, cytokines, myeloperoxidase and reactive oxygen species that causes an increase in vascular permeability and thereby reduces the integrity of the epithelial and the endothelial cells thereby initiating the process of kidney injury (Zhu, Lerman and Lerman 2013). The inflammation of the neutrophils causes activation of the monocytes. The monocytes once activated rapidly migrate to the uninjured portion of the kidney tissues upon leaving the bone marrow and gets effectively differentiated into DCs and occupant macrophages (Tan et al. 2013). Research suggests that the macrophage number increases rapidly after reperfusion and the infiltration is initiated by CCR2 and CX3CR1 signalling models (Zhu, Lerman and Lerman 2013). The inflamed macrophages produce the large amount of ROS and nitrogen intermediates and other inflammatory cytokines that include IL-1β and TNF-α. The different kinds of chemicals produced in response to inflammation of selected neutrophils help in regulating the migration and activation of the other suitable cells that are actively involved in the process of inflammation (Linkermann and Green 2014). The IL-1β and TNF- α thus produced helps in driving a polarized Th1 immune response. The DCs and the monocytes once activated has the ability to activate the naive T-cells by presenting the antigen through the expression of various molecules and production of cytokines (Gomez et al. 2014). Thus, the expression helps in connecting the innate immune response to adaptive immune response.

4. Role of Leukocytes in the pathogenesis of Acute Kidney Injury

According to Molitoirs (2014), neutrophils play a key role in fighting pathogens by the process of phagocytosis. Thus, neutrophil degranulation leads to severe consequences. Once the neutrophils get inflammation during ischemia, the cells keep on releasing a large number of granules along with cytokines, IFN- and IL-17 (Hall 2015). It is also associated with the release of chemokine CXCL1. Thus, the chemicals lead to formation of different kinds of reactive oxygen species within the kidney which in turn causes damage to the renal system. Once the process of ischemia is initiated, the number of macrophages doubles within the kidney while the infiltration of the signal is mediated by CCR2 and CX3CR1pathways. Research suggests that analysis of post-ischemic kidney have led to the identification of many other pro-inflammatory cytokines like IL-6 which are expressed outside the outer medulla. In a recent study by (Li et al. 2012), NK cells play a key role by activating and inhibiting receptors on the surface of their cells. Specific ligands for the NK cells receptors get effectively expressed on the target cells. A modern study revealed Rae-1, an NK cell activating ligand initiates killing of TEC by activation of the NKG2D receptor on the surface of the NK cells (Jang and Rabb 2015).

Dendritic cells play a huge role by transforming matured phenotype recognized by high levels of class-2 major histocompatibility complex. The cells also include a release of various kinds of pro-inflammatory factors and interaction with the NK T cells with the help of CD40 molecule (Molirois 2014). Ysebaert et al. (2000) demonstrated that after ischemia, dendritic cells produce various kinds of cytokines like TNF, IL-6, and RANTES that helps in causing prior depletion of the dendritic cells. IL-12 and IL-23 are the two major components that are mainly produced from activated dendritic cells and their associated cytokines.

