General introduction. General introduction

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1 General introduction 1

2 Chapter 1 Proteinuria is the excretion of proteins into the urine. Presence of abnormal proteinuria, the urinary excretion of abnormal amounts of serum proteins (briefly called proteinuria), is a common indicator for renal disease. Severity of proteinuria correlates with progression of renal disease to renal failure. End stage renal disease (ESRD) in several glomerular diseases is histologically characterized by glomerulosclerosis and tubulo-interstitial fibrosis. Development of proteinuria and glomerulosclerosis is influenced by environmental and genetic factors. The pathogenesis of proteinuria and glomerulosclerosis is not completely known yet. The first aim of this thesis was to obtain better insights in the pathogenesis of congenital and immune-mediated proteinuria. The second aim was to gain information on the pathogenesis of progressive renal disease and on the genetic factors playing a key role in their development. The introductory section of this thesis first leads the reader from macroscopic anatomy to cellular morphology of renal components important for proteinuria and glomerulosclerosis. Next an overview of immunological and non-immune-mediated glomerular damage is given. Then the current hypotheses on development of proteinuria are introduced. The main focus of that section is the explanation of the mechanism of the glomerular filtration barrier, designed to prevent glomerular protein filtration. In addition to proteinuria, several other factors influence development of glomerulosclerosis. Known risk factors and the current hypotheses on pathogenesis of glomerulosclerosis are elaborated on in this introduction. A special form of glomerulosclerosis is primary or idiopathic focal and segmental glomerulosclerosis. Current ideas on pathogenesis and classification of this morphology-based disease are discussed. Because research in humans is hampered by genetic heterogeneity, costs, ethic constraints and availability of tissue, for the work described in this thesis we used two rat models for proteinuria and development of glomerulosclerosis. These models are also described in the introduction. The introductory section concludes with a description of the goals of the studies in this thesis. 8

3 Anatomy Kidney The kidneys are part of the main excretory system of the human body. They regulate homeostasis by excretion of waste products of metabolism. Furthermore, homeostasis is regulated by the kidney through either excretion, or conservation of water and salts and control of body ph. Finally, the kidneys secrete a variety of hormones, including erythropoietin, urodilatin, renin and vitamin D. These hormones contribute amongst others to erythropoiesis, calcium metabolism and blood pressure regulation. 1 Nephron Blood filtration is one of several events that occur in functional units called nephrons. 2 One kidney contains about one million nephrons. Each nephron consists of a glomerulus and a tubular system (Figure 1). Blood enters the kidney through the renal arteries and passes through serial branches (interlobar, arcuate, interlobular) before entering the glomeruli. At the hilus of the glomerulus the afferent arteriole branches and forms a capillary tuft. Within this capillary tuft blood is filtered. Efferent arterioles leave the glomerulus at the hilus and supply the cortical tubular system. Each glomerulus filters about 125 ml/min. That makes a total amount of pre-urine of 180 liters per day. The pre-urine is processed within the tubules. Reabsorption of water and electrolytes and excretion of excessive amounts of molecules takes place within the tubules. The ultrafiltrate runs through consecutively the proximal tubule, thin descending limb, Henle s loop, the thin ascending limb, the thick ascending limb, the distal tubule, and eventually into the collecting tubules. After processing, approximately liters of urine per day is transported into the bladder. Glomerulus Within the glomerulus the blood is filtered. Filtration takes place in the capillary loops and several cell types are involved in maintenance of the glomerular structure and in appropriate filtration. The glomerulus consists of three main cell types: endothelial cells, mesangial cells and epithelial cells. Two types of epithelial cells are present, the visceral epithelial cells, also called podocytes, which line the endothelium and the urinary border, and parietal epithelial cells, which form the capsule surrounding the glomerular tuft. Furthermore, the glomerulus contains extracellular matrix (ECM) and basement membranes that are present between the different cell types. 9

4 Chapter 1 Endothelial cells The glomerular endothelial cells form the capillaries within the glomerulus. These cells are unique in that they are fenestrated. The openings are about nm. Whether these fenestrae have diaphragms has been a persistent controversy. 4-6 Vascular endothelial growth factor (VEGF), mainly produced by podocytes, is thought to play a major role in maturation and maintenance of endothelial cells and of their fenestrae. 7;8 The role of endothelial cells in the glomerular filtration barrier is under debate. 5 Figure 1. Schematic drawing of a nephron. Illustration printed with permission from Elsevier. 3 10

