Glomerular Cell Biology

Structure and Function of the Glomerulus (Renal Corpuscle)

The glomerulus or renal corpuscle is comprised of the glomerular tuft surrounded by Bowman’s capsule and space. The tuft is a specialized microvascular bed which contains three cell types including the fenestrated or sinusoidal glomerular endothelial cells, the visceral epithelial cells known as podocytes, and mesangial cells. The glomerular filtration barrier is made up of the endothelial cells and podocytes together with an intervening glomerular basement membrane, and is the site of formation of the primary urinary filtrate. In the average adult human with normal renal function, 180 L of primary urinary filtrate is formed each day. The filtration barrier must permit free passage of water and small solutes into the urine, while retaining larger macromolecules in the blood. The demands of such “high flux” filtration require that the cell types of the glomerulus exhibit many specialized features, which will be discussed in this chapter.

In all mammals, the renal corpuscle appears as a spherical structure, although its diameter varies to some degree with the size of the organism. In humans, glomeruli are approximately 200 μm in diameter, in elephants 300 μm, in rats 120 μm, and in rabbits 150 μm. A normal human kidney contains approximately one million individual glomeruli.

The central component of the renal corpuscle is composed of a plexus of sinusoidal or fenestrated capillaries that extend into Bowman’s space where the primary urinary filtrate accumulates. The capillary loops are held together by the mesangial cells, and they are covered by a continuous layer of podocytes ( Figure 22.1 ). At the vascular pole, the podocyte layer is continuous with the parietal epithelium of Bowman’s capsule. Cells with intermediate phenotypes (between podocyte and parietal epithelium) can be observed at the transition zone. At the urinary pole of the renal corpuscle, the parietal epithelium is continuous with the epithelium of the proximal tubule ( Figure 22.2c ).

At the site of transition between parietal and visceral epithelium, the afferent and efferent arterioles enter and exit the glomerulus respectively, this is known as the glomerular hilum. After entering, the afferent arteriole branches to form a complex plexus of fenestrated capillaries with loops at the urinary pole. The mesangium is required for proper structure and formation of this plexus, and in its absence only a single ballooned capillary loop forms. The capillary loops come into direct contact with the mesangium at discrete points in a small region known as the juxtamesangial portion. However, the majority of the loops are found within Bowman’s space and are covered entirely by the glomerular basement membrane and podocyte foot processes. This is the surface area across which filtration occurs. The branches of the afferent arteriole give rise to individual vascular lobules within the glomerular tuft; each of these lobules contains its own afferent and efferent capillary with some connections between lobules. After looping at the urinary pole, the efferent capillaries join to form the larger efferent arteriole, which exits the tuft at the glomerular hilum. Along the length of the efferent arteriole, the extraglomerular mesangium is gradually replaced by typical smooth muscle cells ( Figures 22.3 and 22.4 ).

General Description

Glomerular endothelial cells are large, highly flattened cells that form the innermost layer of the glomerular capillary. Peripherally these cells are extremely thin, and the cell body contains the nucleus and all the cell organelles. The peripheral portions of the endothelial cells contain numerous fenestrae, which are 50–100 nm pores that penetrate the cytoplasm ( Figure 22.5 ). The luminal side of endothelial cells is covered by a thick layer consisting of glycoproteins that form “sieve plugs” in the fenestrae and the glycocalyx.

Formation of Glomerular Capillaries

Several transplantation studies have demonstrated that the glomerular capillaries are formed largely by vasculogenesis: endothelial cells are derived from angioblasts believed to be intrinsic to the metanephric mesenchyme. At E12.5, in mice before the formation of immature vasculature, kinase insert domain receptor (Kdr, also known as vascular endothelial growth factor receptor 2 (Vegfr-2) or Flk-1) positive angioblasts are present in the metanephric mesenchyme. In the S-shaped stage of the developing nephron, immature podocytes start expressing vascular endothelial growth factor-A (Vegfa), thereby attracting Kdr-expressing endothelial cells to migrate to the vascular cleft. Transforming growth factor-β1 (Tgfb1) induces apoptosis of the endothelial cells and opens the capillary lumens.