5. Protective Actions of the Regulatory T-Cells

Studies from the past have revealed that the T cell is a family of large cells that contains lymphocytes of different subtypes with different characteristics. In case of ischemic renal injury, T cells primarily recognize the antigens and promote the activity of the inflammatory cells (Kleinschnitz et al. 2013). Based on the context of activation, CD4 T cells differentiates primarily into Th1 cells that produces IFN-ϒ, Th2 cells that produces IL-4 and lastly the Th17 cells that produces IL-17 and other types of Th cells (Kim et al. 2013). While the spectrum of CD3+CD4+ lymphocytes (commonly known as Tregs) helps in suppressing the activation of the inflammatory cells. The transcription factor Fox P3 helps in identifying the Tregs that initiates the production of various kinds of anti-inflammatory cytokines. According to Yang et al. (2015) Tregs activates multiple mechanism system to suppress the inflammation. Souidi, Stolk and Seifert (2013) stated that the T-cells mechanism of action is very simple and it readily helps in suppressing the activity of the ischemic cells by an alternative mechanism. The regulatory T-cells specifically target the dendritic cells through LAG-3-MHC-Class-II interactions in order to stop their maturation (Yang et al. 2015). Kleinschnitz et al. revealed that the regulatory T cells also activate another anti-inflammatory pathway that activates the interaction of CTLA-4 with CD80. Tregs itself helps in promoting their proliferation which in turn increases their ability to protect the kidney from ischemic injury. During ischemia, Treg gets rapidly recruited in the kidney and their interaction with the dendritic cells helps in downregulating the expression of different kinds of constimulatory substances (Burne et al. 2015). Thus, in this way the regulatory T-cells produces an antagonistic effect by inhibiting their own ability to encourage inflammation.

6. Role of Cytokines in protection against Ischemic Renal Injury

The cytokines play a key role in the inflammatory process of ischemic kidney injury, that are produced by the epithelial cells of the renal tubule. Research suggests that the inflammatory cytokines play a crucial role as both mediators of initiating renal injury and immune function (Singh, Singh and Bhatti 2014). While various other studies have revealed that the cytokines have immunomodulatory roles that suppress the development of the renal injury. The Th1 cytokine IFN-gamma limits the progression of the disease by activating a pathway that helps the renal cells to proliferate in a particular manner in order to reduce the inflammation (Malek and Nematbaksh 2015). The cytokines initiate the protection mechanism by up regulating the adhesion molecules of the epithelial cells and thus inhibits uncontrolled proliferation of cells.

In accordance with the study made by Zhao et al. (2014) many cytokines increase the nuclear factor kappa-light-chain enhances that remains within the activated B cells. The activation of this particular molecule further enhances the pro-inflammatory phenotype (Furuichi, Gao and Murphy 2006). Thus, the inflammation of the different kinds of cytokines molecules that remains associated with the process of ischemic kidney injury plays a massive role in activating the T cells which in turn plays a key role in controlling the extent of the disease (Jang and Rabb 2015). Thus, one of the major consequences of the production of cytokines is that the cytokines once inflammated activates the ischemic kidney injury process, but the activation, in turn, plays a key role in generating series of cascade reactions that helps in restoring both the renal tubular and the renal haemodynamic functions.

From the above literature review one can come to the conclusion that a large number of researchers have continued research on ischemic acute kidney injury and different experiments have led to a better understanding of the underlying concepts in association with ischemic kidney injury. However, there are some major drawbacks where more research on the specific area is vastly required. The inflammatory response needs to be studied in more details in order to understand the other pathways that get activated during ischemic kidney injury. The understanding of the pathways will lead to betterment of further investigations that will help the researchers to take better preventive measures arising due to ischemic kidney injury. Other experimental inquiry will help in better understanding of the overall topic that will put light on the role of cytokines and the role of T cells in helping to stop the progress of ischemic kidney failure. In my project I will be using the complement factor C5a to see how it affectsffect on the kidney vasa recta.

METHOD

The number of animals used in the experiment was 4. The animal use in this experiment was provided to me by my supervisor. Live kidneys were immediately Isolated from the animals after it was killed and placed in ice cold Physiological Saline Solution (PSS), bubbled with 95% oxygen/5% carbon dioxide and prepared for slicing. Before the. Each kidney was secured on the slicing block of a vibratome tissue slicer and submerged in a bath of ice cold PSS bubbled with 95% oxygen and 5% carbon dioxide. The PH of this PSS was adjusted to 7.4 using 10M NaOH. The outer cortical dome of the kidney was removed to expose the top of the renal medulla and serial 200mcm thick coronal kidney slices (intact cortex and medulla) were cut. Before reaching the medulla, slices of about 1700mcm were cut. The slices were collected and kept in a holding chamber containing the PSS, bubbled with 95% oxygen and 5% carbon dioxide at room temperature to maintain tissue viability. The live kidney slices were secured in an open-bath chamber using a purpose built platinum slice anchor and transferred to the stage of an upright Olympus microscope. The slices were constantly perfused throughout the duration of the experiment with PSS, 95% oxygen and 5 % carbon dioxide at room temperature. The pericyte on the vasa recta was identified using the bump-on- a log morphology. Real time Video images in the vasa recta diameter were collected every second by attached Rolera XR CCD Camera and recorded using image pro software. The live kidney slice were then perfused with the drug C5a.Time series analysis of the live kidney slice was done using the public domain software imagej. Both pericyte and non pericyte sites were identified on a single vasa recta.