5 Mesangial cells The glomerular capillary loops are held together by the mesangium. The mesangium consists of mesangial cells and ECM. Mesangial cells are mesenchymal cells that contain contractile microfilaments similar to smooth muscle cells. 9 Mesangial cells are bound to the glomerular basement membrane. Mesangial cells play a major role in the maintenance of the structural integrity of the glomerular tuft and in modulating blood flow by relaxation or contraction. 10 Mesangial cells can respond to angiotensin II and can produce prostaglandins, both involved in blood pressure regulation. 11 In addition to blood pressure regulation, mesangial cells play a role in the glomerular immune response. This is supported by the observation that mesangial cells are capable of producing cytokines such as epidermal growth factor and monocyte chemoattractant protein 1 (CCL2). 12 In response to growth factors, such as plateletderived growth factor, mesangial cells can also proliferate. Finally, mesangial cells are capable of taking up filtered macromolecules and immune complexes. 13;14 Glomerular basement membrane The glomerular basement membrane (GBM) is a nm thick network that surrounds the glomerular capillaries. 15 It contains 3 layers: lamina rara interna that is adjacent to the endothelial cells; lamina densa in the middle that consists of type IV collagen, nidogen and laminins; lamina rara externa that is adjacent to the podocyte. The laminae rarae internae and externae contain heparan sulfate proteoglycans. The GBM is negatively charged, which is mainly due to the anionic sites of heparan sulphate and condroitin sulphate side chains of proteoglycans such as perlecan and agrin. The GBM is thought to play a role in charge-selectivity of the glomerular filtration barrier. This view is supported by studies that demonstrate that proteinuria develops when anionic charges are enzymatically removed from the GBM. 16 Furthermore, mice deficient of perlecan were reported to become more prone to develop proteinuria when challenged with an overload of albumin. 17 Laminins also play an important role in the function of the GBM. During glomerular development, laminin 511 (α5β1γ1) is replaced by laminin 512 (α5β1γ2) after birth. 18 Ablation of laminin 511 by knocking out the laminin β2 gene results in proteinuria and neonatal death. 19 Mutations in laminin β2 have been linked to Pierson s syndrome, an early lethal form of congenital nephrotic syndrome. 20 Collagen IV likely provides strength to the GBM. Each collagen IV molecule is composed of 3 alpha chains. Six genetically different alpha chains can be present, α1-α6. During the embryonic development of human glomerular basement membrane, the α1.α1.α2(iv) network appears at the start of early capillary-loop formation, but is gradually replaced by the α3.α4. α5(iv) network in the mature glomerular capillaries or by the α5.α5.α6(iv) network 11

6 Chapter 1 in Bowman s capsule.21 Mutations in the α3, α4 and α5 chain of collagen IV result in hematuria, mild proteinuria and glomerulosclerosis, a syndrome known as Alport s syndrome.22;23 Podocyte Podocytes are differentiated epithelial cells located at the outside of the glomerular tuft. Podocytes have voluminous cell bodies, which bulge into the urinary space. The cells give rise to long primary processes that extend towards the capillary to which they affix by numerous foot processes. These foot processes interdigitate with foot processes of neighboring podocytes, leaving a zipper like structure, which forms 40-nm-wide filtration slits. These slits are bridged by an extracellular structure, known as the slit diaphragm.24 The podocyte foot process is a complex structure that consists of an actin cytoskeleton and a membrane region (reviewed in 25). The cytoskeleton is crucial for the structure of the foot process. It contains a complex of actin-associated molecules. Other molecules such as zonula occludens 1 (ZO-1) and ezrin link the actin cytoskeleton to the surface membrane. The membrane region can be divided into three regions: the basal region, the slit diaphragm region, and the apical region (Figure 2). The basal region contains molecules that link the sole of the foot processes to the GBM. Known molecules in the basal region are dystroglycan and integrin α3β1. The second region is the slit diaphragm. The first molecule identified Figure 2. Schematic view of molecular markers in slit diaphragm and podocyte. Printed with permission from Kerjaschki et al.32 12

7 in the slit diaphragm was nephrin. 26 Furthermore, amongst others neph1, podocin (=NPHS2), FAT 1, p-cadherin, and ZO-1 are present in the slit diaphragm. There is a good body of evidence that the podocyte slit diaphragm resembles an adherenslike intercellular junction, as the slit diaphragm arises from a tight junction during glomerular development and because tight junction-associated molecules such as ZO-1 concentrate along the cytoplasmic surface of the slit diaphragm. 27 The apical region of the foot processes contains negatively charged molecules like podocalyxin. These molecules are thought to play a role in the structure of the podocyte foot processes. 28 Apart from its role in glomerular filtration, the podocyte probably also functions as a structure-stabilizing cell, providing forces to counteract capillary distension. 29 Additionally, podocytes produce the components of the extracellular matrix that make up the GBM, and they provide growth factors like TGF-β and VEGF. 30;31 VEGF is essential for proper function of the endothelial cells. The glomerular filtration barrier The glomerulus filters the blood selectively by permitting the passage of water and electrolytes into the glomerular filtrate, while retarding the passage of cells and protein from the blood. This selective passage is the result of a glomerular filtration barrier. This barrier consists of three layers, the fenestrated endothelium, the glomerular basement membrane and the podocytes, together with the membrane between the interdigitating foot processes, called slit diaphragm (Figure 3). Figure 3. Schematic view of the glomerular filtration barrier. Alb, albumin concentration; ESL, endothelial cell surface layer; GBM, glomerular basement membrane; GFR, the glomerular filtration rate; Q p, the plasma flow rate. Printed with permission from Haraldsson et al

