Chapter Outline
BIOLOGIC FUNCTIONS OF PODOCYTES, 112
ULTRASTRUCTURAL AND MOLECULAR ANATOMY OF PODOCYTES REQUIRED FOR NORMAL STRUCTURE AND FUNCTION, 113
GLOMERULAR DISEASES IN WHICH PODOCYTES ARE THE PRIMARY GLOMERULAR CELL TYPE INJURED, 113
MECHANISMS OF INJURY IN COMMON PODOCYTE DISEASES, 114
Minimal Change Nephropathy, 114
Focal Segmental Glomerulosclerosis, 116
Membranous Nephropathy, 116
Human Immunodeficiency Virus Nephropathy, 116
Diabetic Kidney Disease, 116
RESPONSES BY PODOCYTES TO DISEASE-INDUCED INJURY: LINKING STRUCTURE TO FUNCTION TO CLINICAL FINDINGS, 117
Effacement: A Histologic Change in Podocyte Shape Mediated by the Actin Cytoskeleton, 117
Proteinuria due to Reduced Size and/or Charge Properties, 118
Glomerulosclerosis and Reduced Kidney Function: A Correlation with Depletion in Podocyte Number, 118
EFFECTS OF EXISTING THERAPIES ON PODOCYTES, 119
Glucocorticosteroids, 119
Calcineurin Inhibitors, 119
Anti–B Cell Therapy, 119
Renin Angiotensin Aldosterone System Inhibitors, 119
IDENTIFICATION OF CANDIDATE THERAPEUTIC APPROACHES FOR THE FUTURE, 120
SUMMARY, 120
Normal human urine contains only tiny amounts of protein. The presence of increased amounts of urinary protein (proteinuria), especially albumin (albuminuria), is a cardinal feature of glomerular disease and an important prognostic marker in a wide variety of forms of kidney disease, including the most numerically and economically important form of kidney disease, diabetic nephropathy. Perhaps not so widely appreciated is that albuminuria is also an important independent risk factor for cardiovascular mortality in both diabetic and nondiabetic populations. Therefore the study of albuminuria, the factors that prevent it in the healthy state, the disease mechanisms that lead to its occurrence and its prognostic significance, and, perhaps most importantly, the therapeutic approaches to its modification, all have major clinical and health economic significance.
The healthy kidney limits the amount of albumin passing into the glomerular filtrate by virtue of the selective permeability of the glomerular capillary wall. In a 70-kg adult, approximately 180 L of water and small molecules such as salt, glucose, and amino acids pass relatively freely across the glomerular capillary wall into the primary urine in every 24-hour period. Yet, while allowing this massive permeability to water and small molecules, the glomerular capillary maintains relative impermeability to albumin. The structure of the glomerular capillary wall that serves this selective permeability comprises three interacting components: fenestrated glomerular endothelial cells on the inner (luminal) aspect, podocytes (also called glomerular visceral epithelial cells) on the outer (urinary) aspect of the capillary, and the highly negatively charged glomerular basement membrane (GBM) between these two cellular layers. Each of these three components exerts an important influence on the selective permeability of the glomerular capillary wall to albumin and other proteins, and it is therefore unduly simplistic to think of any of the components in isolation. However, there are structural, scientific, clinical, and therapeutic reasons for focusing on the podocytes as the conductors of this orchestra, which is the purpose of this chapter.
Biologic Functions of Podocytes
The main biologic function of podocytes is to restrict the passage of albumin and other key proteins to the blood space within the glomerular capillaries and to prevent their passage into the extracapillary urinary space. Additional functions include maintaining the shape of the underlying glomerular capillaries to which they adhere, the production of extracellular matrix proteins for the development and likely subsequent maintenance of the GBM, and the production and secretion of survival and angiopathic factors such as vascular endothelial growth factor (VEGF) and angiopoietin for the neighboring glomerular endothelial cells. Alterations to one or more of these functions resulting from glomerular disease–induced injury lead to functional and structural changes that characterize podocyte injury, both clinically and pathologically. These will be discussed later.