The diameter of the vasa recta at both locations were measured every 5 frames for 1200 frames duration (1 frame = 1 second).The slices were super fused with PSS alone for 70 seconds to establish a baseline vessel diameter at pericyte and non-pericyte sites. The slices were exposed to C5A to evoke a change in vasa recta diameter and were then subject to a wash. An average of the first five experiment were taken to represent the resting baseline diameter value and expressed as 100% for both pericyte and non pericyte sites.

All subsequent diameter measurements were calculated and expressed as a percentage of the corresponding baseline value for both pericyte and non pericyte sites. The percentage change in vessel diameter was calculated from the actual vessel diameter measurements which was done throughout the experiments. The statistical significance was determined by calculating the T-test (Crawford et al, 2012).

RESULTS

The animal used in this experiment was killed by cervical dislocation just before the experiment was what was?? perform and immediately perfused in physiological saline solution (PSS). This is an indication that the tubular and vascular cells within the kidney slice were live what was your indicatin that all cells were alive? You didn't test this experimentally bemore specific in what uou are saying. This confirms that the tissue slice were viable for the experiment. Different slices of the tubules ?? Really or do you mean vessels or both? refer to images shown below….were observed in all 8 slices in 3 animals. The pericyte cells in our live kidney slices were also identified to make sure they were viable before the experiment began. The characteristics of the pericytes were determined by analysing large areas in both the inner and outer medulla regions per kidney slice. This was all done when the slices remained submerged in ice no experiments were performed at room temperature cold PSS, bubbled with 95% oxygen ,5% carbon dioxide and at room temperature.

The initial resting internal diameter of the vasa recta capillaries show that it significantly reallyt how much lower state the percentage plus and minus SEM and what is the p value ? lower than the diameter of the non pericyte site. The chemoattractant agent C5a was applied to test its action on vasa recta diameter of both the pericyte and non pericyte sites. This was followed by measuring the diameter of both the pericyte and non pericyte site before, during and after the application of C5a. I was then able to determine any changes in diameter on both sites after the measurements. This information was then used to plot a graph of percentage change in vessel diameter over time. Overall, I realised that over 50% of the pericyte responded by changes in vessel diameter on application of C5a. C5a caused significant vasoconstriction (3-fold) at pericyte sites ( 25.2%) than at non pericyte sites (2.24%).fig of traces(n=11 slices and n=4 animals; p "<"0.05).A representative trace showing the C5a(10ng\ml) evoked changes in vessel diameter at pericyte site is shown in figure…...Similarly, angiotensin II application also indicated a significant vasoconstriction ( approximately 3-fold) at pericyte sites (20.12%) than at non-pericyte sites (6.26%).Fig of traces ( n= 8 slices and N= animals ; p "<" 0.05).Out of the 11 slices produce produced from 4 animals, 5 of the slices evoked pericyte mediated vasa constriction on application of C5a.This gives on average a success rate of approximately 45% what is SEM?. Looking at the 8 slices produced from 6 animals, 7 slices responded and evoked pericyte mediated vasoconstriction on application of Angiotensin II. This gives on average 87.5% success rate. That notwithstanding, C5a caused a significantly greater constriction at pericyte sites compared to that evoked by Angiotensin II.