8 Chapter 1 The glomerular filtration barrier is thought to be size- and charge-selective. 33;34 Size selectivity is partly mediated by functional pores in the spaces between the tightly packed cords of type IV collagen in the GBM. 35 In addition, podocytes are thought to limit filtration by size selectivity. 36 Endothelial cells are not thought to be size selective. 37 Endothelial cells contain a negatively charged glycocalyx which is thought to contribute to the charge selectivity of the glomerular capillary wall. Charge selectivity has been first described by Brenner et al. 38, who used filtration experiments with neutral and negatively charged dextran sulphate. Negatively charged molecules have been found on all three layers of the glomerular filtration barrier. 33 Since then these results have been challenged by others The exact role of all three components in glomerular filtration of proteins needs to be further elucidated. Renal injury Clinical signs of renal damage The kidney faces many potential toxic or injurious agents every day like infectious agents, toxic compounds, antibodies and immune complexes. The resulting damage depends on the type of the agent, the location of the damage in the kidney, and the type of reacting cells. Renal damage can lead to proteinuria, hematuria, nephritic syndrome, nephrotic syndrome, hypertension, and eventually renal insufficiency. A later section of this introduction will deal with the signs, classification, causes, pathogenesis, and consequences of proteinuria in more detail. The nephrotic syndrome is characterized by severe proteinuria, hypoalbuminemia, edema, hyperlipidemia, and hypercoagulation. Proteinuria within the nephrotic syndrome represents the loss of high amounts of albumin in the urine (>3.5 grams/ day). This loss exceeds the compensatory increased albumin synthesis by the liver, which results in hypoalbuminemia (<25 g/l). Hypoalbuminemia causes a decreased capillary colloid-osmotic pressure, leading to extravasation of fluid into the interstitial compartment, resulting in edema. The decreased colloid osmotic pressure also results in an increased synthesis of albumin, lipoproteins and coagulation factors by the liver. This is likely the cause of the development of hyperlipidemia and hypercoagulation. 43 The nephritic syndrome develops as a result of severe inflammation of the glomerulus. It is characterized by proteinuria (>150mg/day), micro- or macroscopic hematuria, and deterioration of renal function. Hematuria is the presence of erythrocytes in the urine. When the urine is normally colored, but erythrocytes are microscopically detected, it is called microscopic hematuria. Macroscopic hematuria 14

9 is the result of major loss of erythrocytes, leading to red or dark brown colored urine. 44 Hypertension can develop in patients with acute glomerular diseases, in vascular diseases like vasculitis, and in chronic renal failure. Hypertension in these diseases is most probably the result of enhanced sodium and water retention, increased activation of the renin-angiotensin-aldosteron-system, increased activation of the sympathetic nervous system, or a combination of these. 45 Renal insufficiency is defined by the loss of renal function. This is characterized by an increase of serum creatinine and a decrease of the glomerular filtration rate (GFR). It can develop acutely or chronically. Acute renal insufficiency is characterized by loss of renal function in a relatively short time period. It can be caused by prerenal, postrenal and intrarenal factors. Chronic renal insufficiency is defined by a slow decline of renal function and loss of nephrons. Because of the slow decline and compensation of the resulting nephrons, clinical signs are present at late stages, when GFR is < 25 ml/min. Glomerular damage can be the result of endothelial, mesangial or epithelial cell damage. Next to a classification based on the affected cell type, renal disease can be subcategorized by the causative agents of the glomerular damage. Glomerular damage can be caused by immunological, infectious, toxic, metabolic, and hemodynamic factors. 2;46 Immunological and non-immunological glomerular injury will be discussed in the next paragraphs. In addition to glomerular damage, tubular interstitial injury can be seen in renal diseases. Tubular damage can result from ischemia, metabolic or toxic effects, and from inflammation due to drug hypersensitivity or infections. For example, infections with BK virus in renal allografts 47 or hypersensitivity to antibiotics such as penicillin 48 can lead to interstitial damage. Furthermore, proteins and cytokines filtered by a damaged glomerulus can directly damage the tubulointerstitial compartment (the cytokine theory). 49 This theory will be explained in a later section of this introduction. Glomerular injury Injury of the glomerulus can be the result of antibody or immune complex deposition at the epithelial compartment, subendothelial compartment, or within the mesangium. As a result, humoral and cellular immune responses are activated. In general, glomerular deposition of immune complexes leads to complement activation and attraction of leukocytes to the kidney. Generation of radical oxygen species (ROS) by both intrinsic renal cells and inflammatory cells induces glomerular damage (reviewed in 50 ). 15

10 Chapter 1 Glomerular endothelial injury Damage to the capillary endothelial cells can lead to proteinuria, hematuria, and a decline in glomerular filtration rate (nephritic syndrome). Postinfectious glomerulonephritis, lupus nephritis, and anti-gbm nephritis are examples of diseases in which glomerular endothelial damage is seen. 51;52 In infectious diseases, bacterial antigens can bind to the GBM subendothelially. Circulating antibodies against these antigens will bind, which results in aggregates along the GBM. 53 In lupus nephritis, DNA fragments can bind to the GBM. Circulating anti-dna-antibodies form immune complexes with these DNA fragments. This is followed by complement activation and influx of inflammatory cells. Anti-GBM nephritis is caused by deposition of autoantibodies against the GBM. 46 Next to immune complex-mediated endothelial damage, other agents can lead to glomerular endothelial damage. For example high glucose 54, glomerular hypertension 55, and calcineurin inhibitors in renal allografts 56, can cause glomerular endothelial damage. Glomerular epithelial injury Antibody deposition at the subepithelial compartment results in complement activation as well. The complement membrane attack complex (MAC, C5b-9) causes damage to the podocyte leading to dedifferentiation, foot process effacement, proliferation, apoptosis, or detachment from the GBM. Since these complement components do not reach the circulation, chemotaxis of inflammatory cells does not take place. Patients typically develop a nephrotic syndrome. Examples of human renal diseases with specific epithelial cell damage are membranous nephropathy, minimal change disease (MCD), and focal and segmental glomerulosclerosis (FSGS). Membranous nephropathy is the most common cause of nephrotic syndrome in adults. MCD is the most frequent cause of epithelial cell damage in children. The pathogenesis of MCD and membranous nephropathy is not known. Epithelial cells in biopsies of patients with MCD are unaffected at light microscopy. However, at electron microscopic level foot processes of the epithelial cells are flattened, a feature that is called foot process effacement. 57 The pathogenesis of FSGS will be discussed later in this introduction. Parietal epithelial cells lie along Bowman s capsule, surrounding the glomerular tuft. These parietal epithelial cells can, unlike visceral epithelial cells, proliferate. Activation and proliferation of these cells is found for example in crescentic glomerulonephritis. 58;59 Glomerular mesangial damage Immune complex deposition in the mesangium can lead to complement activation, followed by activation and proliferation of mesangial cells. This can be the result of 16

