The Pathophysiology of Proteinuria

The Pathophysiology of Proteinuria

C H A P T E R 9 The Pathophysiology of Proteinuria Ton J. Rabelinka, Hiddo J. Lambers Heerspinkb and Dick de Zeeuwb a Department of Nephrology, Leid...

3MB Sizes 0 Downloads 3 Views

Recommend Documents

No documents
C H A P T E R

9 The Pathophysiology of Proteinuria Ton J. Rabelinka, Hiddo J. Lambers Heerspinkb and Dick de Zeeuwb a

Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands, Department of Clinical Pharmacology, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands

b

excretion of these smaller proteins.3,4 This chapter will consider the proteinuria in which albumin is the major portion of the excreted urinary protein. Urine albumin excretion less than 30 mg/day is considered normal. Urinary albumin excretion more than 30 mg/day and less than 300 mg/day is called microalbuminuria. Urine albumin excretion greater than 300 mg/day is called macroalbuminuria.5 Albuminuria is measured in collected urine. The classical and still preferred approach is to collect the urine for 24 hours for analysis. However, for practical reasons the first morning void urine collection is often analyzed. In general the values derived from this specimen have a high correlation with those of the 24-hour collection. However, it is advisable to normalize the albumin concentration for the creatinine concentration in the first morning void urine collection specimen, since that will correct for collection errors in the 24-hour urine and for urine concentration differences in the first morning void. Albumin concentration is currently measured by giant lab-platforms (usually employing nephelometric or immunoturbidimetric techniques). However, in the future patients may monitor their own albumin excretion (similar to glucose or blood pressure measurements) using a point of care device. Such devices are available for measurement of the albumin concentration. Classically, the focus on understanding the pathophysiology of albuminuria has been on ultrastructural alterations in the glomerular basement membrane (GBM) and podocytes. However, recently the endothelium has been recognized as a barrier against albumin filtration into the urine as well. This may explain why CVD may be associated with the presence of

INTRODUCTION The classical view of proteinuria has evolved rapidly over the past several years. Where proteinuria was considered a reflection of renal disease not so long ago, it now is merely implicated as a cause of renal disease, and is becoming a target to treat. This paradigm shift is the subject of much debate among physiologists, pathologists and nephrologists. The glomerulus acts as a size-selective filter for protein filtration. Consequently, tubular fluid contains only proteins of low molecular weight (<60 kD) such as vitamin D-binding protein (DBP) or free retinol-binding protein (RBP), while larger proteins are excluded.1 Albumin, the most abundant plasma protein, is filtered in only very low amounts (1–50 μg/ml). In addition, albumin can be reabsorbed by the tubular epithelium. Consequently, normal total urinary protein excretion in the normal adult should be less than 150 mg/day.2 Higher rates of urinary protein excretion that persist beyond a single measurement should be evaluated. The increased protein leak can be caused by an increase in the circulating levels of several different proteins. For example, one may observe tubular proteinuria consisting of low-molecular weight proteins (less than 25 kD). Such proteinuria usually reflects a systemic disease with overproduction of small peptide fragments that are filtered and may cause tubular damage (such as beta2-microglobulin, immunoglobulin light chains, and polypeptides derived from the breakdown of albumin). In addition, interference with proximal tubular reabsorption, due to tubulointerstitial diseases or genetic mutations, or overproduction of immunoglobulins, such as occurs in myeloma, can lead to increased

P. Kimmel & M. Rosenberg (Eds): Chronic Renal Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-411602-3.00009-3

92

2015 Elsevier Inc. All rights reserved. © 2012

Pathogenesis of Albuminuria

albuminuria. Endothelial dysfunction can also induce specific renal sequelae of albumin leaking through a defective glomerular endothelium. For example, exposure to leaked albumin may induce secondary renal epithelial damage. Albuminuria therefore may not only be a marker of kidney damage, but may also serve as a therapeutic target that should be lowered. (The fewer albumin molecules that leak through the filter, the better the kidneys and the patient will fare.) One may consider drugs that specifically target endothelial “repair” for lowering albuminuria. Finally, if we assume that a leak of albumin in the urine is a consequence of a change in the endothelial layer, it is highly likely that albumin leaks into the vascular wall of other tissues as well. Such leakage can have consequences for the function of these other organs. In particular, generalized vascular albumin permeability might induce chronic inflammation and tissue function loss in other organs as well. This concept may explain the close relationships between albuminuria, renal disease and CVD. Indeed, the presence of microalbuminuria has been shown to be a predictor for the development of renal and cardiovascular complications in patients with and without diabetes.6,7 A generalized, vascular abnormality may also explain why many treatments that halt progression of renal disease are also effective in CVD protection.8

PATHOGENESIS OF ALBUMINURIA In humans approximately 180 liters of glomerular ultrafiltrate are delivered to the proximal tubule each day. Nephrons can endure this filtration workload over a lifetime, underscoring the unique anti-fouling properties of this filtration membrane. The filtration barrier consists of a 3-layered structure, the glomerular endothelium, the glomerular basement membrane (GBM) and the podocytes. Each layer plays a unique and interdependent role in the filtration process. In addition, the mesangial cells contribute to the structural integrity of the filtration barrier and play a permissive role in regulation of glomerular filtration. Each component of the glomerular filtration barrier affects the function of the others, and must be considered individually and collectively in the pathogenesis of albuminuria. The glomerular barrier functions as an integrated system. Electron microscopic studies in the 1960s by Farquhar9,10 demonstrated that in intact glomeruli only about 0.06% of plasma albumin is filtered, implying that the glomerular filter is capable of retaining macromolecules in the circulation while at the same time allowing a large hydraulic conductivity. While tubular reabsorption of albumin may determine whether or not albuminuria finally develops, dysfunction of the

93

glomerular filtration barrier is assumed to play the key role in initiating the development of albuminuria.

The Endothelium The first part of the glomerular barrier that interacts with the flowing blood is the glomerular endothelium. The endothelium is highly fenestrated and its pores are estimated to be around 60–80 nm in diameter.11 These fenestrae do not contain a diaphragm and thus have to be considered real pores.12 While such fenestration greatly facilitates the formation of high volumes of ultrafiltrate, it would also imply loss of macromolecules such as albumin, as in fact has been suggested by some intravital microscopy studies.13 However, as early as 1976 Ryan and Karnovsky demonstrated that under physiological conditions plasma albumin does not penetrate significantly beyond the endothelial layer of the glomerular capillary wall.14 In vivo, the endothelium is covered with a polysaccharide protein gellike structure, the glycocalyx. This layer actively binds plasma proteins and growth factors, and is referred to as the endothelial surface layer (ESL). The composition and biological activity of this surface layer differs in an organ-specific manner. The main constituents of the ESL are protein cores, typically syndecans, with large heparan sulfate side chains (about 60%) or chondroitin sulfate (about 15%) (Figure 9.1).15 Heparan is a so-called glycosaminoglycan (GAG) or mucopolysaccharide that consists of a regular repeating sequence of disaccharide units that are composed of D-glucosamine (or L-iduronic acid) and N-acetyl-Dglucosamine. This polysaccharide is heavily sulfated and thus bears a net negative charge. Plasma proteins such as albumin and orosomucoid and growth factors bind to these negatively charged proteoglycans and glycosaminoglycans.16 The major non-sulfated GAG in the glycocalyx is hyaluronan, which is integrated into the glycocalyx like a mesh and anchors to CD44 on the cell membrane, providing structural cohesion to the ESL. The ESL governs many of the interactions of the flowing blood with the vessel wall such as prevention of clotting (by activating antithrombin III), and migration of immune cells into the vessel wall (by controlling the interaction with leukocyte adhesion molecules) and vascular remodeling (by binding local growth factors such as vascular endothelial growth factor [VEGF] and fibroblast growth factor [FGF]).15 The combination of negatively charged (HS) sulfates and the meshlike structure of the less charged hyaluronan has been proposed to act as a charge selective barrier against albumin filtration through the fenestrae.17 These barrier properties are further increased by local shear forces, which result in increased viscosity of

