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.

PAS stain of glomeruli from E17.5 mouse embryos.

(a): Normal mouse glomerulus. Arrows show normal fold of the basement membrane. (b): Pdgfb ^−/− glomerulus shows a single open aneurysm-like capillary loop without any mesangium (failure of mesangial migration). There is no fold of basement membrane (arrows).

(From Betsholtz, C. (1995). Role of platelet-derived growth factor in mouse development. Int. J. Dev. Biol. 39 : 817–825 with permission.)

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.

A glomerulus showing thrombotic microangiopathy from a patient who received anti-VEGF therapy, showing fragmentation of erythrocytes (FE: arrows) and foamy change (FC: arrow) of endothelium.

(Courtesy of Dr. Laura Barisoni, New York University School of Medicine, New York, NY.)

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.

Tgfb1

Infusion of a neutralizing antibody against transforming growth factor β1 (Tgfb1) to neonatal rats causes a delay in glomerular capillary formation, including the development of fenestrations. In the choroid plexus, neutralization of both Tgfb1 and Vegfa leads to decreased cerebral perfusion, vascular thorombi, and a defect of fenestration, which does not occur when either of these treatments are administered individually. Thus, Tgfb1 also plays a crucial role in the development and maintenance of glomerular endothelial cells, and may work in concert with Vegfa. Additionally, blood levels of soluble endoglin, an antagonist of Tgfb1 are elevated in patients with pre-eclampsia, and correlate with disease severity. Administration of soluble endoglin in combination with sFlt-1 to pregnant rats results in severe pre- eclampsia, including HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets).

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.

Conditional deletion of Angpt1 results in glomerular developmental defects and enhanced diabetic glomerular injury.

(a): A normal glomerulus at embryonic day 17.5 (E17.5). (b): Early conditional deletion of Angpt1 gene at embryonic day 10.5 (midgestation) results in some abnormal glomeruli with single open capillary loops similar to that of Pdgfb-null mouse at E17.5. (c) and (d): Late deletion of Angpt1 at E16.5 doesn’t lead to any immediate phenotype. However, after 20 weeks of streptozotocin-induced diabetes, the diabetic mutant mice that carry Angpt1 deletion show an increase in mesangial matrix expansion and sclerosis (d) compared with that of diabetic controls (c) (LM×1000).

(Courtesy of Dr. Marie Jeansson, Mount Sinai Hospital, Toronto, ON.)

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.

Ephrin-Eph Family

Ephrin-Eph molecules are another family of tyrosine kinase signaling factors that are widely expressed in the developing kidney. In other organs, they are involved in the specification of arteries and veins, as well as in neural development. In the developing kidney, the Ephrin B2 ligand is expressed in podocyte precursors, but later it is expressed by endothelial and mesangial cells. In adults, the EphB4 receptor is mainly expressed in podocytes. Overexpression of the EphB4 receptor causes defects of glomerular arteriolar formation. Deletion of Ephrin B2 from Pdgfrb-expressing pericytes and the mesangial cell population results in abnormal glomerular tuft formation. Pharmacologic inhibition of EphB4 leads to delayed recovery and extended injury of endothelial cells and podocytes in a rat mesangial injury model. Therefore, it seems this family is involved in glomerular tuft formation and maintenance, but the precise mechanism is still unclear.

CXCR4/CXCR7/CXCL12 Axis

Chemokine CXC motif receptor type 4 (CXCR4), CXCR7, and Chemokine CXC motif ligand 12 (CXCL12) are also critical factors in the development and maintenance of glomerular vasculature. Cxcr4 and Cxcr7 are seven-transmembrane G-protein coupled receptors, and Sdf-1/Cxcl12 is their cognate chemokine ligand. A deficiency of Cxcr4 or Cxcl12 leads to a failure of vasculogenesis and embryonic lethality. During renal development, Cxcr4 ^−/− or Cxcl12 ^−/− knockout mice show a ballooning of the glomerular capillary loops which is reminiscent of Pdgfb/Pdgfrb knockout mice. On the other hand, activation of the CXCR4/CXCR7/CXCL12 axis appears to underlie some glomerular diseases, such as rapidly progressive glomerulonephritis (RPGN), diabetic nephropathy, and hemolytic uremic syndrome (HUS). It appears that Cxcl12 is secreted from podocytes or interstitial cells, and acts on Cxcr4 expressed by the endothelial cells to regulate vascular development and function.

Summary

Glomerular endothelial cells form the first perm-selective barrier in the glomerulus. The relationship between their elaborate, fenestrated cell shape and function is maintained by a network of various angiogenic factors. Glomerular endothelial cells also contribute to the charge-selective barrier through the negatively-charged glycocalyx.