11 trapping of immune complexes in the mesangium, binding of specific antibodies to mesangial cells, or indirect binding of immune complexes to mesangial cells via specific receptors. 60 The most common type of mesangioproliferative glomerulonephritis is IgA nephropathy. 61 Patients with IgA nephropathy typically present with nephritic syndrome. Histologically, glomeruli show mesangial expansion and mesangial proliferation. Another example of mesangial damage is diabetic nephropathy, which is a major cause of renal disease and renal insufficiency. Clinically it is characterized by microalbuminuria, later followed by macroalbuminuria and slowly progressive renal insufficiency. Glomerular damage consists of glomerular hypertrophy, mesangial expansion, and thickening of the GBM at early stages. The mesangial expansion sometimes is nodular, a lesion known as a Kimmelstiel-Wilson lesion. The renal damage in diabetic nephropathy is the result of a combination of abnormal glycosylation, hyperfiltration, and hypertension. Chronic hypertension in itself is also injurious to the kidney and can lead to vascular, interstitial, and glomerular damage. Patients typically present with a long history of hypertension, slowly progressive elevation in plasma creatinine concentration and mild proteinuria. 62 Persistent hypertension leads to injury to the nephrons and loss of functional units, resulting in glomerular hyperfiltration and glomerular capillary hypertension. 63 This glomerular hypertension will lead to pressure-induced capillary stretch and glomerular injury. In addition to systemic hypertension, conditions that lead to increased glomerular capillary pressure, such as in diabetes and after renal ablation, can also result in glomerular damage. Proteinuria Epidemiology In healthy people, the concentration of proteins in the urine is low. Proteins in the urine have been identified more than 200 years ago. Several publications in the late 18 th century describe proteinuria, which they found by heating proteinuric urine in a spoon. It was however Richard Bright who first related proteinuria to renal disease. 64 Small molecular weight proteins and small amounts of albumin are filtered by the glomerulus but most of these proteins are reabsorbed by the proximal tubules. This results in less than 150 mg proteins in the urine. The normal excretion of albumin is even less, usually below 30 mg/day (20 μg/min). Persistent albumin excretion between mg/day is called microalbuminuria. Values above 300 mg/day are considered as overt proteinuria or macroalbuminuria. More than 3.5 gram protein excretion per day is called nephrotic range proteinuria, a condition that can lead to 17

12 Chapter 1 nephrotic syndrome. Pathogenesis Proteinuria can be caused by an abnormal glomerular filtration of proteins (glomerular proteinuria) or by a diminished function of the renal tubules resulting in a decreased reabsorption of filtered low molecular weight proteins or an increased excretion of proteins (tubular proteinuria). An example of tubular proteinuria is the Fanconi syndrome. This syndrome can be present in congenital diseases such as cystinosis or Wilson s disease, or can develop later in life secondary to intoxications or for example in Sjögren s disease. 65 In Fanconi syndrome, dysfunction of the proximal tubules leads among others to decreased reabsorption of albumin. This proteinuria mainly consists of low molecular weight proteins and values will rarely exceed 2 g/day. Russo et al. suggest that all cases of albuminuria are the result of defective reabsorption of albumin in the tubules. 66 They hypothesize that albumin filtration is not restricted by the glomerular filtration barrier ( the albumin retrieval hypothesis, reviewed in 67 ). However, the classical view is that most cases of proteinuria are the result of the increased filtration of proteins by the glomerulus. As discussed in the previous section, endothelial cells, the GBM and podocytes contribute to appropriate filtration. Damage to any of these layers of the glomerular filtration barrier can lead to proteinuria. Proteinuria accompanied by glomerular endothelial damage is seen in for example pre-eclampsia. Pre-eclampsia is a potentially lethal complication of pregnancies characterized by hypertension and proteinuria. Pathogenetically, pre-eclampsia is thought to be caused by systemic overexpression of the soluble receptor for VEGF, a factor that is involved in maintenance of the endothelial layer. A decrease in VEGF levels may lead to a dysfunctional glomerular endothelium and to proteinuria. 31;68;69 Secondly, damage to the GBM can lead to proteinuria. Alport s disease, a disease in which a mutation in collagen IV causes dysfunction of the GBM, leads to proteinuria. 23 This has been confirmed in animal models, in which disruption of the collagen and laminin beta chain leads to proteinuria. In all these models changes in endothelial cells and podocytes have been described. 19;22 Finally, in the last decade research on the pathogenesis of proteinuria is mainly focused on the podocyte as the essential cell in maintaining normal glomerular permeability. First, several congenital and acquired proteinuric diseases are accompanied by podocyte or podocyte-associated changes. For example, MCD is associated with subtle podocyte changes seen on electron microscopy, called foot process effacement. 57 In addition, changes in podocyte structure have been described 18