IV. PATHOPHYSIOLOGY

94

9.  The Pathophysiology of Proteinuria

FIGURE 9.1  Top panel shows a cross-sectional overview of the glomerular structure. E: glomerular endothelial cell, G: glycocalyx, PO: podocyte, M: mesangial cell, MM; mesangial matrix. The inset shows a high power view of the endothelial surface layer which consists of glycoproteins (syndecans) that have long heparan sulfate side chains (blue). An important structural component of the glycocalyx is hyaluronic acid (orange), which attaches to the plasma membrane through CD44. The adhesion molecule ICAM is depicted to give a perspective on the dimensions of this endothelial surface layer.

the gel properties of the endothelial surface layer. The Starling filtration equilibrium in non-renal microvasculature is generated by the inability of albumin to permeate the ESL and the resulting oncotic gradients over this layer.18 In support of this concept, acute infusion of high dosages of enzymes that degrade the ESL results in increased fractional albumin clearance, extrapolating this concept to the kidney.19,20 Albumin passage across the endothelium can be observed in almost all the glomeruli after chronic infusion of hyaluronidase, in a dose that leaves the polysaccharide surfaces of the other cells in the glomerulus intact, but predominantly removes the hyaluronan in the endothelial fenestrae. Such albumin passage was never observed in control animals.21

Together these data indicate that the glycocalyx/ ESL acts as a nearly perfect initial barrier to albumin filtration. The presence of such a charge selectivity has recently been debated, as measurements of glomerular sieving coefficients using intravital 2-photon microscopy suggest that such a barrier does not exist.13 However, filtration sieving coefficients measured with these techniques are subject to variations in detector sensitivity, and the nature of the fluorescent probes used. Moreover, genetic strain, age and nutritional status may affect sieving coefficients as well (e.g. aged Fawn Hooded male rats have spontaneous proteinuria). When these facts are taken into consideration, the glomerular sieving coefficient for albumin, when using intravital microscopy, is very low.22 The glycocalyx constitutes the first defense against albumin filtration. Several conditions are associated with modifications or loss of the glycocalyx. One of the earliest changes in the endothelium upon its activation by cytokines or metabolic factors is an alteration in glycocalyx composition. The endothelial glycocalyx markedly changes its surface properties under inflammatory conditions. This initially occurs by adding heparan sulfate to its surface and inducing changes in heparan sulfate composition that facilitate the recruitment of inflammatory cells.23 With sustained inflammation, the production of heparanase is induced in the glomerulus, which may partially degrade the glycocalyx layer, providing another mechanism for localizing and regulating leukocyte recruitment.24 Since glomerular inflammation is associated with loss of glycocalyx dimensions, it is not surprising that glomerulonephritis is often associated with increased glomerular albumin permeability and albuminuria. The direct link between endothelial function and glycocalyx properties may also explain, at least in part, the relationship between cardiovascular risk factors and albuminuria. Normally the endothelium maintains a quiescent phenotype where nitric oxide signaling dominates. Upon exposure to high glucose and lipids, the endothelium switches to redox signaling, becomes activated, and the induction of heparanase and associated glycocalyx changes occurs.25 Such changes may affect the development of vessel wall injury. Glycocalyx changes facilitate lipoprotein and leukocyte transport into the vessel wall, as well as the development of albuminuria.26 This pathophysiology has been best described in diabetes. For example, diabetic Zucker fatty rats have a significant reduction in endothelial cell glycocalyx thickness compared to control rats, which is associated with increased expression of redox stress-induced heparanase. These ultrastructural changes are accompanied by the development of albuminuria.27 Heparanase-knockout mice failed to develop proteinuria and renal damage in response

IV. PATHOPHYSIOLOGY

Pathogenesis of Albuminuria

95

to streptozotocin-induced type 1 diabetes mellitus.24 The role of heparanase was further demonstrated by the observation that treatment of diabetic mice with the specific heparanase inhibitor SST0001 resulted in a lower degree of albuminuria compared to vehicletreated diabetic mice. Finally, patients with type 1 diabetes mellitus show a reduction in systemic glycocalyx volume, which correlates with the presence of microalbuminuria.28

Mesangium Mesangial cells are contractile cells that constitute the central stalk of the glomerulus. On the capillary lumen side, mesangial cells are in direct contact with the glomerular endothelium without an intervening basement membrane, as one typically can observe with pericytes. Mesangial cells are indeed considered to function as specialized pericytes, and are consequently essential to stabilize glomerular endothelial function. Mesangial cells release growth factors and cytokines such as VEGF and angiopoietins adjacent to the endothelium, and engage in gap junction communication with the endothelium.29 Simultaneously, mesangial cell function and survival depends upon signaling from the glomerular endothelium (most notably plateletderived growth factor [PDGF]-B).30 Their contractile properties enable mesangial cells to alter intraglomerular capillary flow and glomerular ultrafiltration surface area and thereby co-regulate single nephron GFR (SNGFR).31 The importance of the mesangium for function of the glomerular filtration barrier is shown by experiments where the mesangium is injured by toxins or antibodies.32 The resulting mesangiolysis invariably results in endothelial injury and proteinuria. However, subtle changes in mesangial function may also affect function of the glomerular filtration barrier and result in the development of proteinuria. For example, murine studies showed the reconstitution of normoglycemic B6 recipient glomeruli with db/db BM-derived mesangial cells were associated with the development of albuminuria and severe glomerular lesions in recipients.33 In the case of primary endothelial injury, widening of the subendothelial space and deposition of proteinaceous material precedes mesangiolytic changes (Figure 9.2). In the case of repetitive endothelial injury this process may lead to the development of lamellated mesangial nodules. These phenomena typically can be observed in disease that primarily affects the endothelium such as diabetes mellitus (Kimmelstiel Wilson lesion) and thrombotic microangiopathy. Once these lesions occur, normal endothelial barrier function is irreversibly hampered, and albumin filtration over the GBM occurs.

FIGURE 9.2  The proposed mechanism of how endothelial function in diabetes and hypertension may lead to mesangial lesions. The top panel shows the normal situation. Endothelial cells and mesangial cells are in close contact. Mesangial cells function as pericytes for the glomerular endothelium. Upon endothelial activation this signaling is disturbed and subendothelial protein deposits may develop. This may result in mesangiolysis and subsequent development of nodular lesions (lower panel).