Integrins

In addition to α3β1 integrin observed in connections between the GBM and mesangial processes, α1β1, α2β1, and fibronectin receptors α5β1 and α8β1 integrins are expressed in the mesangial cells, and are able to activate integrin-linked cell signaling. In the absence of α1 integrin, cultured mesangial cells show decreased proliferation, increased matrix production, and altered intracellular signaling. Mice lacking α1 integrin exhibit more severe Adriamycin and diabetes-induced glomerular injury, highlighting the importance of matrix-related signaling. On the other hand, removal of α2 integrin in mice leads to amelioration of glomerular damage in an Adriamycin and partial renal ablation model. α8 integrin seems to promote adhesion, but inhibits migration and proliferation of mesangial cells in vitro .

Pdgfrb

Pdgfb is secreted from endothelial cells and binds to Pdgfrb expressed in mesangial cells, where it exerts a crucial role in mesangial migration and glomerular tuft formation. Without mesangial cell migration, the looping of glomerular capillaries does not occur. This interaction is discussed further in the endothelial section.

Ephrin B2

As mentioned in the section on the glomerular endothelium, deletion of Ephrin B2 from mesangial cells and pericytes leads to abnormal glomerular tuft formation and reduced numbers of capillary loops. It’s receptor, EphB4 is expressed in podocytes, suggesting an interaction between the mesangium and podocytes.

Laminin

Laminins are secreted as heterotrimers, which are stabilized by disulfide interchain bonding. Each laminin heterotrimer is composed of α, β, and γ chains that combine with each other in nonrandom combinations to form at least 15 different heterotrimers. The laminins are named by their composition; for example: α5β2γ1 is named laminin-521 or LM-521. The heterotrimer structure appears as a “cross” with one longer and three shorter arms. At the COOH end of all α chains is a laminin globular chain (LG) that extends beyond the long arm. This LG domain interacts with cell surface receptors such as integrins and dystroglycans that are expressed by the podocytes.

Interactions between the laminin heterotrimers themselves are mediated by subdomains found in the shorter arms of the “cross.” Laminin also binds to the network of type IV collagen via nidogen, and to agrin, a heparan sulfate proteoglycan of the GBM. The combined network of type IV collagen (see below) and laminin provides mechanical properties to the basement membrane, serving as a scaffold for the placement of other matrix components.

During glomerular formation, laminin trimer compostion changes from LM-111 to LM-511, and finally to LM-521. In the adult, laminins continue to be produced by both endothelial cells and podocytes, as the protein can be identified in the endoplasmic reticula of both cell types.

A series of reports confirm an important role for laminins in GBM function. A mutation of laminin β2 results in Pierson syndrome in humans that is characterized by ocular and neurological symptoms, and congenital nephrotic syndrome. This observation was confirmed using knockout mice of laminin β2 that develop proteinuria even before the onset of visible ultrastructural changes to the podocytes. Recently, the phenotype of the laminin β2 knockout mouse was rescued by overexpression of laminin β1, suggesting redundancy between laminin β1 and β2. Also, deletion of laminin α5 in mice prevents the transition from LM-111 to the mature LM-521 that leads to breakdown of the GBM and failure of glomerular vascularization. A mouse carrying a hypomorphic mutation of laminin α5 also shows glomerular proteinuria, hematuria, and cystic kidneys, suggesting the gene dosage of the laminin α5 chain is crucial for the maintenance of the GBM.

Type IV Collagen

Collagen IV is another major component of the GBM. Similar to other collagens, collagen IV is a trimeric extracelluluar matrix component made up of α chains that are rich in Gly-X-Y amino acid repeats. The type IV collagen family includes six genetically distinct α chains that trimerize with each other in specific combinations to make three types of trimers with each other: (α1) ₂ α2; α3α4α5; and (α5) ₂ α6. Each of these trimers is referred to as a protomer. Protomers are secreted into the extracellular matrix, where they self-polymerize and are subsequently cross-linked by specific enzymes.

Similar to laminin, the composition of type IV collagen trimers undergoes a developmental switch in the GBM. Early in glomerulogenesis (S-shaped stage), the GBM is composed of α2/α1 chains, which are replaced at the capillary loop stage by α3α4α5. Although a basement membrane can form in the absence of type IV collagen its structure is compromised, resulting in variable defects in the filtration barrier depending on the specific genetic mutation. In the adult kidney, the type IV collagen α3α4α5 network is produced only by podocytes.

Mutations in any of the α3, α4 or α5 genes are associated with glomerular disease in patients, emphasizing the key role that type IV collagen plays in glomerular barrier function. The defects can be minimal, as observed in patients with thin basement membrane disease (also known as benign familial hematuria). This disease is inherited in an autosomal dominant fashion, and patients present with isolated microscopic hematuria. As suggested by its name, the GBMs show thinning. Thirty to forty per cent of the patients exhibit mutations in COL4A3 or COL4A4. Although originally assumed to be benign, FSGS has been reported in Cypriot families with this disease.

