How Do Mesangial and Endothelial Cells Form the Glomerular Tuft?

BRIEF REVIEW www.jasn.org

Michael R. Vaughan*† and Susan E. Quaggin*‡§

*Samuel Lunenfeld Research Institute, Mount Sinai Hospital, ‡Institute of Medical Science, and §Department of Medicine and Division of Nephrology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada; and †Division of Nephrology, University of Washington, Seattle, Washington

ABSTRACT The glomerular capillary tuft is a highly intricate and specialized microvascular bed itor cells reside together in a region that filters plasma water and solute to form urine. The mature glomerulus contains known as the aortogonadal mesone- four cell types: Parietal epithelial cells that form Bowman’s capsule, podocytes that phros, where early hematopoiesis takes cover the outermost layer of the glomerular filtration barrier, glycocalyx-coated place. In lower organisms, hematopoi- fenestrated endothelial cells that are in direct contact with blood, and mesangial esis continues to occur in the adult kid- cells that sit between the capillary loops. Filtration begins only after the influx and ney, as in the pronephros of zebrafish. organization of endothelial and mesangial cells in the developing glomerulus. The mammalian kidney develops in Tightly coordinated movement and cross-talk between these cell types is required three successive stages known as the for the formation of a functional glomerular filtration barrier, and disruption of pronephros, the mesonephros, and the these processes has devastating consequences for early life. Current concepts of metanephros; only the metanephros the role of mesangial and endothelial cells in formation of the capillary tuft are gives rise to the definitive adult kidney, reviewed here. and it no longer functions as a site of hematopoiesis. It is still debated J Am Soc Nephrol 19: 24–33, 2008. doi: 10.1681/ASN.2007040471 whether angioblasts migrate into the developing metanephros or arise in situ from a common progenitor. This is an OVERVIEW OF TUFT EMBRYOLOGY ops from the most proximal end of the interesting question given the close Glomerular morphogenesis proceeds renal vesicle that is farthest from the bud geographic and functional relationship through several well-defined stages in tip. Distinct cell types in the glomerulus that these progenitors exhibit during embryonic development, beginning first are first identified in the S-shaped stage, evolution. Experimental data regard- as a renal vesicle, followed by the com- where presumptive podocytes appear as ing the origin of these angioblasts ma-shaped body, S-shaped body, a cap- a layer of columnar-shaped epithelial within the metanephros is discussed in illary loop stage, and then the mature cells. A vascular cleft develops and sepa- a later section. glomerulus (Figure 1). The epithelial rates the presumptive podocyte layer The movement of endothelial pro- components of the glomerulus—the pa- from more distal cells that will form the genitors into the vascular cleft depends rietal epithelial cells and podocytes—de- proximal tubule (Figure 1). It is into this on the expression of angiogenic factors rive from the metanephric mesenchyme. cleft that vascular endothelial cells mi- such as VEGF-A by presumptive podo- 7 8,9 Once inside the developing glo- These mesenchymal cells adjacent and grate followed by mesangial cells. cytes. merulus, endothelial cells proliferate in inferior to the tips of the branching ure- Individual endothelial progenitors, situ and aggregate to form the first capil- teric bud begin to condense at 11.5 d post or “angioblasts,” are easily identified in coitum in the mouse or after 5 wk of ges- the developing kidney because they ex- tation in humans.1,2 This collection of press typical vascular markers such as Published online ahead of print. Publication date cells is known as the pretubular aggregate vascular endothelial growth factor re- available at www.jasn.org. (Figure 1). In response to inductive cues ceptor 2/Flk1 (VEGFR2). At the com- Correspondence: Dr. Susan E. Quaggin, Samuel from the ureteric bud and surrounding ma-shaped stage, these angioblasts hug Lunenfeld Research Institute, Mt. Sinai Hospital, Uni- the outside of the developing nephron versity of Toronto, 600 University Avenue, Toronto, stroma, the aggregates undergo a mesen- Ontario M5G 1X5, Canada. Phone: 416-586-4800; chymal-to-epithelial transition forming and then “stream” into the vascular Fax: 416-586-8588; E-mail: [email protected] cleft (Figure 1). Much earlier in devel- the renal vesicle and then the comma- Copyright © 2008 by the American Society of shaped body.3–6 The glomerulus development, angioblasts and renal progen- Nephrology