You need to expand this section as discussed previously each data set should be explained time to onset of max effect, whether effect is reversible, how many experiments actually worked, state this as your % of successful experiments. Compare numericaly the effect of C5a to AngII how much bigger is the max constriction….what % and is this statistically significant?

Also be very careful of text formatting issue spacing is often incprrect and this is unacceptable

DIC Imaging and corresponding trace of pericyte mediated constriction by C5a (Figure 1) and Angiotensin II (figure 2)

pre-drug exposure (i) during super fusion of drug (ii) post washout of ANG II super fused with control PSS solution as shown (iii).Application of ANG II causes a decrease in vasa recta diameter at the pericyte site.

DISCUSSIONS

The present study investigates the role of innate immune response in ischaemic acute kidney injury. It shows how the complement factor c5a can cause Ischaemic acute kidney injury by its effect on vasa recta capillaries. The result indicates that it`s effect is mediated by pericytes cells lining the vasa recta in the medulla of the kidney. Moreover, in the absence of an in vivo method to study the role of pericyte mediated vasa recta constriction, this study has also demonstrated that the in vitro live kidney slice model is an ideal technique. As such, it has shown to be a valuable method to use by many researchers. Why is ideal – you must back up all statements made with some form of explanation….

To maintain the viability of the tissue, the slices were continuously perfused with PSS, 95% of oxygen and 5% of carbon dioxide and at room temperature. Out of the 11 slices from the 4 animals, 5 of them indicated significant pericyte mediated vasa recta constriction on application of C5a. In a similar manner, 7 out of 8 slices responded on application of ANG II. These findings confirm the viability of the kidney slice for physiological experiments

This study has shown that the mean maximum constriction evoked by C5a is 23.8 (n=4 animals and n=8 slices) and the mean maximum constriction evoked by ANG II is 20.1(n=6 animals and n=8 slices). From the standard error of the means these should be shown next to % changes where ever stated and the standard deviations t-test were done. The p values for both ANG II and C5a were 0.0011 and 0.018 respectively. The two values were statistically significant as they were both less than 0.05%. Therefore I can confidently say that the constriction evoked by both Angiotensin II and C5a application on the vasa recta was mediated by pericytes cells and not due to any other factor. This is strongly supported by time matched control experiment carried out (fig 9). When a tissue slice was perfused with just PSS, it was realised that there was no contraction mediated at the pericyte site. Thus the constriction revoked by C5a is mediated by pericyte.

These findings demonstrate that C5a causes pericyte mediated constriction of in situ vasa recta capillaries as compared to the constriction at the non pericyte site. Similarly, Angiotensin II causes pericyte mediated constriction of the vasa recta capillaries. The results are broadly consistent with related experimental studies carried out using angiotensin II (Crawford et al., 2012). However, the constriction observed with the application of C5a is greater( 23.8%) than the pericyte mediated constriction of vasa recta evoked by the Angiotensin II (20.1%).This indicates that the magnitude of constriction mediated by C5a is greater than that of other endogenous vasoactive mediators such as angiotensin II. This is not perhaps surprising given that the endogenous angiotensin II is produced almost consistently in the body in response to decrease in blood sodium concentration, whereas C5a is often produced by the complement system in an event of an inflammatory response. Hence, C5a is a more potent vasa constrictor than ANG II.