13 in several renal diseases like diabetic nephropathy, FSGS, and IgA nephropathy. 32;70;71 Disruption of the anatomical relationship between adjacent podocytes and between podocytes and the GBM is one of the earliest morphological features seen in proteinuric diseases. However, it is unclear whether podocyte changes are the cause or the result of proteinuria. In addition to changes of the podocyte structure, the podocyte plays a key role in the function of the GBM and the endothelial layer by producing growth factors like VEGF. 72 Changes in these growth factors may contribute to development of proteinuria as well. Genetic predisposition Genetic factors play an important role in development of renal disease and proteinuria. Genetic predispositions are found in both congenital and acquired renal diseases. In recent years several mutations in genes expressed in glomerular cells and especially in podocytes are found in congenital and early onset proteinuria. 73 For example mutations in nephrin, a molecule required for a functional slit diaphragm, are found in patients with nephrotic syndrome of the Finnish type. Mutations in nephrin lead to a dysfunctional slit diaphragm, resulting in foot process effacement. 26 Furthermore, mutations in NEPH 1, α-actinin-4, and NPHS2 have been described in patients with nephrotic syndrome In addition, the earlier mentioned Alport s disease is caused by a congenital mutation in collagen IV. 23 Moreover, familial FSGS has been associated with loci on chromosomes such as 11q21-q22 78 and 1q Responsible genes located on these loci have not been identified yet. Apart from its role in congenital or early onset proteinuria, genetic factors play an important role in acquired renal diseases. They influence both severity of proteinuria and progression of renal disease. For example, diabetic nephropathy is associated with loci on chromosomes 10 and 18 80;81, and susceptibility to develop familial IgA nephropathy has been linked to loci on chromosomes 2, 4, 6, and Animal models have been helpful in detecting the role of genetics in several renal diseases. Knock-out models for genes expressed in the glomerular filtration barrier have been used to investigate the role of several proteinuria-associated molecules In addition, several animal models for spontaneous proteinuria have been used Finally, differences in proteinuria in experimental models between inbred strains have been helpful in detecting susceptibility genes for the severity of proteinuria. 93;94 Linkage analyses between strains that are resistant or prone to development of proteinuria, have brought about several chromosomal regions linked to the development of proteinuria. These chromosomal regions are called quantitative trait loci (QTL). For example QTL on chromosomes 6 and 8 have been linked to proteinuria in Munich Wistar Frömter rats

14 Chapter 1 Consequences of proteinuria Proteinuria is a common feature of many renal diseases. Proteinuria itself is an independent risk factor for a decline in GFR and for the development of ESRD. 95 Even an increase in albuminuria within the normal range is associated with the development of overt proteinuria in type 2 diabetes. 96 Several theories have been postulated about the role of proteinuria in progression of renal disease. 97 First, filtered albumin itself may damage tubular cells. The high metabolism of tubular cells needed to reabsorb as much as albumin as possible, could lead to toxic effects. Furthermore, other molecules such as cytokines, complement, growth factors, and lipids filtered by the glomerulus are thought to injure the tubular cells. This injury leads to tubular apoptosis, an inflammatory response, and fibrosis. Progression of renal disease was thought to start with podocyte damage. This will be discussed in the next section. Moreover, proteinuria is also an independent risk factor for development of cardiovascular diseases in diabetic and non-diabetic renal disease Patients with microalbuminuria and a normal or small reduced glomerular filtration rate have more frequent coronary events and death. 101;102 Reduction in albuminuria translated in less cardiovascular events. 103 Endothelial dysfunction, a diminished number of circulating bone marrow-derived cells, abnormalities of apolipoproteins, insulin resistance, and other factors are thought to contribute to the development of cardiovascular events in patients with albuminuria (reviewed in 104 ). Progression of renal disease Epidemiology and risk factors In the United States of America about 4.3% of the population has stage 3 renal insufficiency (creatinine clearance 50ml/min). 105 However, this percentage is increasing worldwide. Outcome in renal disease is heterogeneous. Several factors contribute to development of ESRD (Table 1). The type of renal disease and the severity of disease predicts the chance of developing ESRD. As described in the previous section, proteinuria is an independent risk factor for progressive renal insufficiency. Furthermore, ethnicity, number of nephrons, age, and gender contribute to susceptibility to progression. 106 Interestingly, males are more prone to develop ESRD than females. Possible explanations for this feature are specific gender-related differences in renal hemodynamics, alterations in the renin-angiotensin system, and modulating effects of sex hormones The presence of testosterone worsens the outcome of renal disease, whereas estrogens seem to be protective in several experimental models Estrogen and androgen receptors within the glomerulus 20