Ultrastructural and Molecular Anatomy of Podocytes Required for Normal Structure and Function
Structure
The complex podocyte ultrastructure comprises a cell body, from which extend long branching cellular processes. Major processes give rise to interdigitating secondary processes, which are arranged like the teeth of a zipper ( Figure 4.1 ). These minor processes end in foot processes, which attach podocytes to the underlying GBM. The gaps between these secondary processes form specialized and modified tight junctions called the slit diaphragms, through which glomerular filtration occurs (see Figure 4.1 ). This structure is analogous to a molecular sieve, which limits the passage of macromolecules based on size, in which the diameter of the sieve (40 nm) is smaller than that of albumin. Foot processes are actively motile due to a well-organized and abundant actin cytoskeleton that is in direct communication with the slit diaphragm (see later). Multiple podocyte-specific proteins in the slit diaphragm enable this structure to serve several functions ( Figure 4.2 ). They include being a size, charge, and shape macromolecular filter to proteins (see later), anchoring the filter to the GBM, and communicating with the actin cytoskeleton in the foot processes to process cues appropriate for the regulation of podocyte shape and polarity.
Slit Diaphragm Proteins
Several classes of proteins make up the structure of the slit diaphragm, and each is required to accomplish specialized and diverse biologic functions in health (see Figure 4.2 ). Perhaps the best known is the transmembrane protein nephrin, which spans the slit diaphragm across adjacent foot processes, giving rise to the characteristic zipper structure that participates in homotypic intercellular interactions. Podocin helps anchor nephrin to the plasma membrane, whereas the family of Neph proteins ensures the cis -configuration of nephrin for proper function. Nephrin, and likely additional slit diaphragm proteins, is further anchored to the actin cytoskeleton by the scaffold proteins CD2-associated protein (CD2AP), zonula occludens 1 (ZO-1), and membrane-associated guanylate kinase, WW and PDZ domain–containing protein 2 (MAGI-2), and the actin-binding proteins α-actinin-4 and Ras activating-like protein IQGAP1 (IQGAP1). In addition, transient receptor potential cation channel, subfamily C, member 6 (TRPC6) and TRPC5 are cationic sensing channels at the slit diaphragm. The protein structure of nephrin, and particularly its high number of intracytoplasmic tyrosine residues, strongly suggests a signaling function for this protein. It has become apparent that slit diaphragm proteins play a key role in signaling to the podocyte actin cytoskeleton and in turn control the shape and structural integrity of the podocyte. The slit diaphragm complex communicates actively with the rest of the podocyte to convey mechanical pressures and other signals to adapt to change. Phosphorylation of nephrin by Src family tyrosine kinases plays a role in signal transduction via phosphatidylinositol-3-kinase (PI3-K), Akt, and other pathways.
Cytoskeleton
The podocyte cytoskeleton ensures cell contractility, cell shape, and polarity. Foot processes contain long actin fiber bundles that run cortically and contiguously to link adjacent podocytes. The cell body and major and secondary processes contain vimentin-rich intermediate filaments that assist in maintaining cell shape and rigidity, and large microtubules form organized structures along major and minor processes. Actin, α-actinin, and myosin form a contractile system in podocyte foot processes, and, along with the microtubule system, anchor podocytes to specific matrix proteins in the underlying GBM by way of integrins, vinculin, and talin. Several Rho GTPases also regulate podocyte contractility ; actin is also regulated by podocyte-specific proteins, such as synpaptopodin and actinin-4, which are actin-binding proteins. Studies have shown that this well-orchestrated actin- and microtubule-cytoskeleton accounts for not only the shape of podocytes, but the ability to migrate.
Glomerular Diseases in Which Podocytes are the Primary Glomerular Cell Type Injured
Certain types of proteinuric glomerular diseases are characterized by primary injury of podocytes. The three leading primary glomerular diseases (defined as a kidney-specific disease) are focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), and minimal change disease (MCD). “Nontraditional” podocyte diseases include diabetic kidney disease, amyloidosis, Fabry’s disease, membranoproliferative glomerulonephritis and postinfectious glomerulonephritis. The inciting causes of each podocyte disease differ, and therefore each disease affects podocytes in different ways; in turn, the response to injury in each disease differs, leading to different histologic and clinical manifestations ( Table 4.1 ). Yet, regardless of the inciting causes and their mediators of podocyte injury, several common clinical and pathologic responses occur, which will be highlighted later.