Real time measurements of the changes in diameter was done and it was found that the vessel diameter changes evoked by C5a and ANG II was not achieved with all the tissues. This occurred in about 45% of all the tissues perfusion with c5a and 38% of the tissues perfused with ANG II. The lack of response in some pericyte sites could have been because of their death due to the lengthy duration of the experiment even though they were constantly in PSS and as such, the tissue started losing its viability. On the other hand, the minimal contraction which was observed in some of the non pericyte sites of the tissue slices might have come from some hidden pericytes which had not been identified. However, Crawford et al (2012) came out with a similar conclusion as some pericytes did not respond with ANG II perfusion on isolated and in situ retinal pericytes. The suggestion here is that all Pericyte do not constrict in a uniform manner as a syncytium. This is because it might result in a significant vasoconstriction and reduction of medullary blood flow which will be quite profound and extremely dangerous to the already borderline hypoxic tissue. Hence ,it is believed that the pericyte act to regulate vasa recta diameter in a way that is offering uniform distribution of blood flow and fine-tuning of blood flow as oppose to causing complete cessation of blood flow (Crawford et al,2012).

The representative traces evoked by ANG II (Figure 7) concur with other studies which show that Angiotensin II evoked changes in vessel diameter were reversible as the vessel returned to about 95% of their original resting diameter baseline following it`s washout. This property is consistent with previous reports that ANG II significantly reduce blood flow in the medulla (Pallone et al, 2003). On the other hand C5a pericyte mediated vasa recta response as seen from most of my tissue slices analysis, 80% appears irreversible. Reversibility was only witnessed from about 20% of the tissues. The most likely explanation to this finding is that the duration the experiment was being carried out was short since I never had much time. I can suggest increasing the duration of the experiment from 1200 seconds to about 3000 seconds would have resulted to some degree of reversibility in most of the slices. By looking at a physiological system, the longer it takes for the diameter of the vasa recta to retreat to its normal baseline decreases the flow of blood and movement of RBCs propagating ischaemic acute kidney injury. This might also results to hypoxia if the vessel remain in that state for too long.

Despite the greater percentage in reversibility evoked by ANG II, some of the vessels (20%) started to dilate towards their resting diameter baseline before ANG II washout.This is an indication of possible sensitisation of the Angiotensin AT 1 receptors. This observation is similar to what Zhang et al (2005) saw with their experiments with rats. It was seen that the sensitisation was maybe due to the release of nitric oxide after an isolated DVC of a rat was super perfused with ANG II .Sensitisation causes a decrease in number of receptors on the cell surface since they become internalised

From figure one trace above, as observed from about 10% of my slices, after C5a has been washed out, the tissue contraction continues for a while before it becomes stabilised. The possible suggestive explanation for this maybe because the agent (C5a) required more time to exert it`s effect. This meant the contraction would have continued had it been the drug was not washed out. Such an effect in a physiological system will tend to decrease blood flow and movement of RBCs to the kidney as the diameter of the vasa recta keeps on reducing. This will propagate ischaemic acute kidney injury. Hence this will produce a detrimental effect especially when we have continuous inflammation. On the other hand, the effect mediated by ANG II on the kidney is regulated by hormones and can thus be terminated at any instant.

The main limitation of this study is that it was performed in vitro. With an in vivo studies there are other parameter which can alter the outcome of such experiment. Hence with an in vitro study it will be easy to come up with false positive conclusions really – how so???. For example, if these two agents were in a physiological system ,there will be a possibility of both having a combined effect – explain this more. This can be seen in a situation where C5a causes vasoconstriction at the in situ vasa recta capillaries, reducing the subsequent flow of blood to the kidney. This will result in a decrease in blood sodium levels which will trigger the production of ANG II which will result in even more constriction .Moreover, there are other vasoactive agents which can act to counteract this effect in an in vivo study as oppose to an in vitro study. Therefore, the future direction to this studies is to look at this in vivo which is extremely challenging.

CONCLUSION

The hypothesis was supported as the results produced are statistically significant, C5a (P = 0.018) and ANG II (P = 0.0011).This indicates that both C5a and Angiotensin II causes pericyte-mediated constriction of in situ vasa recta capillaries. The magnitude of constriction mediated by C5a is greater than that of other endogenous vasoactive mediators such as ANG II. This was justified by time matched control experiment. Hence, the effects produced to the kidney by these agents in collaboration with other endogenous mediators can result in ischaemic acute kidney injury.

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