15 are thought to mediate the induction or prevention of glomerulosclerosis. 115;116 The exact influence of sex hormones on the severity of renal disease and progression to ESRD needs further investigation. Other risk factors are hyperfiltration, obesity, increased serum creatinine, and chronic renal damage in renal biopsy. 117 The impact of each risk factor is disease-dependent. Risk factors for development of chronic kidney disease Age (older) Hyperfiltration states Histology Gender (male) Diabetes mellitus Renal disease Ethnicity (black) Hypertension Tubular atrophy Metabolic syndrome Obesity Low nephron number Proteinuria High protein intake Glomerulosclerosis Dyslipidemia Low nephron number Glomerular hypertrophy Serum creatinine Anemia Table 1. Risk factors for development of chronic kidney disease and end-stage renal disease. Symptoms and treatment Progression of renal disease is defined by persistent deterioration of the GFR. Progression is categorized in 5 stages (Kidney Disease Outcome Quality Initiative guidelines). A GFR <15 ml/min is defined as kidney failure (stage 5). Progressive renal insufficiency can be accompanied by proteinuria, anemia, and hypertension. As proteinuria, dyslipidemia, and hypertension are risk factors for development of ESRD, treatment is now focused on ameliorating these features. Although only a small percentage of patients with a renal disease develops ESRD and progression can be limited by the available drugs, still the number of patients with ESRD is increasing worldwide and new treatment modalities are necessary. Better insight in the pathogenesis of progressive renal disease is required to develop new treatment options. Pathogenesis Progression of renal diseases is histologically characterized by glomerular damage, interstitial fibrosis and tubular atrophy. Regardless of the underlying glomerular disease, glomeruli change by inducing fibrosis and by attaching to Bowman s capsule. This process has been thoroughly described by Kriz et al. in several experimental models The process starts with damage to the podocytes. Damaged or apoptotic podocytes detach from the glomerular basement membrane, leading to a naked 21

16 Chapter 1 GBM. When this naked GBM binds to Bowman s capsule two sequences occur. First, deposition of ECM occurs within the glomerular segment that is attached to Bowman s capsule, leading to glomerulosclerosis. In addition, a misdirected filtration of serum proteins into the tubulointerstitial space instead of into the urinary space is proposed to cause inflammation and fibrosis of the tubulointerstitial compartment. Of note, proteinuria itself is also thought to cause injury to the tubules. Evidence for a key role for podocyte depletion in development of glomerulosclerosis has been found by many groups. In several rat models of spontaneous proteinuria and progressive glomerulosclerosis a reduced number of podocytes preceded the development of glomerulosclerosis. 121 More direct evidence was given by elegant experiments in which dose-dependent podocyte depletion was induced with an injection of diphtheria toxin in rats expressing the human diphtheria toxin receptor transgene. 122 Depletion of <20% of the podocytes caused transient proteinuria and mesangial expansion, loss of 20 to 40% of the podocytes resulted in persistent proteinuria and FSGS but no progressive decline in renal function, and finally loss of >40% of the podocytes resulted in progressive glomerular failure. Next to an absolute podocyte reduction, a relative reduction in podocytes is seen in renal diseases in which glomerular hypertrophy takes place. For example, in diabetic nephropathy and after nephrectomy, hyperfiltration of the glomeruli causes enlargement of the glomeruli. Because glomerular podocytes are unable to proliferate, a decreased podocyte to GBM ratio develops, leading to the above described mechanism of glomerulosclerosis. 122;123 Tubulointerstitial fibrosis and glomerulosclerosis result from accumulation of ECM molecules in response to renal injury. 124 Both interstitial ECM deposition and the deposition of periodic acid-schiff-positive ECM in the glomeruli highly correlate with renal function. 125;126 The network of ECM includes fibronectin and the collagens I, III, and IV. Collagen I and collagen III are the main components of renal fibrotic lesions 127, and fibronectin is one of the main ECM components in glomerulosclerotic lesions. 128 Excessive ECM protein deposition in the kidney may result from increased mrna synthesis of ECM molecules, from decreased degradation of ECM proteins by reduced activity of matrix metalloproteases (MMPs) or by a reduced ability of MMPs to degrade ECM proteins that have undergone posttranslational modifications, or from a combination of these factors. Genetics and progressive renal disease Genetic factors contribute to the risk of developing progressive glomerulosclerosis. The observations that males are more prone to progress than females and that some families are more susceptible to develop glomerulosclerosis, suggest that genetic 22

17 factors contribute to the susceptibility to disease progression and to the rate of this progression. One example is diabetic nephropathy. Familial clustering in diabetic nephropathy has been found 129 and polymorphisms in candidate genes have been identified, although replication of these data was difficult. In the last decade loci on human chromosomes 10 and 18 were associated with diabetic nephropathy. 130;131 Although much effort was put into finding genes involved in progressive renal disease in humans, only few have been identified. Therefore, and because genetic research in humans is hampered by heterogeneity and high costs, research is partly directed to experimental models. Whole genome linkage analyses in Munich-Wistar-Frömter (MWF), Dahl salt-sensitive (Dahl S), and Fawn-hooded hypertensive (FHH) rats, have identified multiple QTL, that are involved in the development of hypertensionmediated glomerulosclerosis. 89;92;93 These rat strains are all inbred strains that display hypertension and nephrosclerosis with aging as an inherited trait. In addition, a QTL on rat chromosome 2 (RNO2) has been linked to progressive albuminuria and renal damage in mice. 132 Focal and segmental glomerulosclerosis In 1957, Rich described a focal and segmental sclerosis of glomeruli in an autopsy study of patients with nephrotic syndrome. 133 Later Habib et al. defined the histological features of this glomerulopathy and proposed the term focal and segmental hyalinosis and sclerosis. 134 FSGS is defined as a clinical-pathologic syndrome, characterized by proteinuria, usually of the nephrotic range, associated with focal and segmental glomerular sclerosis and foot process effacement. The pattern of glomerulosclerosis is focal, involving a subset of glomeruli, and segmental, involving a portion of the glomerular tuft. As the disease progresses glomerular lesions become more diffuse and global. Clinical symptoms Patients with FSGS present with proteinuria, usually in the nephrotic range. Many patients also present with hypertension (45-65%) and microscopic hematuria (30-50%). 135 The level of kidney function varies between patients. When proteinuria does not respond to corticosteroids, FSGS is more likely to be present than MCD, a disease in which patients also present with nephrotic range proteinuria and foot process effacement, but without glomerulosclerosis. The renal prognosis is better in patients with MCD compared to FSGS. The clinical presentation, response to treatment, and rate of progression to renal insufficiency vary enormously. About 30-50% of all patients with FSGS develop end-stage renal disease. 135;136 After renal transplantation, 23