Fenestrae and Diaphragms

Ultrastructural analysis demonstrates that diaphragms are often observed in fenestrae of glomerular endothelial cells in rodent embryos. Diaphragmed fenestrae are formed in the S-shaped stage, and then the diaphragms disappear from the capillary loop stage onwards. The main component of the fenestration diaphragm is type II transmembrane glycoprotein plasmalemmal vesicle-associated protein-1 (Pv-1), but its precise function is still unknown. Fenestrae in adult glomerular endothelial cells do not have diaphragms ( Figure 22.5 ); however, fenestrae bridged by diaphragms can be found along the intraglomerular segment of the efferent arteriole and its derivatives. Diaphragms can also be observed in a drug-induced nephritis model, suggesting that diaphragms are required in the development and remodeling of fenestrations, thereby compensating for the immaturity of the barrier function in these settings.

Glycocalyx/Sieve Plugs

The luminal membrane of endothelial cells is covered by a highly negatively-charged layer called the endothelial surface layer (ESL). The relatively dense, membrane-associated part of this layer is called the glycocalyx, and the larger, less compact component is known as the endothelial cell coat ( Figure 22.6 ). The main components of the ESL are glycoproteins, glycoaminoglycans (GAGs), and membrane-associated and secreted proteoglycans. Ultrastructural examinations with sophisticated specialized fixation techniques have revealed that this layer also fills the fenestrae with slit diaphragm-like “sieve plugs”. The thickness of the glycocalyx is estimated to be 50–100 nm, and that of the loose endothelial cell coat is considered to be 200–400 nm. The relative importance of the ESL and sieve plug in the glomerular filtration barrier is still controversial.

Functional Maintenance of Glomerular Endothelial Cells: Insights from Studies of Angiogenic Factors

A number of factors are involved in the maintenance of glomerular endothelial structure and function, and coordinate an elaborate cross-talk between endothelial cells and other cell types of the glomerulus ( Figure 22.7 ).

Pdgfb/Pdgfrb

Platelet-derived growth factor B (Pdgfb) is secreted from endothelial cells and binds its receptor (Pdgfrb) on mesangial cells. Pdgfb or Pdgfrb knockout mice have a defect in mesangial migration and a single, dilated glomerular capillary loop ( Figure 22.8 ). Endothelial specific deletion of Pdgfb results in the same phenotype as that seen in mice with a systemic knockout. Also, this paracrine system requires retention of the ligands in the pericellular space, because mutants with deletion of the Pdgfb retention motif demonstrate delayed mesangial migration and, later on, proteinuria and glomerulosclerosis.

Vegfa

Vegfa facilitates the formation of fenestrae in cultured glomerular endothelial cells. Podocytes produce large amounts of Vegfa that can bind to Kdr on endothelial cells. Cell-selective deletion of Vegfa from podocytes demonstrates that Vegfa signaling is required for formation and maintenance of the glomerular vasculature, its fenestrated phenotype and the filtration barrier. Mice treated with soluble fms-related tyrosine kinase-1 (sFlt-1, discussed below), a decoy receptor of Vegfa, show striking attenuation of endothelial fenestration, highlighting the necessity for Vegfa in the maintenance of fenestration ( Figure 22.5 ). In mutant mice that carry a podocyte-specific gene deletion of Vegfa , a few endothelial cells migrate into the developing glomeruli but they fail to develop fenestrations and rapidly disappear, causing renal failure and neonatal death. The deletion of one allele of the Vegfa gene from podocytes leads to a glomerular defect known as endotheliosis, characterized by endothelial swelling and loss of fenestrations – a universal feature found in thrombotic microangiopathies. Overexpression of the major angiogenic Vegfa 164 isoform in podocytes results in collapse of the glomerular tuft. Additionally, patients receiving anti-VEGF therapy may develop proteinuria due to thrombotic microangiopathy (TMA) of the glomerulus with prominent endotheliosis ( Figure 22.9 ). Indeed, deletion of Vegfa in mature podocytes of adult mice leads to TMA. Taken together, these results indicate an indispensable role for Vegfa in the development, maintenance, and function of the glomerular vasculature and filtration barrier. They also highlight the importance of Vegfa paracrine signaling from the podocyte to Kdr on glomerular endothelial cells.

sFlt-1 is an alternatively spliced soluble form of VEGF receptor 1 (VEGFR-1)/Flt-1, and binds to VEGF as a decoy, thereby acting as a potent inhibitor of VEGF activity. Treatment of mice with adenoviral-induced sFlt-1 leads to a massive reduction of endothelial fenestrae ( Figure 22.5b ). sFlt-1 blood levels are elevated in patients with pre-eclampsia and administration of sFlt-1 to pregnant rats causes hypertension and proteinuria with histological glomerular endotheliosis. The endothelium is the most common glomerular region affected in pre-eclampsia, suggesting a functional role for sFlt-1 in the function of the glomerular endothelium. A recent study also implicated sFlt-1 in the pathogenesis of PR3-ANCA-associated vasculitis affecting the glomeruli.