24 ISSN : 1046-6673/1901-24 J Am Soc Nephrol 19: 24–33, 2008 www.jasn.org BRIEF REVIEW lary loops. Rather than using a process men that develops only later through se- lial cells differentiate, becoming flat- called “sprouting angiogenesis,” lective apoptosis of endothelial cell sub- tened, and acquire a fenestrated mor- whereby a vessel with a lumen invades a sets.10 This process of lumenation is phology. The fenestrae are pores lined by tissue, glomerular precapillary cords dependent on TGF-␤ signaling.11 As glo- plasma membrane that pass through the form as a result of homotypic interac- meruli mature, the cords become lume- endothelial cell. Unlike other fenestrated tions between adjacent endothelial cells. nal and the initial capillary loop divides endothelial cells found in pituitary and Initially, these capillary cords lack a lu- into six to eight loops. Residual endothe- endocrine organs, the majority of glo-

Figure 1. Migration of endothelial cells into the developing glomerular tuft. (Top) An embryonic day 12.5 mouse metanephros is outlined in black. Endothelial cells express a VEGFR2-GFP transgene and stain brown. The glomerulus develops from a pretubular aggregate (agg) that forms immediately adjacent and below the tips of the ureteric buds (ub). These aggregates derive from metanephric mesenchymal cells that have been induced to condense and epithelialize by signals produced in the ureteric buds. (Middle and Bottom) VEGFR2-positive cells are seen to “hug” the developing comma-shaped–stage nephron. The s-shaped stage is defined by the presence of a layer of podocyte precursors (presumptive podocytes) (po) and a vascular cleft. Endothelial cells seem to be streaming into this cleft from the Metanephric mesenchyme (MM). At the capillary loop stage, pockets of endothelial cells sit right next to the podocytes, and mesangial cells are soon found inside a single capillary loop. By the maturing stage, capillary lumens are beginning to form and a large population of mesangial cells is present. Schematic diagrams of each developmental stage are shown above the photomicrographs. Podocytes have been digitally colorized for identification (pink). Adapted from Saxen.1 pa, parietal epithelial cell; po, podocyte; me, mesangial cell; cap, capillary loop.

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of mesangial cells (known as mesangiolysis) results in dilation of the glomerular capillary loops. Although it is clear that mesangial cells are required for this looping, the specific mesangial factors guid- ing such events have not been identified.