The same mutations in COL4A3 and COL4A4 inherited in a homozygous fashion as an autosomal recessive disease result in a more severe disease known as Alport’s syndrome, a hereditary basement membrane disease associated with progressive glomerulopathy that leads to renal failure, and is associated with deafness and ocular abnormalities. The commonest form of Alport syndrome is X-linked, caused by mutations in the α5 chain ( Figure 22.13 ). Goodpasture syndrome, an autoimmune disease characterized by glomerulonephritis and lung hemorrhage, is mediated by antibodies against the type IV collagen α3 chain.

Transmission electron micrograph of the GBM in a patient with Alport syndrome.

Thickening, splitting and lamination of the GBM can be seen (TEM×14,900).

(From Rumpelt, H. J. (1987). Alport’s syndrome: Specificity and pathogenesis of glomerular basement membrane alterations. Pediatr. Nephrol. 1 : 422–427, with permission.)

Intriguingly, recent studies have shown that a mutation of the type IV collagen α1 chain causes hereditary angiopathy with nephropathy, aneurysms, and muscle cramps (HANAC syndrome); the renal manifestations are characterized by hematuria and large bilateral cysts. A rodent model of this mutation leads to defects of the GBM, suggesting a functional role for the α1 chain in the GBM, despite the prominence of a3a4a5 trimers in mature type IV collagen.

Nidogen

Nidogen-1 and -2 (previously known as entactin-1 and entactin-2) are virtually ubiquitous basement membrane glycoproteins. Nidogen-1 binds both laminin and type IV collagen, suggesting that it is involved in forming the GBM. However, it was demonstrated that a single knockout of either nidogen-1 or nidogen-2 in mice results in no GBM phenotype. Although deletion of both nidogen genes results in perinatal lethality, suggesting some overlapping roles of the two genes, the glomerular basement membrane still forms apparently normally. The exact role and requirement of nidogen in the GBM is not yet known.

Proteoglycans

Heparan sulfate proteoglycans of the GBM are concentrated in the laminae rarae internae and externae, and provide an electronegative charge to the GBM. The major proteoglycans of the GBM are heparan sulfate proteoglycans; most prominent is agrin, but perlecan is also present. Although classical studies suggested that the negative charge of the GBM was essential to retard passage of neutral and negatively-charged macromolecules across the barrier, this model has been challenged due to results obtained from genetically modified mice. Podocyte-specific knockout mice for agrin and/or perlecan, either in isolation or together, does not result in proteinuria or overt filtration defects. Current thoughts on the glomerular charge barrier are discussed later in the chapter.

Wt-1

The transcription factor Wt-1 is first expressed in the metanephric mesenchyme, which contains progenitors of the tubular and glomerular epithelium. As renal development continues, its expression becomes restricted to the renal vesicle, comma, and the S-shaped body. Later on, its expression is observed in immature and mature podocytes, which persists throughout life.

Wt-1 is a transcription factor with four zinc fingers that can bind both DNA and RNA. There are four major splice variants of Wt-1 mRNA, which may be responsible for its variety of functions in development and normal physiology. Embryonic loss of Wt-1 in mice leads to complete renal and gonadal agenesis, defects in the epicardium, lack of sub-epicardial mesenchymal cells, and adrenal agenesis, suggesting its important role in multiple organs.

Several mouse models underscore the importance of Wt-1 function during nephrogenesis and in podocytes. Postnatal deletion of the Wt-1 gene results in glomerulosclerosis with loss of podocyte foot processes and molecular markers of differentiation such as nephrin, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat mass, and failure of erythropoiesis. In Wt-1 knockout mice, restitution of Wt-1 expression using a human derived Wt-1-YAC transgene leads to a rescue of cardiac defects, but incomplete glomerular development. Furthermore, Wt-1 haploinsufficient mice develop adult onset mesangiosclerosis and glomerulonephritis.

In humans, Wt-1 mutations are associated with two glomerulopathies: Denys–Drash syndrome (DDS) and Frasier syndrome (FS), which can both present early in life and cause abnormal glomerular development. DDS is caused by heterozygous mutations of the Wt-1 gene that predominantly affect the zinc finger regions in exons 8 (zinc finger II) and 9 (zinc finger III), and directly interfere with the DNA-binding capacity of Wt-1. The characteristic clinical picture of DDS is a triad of congenital nephrotic syndrome, XY pseudohermaphroditism, and Wilm’s tumor. The characteristic renal lesion in these patients is diffuse mesangiosclerosis due to increased matrix deposition on the vascular side of the glomerular basement membrane. Expression of the DDS mutation in mice, either in podocytes or systemically, leads to various glomerular lesions similar to those of humans. Wt-1 is expressed in podocytes, which are found on the opposite side of the glomerular basement membrane (GBM) to the mesangium. The glomerular lesion in DDS therefore highlights the importance of interactions that occur between podocytes on one side of the GBM and endothelial cells or mesangial cells on the other side of the GBM. Frasier syndrome (FS) is characterized by focal segmental glomerular sclerosis, male-to-female sex reversal, but no tumors. It results from a dominant mutation that causes an inability to include an alternatively spliced lysine-threonine-serine (KTS) sequence after the third zinc finger of Wt-1 . Interestingly, mice engineered to exclusively express the variant that is +KTS or −KTS display normal induction of the metanephric mesenchyme by the ureteric bud, but have malformed glomeruli. These findings suggest different roles for the alternatively spliced variants of Wt-1 in glomerular development and maintenance.