The Glomerular Basement Membrane The GBM is an amorphous, 300- to 350-nm-thick extracellular structure that previously has been considered to be the primary size- and charge-selective macromolecular filter. The normal GBM is composed of laminin-521 (α5β2γ1), type IV collagen α3α4α5, nidogen and heparin sulfate proteoglycan (HSPG; primarily agrin). Podocytes are considered the primary source of the GBM components laminin β2 and the collagen α3α4α5 (IV) network.34,35 Collagen IV is the major structural component of the GBM. Collagen IV forms a highly cross-linked network to which the other components of the GBM can attach. There are six collagen IV isoforms, α1–α6. Each isoform forms a triple helix structure with two other isoforms to

IV. PATHOPHYSIOLOGY

96

9.  The Pathophysiology of Proteinuria

generate the collagen IV network. The collagen IV α1/ α2 network is essential for the maintenance of normal basement membrane integrity and function under conditions of increased mechanical demands during early nephron development.36 The collagen IV α3/α4/α5 network is essential for long-term maintenance of glomerular structure and function.37 In patients with Alport syndrome, which is caused by mutations in one of the genes of the collagen IV α3/α4/α5 network, the GBM is more susceptible to proteolysis, and may ultimately deteriorate.38 Such deterioration leads to hematuria, and eventually to albuminuria and progressive kidney disease. Laminins are extracellular matrix glycoproteins that self-assemble into a network, or interact with other glycoproteins or integrin receptors. The presence of laminins is critical for the structural assembly of the GBM and for cell–matrix interactions. Laminin-521, the major laminin in the mature GBM,39 is essential for normal glomerular filtration, as shown by the observation that both podocyte-specific laminin α5- and laminin β2-knockout mice develop proteinuria.40,41 Moreover, patients with Pierson syndrome, a disease caused by mutations in the laminin β2 gene, also develop proteinuria.34,42 The development of proteinuria in laminin β2-knockout mice can be abrogated by overexpression of laminin β1, a structurally similar homolog of laminin β2, in podocytes,43 underscoring the intricate relationship between podocytes and the GBM. Interestingly, the glomerular ultrastructure of proteinuric 1-week-old Lamb2-null mice reveals intact podocyte foot processes and slit diaphragms, suggesting a role for the GBM as an independent and indispensible filtration barrier to plasma proteins.41 The nidogen family consists of two members: nidogen 1 and nidogen 2. Both proteins are major components of all basement membranes. Nidogens can bind to both collagen IV and laminin, and are considered to form ternary complexes between collagen IV and laminin.44,45 Loss of one nidogen does, however, not result in proteinuria and basement membrane structures appear normal in murine studies.46,47 The GBM has three layers that can be demonstrated ultrastructurally. These are the lamina rara interna, the lamina densa, and an outermost lamina rara externa. The lamina rara interna and lamina rara externa are rich in anionic sites composed of richly sulfated glycosaminoglycans, particularly heparan sulfate. This polysaccharide is attached to a core protein, which in the case of the GBM is predominantly agrin. For a long time it was assumed that the presence of negatively charged heparin sulfates (HS) in the GBM was essential for the charge-selective permeability of the glomerular filtration barrier. In support of this concept, removal of GAGs in the GBM by perfusion of bacterial

GAG-degrading enzymes led to the passage of albumin,48 while administration of anti-HS(PG) antibodies in rats led to the development of albuminuria.49 Finally, in glomerular diseases, such as diabetic nephropathy, systemic lupus erythematosus, and membranous glomerulopathy, decreased expression of HS in the GBM was observed.50,51 However, when investigating negatively charged and neutral Ficoll filtration on isolated GBMs, there appeared to be no such charge barrier.52 Mice with podocytes lacking the predominant core protein agrin did not develop proteinuria and had normal glomerular architecture, despite the fact that they lacked the majority of anionic sites in the GBM.53 Mice lacking other combinations of glomerular HSPGs, such as perlecan and collagen XVIII, or agrin and perlecan, developed neither glomerular abnormalities nor proteinuria.54,55 Smithies elegantly argued that the GBM can be considered to behave like a gel, and that thus albumin transport through the GBM is mainly determined by diffusion.56 The GBM is a critical component of the glomerular filtration barrier as it provides a scaffold that supports the physiological function of the glomerular endothelium and podocytes. Severe structural abnormalities of the GBM result in enhanced albumin leakage. Under normal conditions the GBM probably allows diffusive albumin transport and does not seem to constitute a major charge barrier. The hydraulic conductance of the GBM accounts, however, for most of the fluid restriction of the intact glomerular barrier.57 The GBM should therefore be considered an integral and essential component of the glomerular filtration barrier.

The Podocyte Podocytes are highly specialized epithelial cells that cover the outside of the glomerular capillary. Podocytes have a cell body with numerous primary, secondary and tertiary extensions, called foot processes. Adjacent foot processes are interconnected by slit diaphragms, whose main function is probably to act as mechanosensors and to form the final barrier to filtration (Figure 9.3). In 1998, Kestilä et  al.58 isolated the gene mutated in congenital nephrotic syndrome of the Finnish type (CNF, NPHS1), a rare autosomal recessive disease characterized by massive nonselective proteinuria at birth and lack of a podocyte slit diaphragm. The disease gene was shown to encode a novel protein named nephrin, which in the kidney is solely located in glomerular podocytes. Inactivation of the gene in mice leads to massive proteinuria, absence of a slit diaphragm, and neonatal death,59 recapitulating the human disease. These observations have led to the discovery of a network of plasma-membrane proteins in the slit diaphragm. The presence of nephrin, Neph1,

IV. PATHOPHYSIOLOGY

Pathogenesis of Albuminuria

FIGURE 9.3  The top panel shows an overview of the glomerular structure. In the inset a schematic cross-sectional drawing is given of the regulation of podocyte function and maintenance of the slit diaphragm. Proteins where mutations or deletions result in proteinuria have been indicated (for further explanation see text). E: glomerular endothelial cell, G: glycocalyx, PO: podocyte, M: mesangial cell, MM; mesangial matrix; TrpC6, transient receptor potential C6. NSCC, nonselective cation channel.

podocin, TRPC6 and FAT1 are essential for normal glomerular filtration.60–62 The molecular structure of the slit diaphragm has been proposed to act as a true barrier against filtration of large molecules. Using electron tomography, winding molecular strands that cross the filtration in a zipper-like pattern that form pores of the size of albumin or smaller can be observed.63 The slit diaphragm is closely linked to the podocyte actin cytoskeleton by linker proteins such as CD2associated protein (CD2AP) and the non-catalytic region of tyrosine kinase (NCK) proteins.64,65 This highlights another function of the podocytes, balancing the distending transmural pressure gradient over the capillary. From this perspective the slit diaphragm can be considered a mechanosensor that controls the interaction with neighboring podocytes. Mutations in these

97

proteins can result in albuminuria, underscoring the fact that the integrity of the slit diaphragm is dependent upon its association with the cytoskeletal cellular machinery. Podocytes adhere to the GBM via adhesion protein complexes such as dystroglycans and by an α3β1integrin complex. Disruption of the podocyte-GBM adhesion is required for movement of the podocytes, as can be observed in effacement. In podocytes, integrins localize exclusively to the basal membrane. Integrins link to the actin cytoskeleton of the podocytes. The associated outside-in signaling is essential for podocyte function and survival, as is illustrated by α3-integrin knockout mice which develop congenital nephrotic syndrome.66 These experiments also illustrate how the GBM acts as a vital scaffold for podocyte function. There are at least two different mechanisms by which podocytes can become involved in, and contribute to, the development and extent of albuminuria. Haraldsson and Deen67 showed elegantly that the most selective part of a multi-layered passive filter cannot be in the last layer (that is, the slit diaphragm), because retention and accumulation of albumin would occur within the filter immediately in front of the slit diaphragm and lead to clogging. As we argued before, the endothelium appears to act as the primary filtration barrier. Upon endothelial activation, albumin starts to leak through the GBM and the podocytes are subsequently exposed to albumin. Interestingly, podocytes are equipped with a megalin-cubulin system,68 as well as scavenger receptors such as the receptor for advanced glycation end products,69 suggesting that podocytes may endocytose albumin. In the case of diabetes mellitus, podocytes may also be exposed to chemically modified and glycated albumin. Blocking uptake by these scavenger receptors reduces podocyte injury. Experimental data suggest that endocytosed albumin induces a mesenchymal transformation in podocytes, with loss of slit diaphragm proteins and induction of desmin.21,70 From this perspective, changes in podocyte structure can be regarded as a response to injury (Figure 9.4). Secondly, elegant murine studies have unambiguously demonstrated that normal podocyte function is required for the integrity of the glomerular filtration barrier. Podocyte-specific deletions of genes that control its phenotype lead to loss of the glomerular endothelial phenotype and the development of proteinuria. This has been best demonstrated for the paracrine regulation of components of the VEGF-axis by podocytes. Such gene deletion not only results in podocyte effacement but also in the disappearance of the glomerular endothelial fenestration.71,72 This is clinically exemplified by pre-eclampsia, a severe proteinuric kidney disease that occurs during pregnancy, where a circulating soluble