ORIGIN OF ENDOTHELIAL AND MESANGIAL CELLS

Conflicting views exist regarding the origin of glomerular endothelial and mesangial cells. Although the metanephric mesenchyme contains VEGFR-2–positive angioblasts from early stages of development, cross-species transplanta- Figure 2. Mesangial cells in the early glomerular tuft. Mesangial cells (green) express tion studies suggest that extrarenal cells desmin, whereas podocytes (red) express WT1. Vascular ␣-SMA, another marker of also contribute to glomerular vascula- mesangial cells, is expressed after desmin. Figure courtesy of J. Miner, PhD, Washington ture. This is perhaps not surprising, University, St. Louis. given the ability of donor endothelial cells to “hook up” with host vasculature merular fenestrae in adult kidneys lack cells are identified by their expression of under very artificial circumstances (Hel- typical diaphragms associated with the various markers, including Thy1.1 (in mutt Augustin, DVM, PhD, Joint Re- PV1 protein.12 However, they are rats), desmin, ␣-smooth muscle actin search Division Vascular Biology of the bridged by an electron-dense complex (␣-SMA), and the PDGF beta receptor Medical Faculty Mannheim, University that is referred to by some but not all (PDGFR␤) (Figure 2).16,17 The subse- of Heidelberg and the German Cancer groups as a “diaphragm.”13,14 Exactly quent looping of glomerular capillaries Research Center, Germany, personal how this diaphragm forms during devel- will not proceed in the absence of mesan- communication, March 2007); for ex- opment is unclear, but it is thought to gial cells or in glomeruli with defects in ample, human endothelial cells trans- arise from proteoglycans and other pro- basement membrane that prevent adher- planted under the skin of the mouse can teins produced by differentiating glo- ence of mesangial cells.17,18 This demon- form functional vessels that connect to merular endothelial cells. Disruption of strates that mesangial cells play a key murine vessels. What is also intriguing this barrier through injection of enzymes morphogenetic role in forming the cap- about kidney transplantation experi- (hyaluronidase, heparinase, and chon- illary tuft. In the prevailing model, mes- ments is the variability in host versus do- droitinase) results in changes in glomer- angial cells split a single vessel loop that nor contribution, which can range from ular permeability.15 extends into Bowman’s space by forming only a few cells to the majority of After the initial influx of endothelial a capillary tuft with multiple, parallel cells.19–23 The factors that permit host cells, mesangial cells enter the glomeru- branches known as “intussusceptive” cells to contribute more efficiently to lus, forming a stalk, or “core,” of cells splitting of vessels (Figure 3). Mesangial glomerular vasculature than donor cells around the early, single capillary loop. cells also provide a mechanical function under certain circumstances are not During nephrogenesis, these mesangial for preservation of capillary loops, as loss known but may depend on the location of the transplant site, the fetal age of the transplanted kidney, or other interspe- cies differences. Local environment also seems more important for vasculogen- esis in the kidney than in some other organs. Although many parts of the embryonic kidney grow and develop in explant cultures (e.g., branching of the ureteric Figure 3. Intussusceptive splitting of vessels by mesangial cells. PDGF-B is produced by bud, early nephron formation), the renal 24 glomerular endothelial cells and promotes recruitment of mesangial cells that express the vasculature never develops ex vivo. This PDGFR␤. Mesangial cells split a single vessel loop that extends into Bowman’s space into is in striking contrast to the strong vascu- a capillary tuft with multiple, parallel branches. Figure courtesy of C. Betsholtz, PhD, lar development and migration seen in Karolinska Institute, Stockholm, Sweden. heterologous mesonephric-gonad ex-