Angiopoietins

Another family of angiogenic factors required for the development and homeostasis of glomerular endothelial cells is the Angiopoetin–Tek signaling system. Angiopoietin 1 (Angpt1) and Angiopoietin 2 (Angpt2) are ligands for Tek tyrosine kinase (Tek/Tie-2). Angpt1 binds to the Tek receptor expressed on endothelial cells, and causes its phosphorylation. This signal leads to enhanced survival of endothelial cells, stabilization of the endothelial cell-to-cell connection, and reduced permeability. Angpt2 is considered to be a competitive antagonist of Angpt1 by binding Tek but not activating any intracellular signaling. There is some data, however, that suggests that Angpt2 can activate Tek signaling under certain conditions. Angpt1, Angpt2 and Tek are all expressed in developing kidneys. Angpt1 is expressed widely in the condensing mesenchyme in the developing kidney and its derivatives, and in mature podocytes. Angpt2 shows a more restricted expression pattern, localizing to endothelial cells, pericytes, smooth muscle cells of cortical and large blood vessels, and immature mesangial cells. Tek is expressed both in mature and immature glomerular endothelial cells. Angpt1 conventional knockout mice die at embryonic day 12.5, thus precluding any analysis of its role in the glomerular vasculature. In mouse metanephric organ culture, recombinant Angpt1 enhances the growth of interstitial capillaries. A recent report demonstrated that Angpt1 treatment of isolated rat glomeruli reduced vascular permeability and increased the depth of the glycocalyx layer. Cell type-specific/inducible knockout approaches, however, have revealed a crucial role for Angpt1 in glomeruli. Deletion of Angpt1 at E10.5 leads to a single, dilated glomerular capillary loop without mesangial migration in a portion (~10%) of glomeruli that is reminiscent of the Pdgfb/Pdgfrb mouse mutant phenotype ( Figure 22.10a,b ). Although deletion of Angpt1 after E13.5 doesn’t cause any immediate vascular phenotype, streptozotocin-induced diabetic mice with global or glomerular-specific Angpt1 deletion develop increased urinary albumin excretion, severe mesangial expansion, glomerular sclerosis, and early mortality ( Figure 22.10c,d ). Another report also demonstrated that treatment of diabetic mice with Angpt1 recombinant protein is protective for renal function. Angpt1 is therefore dispensable in quiescent, mature glomeruli, but is essential in development and in the vascular response to injury.

Angpt2 knockout mice are briefly viable in the post-natal period, and exhibit increased pericyte coverage of peritubular capillaries. The mice die soon after birth however, precluding analysis of the role of Angpt2 in more mature capillary beds. Angpt2 overexpression in podocytes causes proteinuria and podocyte apoptosis in formed glomeruli.

Angiopoietin ligands seem to function in concert with Vegfa. In Vegfa-rich conditions, Vegfa and Angpt2 work together to promote sprouting. The precise degree of cross-talk between these pathways is still under investigation.

Glomerular Endothelial Cells as a Source of Vasoregulators

Glomerular endothelial cells produce both nitric oxide, a vasodilator, and endothelin-1, a vasoconstrictor. Nitric oxide is produced by NO synthases. Both endothelial and inducible NO synthases are expressed by glomerular endothelial cells in vitro and in vivo . eNOS expression and activation is at least partially influenced by Vegfa. In many rodent disease models and human patients with kidney diseases, overproduction of NO and its derivatives has been observed. It is considered that excessive NO production generated by inducible NO synthase (iNOS) results in glomerular injury, whereas NO generated from endothelial NO synthase (eNOS) is protective by preserving endothelial survival and function. In diabetic patients, eNOS expression is increased in renal endothelial cells, whereas iNOS expression is preferentially upregulated in inflammatory cells. The degree of eNOS expression is related to the severity of the glomerular lesion and proteinuria. Diabetic eNOS knockout mice develop more severe glomerular lesions and greater albuminuria. In addition, excretion of NO-related products is often reduced in diabetic patients with nephropathy.