18 Chapter 1 the incidence of recurrence is between 20% and 30% In recurrent FSGS many patients present with proteinuria within a few weeks after transplantation. Once the first renal graft fails due to recurrence, in about 80% of the second allografts FSGS recurs. Etiology FSGS can be divided into primary and secondary forms of FSGS. In most cases of FSGS no etiological agent was determined. This is called primary or idiopathic FSGS. A characteristic of primary FSGS is that podocytes are damaged in a global and diffuse manner. Also in glomerular areas where no glomerulosclerosis is present in light microscopy, foot process effacement can be observed. In a minority of patients a recognized etiologic association is found (secondary FSGS). They include genetic mutations in podocyte-associated molecules such as a-actinin-4, podocin and WT- 1 75;76;140, viruses such as Human Immunodeficiency Virus and Parvovirus B19 141;142, and drug-induced FSGS. 143 Furthermore, primary FSGS should be differentiated from FSGS as a result of maladaptation of the kidney. 144 For example reduced renal mass, as in unilateral renal agenesis, surgical ablation or in advanced renal disease, can lead to glomerular hypertrophy of the remaining nephrons and subsequently to glomerulosclerosis that resembles primary FSGS. Furthermore, hemodynamic stress to the nephron as is seen in hypertension or vaso-occlusive processes, can lead to FSGS. In primary FSGS, a circulating factor is thought to cause proteinuria. Several observations support this hypothesis. First, rapid onset of proteinuria following renal transplantation suggests a role for a circulating factor. 145 Second, plasmapheresis shortly after transplantation reduces the risk of recurrent FSGS. 146 Third, injection of plasma or fractions of plasma from patients with recurrent FSGS may cause proteinuria or albuminuria in experimental animals. 147;148 Recently, mutations in the soluble urokinase receptor have been identified to cause proteinuria in patients with FSGS. 149 Morphological classification FSGS is a disease with many different forms in terms of clinical features, outcome, and morphology. 150 Based on glomerular morphology, D Agati et al. recently proposed a classification of FSGS variants, termed the Columbia classification, that distinguishes five variants of FSGS. 151 Examples of all variants are shown in Figure 4. 1) Tip lesion variant. This variant requires at least 1 segmental lesion involving the tip domain. The tip domain is the outer 25% of the glomerular tuft next to the origin of the proximal tubule. Patients with this variant typically present with nephrotic 24

19 tip FSGS cellular FSGS collapsing FSGS perihilar FSGS FSGS NOS Figure 4. Typical glomerular morphology of the five FSGS variants according to the Columbia classification. Published with permission from Stokes et al

20 Chapter 1 range proteinuria and usually have a good response to treatment. Only few patients progress to ESRD. This lesion is sometimes regarded as MCD like lesion, as the prognosis is similar ) Cellular variant. This variant is characterized by the finding of at least one glomerulus with segmental endocapillary hypercellularity occluding the capillary lumina. The cellular lesion is the least common morphological variant of FSGS. 153;154 3) Collapsing variant. Histologically, glomeruli in this variant show collapse of the glomerular tuft concomitant with epithelial cell hypertrophy and hyperplasia. This variant is associated with severe proteinuria at presentation and a rapid decline in renal function. 154 The collapsing variant is associated with several diseases like infections (HIV and parvovirus B19) 141;142, autoimmune diseases (Systemic Lupus Erythematosis) 155, malignancies (multiple myeloma) 156, genetic disorders (sickle cell anemia) 157, drug exposure (Pamidronate) 143, and posttransplantation conditions (de novo or recurrent collapsing FSGS). 158;159 Research of this variant of FSGS in renal allografts is hampered by the occurrence of the de novo collapsing variant. 4) Perihilar variant. This variant is characterized by segmental glomerular lesions predominantly located at the vascular pole. This variant is present in about 25% of all cases 154;160 and is more common in adults than in children. 161 It is associated with secondary forms of FSGS mediated by glomerular hyperfiltration. 62;162 It has been hypothesized that hypertension-induced glomerulosclerosis occurs in the perihilar regions due to greater filtration pressure in the more proximal portions of the glomerular capillary loops ) FSGS not otherwise specified (FSGS NOS). FSGS NOS is defined by at least 1 glomerulus with segmental increase in matrix which obliterates the capillary lumina. For this variant typical lesions seen in all other variants must be excluded. This variant is the most common morphological form of FSGS. All four previous described forms may evolve into this variant when disease progresses. 151 Although progression to renal failure in FSGS is predicted by the response to therapy, this new classification could shed more light on the pathogenesis of the several morphological features. Some variants are more prone to progress to renal failure. For example, presence of collapsing FSGS has a worse renal prognosis than tip lesion FSGS. 154 The role of this classification in primary FSGS and recurrence FSGS inallografts needs to be further investigated. Genetics in focal and segmental glomerulosclerosis In recent years several autosomal dominant and recessive forms of FSGS have been described. Many mutations in podocyte-associated molecules are found in congenital forms of FSGS (reviewed in 164 ). Dependent on the mutated gene or the specific 26