26 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 24–33, 2008 www.jasn.org BRIEF REVIEW plants. The reasons for these differences um.29 Similar to native mesangial cells, angial cells at 14.5 d post coitum results are not understood.25 these reconstituted mesangial cells re- in arrested glomerular development with Similar to endothelial cells, the origin sponded to angiotensin II, suggesting a reduced number of capillary loops.34 of the mesangial cell precursors remains that they are functional and not simply Genetic deletions of other vascular growth a mystery, although cells from within the trapped bone marrow–derived cells. factor ligands are not yet known because metanephric mesenchyme always con- Male-to-male transplant studies revealed standard null mice for various vascular tribute to the mesangium in cross-spe- a single y-chromosome in transplanted growth factor receptors all produce lethal- cies transplant experiments.19,20 Mesan- mesangial cells, thereby demonstrating ity during embryogenesis that precludes gial molecular markers have also been that these cells were not merely the result analysis of kidney phenotypes. However, it observed within developing glomeru- of cell fusion. is predicted that these vascular growth fac- lar arterioles,17 confirmed by lineage tors also play important roles in glomeru- tagging experiments in transgenic mice lar endothelial cell proliferation and re- that express Cre-recombinase from the GENETIC BASIS OF ENDOTHELIAL modeling as in other vascular beds; for endogenous renin locus.26 Growth fac- AND MESANGIAL CELL example, standard null mice for VEGFR2/ tors such as PDGF-B that are produced RECRUITMENT Flk1 fail to make intact blood vessels as a by glomerular endothelial cells are ma- result of loss of endothelial cells, whereas jor signals for mesangial cell recruit- Gene targeting experiments in mice and null mice for VEGFR1/Flt1 (the decoy re- ment and is discussed next. Interest- genetic studies in humans demonstrate ceptor for VEGFR1) exhibit overgrowth of ingly, the fact that mesangial cells an essential role for molecular cross-talk endothelial cells that prevents proper mod- appear after the influx of endothelial between cells in the glomerular compart- eling of the vasculature and death of the cells suggests that the endothelial cells ments during the formation of the tuft fetus. Because both of these tyrosine kinase may control glomerular development (Table 1). The best understood examples receptors for VEGF-A are also expressed in through factors such as PDGF-B. of this cross-talk involve growth factor all endothelial cells of the kidney, it is likely Alternative sources for mesangial cells signaling pathways, although mutations that they are required for growth and re- have been proposed by work in models of in a number of genes expressed by podo- modeling of the renal vasculature. Tie1, glomerular disease. Mesangiolysis, the cytes (e.g., transcription factors) also another vascular tyrosine kinase receptor, most extreme form of mesangial dam- have profound effects on both glomeru- may play a unique role in developing glo- age, results in loss of mesangial cells, de- lar endothelial and mesangial cells. merular endothelia. Tie1 null mice die dur- struction of the mesangial cell matrix, ing embryonic life as a result of loss of in- and ballooning of the capillary loop. In Podocyte and Mesangial Cell– tegrity of the endothelium followed by the anti-Thy1.1 model of mesangiolysis, Derived Cues in Vascular Signaling hemorrhage. To overcome this early de- the mesangial compartment is regener- Pathways fect, chimeric mice were generated from ated by cells recruited from the juxtaglo- During glomerular development, podo- embryonic stem cells that lacked a func- merular apparatus, also referred to as the cytes produce a number of angiogenic tional vascular receptor tyrosine kinase extraglomerular mesangium.27 Other growth factors, including VEGF-A, Tie1. Tie1 null cells from this chimera con- animal studies demonstrate that bone VEGF-C, angiopoietin-1, and ephrinB2, tributed to the endothelium within various marrow–derived cells can replenish whereas adjacent endothelial cells ex- vascular beds but were unable to contrib- mesangial cells.28 For investigation of the press their cognate receptors.9,30–33 Dele- ute to the glomerular vasculature, suggest- contribution of bone marrow–derived tion or overexpression of VEGF-A selec- ing an important and possibly unique role cells with mesangiolysis, crude prepara- tively within podocytes, for example, for Tie 1 in the glomerulus.35 Interestingly, tions of donor bone marrow cells from results in dramatic defects in endothelial no ligand for Tie1 has yet been identified, rats that express enhanced green fluores- cell in-growth, proliferation, survival, so whether one is produced by glomerular cence protein in all tissues were trans- and differentiation in glomeruli.9 Capil- cells is unclear. planted into wild-type recipients. The lary ghosts are observed in glomeruli de- The Notch signaling pathway is an- wild-type rats with “fluorescence- ficient in VEGF-A (Figure 4), and pro- other key regulator of vascular develop- tagged” bone marrow were then given gressive decreases in the levels of ment. The vessels in developing mouse anti-Thy1 antibody to induce mesangi- VEGF-A lead to more profound defects retina respond to levels of VEGF-A olysis. After mesangial injury, tagged in adjacent endothelia, emphasizing the through specialized “tip” cells whose fate bone marrow cells contributed up to 7% importance of this paracrine signaling is determined by ␦-like 4-Notch1 signal- of the reconstituted population of mes- pathway. ing.36 Members of the Notch signaling angial cells. More recently, clones of pu- Although ephrinB2 is seen at the S- pathway are dynamically expressed dur- rified hematopoietic bone marrow stem shaped stage in developing podocytes, its ing glomerular development,37 and glo- cells (defined as linϪ/Sca1ϩ/c-kitϩ/ expression rapidly switches to the endo- meruli from mice that are homozygous CD34Ϫ) expanded ex vivo were found on thelial and mesangial compartments. for a hypomorphic Notch2 allele arrest at transplantation to populate the mesangi- Deletion of ephrinB2 from mouse mes- the capillary loop stage or form defective