While the contribution of Wt-1 to renal development and maintenance is undeniable, its precise role is still being elucidated. Human and animal models seem to indicate it plays a role in regulating the expression of a number of developmental genes. During early renal development the regulatory gene Pax-2 is expressed in the induced metanephric mesenchyme and ureteric bud, but is absent in the mature renal epithelium. In podocytes, the expression of Wt-1 coincides with a downregulation of Pax-2, suggesting a role for Wt-1 in transcriptional repression of this gene. Indeed, in patients with DDS, downregulation of Wt-1 was associated with increased Pax-2 expression ; however, it is unclear if this increased Pax-2 expression is pathogenic. Recent advances in high-throughput technology for assessing transcription factor activity have elucidated a number of other Wt-1 regulated genes that are known to play a role in renal development, including Six2, Bmp7, Sall1, and HeyL.

Wt-1 also plays a role in maintaining podocyte homeostasis beyond development. The essential podocyte slit diaphragm protein nephrin has been identified as a transcriptional target of Wt-1, and is downregulated in the glomeruli of mice lacking the Wt-1(−KTS) splice variant. Furthermore, Wt-1 modulates the podocyte glycocalyx through transcriptional regulation of Podocalyxin and 6-O-endosulfatases Sulf1 and Sulf2, which play a role in maintaining the charge of the glomerular filtration barrier, podocyte foot process separation, and bioavailability of essential growth factors such as vascular endothelial growth factor-α (Vegfa) and fibroblast growth factor (Fgf)-2. In addition to post-translational regulation of Vegfa bioavailability, Wt-1 can also directly regulate expression of Vegfa, which is essential to the maintainenance of the glomerular vasculature. Given this wide range of regulatory functions, it is still unclear what is responsible for the pathology observed in DDS and FS patients, but it is likely a convergence of a number of these signaling networks.

Lmx1b

Lmx1b encodes a Lim-domain protein that is mutated in Nail–Patella syndrome. In mice lacking Lmx1b, podocytes retain their immature cuboidal phenotype, fail to form foot processes or endothelial fenestrations, and have a split glomerular basement membrane. These findings are associated with a concomitant downregulation of Vegfa, Synaptopodin, Nphs2, type III collagen α4 (Col3a4), and type IV collagen α4 (Col4a4). Lmx1b-binding elements in the Nphs2 and Col4a4 promoter regions suggest that this occurs through direct transcriptional regulation.

Tcf21

Tcf21 (also known as Pod1/Capsulin/Epicardin) encodes a basic helix-loop-helix transcription factor that is highly-expressed in the developing kidney, lung, intestine, and pancreas at sites of mesenchymal–epithelial interaction. In the developing mouse kidney, it is expressed in the condensing mesenchyme, and knockdown of Tcf21 expression in renal explants causes decreased mesenchymal condensation and ureteric bud branching. Subsequently, Tcf21 expression is restricted to the primitive podocytes of the S-shaped bodies, and genetic deletion of Tcf21 in mice leads to failure of podocyte terminal differentiation ( Figure 22.16 ). Tcf21 ^−/− knockout mice have a marked reduction in glomerular number, and the remaining glomeruli are developmentally arrested between S-shaped body and capillary loop stages.

Transmission electron micrograph of podocyte foot processes at E18.5.

Normal mouse podocytes show organized foot processes assembly (a), whereas Tcf21 null mouse podocytes demonstrate defects of foot process development (b).

Kreisler

Kreisler (Mafb) encodes a basic domain leucine zipper transcription factor that is expressed in podocytes at the capillary loop stage of glomerular development. Mice with a homozygous enu mutation of Kreisler show a similar but milder phenotype to those with a Tcf21 deletion. They are born with glomeruli arrested at the capillary loop stage, and podocytes that adhere to the GBM but fail to form foot processes. Kreisler mutants, however, express Tcf21, suggesting that Tcf21 is either upstream of Kreisler in podocyte development or acts by a different mechanism.

Foxc2

Foxc2 is a winged helix transcription factor that was identified during a screen of enriched genes in mRNA isolated from glomeruli at different stages of development. Foxc2 is first expressed in putative podocytes during the comma shaped body stage of glomerular development, and as such is the earliest known podocyte marker. Glomeruli from Foxc2 ^−/− mice display aberrant podocyte foot process formation, mesangial cell clustering at the base of the glomerular tuft, and swollen endothelial cells lacking fenestrae, similar to Tcf21 ^−/− mutant mice. In these mice, the endothelial and mesangial defects are thought to be secondary to the podocyte defects. Gene array of Foxc2 ^−/− glomeruli identified over 700 differentially regulated genes. Notably, however, there was a strong downregulation of Nphs2, Col4α3, and Col4α4, which are fundamental to slit diaphragm assembly and GBM formation.