| Broad Classification of Podocyte Diseases | Disease | Mediators and Mechanisms of Inciting Injury to Podocytes |
|---|---|---|
| Traditional podocyte diseases—podocytes are considered the primary cell injured in disease | Focal segmental glomerulosclerosis |
|
| Membranous nephropathy |
| |
| Minimal change disease |
| |
| Nontraditional podocyte diseases—podocytes considered injured as the primary or secondary cell by the disease process | Diabetic nephropathy |
|
| Mesangioproliferative glomerulonephritis | ||
| Amyloidosis | ||
| Fabry’s disease | ||
| Postinfectious glomerulonephritis |
Mechanisms of Injury in Common Podocyte Diseases
The following is a brief overview of how each proteinuric glomerular disease leads to podocyte injury. Readers are referred to a more general discussion of these glomerular diseases in Chapter 32 .
Minimal Change Nephropathy
The fact that the podocyte is the only glomerular cell that is structurally altered in MCD has led to the assumption that this disease is a “podocytopathy.” There are certainly changes in expression patterns of podocyte-specific genes, but it is difficult to be sure whether these are cause or effect. Unfortunately, there are no satisfactorily specific animal models of MCD: administration of puromycin aminonucleoside causes similar morphologic changes, but puromycin aminonucleoside is a cellular toxin that is not specific for podocytes. Administration of the anti-nephrin monoclonal antibody 5-1-6 in rats rapidly induces proteinuria with the glomerular structure initially remaining normal. Perhaps the most exciting insights concern the production by podocytes of a hyposialylated version of a protein called angiopoietin-like 4, which seems to mediate proteinuria in rats overexpressing this protein selectively in their podocytes. The proteinuric animals had normal glomerular morphologic characteristics by light microscopy. A later publication from the same group suggests that the normal form of angiopoietin-like 4 may actually be antiproteinuric but may explain the hypertriglyceridemia that is typically seen in nephrotic syndrome. These observations strengthen the link between podocyte injury and MCD, but a direct causal link is not yet proven.
Focal Segmental Glomerulosclerosis
The causes or mechanisms underlying FSGS are broadly considered as hereditary or congenital and sporadic or acquired in nature. Study of podocytes received new impetus in the late 1990s when the positional cloning of the gene responsible for congenital nephrotic syndrome of the Finnish type led to the identification of the archetypal podocyte-specific protein, nephrin. This was rapidly followed by the identification of other proteinuric diseases linked to podocyte-specific single-gene disorders, including those affecting podocin, Wilms’ tumor 1, CD2AP, α-actinin-4, TRPC6, and phospholipase Cε 1 (PLCE1). In each of these conditions it is generally accepted that proteinuria results directly from the disruption of these constitutively expressed genes in the podocyte, leading to FSGS. However, all these genetic disorders are rare, and a key question for practicing nephrologists is whether podocyte-specific gene mutations or polymorphisms play a role as predisposing factors for the much more common “sporadic” forms of proteinuric disease.
There is abundant evidence that podocyte injury and depletion lead to classical FSGS in experimental models (reviewed by D’Agati). In human disease an opportunity to study the very earliest features of FSGS is afforded by studies of recurrence of this disease in transplanted kidneys: changes in podocytes can be seen in reperfusion biopsies and are predictive of full-blown FSGS recurrence. An example of bringing the knowledge of podocyte biology to therapeutic application is afforded by the studies of the expression by podocytes of the protein B7-1 (also known as CD80, a protein originally thought to have its major role as a lymphocyte costimulatory protein). Upregulation of B7-1 on podocytes has been shown to be key to the development of nephrotic syndrome in various animal models, and this led to the experiment in which abatacept, a fusion protein that targets B7-1, was used in the treatment of five patients with FSGS and led to marked reductions in proteinuria. Another area of excitement in relation to FSGS concerns the possible role of a circulating mediator called soluble urokinase-type plasminogen activator receptor (suPAR), which could also explain the recurrence of FSGS in a proportion of kidney transplants.