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Table 1. Genes involved in endothelial and mesangial recruitment and formation of the capillary tuft Gene Disruption Glomerular Cell Expression Glomerular Tuft Phenotype PDGF-B17: Standard null and endothelial cell Endothelial cells Failure of mesangial cell in-growth; ballooned capillary selective deletion of PDGF-B PDGFR␤: Standard null allele Mesangial cells Similar to PDGF-B VEGF-A9,57 Podocytes podocyte-specific KO (100% loss) Failure of endothelial cells to migrate into cleft, decreased survival, no fenestrations podocyte-specific KO (Ϫ75% VEGF-A) Loss of endothelial cells within 1 to 3 d after maturation, followed by massive mesangiolysis podocyte-specific KO (Ϫ50% VEGF-A) Swollen endothelial cells; endotheliosis followed by loss of endothelial cells EphrinB234: Pericyte/mesangial-specific null Podocyte precursors in S-shaped body; Fewer capillary loops allele changes to endothelial cell and mesangial cells at capillary loop GPR442 Vasculature, glomerular cell type not No vascular ␣-SMA–positive mesangial cells in the reported glomeruli Tie158: Chimeric analysis Endothelial cells Tie null cells cannot contribute to the glomerular microvasculature Pod1/tcf2145,51: Standard null allele Podocytes Poorly developed capillary loop and reduced in- growth of mesangial cells; no or fewer fenestrations, podocyte defects Lmx1b46–48: Standard null allele Nail-patella syndrome in patients; no fenestrations in glomerular endothelial cells Foxc239: Standard null allele Similar to Pod1/tcf21 ␣3 integrin44,59: Standard null allele, podocyte- Podocytes Fewer capillary loops, poorly developed mesangium, specific null allele podocyte defects Laminin ␣5 standard null allele18,52 Podocytes? Arrested glomerular development; avascular; endothelial and mesangial cells are extruded from the glomerulus chimeric laminin pseudo Љknock-insЉ18 Mesangial cells cannot adhere to the GBM; ballooning of capillaries WT160,61: Human mutations Podocytes Denys-Drash syndrome; gonadal and renal dysgenesis, mesangial sclerosis LAMB254,62: Human mutations GBM produced by podocytes? Pierson syndrome; diffuse mesangial sclerosis PLCe155 Podocytes truncating mutations in patients Diffuse mesangial sclerosis missense mutations in patients FSGS glomeruli with aneurysmal dilation of this property. Rather, it is the capacity of clearly insufficient. Several groups are the capillaries. These glomeruli consist of the glomerular endothelia to withstand actively looking for glomerular genes a disorganized clump of cells that express or provide high permeability to water and/or profiles of glomerular genes that some podocyte markers but lack mesan- and small solutes that is so unusual. This dictate the unique properties of these en- gial and endothelial cell markers. Levels is accomplished in part by the presence dothelial cells. To date, a number of in- of VEGF-A are reduced in the glomeruli of fenestrations. Glomerular fenestra- teresting candidate genes (e.g., chloride of these mice, which may explain the de- tions have many similarities to fenestra- CN5, EHD3) that are expressed selec- fect in endothelial and mesangial cell re- tions found in endothelial cells of other tively in glomerular endothelial cells cruitment. organs, except for the absence of dia- have been identified by a serial analysis of Glomerular endothelial cells exhibit phragms. Perhaps the most similar endo- gene expression (SAGE) or array analy- shared but also unique properties with thelial cells are found in the liver sinu- sis; however, their roles in glomerular other vascular endothelia. It is perhaps soids, which also lack diaphragms. development in vivo are not yet not surprising that macromolecules such What regulates formation of the fen- known.38,39 Identification of these as albumin are primarily prevented from estrations? Although VEGF production unique transcripts should provide im- crossing the endothelium (although this by podocytes is necessary for mainte- portant clues to specialized features of is an area of controversy), because endo- nance of fenestrations as shown in the the endothelium and ultimately will al- thelium throughout the body possesses podocyte-selective null models, it is low us to develop useful genetic tools to

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manipulate gene expression selectively within the glomerular endothelium. This is relevant because many glomerular diseases are thought to result from damage to the glomerular endothelium; this is not contested in diseases such as throm- botic microangiopathy but is debated in diseases such as diabetic nephropathy. The generation of glomerular endothelial-specific Cre mouse lines will permit researchers to address these questions genetically. These mouse tools will also permit researchers to dissect the importance of the glomerular endothelium in generating and maintaining the perms- elective properties of the glomerular filtration barrier, which is still hotly debated.