Notch

The Notch family includes four well-conserved genes that encode transmembrane receptors involved in cell fate specification and development from invertebrates to mammals. Notch is fundamental in the segmentation of the metanephric mesenchyme into 20 functionally-distinct cell types segregated into different compartments along the nephron.

During normal development, Notch2 is expressed in condensing structures of the metanephric mesenchyme, such as the metanephric vesicles, comma-shaped bodies, and S-shaped bodies, but is eventually restricted to differentiating podocytes in more mature glomeruli. Jag1 is the Notch2 ligand thought to be important for glomerular differentiation. During development, it is expressed in renal vesicles, comma-shaped bodies, and S-shaped bodies. In more mature glomeruli it then localizes to the inner region of the glomerular tuft to endothelial and/or mesangial cells. Mice homozygous for a hypomorphic mutation in the Notch2 gene exhibit hypoplastic kidneys, and an arrest of glomerular development prior to the capillary loop stage. A conditional inactivation of Notch2 from nephron progenitor cells results in a more severe “distal tubule only” phenotype, with a complete failure of glomerulogenesis and proximal tubule formation.

In humans, mutations in Jag1 or Notch2 cause Alagille syndrome, an autosomal dominant disorder characterized by the presence of cholestatic liver disease, cardiac disease, ocular abnormalities, skeletal abnormalities, and characteristic facial features. A large proportion of Alagille patients also develop renal disease characterized by glomerular lesions, cystic kidneys, and ultimately renal failure. Taken together these studies suggest an important role for Notch signaling in establishing the proximal–distal orientation of the nephron, and segmentation of the proximal structures.

Recent evidence also reports a crucial role for the Notch pathway in the maintenance of mature podocytes. In patients with a variety of proteinuric nephropathies, including diabetic nephropathy and FSGS, activation of the Notch pathway was observed in podocytes. Furthermore, upregulation of Notch expression in podocytes of transgenic mice results in severe proteinuria and glomerulosclerosis with dedifferentiation of podocytes.

Nephrin

While this first description of the SD shed light on the filtration function of podocytes, the molecular composition of the SD remained poorly-defined. However, the discovery that a mutation in the NPHS1 gene causes Congenital Nephrotic syndrome of the Finnish variety suggested that dysregulation of SD structure can cause glomerular disease. This disease is characterized by massive proteinuria in utero , lack of a slit diaphragm, and abnormal foot process formation. The NPHS1 gene encodes a 180 kDa transmembrane protein of the immunoglobulin superfamily called nephrin that is expressed in the glomerular podocyte, and localizes specifically to the SD. These findings defined the importance of nephrin to the formation and maintenance of a normal SD, and led to the postulation of a “zipper-like model” of nephrin assembly in the SD. Accordingly, it is considered that the SD is the principle structure of the glomerular filtaration barrier and nephrin is its main component. Subsequent work, however, has also elucidated an important role for nephrin as a mediator of actin cytoskeletal organization by binding Src homology domain SH2/SH3 containing Nck adaptor proteins. The cytoplasmic tail of nephrin contains three tyrosine-aspartic acid-x-valine (YDxV) residues which, when phosphrylated by Src family kinases, recruit the SH2 Nck adaptor proteins and induce local actin polymerization ( Figure 22.17 ). Following the identification of nephrin, intensive research has led to the discovery of several other transmembrane proteins that participate in the formation of the slit diaphragm ( Figure 22.18 ).

Transmission electron micrograph of distinct mouse podocyte foot-processes present at 4 days of birth (a), but absent in Nck knockout mice (b). (c)–(g): Cellular immunostaining showing co-localization of Nck2, nephrin at the actin tail. Nck-nephrin interaction is required for nephrin-dependent actin reorganization (Green: nephrin; purple: Nck2; red: palloidin=actin). (d), (e), and (f) show actin, nck2, and nephrin staining, respectively.

((a) and (b): From Jones, N., et al. (2006). Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 440 : 818–823, with permission; (c)–(g): Courtesy of Drs. Tony Pawson and Nina Jones.)

Schematic diagram of the podocyte foot process and slit diaphragm with associated molecules.

Podocin

NPHS2 is a gene that is mutated in some forms of steroid-resistant nephrotic syndrome that cause early onset proteinuria, and focal and segmental glomerulosclerosis. This gene encodes a protein called podocin, which associates in podocyte lipid rafts with nephrin and another SD component, CD2AP, via its C-terminal domain. Nephrin is thought to contribute directly to the formation of the SD, while podocin and CD2AP are thought to mediate its connection to the podocyte actin cytoskeleton. Mouse models in which any of these three components are disrupted lead to a congenital nephrotic syndrome.