Endothelin-1 is a potent vasoconstrictor which binds to one of two receptors: endothelin receptor type A (Ednra); and endothelin receptor type B (Ednrb). Binding of endothelin-1 to Ednra results in vasconstriction, while binding to Ednrb causes vasodilation. In glomerular endothelial cells, Ednrb is dominant, whereas Ednra is expressed by mesangial cells.

Glomerular endothelial cells are also involved in the renin–angiotensin–aldosterone system (RAAS). They express angiotensin-converting enzyme (ACE) and produce angiotensin II. The relative contribution of the glomerular endothelium compared to the systemic endothelium with regard to angiotensin II production, however, is still unknown. Furthermore, the potential role of angiotensin receptors in endothelial cells is also unknown.

Description

Mesangial cells are irregularly shaped cells which extend processes from their cell body towards the glomerular basement membrane (GBM). The “mesangium” refers to the mesangial cells together with the mesangial matrix they produce ( Figures 22.11 and 22.12 ). Mesangial cells provide structural support to the glomerular tuft, produce and maintain mesangial matrix, communicate with other glomerular cells by secreting soluble factors, and may contribute to the glomerular capillary flow via their contractile properties.

Mesangial Cells Provide Structural Support to the Glomerular Tuft

The mesangium forms the central core of the glomerular tuft. The processes which they extend towards the GBM are densely populated by bundles of actin, myosin, and β-actinin microfilaments. These processes attach directly or by interposition of microfibrils to the GBM. They also extend underneath glomerular endothelial cells toward the mesangial angles of the GBM, anchoring two opposing mesangial angles together through their microfilament bundles ( Figure 22.12b ). These microfilament bundles cross the mesangial cells to tether opposing parts of the GBM through α3β1 integrin and the basal cell adhesion molecule (BCAM) glycoprotein, which bind laminin α5 in the GBM. These structures are believed to supply protection from hydraulic pressure by providing inward-directed tension.

Mesangial Cells Produce and Maintain Mesangial Matrix

The mesangial matrix fills the remaining spaces between the mesangial cells and the perimesangial glomerular basement membrane (GBM, for review see ). This matrix is composed of a diverse array of common matrix proteins including collagens type III, IV, V, and VI; heparan sulfate proteoglycans including biglycan and decorin ; and the elastic fiber proteins fibronectin, laminin, entactin, and fibrillin-1, among others. Fibronectin is the most abundant of these, and is associated with microfibrils which network to form the basic ultrastructure of the matrix. These microfibrils are unbranched and non-collagenous with a diameter of 15 nm, and form a dense three-dimensional network that contributes to the anchoring of mesangial cells to the GBM. It is thought that these microfibrils allow for the transmission of mesangial cell contractile forces to the GBM.

Mesangial phenotypic changes are a hallmark of certain glomerular diseases such as diabetic nephropathy. This condition is characterized by glomerular sclerosis due to an accumulation of mesangial matrix and thickening of the GBM. The sclerotic lesion contains an abundance of type IV collagen normally present in the glomerulus, but also contains types I and III collagen which are usually absent but are produced by injured mesangial cells.

Signaling Molecules Involved in Mesangial Cell Biology

Other Secretary Molecules and Receptors

Mesangial cells produce, and are also influenced by, many growth factors including Tgfb1, connective tissue growth factor (Ctgf), insulin like growth factor (Igf), fibroblast growth factor (Fgf), and hepatocyte growth factor (Hgf). Among these factors, Hgf antagonizes the pro-fibrotic actions of Tgfb1, whereas the other factors are upregulated by mesangial cells in disease conditions including diabetes or Thy1.1 nephritis, and facilitate glomerular matrix accumulation. Vasoactive factors such as angiotensin II and endothelins promote mesangial proliferation, and this effect may be mediated by transactivation of the Egf receptor. However, because of the lack of effective tools for deleting genes specifically from mesangial cells, the precise functions of these factors in normal physiology remain to be determined.