IV. PATHOPHYSIOLOGY

98

9.  The Pathophysiology of Proteinuria

Normal

Endothelial activation

Result

FIGURE 9.4  Proposed mechanism for the development of albuminuria in the setting of endothelial dysfunction and endothelial activation. The top panel shows the normal situation with a negatively charged glycocalyx. The middle panel shows upon endothelial cell activation, through redox sensitive signaling mechanisms, the endothelial glycocalyx is shed and albumin can pass the endothelial layer. Podocytes are subsequently exposed to modified albumin and the normal podocyte–endothelial signaling is disturbed. The lower panel shows podocytes take up albumin and undergo transformational changes that may result in podocyte effacement and podocyte drop-off.

form of VEGF receptor 1 (sFlt-1, also known as VEGFR-1) scavenges and neutralizes VEGF in the vasculature of the mother. sFlt-1 is secreted from the placenta and binds podocyte-derived VEGF at the glomerular filtration barrier, resulting in albuminuria, severe endothelial

damage and hypertension.18 Therefore, primary injury of podocytes, either by genetic mutations (Table 9.1) or immunological injury, results in severe derangement of the integrity of the glomerular filter and the development of macroalbuminuria (Figure 9.5). There is a clear relationship between podocyte injury and development and progression of CKD. Podocytes adhere to the GBM only by their foot processes, which makes them susceptible to detachment, while being exposed to shear from the flow of ultrafiltrate. It is assumed that podocytes are terminally differentiated cells with no replicative potential. This makes recent observations that demonstrate that podocytes can move along the GBM, to cover areas where other podocytes have dropped off, to repair glomerular integrity, of great relevance.73 Such migration of podocytes is associated with loss of foot processes, also referred to as foot process effacement (FPE). Such findings led to the idea that FPE causes proteinuria. While FPE is associated with loss of slit diaphragm function, it is unclear how foot processes fused to sheets of continuous cytoplasm covering the GBM should increase glomerular permeability. It has been proposed that the migration and effacement of podocytes serves to limit the proteinuria that develops as a consequence of areas where podocytes have dropped off.74 Shedding of podocytes is a common phenomenon in glomerular diseases.75,76 The majority of podocytes that were shed in experimental models appear viable and display FPE,74 suggesting they have dropped off the GBM in the repair response. Once a GBM remains denuded, the glomerular barrier is severely disrupted, parietal epithelial cells may attach to these areas and the glomerular structure is usually lost.77 Together the severity of podocyte injury and its sequelae represent a final, and largely irreversible pathway that determines loss of glomerular structure, and hence CKD. The importance of severe podocyte injury is underscored by experiments in transgenic rats which express the human diphtheria toxin receptor (hDTR) on podocytes.78 When injected with diphtheria toxin selective injury to the podocytes follows. Depending on the magnitude of the initial injury, determined by the dose or the frequency of the administered toxin, there is either recovery or progressive scarring of the renal glomerulus.

Tubular Albumin Transport Ultrafiltered albumin, whatever the total amount in the lumen of the initial proximal tubule under physiologic conditions may be, is reabsorbed, because normal urine is virtually free of albumin. Albumin reabsorption takes place in the proximal tubule via receptor-mediated endocytosis. Two

IV. PATHOPHYSIOLOGY

TABLE 9.1 Genetic Mutations that Lead to Proteinuria Gene/Protein

Human/Animal

Disease/Phenotype

NPHS1/Nephrin

Human/mouse

FP Effacement/Proteinuria

NPHS2/Podocin

Human

FP Effacement/Proteinuria

PLC-epsilon 1

Human/Zebra Fish

Diffuse Mesangial Sclerosis (DMS)

NcK

Mouse

FP Formation Defect

Fyn

Mouse

Subtle Changes in FP

Fyn/Yes Combined

Mouse

FP Effacement/Proteinuria

Kirrel/Neph1

Mouse

FP Effacement/Proteinuria

TRPC6

Human

FSGS

Combined CD2ap/Fyn

Mouse

FSGS

ACTN4/Alpha-Actinin4

Human/Mouse

FSGS

NotchI Transgenic

Mouse

FSGS

NFAT Transgenic

Mouse

FSGS

uPAR

Mouse

NA

Focal Adhesion Kinase

Mouse

NA

CDQ2/Coenzyme Q10

Human

FSGS

HGF/C-met

Mouse

Normal

aPKC Lambda/Iota

Mouse

FSGS/FP Effacement/Proteinuria

PDSS2/Prenly Diphosphate Synthase Subunit 2

Human/Mouse

FP Effacement/Proteinuria

Synaptopodin

Mouse

Normal

Cfl 1/Cofilin 1

Mouse/Zebra Fish

FP Effacement/Proteinuria

Sidekick Transgene

Mouse

FSGS

INF2

Human

FSGS

ATG5

Mouse

Glomerulosclerosis/Proteinuria

PI3KcII

Mouse

DMS/FP Effacement/Proteinuria

EP4 Receptor Transgenic

Mouse

Proteinuria

AT1 Transgenic

Mouse

FP Effacement

Beta Catenin

Mouse

NA

Podocalyxin Transgenic

Mouse

FP Effacement Defect

FAT1

Mouse

FP Effacement/Proteinuria

GLEPP1

Mouse

Broadening of FP/No Proteinuria

VEGF Transgenic

Mouse

Collapsing Glomerulopathy

VEGF Heterozygous Deletion

Mouse

Endotheliosis/Proteinuria

Lamb2/Laninin β2

Human/Mouse

DMS/FSGS (Pierson Syndrome)

Beta 1 Integrin

Mouse

Proteinuria

Integrin Alpha 3 Subunit

Mouse

Disorganized GBM/FP Formation Defect/Proteinuria

Integrin Linked Kinase

Mouse

GBM Alteration/FSGS

Cubulin

Mouse

Normal/Minimal Proteinuria

Megalin

Mouse

LMW Proteinuria

WT1

Human

Denys Drash and Frasler Syndrome

FTIP

Mouse

Proteinuria

LMX1B/LIM Homeobox Transcription Factor

Human

Nail Patella Syndrome

SMARCALI

Human

FSGS/Steroid Resistant NS

PODOCYTES

GLOMERULAR BASEMENT MEMBRANE

TUBULES

TRANSCRIPTION FACTORS

Abbreviations: DMS: diffuse mesangial sclerosis; FP: foot process, FSGS: focal segmental glomerulosclerosis, GBM: glomerular basemement membrane, LMW: low molecular weight, NA: not available. Adapted from Reference62.