Neph Proteins

The C-terminal domain of podocin also interacts with another group of three immunoglobulin superfamily transmembrane proteins called Neph1, 2, and 3 that bear significant homology to nephrin. This family is defined by their well-conserved cytoplasmic tail with a centrally located tyrosine residue required for interacting with podocin. The extracellular domain of Neph1 interacts with nephrin in the SD, and is essential for the regulation of glomerular perm-selectivity. In addition to podocin, the C-terminal (intracellular) domain binds the tight junction protein-1 (Tjp-1/ZO-1), which in turn tethers it to the podocyte actin cytoskeleton. Like nephrin, Neph1 plays a role in intracellular signaling, and is tyrosine phosphorylated at the SD, particularly in certain disease models. Mutations in Neph1 result in proteinuria and perinatal lethality in mice, but the phenotype is less dramatic than that observed in nephrin knockout mice, suggesting that nephrin may be more crucial to maintain the SD. This Nephrin-centric view of the slit diaphragm was called into question by a recent study showing that chickens and developing chicks lack nephrin, but express all three Neph proteins. Ultrastructurally, however, chickens are still able to assemble a SD in the glomerular filtration barrier. These SDs lacking nephrin still express P-cadherin and the large protocadherin Fat-1, suggesting that heterophilic interactions between Neph proteins and these other cadherins may be sufficient to allow SD assembly.

Cadherin and Catenins

In addition to these atypical junctions, the slit diaphragm contains adherens junction proteins P-cadherin, and α, β, γ catenins. P-cadherin is a transmembrane protein, and the extracellular domain is thought to contribute to slit diaphragm formation, while its cytoplasmic tail connects to β or γ catenin. Linkage of this complex to the actin cytoskeleton is believed to occur through an interaction between α catenin and Tjp-1/ZO-1 or α-actinin-4, both of which can directly bind actin.

The occludens protein Tjp-1/ZO-1 is expressed specifically at the points of insertion of the slit diaphragms in mature glomeruli. Tjp-1/ZO-1 appears early on in podocyte development when the apical junctional complexes between podocytes are composed of typical tight and adherens junctions, and persists as these junctional complexes migrate to the basolateral side and ultimately form the SDs. These findings suggest that tight junction proteins play a role in podocyte SD development and function, independent of their ability to form tight junctions.

α -Actinin-4

In humans, mutations in α-actinin-4 result in an autosomal dominant familial FSGS. In vitro the mutant form of α-actinin-4 binds F-actin more strongly than wild-type. This is thought to reduce the podocytes’ ability to respond dynamically to the hydrostatic pressure required for normal glomerular filtration, and ultimately leads to podocyte injury. In addition, α-actinin-4 is thought to mediate the interaction between the actin cytoskeleton and integrins to regulate podocyte adhesion to the glomerular basement membrane.

FAT1

FAT1 is another member of the cadherin superfamily expressed in the podocyte at the SD, and has 34 tandem cadherin-like extracellular repeats. With its large extracellular domain, FAT1 is a major molecular component of the SD, and is required for normal foot process formation as FAT1 knockout mice lack SDs. Because of its localization to the cell–cell contact sites and tips of cellular processes, FAT1 may be involved in the initial steps of cell–cell interaction between podocytes.

Rho GTPases

Given the actin rich nature of the podocyte cytoskeleton, it is not surprising that Rho GTPases – master regulators of cytoskeletal dynamics – are important in podocyte biology. At the leading edge, Rac1 and Cdc42 promote lamellipodia and fillipodia formation, thus enhancing cell motility. In contrast, RhoA promotes a contractile phenotype by inducing formation of actin-myosin stress fibers. In this sense, it is believed that a balance between the opposing activities of RhoA and Cdc42/Rac1 regulates podocyte cytoskeletal dynamics. Both overexpression and inhibition of these small Rho GTPases in podocytes causes glomerular injury in mouse models. Podocyte-specific deletion of Cdc42 leads to a congenital nephropathy in transgenic mice by impairing actin polymerization at sites of nephrin clustering ( Figure 22.19 ), while RhoA deletion does not result in proteinuria. By contrast, overexpression of RhoA in podocytes results in an FSGS phenotype in mice.

Scanning ((a) and (b)) and transmission ((c) and (d)) electron micrograph of murine podocytes.

(a) and (c): Normal appearance of podocytes. (b) and (d): Podocytes lacking Cdc42 show extensive effacement of foot process at postnatal day 5 ((a), (b)×11, 000; (c), (d)×27,000).

(From Scott, R. P., et al. (2012) Podocyte-specific loss of Cdc42 leads to congenital nephropathy. J. Am. Soc. Nephrol. 23 (7): 1149–1154 with permission.)