21 mutation within the gene, the severity of the disease and the age of onset differ. 165 In addition, it is thought that about 18% of all FSGS cases are familial disease. 166 Still, in many families with FSGS no responsible gene has been found. Animal models for proteinuria and focal and segmental glomerulosclerosis In order to understand the mechanisms of proteinuria and progression of renal disease, research has focused on observations in humans. However, research in humans is hampered by genetic heterogeneity, variation in environmental factors, high costs, and ethical problems. Research in animal models overcomes most of these issues. Especially the pathogenesis and sequence of events can be investigated more easily. The fact that many relevant genes are conserved between species, makes animal models more attractive as a surrogate for the human situation. Several animal models can be used in renal research. We used two types of animal models. In the first model, differences in susceptibility of spontaneous development of proteinuria and glomerulosclerosis were observed. Secondly, substrain-related differences in proteinuria and progressive glomerulosclerosis were examined in an antibody-induced rat model for mesangial damage. Munich-Wistar-Frömter rat A unique model of development of spontaneous proteinuria and progressive glomerulosclerosis is the Munich-Wistar-Frömter rat (MWF). This rat strain shows a gender-specific congenital renal phenotype. Male MWF rats have an inherited deficit in the number of nephrons and spontaneously develop albuminuria and mild hypertension followed by overt proteinuria, FSGS, and renal failure at an older age. 167;168 In contrast, although female MWF rats demonstrate a similar reduction in the total number of nephrons and exhibit mild albuminuria and hypertension, they do not develop overt proteinuria or FSGS Overt proteinuria in male MWF coincides with glomerular hypertrophy 121, and redistribution of the slit diaphragm proteins zonula occludens-1 and nephrin. 172 Progression to glomerulosclerosis is preceded by a decrease of the number of podocytes. 121 Linkage analysis has shown loci on chromosomes 6 and 8 that are linked to development of albuminuria in MWF. 89 The mechanisms of development of proteinuria and the role of glomerular hypertrophy are not completely known yet. Previous research has shown that glomerular size and glomerular capillary pressures are identical between male and female MWF. 173 However, the single nephron glomerular filtration rate is increased in males compared to females. This spontaneous model of proteinuria makes investigation 27

22 Chapter 1 of gender effect in the development of proteinuria and glomerulosclerosis possible. Furthermore, the role of glomerular hyperfiltration can be determined. Investigation of glomerular and podocyte-associated changes before and during development of proteinuria may shed new light on the pathogenesis of this model. Anti-Thy-1 glomerulonephritis The second model is the anti-thy-1 glomerulonephritis model. This model is frequently used for investigation of acute inflammation-induced mesangial damage and proliferation. The model is induced by injecting antibodies against Thy 1.1, a transmembrane protein expressed by mesangial cells in the rat. Several anti-thy-1 antibodies are available. All our experiments were performed with the ER4 mouse IgG2a monoclonal antibody. 174 Binding of this antibody to the mesangial cells results in immediate complement activation. Only few hours after induction, deposition of complement factors C3, C9, and C5-C9 complex can be found. 174;175 Complement activation is further accompanied by increased intraglomerular coagulation, represented by deposition of fibrinogen and platelet. This acute injury leads to lyses of mesangial cells. Thereafter, an influx of inflammatory cells (polymorphic nuclear cells and monocytes) is found. 176 After a few days glomerular capillary ballooning, called microaneurysms, can be observed accompanied by mesangial cell proliferation and glomerular hypercellularity. In most rat strains microaneurysms are filled up by proliferating cells and ECM, and apoptosis of the mesangial cells occurs. 177;178 The histological damage after one injection of anti-thy-1 antibody is accompanied in most rat strains by massive proteinuria, reaching maximal levels at days 2-7 and gradually decreasing to normal levels thereafter. Glomerular lesions usually resolve within 3 weeks of time. Progression to glomerulosclerosis within the anti-thy-1 glomerulonephritis model can be induced when anti-thy-1 antibodies are repetitively injected or when the glomerulonephritis is induced after unilateral nephrectomy. 179;180 In one particular substrain of Lewis rats, Lewis/Maastricht, a single injection of anti-thy-1 antibody does lead to progressive glomerulosclerosis. This progressive glomerulosclerosis is preceded by a transient proteinuria, as seen in other rat strains in which glomerular damage resolves. In contrast to Lewis/ Maastricht, Lewis/Møllegard rats show a mild disease after induction of anti-thy-1 glomerulonephritis. Although Lewis/Møllegard rats develop full blown glomerular damage, no proteinuria is observed and glomerular damage resolves within 4 weeks. 181 The anti-thy-1 glomerulonephritis model in Lewis substrains provides a useful tool to investigate the pathogenesis and genetics of proteinuria and progression to glomerulosclerosis after acute glomerular damage. 28