100

9.  The Pathophysiology of Proteinuria

FIGURE 9.5  Proposed mechanisms by which primary epithelial injury leads to proteinuria. Genetic mutations of proteins that are required for podocyte function or direct antibody mediated injury of podocytes may lead to podocyte effacement and dysfunction of the normal signaling between podocytes and endothelium. Consequently, the endothelium loses its highly specialized phenotype with fenestrations and the covering endothelial surface layer, the glycocalyx. This will result in albumin leakage through endothelium and disturbed slit diaphragm function. Together this results in loss of glomerular integrity (lower panel).

receptors, cubilin and megalin, have been identified as being involved in albumin uptake. Cubilin, also known as the intrinsic factor cobalamin receptor, is a peripheral membrane protein (approximately 460  kD). Megalin binds cubilin with high affinity and contributes to the

internalization of cubilin-ligand complexes.1,79–81 The normal function of cubilin is also dependent on the transmembrane protein amnionless (AMN), which colocalizes with cubilin and is essential for the trafficking of cubilin to the apical membrane.82 Megalin is a large transmembrane protein (approximately 600  kD) that belongs to the LDL receptor family. Megalin binds and mediates the endocytosis of a large and highly diverse group of ligands, including plasma proteins, peptides, enzymes, vitamin-binding proteins, hormones, and hormone-binding proteins, as well as drugs and toxins. Some of the ligands are shared with cubilin, while others are specific for either megalin or cubilin.83 Albumin binds both cubilin84 and megalin.85 Tubular uptake of albumin is markedly decreased in conditional cubilin, megalin, or combined double knockout mice,81,86 in dogs with cubilin dysfunction due to mutations in AMN,84 and in humans with mutations in the cubilin gene CUBN.87 However, while this resulted in a six-fold increase in urinary albumin excretion compared to a baseline excretion of 30 mg/day, overall net  albumin excretion was still very low (in the microalbuminuria range) compared to the amount of albuminuria that can be observed in patients with the nephrotic syndrome.81 Under normal circumstances, the tubular concentration of albumin therefore is below the level that saturates this retrieval system. This means that even if some albumin passes through the glomerular filter, only very little albumin is excreted in the urine. This is illustrated in experiments where the glycocalyx is disrupted and substantial albumin passage through the endothelium and the GBM can be demonstrated.21 In the short term most of this albumin was reabsorbed by epithelial cells, and no net  albuminuria developed. Interestingly, this only concerns the handling of albumin, as other macromolecules such as infused large dextrans pass through the glomerular filter and do reach the urine,21 underscoring the unique way that albumin is handled by the kidney. Finally, when considering net  albuminuria, the SNGFR has to be taken into account as well. SNGFR is a prime factor determining the concentration in the proximal tubules of macromolecules such as albumin. However, under various physiological and pathological circumstances in which SNGFR is substantially less than normal, the tubular concentration of albumin can increase sufficiently to exceed the saturation level for albumin tubular reabsorption. The albuminuria which then occurs is reversible, and does not imply abnormalities in either the GBM or in the proximal tubules.56

Tubular Handling of Non-Albumin Proteins The tubular endocytotic machinery not only plays a role in albumin reabsorption, but is also involved

IV. PATHOPHYSIOLOGY

101

Conclusion

in uptake of low-molecular weight proteins. Several renal syndromes have been identified that – in the absence of glomerular disease – are predominantly characterized by reduced tubular reabsorption of lowmolecular weight proteins. These syndromes include Imerslund-Gräsbeck syndrome (IGS), Dent’s disease, Lowe syndrome, Donnai-Barrow syndrome (DB/FOAR syndrome) and cystinosis. In IGS and DB/FOAR syndrome, proteinuria is caused by mutations in the cubilin-AMN and megalin receptor complex. In cystinosis and Dent’s disease, the defective proteins have been identified as a lysosomal cystine transporter (cystinosin) and an endosomal CL−/H+ exchanger (CLC-5). Lowe’s syndrome is characterized by a mutation in the OCRL 1 gene which affects targeting of lysosomal enzymes.88

RENAL CONSEQUENCES OF ALBUMINURIA When podocytes are loaded with albumin they may display a response to injury, in particular in the setting of metabolic conditions such as diabetes mellitus, where albumin is glycated.89 The podocytes endocytose albumin and subsequently lose their cytoskeletal organization, as reflected by loss of synaptopodin and nephrin, and may display a migratory phenotype. Recent studies have linked this podocyte response to the development of glomerulosclerosis.73 Recently, there has been extensive debate regarding whether tubular albumin reabsorption and the subsequent lysosomal degradation may result in tubular injury (so-called protein overload injury).90,91 In vitro studies show that overload of albumin exerts cytotoxic effects on proximal and distal tubular cells, by activating a wide array of intracellular signaling pathways such as extracellular signal-regulated kinases (ERK), nuclear factor kappa-light-enhancer of activated B cells (NF-κB), and protein kinase C (PKC),92–97 which induce the release of inflammatory (monocyte chemotactic protein-1, regulated on activation, normal T cell expressed and secreted or RANTES),93,98,99 vasoactive (reactive oxygen species, endothelin),100–102 and fibrotic (transforming growth factor-β, collagens) substances,103–106 causing interstitial damage and ultimately leading to irreversible renal deterioration. Moreover, albumin overload may also cause cellular apoptosis107,108 leading to decreased nephron functionality. Besides albumin itself, substances bound to albumin, such as free fatty acids, other proteins, or glycated albumin, can act as profibrotic and pro-inflammatory stimuli in the tubule and aggravate renal damage provoked by albuminuria.109 Also, in vivo, excessive tubular reuptake of albumin stimulates a wide array of cytotoxic signals which affect the interstitium, fibroblasts and nearby blood vessels, which have been suggested

to cause tubulointerstitial dysfunction, fibrosis, volume expansion, and hypertension leading to worsened renal survival.110–112 The relevance of these findings was challenged by Theilig and colleagues, who used an elegant crerecombinase induced deletion of megalin in mice. They found a mosaic deletion of megalin and thus were able to observe the renal response both in the presence and absence of tubular albumin reabsorption in the same kidney.113 Upon induction of glomerular injury, the tubules that could reabsorb albumin demonstrated increased expression of pro-inflammatory cytokines and TGF-beta. However, the development of tubulo­ interstitial injury was entirely related to the extent of glomerular injury and not to tubular activation. Notions challenging a possible pathogenic relationship between albumin and the tubular epithelium are also supported by clinical observations in patients with minimal change glomerular disease who may have massive amounts of albuminuria for prolonged periods without signs of tubulointerstitial inflammation or development of decrements in GFR. These findings could be reconciled if one takes into account the background disease. If albuminuria occurs in the setting of renal inflammation, the epithelial response to albumin endocytosis may differ from that in non-inflammatory renal diseases. In addition, the toxicity of albumin and its ability to engage with alternative scavenger receptors that have been linked to inflammation (such as RAGE) may depend on glycation and binding of cytokines and lipid moieties.

CONCLUSION The development of albuminuria is probably a multistep process where initially loss of the endothelial barrier function may play a critical role. Endothelial activation and subsequent shedding of the glycocalyx surface allows albumin to penetrate the subpodocyte space. Podocytes may then take up albumin through scavenger receptors and display actin skeleton rearrangements and injury. Compensatory tubular reabsorption and the accompanying inflammatory responses may further contribute to the structural interstitial damage that has been associated with albuminuria. These combined changes may lead to progressive renal functional loss. While research tends to use a reductionist approach in this field, it should also be noted that the function of each of the components of the glomerular filtration barrier is dependent upon the others. The glomerular endothelium needs an instructive scaffold (the GBM that has been produced by the podocyte), and signaling molecules from pericapillary cells such as the

IV. PATHOPHYSIOLOGY

102

9.  The Pathophysiology of Proteinuria

mesangium and podocytes to maintain its very specialized and unique phenotype. At the same time, podocytes cannot survive without a properly functioning endothelium, as illustrated by experiments where the endothelial nitric oxide system is deficient in diabetes mellitus, and subsequent podocyte injury develops.114 Altogether, the sequence of renal events that lead to the consistent appearance of albumin in the urine invariably points towards serious alterations of the renal ultrastructure. Therefore the presence of albuminuria always warrants a diagnostic evaluation to identify and treat underlying disease. Moreover, given the renal epithelial response to glomerular albumin permeability in certain diseases, albuminuria may serve as a therapeutic target.