Recently, dysregulation of Rho-GTPase signaling has also been linked to human glomerular disease. ARHGAP24 is a RhoA-activated Rac1 GTPase-activating protein (Rac1-GAP); a mutation that impairs Rac1-GAP activity results in FSGS in humans. Two recent reports also showed that mutations in the Inverted Formin 2 ( IFN2 ) gene result in autosomal dominant FSGS. INF2 interacts with other diaphanous related formins such as Cdc42; disease causing mutations in INF2 result in decreased CDC42 targeting to the plasma membrane, and impaired actin polymerization and depolymerization in podocytes.

α 3 β 1 Integrin

α3β1 integrin is a heterodimeric cell adhesion receptor with specificity for collagen types I and VI, laminins, fibronectin, and nidogen. α3β1 integrin is highly-expressed on the basolateral surface of mature and developing podocytes, and plays an important role in the development and maintenance of podocyte foot processes. Mice with a targeted mutation of the α3 integrin gene lack podocyte foot processes, and have a reduced number of capillary loops. Furthermore, the podocyte-derived basement membrane fails to fuse with the endothelium-derived basement membrane and becomes fragmented and disorganized. A recent paper reported three paediatric patients who have mutations in ITGA3 , which encodes α3 integrin; they presented with congenital nephrotic syndrome and severe basement membrane abnormalities. Taken together, these findings indicate that α3β1 integrin is not only a receptor for GBM components, but is also required for its development and maintenance. Similarly, podocyte specific ablation of the β1 integrin gene in mice results in massive proteinuria, abnormal capillary morphogenesis, podocyte foot process effacement, and podocyte apoptosis.

α v β 3 Integrin

While α3β1 integrin is the primary integrin expressed in podocytes, more recent work has identified a role for αvβ3 integrin in podocyte homeostasis. αvβ3 integrin is a vitronectin receptor that localizes to podocyte foot processes, and is predominantly associated with non-lipid raft fractions of the cell membrane. Genetic deletion of β3 integrin does not result in an overt renal phenotype, but these mice are protected from LPS-induced proteinuria, suggesting that αvβ3 integrin activation may play a role in the pathogenesis of proteinuria in the setting of glomerular injury. Further work by Wei et al. showed that activation of β3 integrin by soluble urokinase receptor (suPAR) may occur in primary focal segmental glomerular sclerosis; the authors suggest that suPAR may be the circulating factor responsible for recurrence of FSGS after transplant. While these data suggest that αvβ3 integrin activation may injure the podocyte, other work shows that αv integrins act as receptors for osteopontin, which is a protective factor in stretch-induced podocyte injury.

Dystroglycan

Integrins are not the only adhesion proteins to be expressed in podocyte foot processes. The dystrophin–glycoprotein complex (DGC) is a group of proteins that includes α- and β-dystroglycan, utrophin, and dystrophin, and plays a central role in stabilizing skeletal muscle cell membranes by tethering the cytoskeleton to the basement membrane components laminin, perlecan, and agrin. α- and β-dystroglycan have also been localized to podocyte foot processes, and their expression is diminished in several mouse models of glomerular disease and human minimal change disease. However, recent studies have shown that genetic deletion of dystroglycan from podocytes in mice does not result in a glomerular phenotype or increased susceptibility to injury, suggesting that integrins may be the primary functional extracellular matrix receptors in the podocyte.

Integrin-Linked Kinase

Given the central, non-redundant role of α3β1 integrin in the development and maintenance of the glomerular filtration barrier, it is of interest to identify its interacting proteins in podocytes. Integrin-linked kinase (ILK) is a serine/threonine kinase with kinase-dependent and -independent functions that interact with the cytoplasmic domains of β1 and β3 integrins. Detailed molecular studies have elucidated a role for ILK in podocyte morphology and health. ILK forms a complex with Pinch1 and α-Parvin to regulate matrix adhesion, foot process formation, and inhibit podocyte apoptosis. ILK also forms a ternary complex with α-actinin-4 and α3β1 integrin, providing a link between the GBM, the actin cytoskeleton, and nephrin in the SD. ILK was first identified as a potential mediator of glomerular disease by two groups showing that it is upregulated in glomeruli of patients with diabetic nephropathy, congenital nephrotic syndrome of the Finnish type, and two proteinuric mouse models. Indeed, activation of ILK in vivo using a rodent model of puromycin-associated nephropathy, and in vitro by overexpression of a kinase-active ILK transgene, caused activation of β-catenin, podocyte detachment and apoptosis, and transcriptional repression of the SD components P-cadherin and Cd2ap. However, while ILK activation may contribute to podocyte injury in disease, it also plays a fundamentally important role in normal podocyte physiology. Mice with a podocyte-specific deletion of the ILK gene appear normal at birth, but develop focal segmental glomerular sclerosis with GBM thickening, and podocyte foot process effacement characterized by an aberrant distribution of α-actinin-4 and nephrin. The deletion of ILK causes an upregulation of focal adhesion kinase (FAK), a non-receptor tyrosine kinase which is involved in focal adhesion turnover, cell spreading, and motility. This result is supported by recent work showing that FAK is activated in LPS and anti-GBM models of podocyte injury, and genetic deletion of FAK protects mice from proteinuria.