Membranous Nephropathy
The subepithelial (subpodocyte) location of the immune deposits in MN and analogies with the representative animal model of passive Heymann nephritis have led to attempts to identify podocyte antigens that are targets of the immune response. Ronco and Debiec identified a podocyte antigen (neutral endopeptidase) that is the target of an alloimmune response in neonatal MN but does not seem to explain idiopathic MN. The identification of autoantibodies to the M-type phospholipase A 2 receptor (PLA 2 R) by Beck and colleagues in approximately 70% of patients with idiopathic MN represents a very significant advance. PLA 2 R is expressed by human podocytes, but its role in podocyte biologic processes and the causative link between anti-PLA 2 R autoantibodies and the pathologic condition of MN require further study.
Human Immunodeficiency Virus Nephropathy
Another acquired renal disease in which study of podocytes has been very informative is human immunodeficiency virus (HIV) nephropathy. Podocytes may be directly infected by HIV-1, though because they lack the cellular receptors for HIV that have been identified in lymphocytes, other mechanisms for uptake of HIV viral proteins may be involved. Presence of HIV viral proteins leads to upregulation of the key mediator VEGF and its receptor. Addition of exogenous VEGF to cultured podocytes leads to proliferation and dedifferentiation as seen in HIV nephropathy. HIV alters the shape of podocytes by disrupting the actin cytoskeleton. The kidney is known to act as a reservoir for HIV, and the podocyte may be a cell that is not easily accessible to antiviral defense mechanisms. Thus improved understanding of the relationship between HIV and the podocyte may be vitally important to the future success of anti-HIV therapeutic strategies.
Diabetic Kidney Disease
Diabetic kidney disease, extensively covered in other parts of this edition, is the numerically and economically most important form of progressive kidney disease worldwide. The role of the podocyte therein has been the topic of much exciting literature. Podocyte number is a better predictor of prognosis in diabetic nephropathy than GBM thickness, mesangial proliferation or sclerosis, or any other feature of glomerular injury. Podocyte abnormalities are detectable very early in the disease. It seems that VEGF is key to understanding the importance of the podocyte in diabetic nephropathy with chronic hyperglycemia leading to excessive production of VEGF by the podocyte and an abnormal crosstalk with the nitric oxide pathway. There may also be a link with insulin responsiveness of podocytes: insulin stimulates the production of VEGF by podocytes, and the selective deletion of the insulin receptor from podocytes leads to progressive kidney disease with many features in common with diabetic nephropathy even though these animals are not diabetic.
Responses by Podocytes to Disease-Induced Injury: Linking Structure to Function to Clinical Findings
The discussion of how podocytes may be injured in the common individual proteinuric glomerular diseases leads to the question “How does podocyte injury translate into the characteristic clinical findings in these diseases?” This requires that we focus on how the responses to podocyte injury lead to the characteristic histopathologic changes seen on renal biopsy, proteinuria (the clinical signature of podocyte injury), and glomerulosclerosis (which leads to reduced kidney function and progression of the disease).
Effacement: A Histologic Change In Podocyte Shape Mediated by the Actin Cytoskeleton
The podocyte depends for its normal functions on maintenance of the complex architecture of interdigitating secondary cell processes. Anything that disrupts this will lead to damage to the selective glomerular filter, with proteinuria being the demonstrable clinical consequence. Regardless of the underlying disease, a characteristic and almost predictable response to podocyte injury is a change in the shape of podocytes, called effacement ( Figure 4.3 ). On examination by electron microscopy, effaced podocytes appear flattened and even fused (although the latter does not happen functionally). Numerous studies have shown that effacement is an active process, due to changes in the actin cytoskeleton of the podocyte, which forms the “backbone” of these highly specialized cells. Further evidence that effacement is an active process is that in some instances it can be reversed, such as in treatment-responsive patients with MCD. There has been debate whether effacement per se causes proteinuria, because proteinuria due to podocyte damage can occur independent of this change in shape. The relationship between podocyte foot process effacement and proteinuria has been questioned, and it is clear that there is still much to learn about this long-recognized but still poorly understood ultrastructural phenomenon. Confusingly, effacement has also been reported (in the absence of proteinuria) in the protein-malnutrition state kwashiorkor, suggesting that it may be a feature of hypoalbuminemia rather than of proteinuria per se. However, it is the view of the authors that effacement is a manifestation of serious podocyte injury, and that this histologic finding implies changes in either slit diaphragm proteins, actin binding and regulating proteins, podocyte attachment to the GBM, and/or other events. Therefore teasing out precisely the biologic role of effacement in the development and maintenance of proteinuria may not be important.