References 1. Christensen EI, Verroust PJ, Nielsen R. Receptor-mediated endocytosis in renal proximal tubule. Pflugers Arch 2009;458:1039–48. 2. Maack T, Park CH, Camargo MJF. In: Seldon DW, Giebisch G, editors. Renal filtration, transport and metabolism of proteins in the kidney. New York: Raven Press; 1992. p. 3005–38. 3. Amer H, Lieske JC, Rule AD, Kremers WK, Larson TS, Franco Palacios CR, et  al. Urine high and low molecular weight proteins one-year post-kidney transplant: relationship to histology and graft survival. Am J Transplant 2013;13:676–84. 4. Sanders PW. Light chain-mediated tubulopathies. Contrib Nephrol 2011;169:262–9. 5. K/DOQI. Clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis 2002;39:S1–266. 6. Matsushita K, van der Velde M, Astor BC, Woodward M, Levey AS, de Jong PE, et  al. Association of estimated glomerular filtration rate and albuminuria with all-cause and cardiovascular mortality in general population cohorts: a collaborative metaanalysis. Lancet 2010;375:2073–81. 7. Ruggenenti P, Porrini E, Motterlini N, Perna A, Ilieva AP, Iliev IP, et  al. Measurable urinary albumin predicts cardiovascular risk among normoalbuminuric patients with type 2 diabetes. J Am Soc Nephrol 2012;23:1717–24. 8. Ibsen H, Olsen MH, Wachtell K, Borch-Johnsen K, Lindholm LH, Mogensen CE, et  al. Reduction in albuminuria translates to reduction in cardiovascular events in hypertensive patients: losartan intervention for endpoint reduction in hypertension study. Hypertension 2005;45:198–202. 9. Farquhar MG, Palade GE. Glomerular permeability. II. Ferritin transfer across the glomerular capillary wall in nephrotic rats. J Exp Med 1961;114:699–716. 10. Farquhar MG, Wissig SL, Palade GE. Glomerular permeability. I. Ferritin transfer across the normal glomerular capillary wall. J Exp Med 1961;113:47–66. 11. Deen WM, Lazzara MJ, Myers BD. Structural determinants of glomerular permeability. Am J Physiol Renal Physiol 2001;281:F579–96. 12. Lea PJ, Silverman M, Hegele R, Hollenberg MJ. Tridimensional ultrastructure of glomerular capillary endothelium revealed by high-resolution scanning electron microscopy. Microvasc Res 1989;38:296–308. 13. Comper WD, Russo LM. The glomerular filter: an imperfect barrier is required for perfect renal function. Curr Opin Nephrol Hypertens 2009;18:336–42.

14. Ryan GB, Karnovsky MJ. Distribution of endogenous albumin in the rat glomerulus: role of hemodynamic factors in glomerular barrier function. Kidney Int 1976;9:36–45. 15. Weinbaum S, Tarbell JM, Damiano ER. The structure and function of the endothelial glycocalyx layer. Annu Rev Biomed Eng 2007;9:121–67. 16. Friden V, Oveland E, Tenstad O, Ebefors K, Nyström J, Nilsson UA, et  al. The glomerular endothelial cell coat is essential for glomerular filtration. Kidney Int 2011;79:1322–30. 17. Henry CB, Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan. Am J Physiol 1999;277:H508–14. 18. Levick JR, Michel CC. Microvascular fluid exchange and the revised Starling principle. Cardiovasc Res 2010;87:198–210. 19. Jeansson M, Haraldsson B. Glomerular size and charge selectivity in the mouse after exposure to glucosaminoglycan-degrading enzymes. J Am Soc Nephrol 2003;14:1756–65. 20. Jeansson M, Haraldsson B. Morphological and functional evidence for an important role of the endothelial cell glycocalyx in the glomerular barrier. Am J Physiol Renal Physiol 2006;290:F111–6. 21. Dane MJ, van den Berg BM, Avramut MC, Faas FG, van der Vlag J, Rops AL, et al. Glomerular endothelial surface layer acts as a barrier against albumin filtration. Am J Pathol 2013;182:1532–40. 22. Sandoval RM, Wagner MC, Patel M, Campos-Bilderback SB, Rhodes GJ, Wang E, et al. Multiple factors influence glomerular albumin permeability in rats. J Am Soc Nephrol 2012;23:447–57. 23. Wang L, Fuster M, Sriramarao P, Esko JD. Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. Nat Immunol 2005;6:902–10. 24. Gil N, Goldberg R, Neuman T, Garsen M, Zcharia E, Rubinstein AM, et  al. Heparanase is essential for the development of diabetic nephropathy in mice. Diabetes 2012;61:208–16. 25. Rabelink TJ, de Boer HC, van Zonneveld AJ. Endothelial activation and circulating markers of endothelial activation in kidney disease. Nat Rev Nephrol 2010;6:404–14. 26. van den Berg BM, Spaan JA, Vink H. Impaired glycocalyx barrier properties contribute to enhanced intimal low-density lipoprotein accumulation at the carotid artery bifurcation in mice. Pflugers Arch 2009;457:1199–206. 27. Kuwabara A, Satoh M, Tomita N, Sasaki T, Kashihara N. Deterioration of glomerular endothelial surface layer induced by oxidative stress is implicated in altered permeability of macromolecules in Zucker fatty rats. Diabetologia 2010;53:2056–65. 28. Nieuwdorp M, Mooij HL, Kroon J, Atasever B, Spaan JA, Ince C, et al. Endothelial glycocalyx damage coincides with microalbuminuria in type 1 diabetes. Diabetes 2006;55:1127–32. 29. Schlondorff D, Banas B. The mesangial cell revisited: no cell is an island. J Am Soc Nephrol 2009;20:1179–87. 30. Levéen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C. Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev 1994;8:1875–87. 31. Stockand JD, Sansom SC. Glomerular mesangial cells: electrophysiology and regulation of contraction. Physiol Rev 1998;78:723–44. 32. Morita T, Yamamoto T, Churg J. Mesangiolysis: an update. Am J Kidney Dis 1998;31:559–73. 33. Zheng F, Cornacchia F, Schulman I, Banerjee A, Cheng QL, Potier M, et  al. Development of albuminuria and glomerular lesions in normoglycemic B6 recipients of db/db mice bone marrow: the role of mesangial cell progenitors. Diabetes 2004;53:2420–7. 34. Miner JH, Go G, Cunningham J, Patton BL, Jarad G. Transgenic isolation of skeletal muscle and kidney defects in laminin beta2