Tetraspanin CD151

CD151 is a tetraspanin family protein with affinity for α3β1 integrin that is expressed abundantly in the glomerulus. While CD151 is not absolutely required for α3β1 integrin binding to the extracellular matrix, it stabilizes the active conformation of α3β1 integrin and strengthens this interaction. A nonsense mutation in CD151 causes hereditary nephropathy in patients characterized by a splitting and thickening of the GBM, along with pretibial epidermis bullosa, sensorineural deafness, and thalassemia. A podocyte specific knockout mouse model recapitulates this renal phenotype. Mechanistically, CD151 causes a redistribution of α3β1 integrin at the interface between the podocyte and GBM, increasing its binding affinity for laminin-511/521. These findings suggest that CD151 may strengthen the adhesion of podocytes to the GBM, protecting them from higher glomerular pressures.

Podocalyxin

Podocalyxin is a CD34-related sialomucin protein that is highly-expressed by podocytes and also by mesothelia, vascular endothelial cells, hematopoietic stem cells, and platelets. Mice with a conventional knockout of the podocalyxin gene die within the first 24 hours of life from renal failure. Importantly, podocyte foot processes do not form, and intercellular junctions between adjacent foot processes are abnormal and appear immature. These data provide functional evidence that podocalyxin is a key molecule in podocyte development. Cell biologic experiments have shown that podocalyxin interacts with the Na ⁺ /H ⁺ exchanger regulatory factor 2 (NHERF2) and phosphorylated ezrin in a complex, connecting it to the actin cytoskeleton of the podocyte foot process. Disruption of this interaction results in nephrotic syndrome. Podocalyxin appears to regulate foot process architecture through activation of the small Rho-GTPase, RhoA, mediated by its interaction with the NHERF/Ezrin complex.

mTOR

The mechanistic target of rapamycin (mTOR) is an evolutionarily-conserved serine-threonine kinase that interacts with regulatory associated protein of mTOR (Rptor) or Rptor-independent companion of MTOR (Rictor) to form mTORC1 and mTORC2 complexes, respectively. mTORC1 is a key regulator of cellular metabolism, including protein translation, ribosomal biogenesis, cell growth and proliferation, and suppression of autophagy in response to amino acids, growth factors, and elevated cellular ATP levels. mTORC2 is regulated primarily by growth factors to promote actin cytoskeletal rearrangement, cell survival, and cell cycle progression. Rapamycin is an mTOR inhibitor that is used clinically and is thought to specifically inhibit mTORC1 function. In certain cell types including the podocyte, however, chronic inhibition of mTORC1 by rapamycin also results in downregulation of mTORC2 functions. The importance of mTOR in podocyte biology was first suggested by the clinical observation that rapamycin causes proteinuria.

Deletion of mTOR itself or R ptor in podocytes of mice results in proteinuria. Conversely, ectopic mTORC1 activation in mouse podocytes, accomplished by deletion of its suppressor Tsc1 , results in kidney disease with many of the features of diabetic nephropathy (DN), including podocyte hypertrophy and loss and proteinuria, and is attributed to endoplasmic reticulum stress. Although mTORC1 appears to be crucial for podocyte function, loss of Rictor from podocytes of mice does not result in a phentoype.

Given its central role in cellular metabolism, mTOR is likely to play multiple roles in podocyte biology. Regulation of autophagy is one pathway regulated by mTOR that appears to be crucial for podocyte function. Autophagy is a lysosomal-dependent cellular survival response to starvation or lack of growth factors in which cells degrade cellular constituents from proteins to entire organelles, such as mitochondria, in order to provide a supply of nutrients under conditions of stress. A basal level of autophagy is, however, necessary to remove damaged organelles, excessive lipids, and long-lived or misfolded proteins. The basal level of autophagy appears to be increased in podocytes, and it has been suggested that autophagy may be required to protect this long-living cell from injury. In keeping with this model, deletion of Atg5, a key component of the autophagic pathway, results in late onset of glomerular disease in mice at 20 to 24 months of age due to the accumulation of damaged organelles and ubiquitinated protein complexes. In contrast, deletion of mTOR from podocytes results in disruption of autophagic flux, with subsequent accumulation of autophagolysomes in the podocyte ( Figure 22.20 ). Clinically, dysregulation of autophagy is observed in patients with lysosomal storage diseases such as Fabry’s disease, Aspartilglucoseaminuria or Scheie’s disease, where the inability to acidify lysosomes causes a failure of lysosomal reformation ; these patients are prone to developing proteinuria, providing additional support that the autophagic pathway is clinically relevant.

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