IV. PATHOPHYSIOLOGY

REFERENCES

mutant mice: implications for Pierson syndrome. Development 2006;133:967–75. 35. Abrahamson DR, Hudson BG, Stroganova L, Borza DB, St John PL. Cellular origins of type IV collagen networks in developing glomeruli. J Am Soc Nephrol 2009;20:1471–9. 36. Pöschl E, Schlötzer-Schrehardt U, Brachvogel B, Saito K, Ninomiya Y, Mayer U. Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development. Development 2004;131:1619–28. 37. Harvey SJ, Zheng K, Sado Y, Naito I, Ninomiya Y, Jacobs RM, et  al. Role of distinct type IV collagen networks in glomerular development and function. Kidney Int 1998;54:1857–66. 38. Hudson BG. The molecular basis of Goodpasture and Alport syndromes: beacons for the discovery of the collagen IV family. J Am Soc Nephrol 2004;15:2514–27. 39. Miner JH. Renal basement membrane components. Kidney Int 1999;56:2016–24. 40. Goldberg S, Adair-Kirk TL, Senior RM, Miner JH. Maintenance of glomerular filtration barrier integrity requires laminin alpha5. J Am Soc Nephrol 2010;21:579–86. 41. Jarad G, Cunningham J, Shaw AS, Miner JH. Proteinuria precedes podocyte abnormalities in Lamb2−/− mice, implicating the glomerular basement membrane as an albumin barrier. J Clin Invest 2006;116:2272–9. 42. Zenker M, Aigner T, Wendler O, Tralau T, Müntefering H, Fenski R, et al. Human laminin beta2 deficiency causes congenital nephrosis with mesangial sclerosis and distinct eye abnormalities. Hum Mol Genet 2004;13:2625–32. 43. Suh JH, Jarad G, VanDeVoorde RG, Miner JH. Forced expression of laminin beta1 in podocytes prevents nephrotic syndrome in mice lacking laminin beta2, a model for Pierson syndrome. Proc Natl Acad Sci USA 2011;108:15348–53. 44. Fox JW, Mayer U, Nischt R, Aumailley M, Reinhardt D, Wiedemann H, et  al. Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV. EMBO J 1991;10:3137–46. 45. Miosge N, Sasaki T, Timpl R. Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice. Matrix Biol 2002;21:611–21. 46. Murshed M, Smyth N, Miosge N, Karolat J, Krieg T, Paulsson M, et al. The absence of nidogen 1 does not affect murine basement membrane formation. Mol Cell Biol 2000;20:7007–12. 47. Schymeinsky J, Nedbal S, Miosge N, Pöschl E, Rao C, Beier DR, et  al. Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice. Mol Cell Biol 2002;22:6820–30. 48. Kanwar YS, Linker A, Farquhar MG. Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion. J Cell Biol 1980;86:688–93. 49. van den Born J, van den Heuvel LP, Bakker MA, Veerkamp JH, Assmann KJ, Berden JH. A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats. Kidney Int 1992;41:115–23. 50. Shimomura H, Spiro RG. Studies on macromolecular components of human glomerular basement membrane and alterations in diabetes. Decreased levels of heparan sulfate proteoglycan and laminin. Diabetes 1987;36:374–81. 51. van den Born J, van den Heuvel LP, Bakker MA, Veerkamp JH, Assmann KJ, Weening JJ. Distribution of GBM heparan sulfate proteoglycan core protein and side chains in human glomerular diseases. Kidney Int 1993;43:454–63. 52. Bolton GR, Deen WM, Daniels BS. Assessment of the charge selectivity of glomerular basement membrane using Ficoll sulfate. Am J Physiol 1998;274:F889–96.

103

53. Harvey SJ, Jarad G, Cunningham J, Rops AL, van der Vlag J, Berden JH, et  al. Disruption of glomerular basement membrane charge through podocyte-specific mutation of agrin does not alter glomerular permselectivity. Am J Pathol 2007;171:139–52. 54. Goldberg S, Harvey SJ, Cunningham J, Tryggvason K, Miner JH. Glomerular filtration is normal in the absence of both agrin and perlecan-heparan sulfate from the glomerular basement membrane. Nephrol Dial Transplant 2009;24:2044–51. 55. Rossi M, Morita H, Sormunen R, Airenne S, Kreivi M, Wang L, et  al. Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidney. EMBO J 2003;22: 236–45. 56. Smithies O. Why the kidney glomerulus does not clog: a gel permeation/diffusion hypothesis of renal function. Proc Natl Acad Sci USA 2003;100:4108–13. 57. Daniels BS, Hauser EB, Deen WM, Hostetter TH. Glomerular basement membrane: in vitro studies of water and protein permeability. Am J Physiol 1992;262:F919–26. 58. Kestilä M, Lenkkeri U, Männikkö M, Lamerdin J, McCready P, Putaala H, et al. Positionally cloned gene for a novel glomerular protein–nephrin–is mutated in congenital nephrotic syndrome. Mol Cell 1998;1:575–82. 59. Putaala H, Soininen R, Kilpelainen P, Wartiovaara J, Tryggvason K. The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet 2001;10:1–8. 60. Tryggvason K, Pettersson E. Causes and consequences of proteinuria: the kidney filtration barrier and progressive renal failure. J Intern Med 2003;254:216–24. 61. Patrakka J, Tryggvason K. Molecular make-up of the glomerular filtration barrier. Biochem Biophys Res Commun 2010;396:164–9. 62. Garg P, Rabelink T. Glomerular proteinuria: a complex interplay between unique players. Adv Chronic Kidney Dis 2011;18:233–42. 63. Wartiovaara J, Ofverstedt LG, Khoshnoodi J, Zhang J, Mäkelä E, Sandin S, et  al. Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography. J Clin Invest 2004;114:1475–83. 64. Yuan H, Takeuchi E, Salant DJ. Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton. Am J Physiol Renal Physiol 2002;282:F585–91. 65. Jones N, Blasutig IM, Eremina V, Ruston JM, Bladt F, Li H, et al. Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 2006;440:818–23. 66. Kang YS, Li Y, Dai C, Kiss LP, Wu C, Liu Y. Inhibition of integrin-linked kinase blocks podocyte epithelial-mesenchymal transition and ameliorates proteinuria. Kidney Int 2010;78:363–73. 67. Haraldsson B, Nystrom J, Deen WM. Properties of the glomerular barrier and mechanisms of proteinuria. Physiol Rev 2008;88:451–87. 68. Prabakaran T, Christensen EI, Nielsen R, Verroust PJ. Cubilin is expressed in rat and human glomerular podocytes. Nephrol Dial Transplant 2012;27:3156–9. 69. Guo J, Ananthakrishnan R, Qu W, Lu Y, Reiniger N, Zeng S, et  al. RAGE mediates podocyte injury in adriamycin-induced glomerulosclerosis. J Am Soc Nephrol 2008;19:961–72. 70. Morigi M, Buelli S, Angioletti S, Zanchi C, Longaretti L, Zoja C, et al. In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies. Am J Pathol 2005;166:1309–20. 71. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, et al. Glomerular-specific alterations of VEGF-A expression lead to

IV. PATHOPHYSIOLOGY

104

9.  The Pathophysiology of Proteinuria

distinct congenital and acquired renal diseases. J Clin Invest 2003;111:707–16. 72. Jin J, Sison K, Li C, Tian R, Wnuk M, Sung HK, et  al. Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function. Cell 2012;151: 384–99. 73. Peti-Peterdi J, Sipos A. A high-powered view of the filtration barrier. J Am Soc Nephrol 2010;21:1835–41. 74. Kriz W, Shirato I, Nagata M, LeHir M, Lemley KV. The podocyte’s response to stress: the enigma of foot process effacement. Am J Physiol Renal Physiol 2013;304:F333–47. 75. Weil EJ, Lemley KV, Yee B, Lovato T, Richardson M, Myers BD, et al. Podocyte detachment in type 2 diabetic nephropathy. Am J Nephrol 2011;33(Suppl. 1):21–4. 76. Yu D, Petermann A, Kunter U, Rong S, Shankland SJ, Floege J. Urinary podocyte loss is a more specific marker of ongoing glomerular damage than proteinuria. J Am Soc Nephrol 2005;16:1733–41. 77. Smeets B, Uhlig S, Fuss A, Mooren F, Wetzels JF, Floege J, et al. Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells. J Am Soc Nephrol 2009;20:2604–15. 78. Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, et  al. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J Am Soc Nephrol 2005;16:2941–52. 79. Moestrup SK, Kozyraki R, Kristiansen M, Kaysen JH, Rasmussen HH, Brault D, et  al. The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalinbinding peripheral membrane protein with homology to developmental proteins. J Biol Chem 1998;273:5235–42. 80. Seetharam B, Christensen EI, Moestrup SK, Hammond TG, Verroust PJ. Identification of rat yolk sac target protein of teratogenic antibodies, gp280, as intrinsic factor-cobalamin receptor. J Clin Invest 1997;99:2317–22. 81. Amsellem S, Gburek J, Hamard G, Nielsen R, Willnow TE, Devuyst O. Cubilin is essential for albumin reabsorption in the renal proximal tubule. J Am Soc Nephrol 2010;21:1859–67. 82. Fyfe JC, Madsen M, Højrup P, Christensen EI, Tanner SM, de la Chapelle A, et  al. The functional cobalamin (vitamin B12)intrinsic factor receptor is a novel complex of cubilin and amnionless. Blood 2004;103:1573–9. 83. Christensen EI, Birn H, Storm T, Weyer K, Nielsen R. Endocytic receptors in the renal proximal tubule. Physiology (Bethesda) 2012;27:223–36. 84. Birn H, Fyfe JC, Jacobsen C, Mounier F, Verroust PJ, Orskov H, et  al. Cubilin is an albumin binding protein important for renal tubular albumin reabsorption. J Clin Invest 2000;105:1353–61. 85. Cui S, Verroust PJ, Moestrup SK, Christensen EI. Megalin/ gp330 mediates uptake of albumin in renal proximal tubule. Am J Physiol 1996;271:F900–7. 86. Weyer K, Storm T, Shan J, Vainio S, Kozyraki R, Verroust PJ, et  al. Mouse model of proximal tubule endocytic dysfunction. Nephrol Dial Transplant 2011;26:3446–51. 87. Storm T, Emma F, Verroust PJ, Hertz JM, Nielsen R, Christensen EI. A patient with cubilin deficiency. N Engl J Med 2011;364:89–91. 88. Nielsen R, Christensen EI. Proteinuria and events beyond the slit. Pediatr Nephrol 2010;25:813–22. 89. Doublier S, Salvidio G, Lupia E, Ruotsalainen V, Verzola D, Deferrari G, et al. Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II. Diabetes 2003;52:1023–30. 90. Birn H, Christensen EI. Renal albumin absorption in physiology and pathology. Kidney Int 2006;69:440–9.

91. Gekle M. Renal tubule albumin transport. Annu Rev Physiol 2005;67:573–94. 92. Reich H, Tritchler D, Herzenberg AM, Kassiri Z, Zhou X, Gao W, et  al. Albumin activates ERK via EGF receptor in human renal epithelial cells. J Am Soc Nephrol 2005;16:1266–78. 93. Wang Y, Rangan GK, Tay YC, Wang Y, Harris DC. Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor kappaB in proximal tubule cells. J Am Soc Nephrol 1999;10:1204–13. 94. Lee EM, Pollock CA, Drumm K, Barden JA, Poronnik P. Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells. Am J Physiol Renal Physiol 2003;285:F748–57. 95. Drumm K, Bauer B, Freudinger R, Gekle M. Albumin induces NF-kappaB expression in human proximal tubule-derived cells (IHKE-1). Cell Physiol Biochem 2002;12:187–96. 96. Dixon R, Brunskill NJ. Activation of mitogenic pathways by albumin in kidney proximal tubule epithelial cells: implications for the pathophysiology of proteinuric states. J Am Soc Nephrol 1999;10:1487–97. 97. Morigi M, Macconi D, Zoja C, Donadelli R, Buelli S, Zanchi C, et  al. Protein overload-induced NF-kappaB activation in proximal tubular cells requires H(2)O(2) through a PKC-dependent pathway. J Am Soc Nephrol 2002;13:1179–89. 98. Wang Y, Chen J, Chen L, Tay YC, Rangan GK, Harris DC. Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein. J Am Soc Nephrol 1997;8:1537–45. 99. Zoja C, Donadelli R, Colleoni S, Figliuzzi M, Bonazzola S, Morigi M, et  al. Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation. Kidney Int 1998;53:1608–15. 100. Whaley-Connell AT, Morris EM, Rehmer N, Yaghoubian JC, Wei Y, Hayden MR, et al. Albumin activation of NAD(P)H oxidase activity is mediated via Rac1 in proximal tubule cells. Am J Nephrol 2007;27:15–23. 101. Zoja C, Morigi M, Figliuzzi M, Bruzzi I, Oldroyd S, Benigni A, et  al. Proximal tubular cell synthesis and secretion of endothelin-1 on challenge with albumin and other proteins. Am J Kidney Dis 1995;26:934–41. 102. Vlachojannis JG, Tsakas S, Petropoulou C, Goumenos DS, Alexandri S. Endothelin-1 in the kidney and urine of patients with glomerular disease and proteinuria. Clin Nephrol 2002;58:337–43. 103. Wohlfarth V, Drumm K, Mildenberger S, Freudinger R, Gekle M. Protein uptake disturbs collagen homeostasis in proximal tubule-derived cells. Kidney Int Suppl 2003:S103–9. 104. Stephan JP, Mao W, Filvaroff E, Cai L, Rabkin R, Pan G. Albumin stimulates the accumulation of extracellular matrix in renal tubular epithelial cells. Am J Nephrol 2004;24:14–19. 105. Goumenos DS, Tsakas S, El Nahas AM, Alexandri S, Oldroyd S, Kalliakmani P, et al. Transforming growth factor-beta (1) in the kidney and urine of patients with glomerular disease and proteinuria. Nephrol Dial Transplant 2002;17:2145–52. 106. Diwakar R, Pearson AL, Colville-Nash P, Brunskill NJ, Dockrell ME. The role played by endocytosis in albumin-induced secretion of TGF-beta1 by proximal tubular epithelial cells. Am J Physiol Renal Physiol 2007;292:F1464–70. 107. Koral K, Erkan E. PKB/Akt partners with Dab2 in albumin endocytosis. Am J Physiol Renal Physiol 2012;302:F1013–24. 108. Tejera N, Gómez-Garre D, Lázaro A, Gallego-Delgado J, Alonso C, Blanco J, et  al. Persistent proteinuria up-regulates angiotensin II type 2 receptor and induces apoptosis in proximal tubular cells. Am J Pathol 2004;164:1817–26.

IV. PATHOPHYSIOLOGY

REFERENCES

109. Pollock CA, Poronnik P. Albumin transport and processing by the proximal tubule: physiology and pathophysiology. Curr Opin Nephrol Hypertens 2007;16:359–64. 110. Chen L, Wang Y, Tay YC, Harris DC. Proteinuria and tubulo­ interstitial injury. Kidney Int Suppl 1997;61:S60–2. 111. Johnson DW, Saunders HJ, Baxter RC, Field MJ, Pollock CA. Paracrine stimulation of human renal fibroblasts by proximal tubule cells. Kidney Int 1998;54:747–57. 112. Burton C, Harris KP. The role of proteinuria in the progression of chronic renal failure. Am J Kidney Dis 1996;27:765–75.

105

113. Theilig F, Kriz W, Jerichow T, Schrade P, Hähnel B, Willnow T, et  al. Abrogation of protein uptake through megalin-deficient proximal tubules does not safeguard against tubulointerstitial injury. J Am Soc Nephrol 2007;18:1824–34. 114. Yuen DA, Stead BE, Zhang Y, White KE, Kabir MG, Thai K, et al. eNOS deficiency predisposes podocytes to injury in diabetes. J Am Soc Nephrol 2012;23:1810–23.

IV. PATHOPHYSIOLOGY