Growth Factors and Endothelial- Derived Cues As outlined, podocytes and mesangial Figure 4. Transmission electron micrographs of the glomerular filtration barrier. Wild- cells provide molecular signals to the de- type mouse (left) and transgenic mouse (right) with the latter showing selective deletion veloping glomerular vasculature. In a re- of VEGF-A from the podocytes (po). Podocytes are seen in both, but the endothelium (en) ciprocal manner, glomerular capillaries is entirely missing from the null mouse, leaving a “capillary ghost.” Immunostaining for deliver blood, nutrients, and mechanical WT1 (podocytes [green]) and platelet-endothelial cell adhesion molecule (endothelial and molecular signals to adjacent part- cells [red]) confirms the absence of capillary wall or endothelium in VEGF-A null mouse. ners in the tuft. The first example of this (Reproduced with permission from JCI.) cross-talk during glomerular development was provided by the dramatic phenotype in PDGF-B and PDGFR␤ null mice.17,40 In the glomerulus, endothelial cells express PDGF-B, whereas mesangial cells express its receptor. In PDGFR␤ null mice, capillary tufts fail to develop and completely lack mesangial cells. Ul- trastructurally, the glomeruli in these null mice contain only a few distended or ballooned capillary loops in which the basement membrane and overlying podocytes are juxtaposed against Bow- man’s capsule, with a loss of Bowman’s space (Figure 5). Endothelial cells de- leted conditionally of PDGF-B were shown to have a similar ballooned capillary phenotype, confirming the presence of a paracrine signaling loop from the glomerular endothelia to mesangium.41 More recently, deletion of the G protein–coupled receptor, GPR4, from mice resulted in glomeruli with fewer mesan- Figure 5. Glomerular phenotype in PDGFR-␤ receptor null mice. Mesangial cells do not gial cells. Although tortuous and dilated migrate into the capillary tuft, resulting in a single balloon-like capillary loop. (Figure 5 blood vessels with hemorrhaging are courtesy of C. Betsholtz.) found throughout the GPR4 null neo-

J Am Soc Nephrol 19: 24–33, 2008 Formation of Glomerular Tuft 29 BRIEF REVIEW www.jasn.org nates, the glomerular capillary tuft ap- However, differentiation of specialized in mesangial cell recruitment, and pears well formed. Interestingly, vascular podocyte features, such as slit dia- podocyte abnormalities (Figure 6). Mi- ␣-SMA–positive cells, a late marker of phragms, was not described in these croarray and candidate gene analysis mesangial differentiation, were reduced.42 mutants. It seems likely that endothe- shows that levels of type IV collagen (a Given the fairly normal-appearing glomer- lial cells produce signals that are re- component of the glomerular base- ular architecture, it will be important to quired for maintenance, survival, and ment membrane [GBM]) and podocin know whether earlier markers of mesangial differentiation of podocytes. In podo- are reduced in glomeruli from mice cell differentiation are expressed (e.g., cyte-selective VEGF-A null mice, al- lacking any of the transcription factors PDGFR␤, desmin) and whether the mes- though podocytes are present, ultra- listed.39,48–51 Given the similar renal angial cell lineage is present but remains structural abnormalities such as phenotypes and expression profiles, undifferentiated. GPR4 is expressed in the abnormal slit diaphragms are present these genes may regulate the same tran- vasculature and kidney and functions as an as well (Figure 4). scriptional pathways. extracellular pH sensor, suggesting that Null mice have also demonstrated physiologic changes within the vascular Transcription Factors, Integrins, that matrix proteins in the GBM, such microenvironment are determinants of and Matrix Proteins as laminins, are critical for glomerular maturation. Several other genes expressed by podo- maturation. Laminin ␣5 null embryos Is differentiation of a mature glo- cytes are required for proper formation show extrusion of endothelial and mes- merular endothelium also required for of glomerular capillary loops and mesangial cells from glomeruli that appear terminal differentiation of the podo- angium. These genes encode ␣3 inte- constricted with a complete absence of cyte lineage? The data are conflicting. grin and four transcription factors: vasculature.52 The developmental Studies from zebrafish show that endo- Pod1 (Tcf21/capsulin/epicardin), switch between laminin chains in the thelial cells are not required for deter- Lmx1b, Foxc2, and Kreisler.39,44–47 GBM, from ␣1to␣5, is required for mination of the podocyte cell lineage Glomeruli from null mice for each of adhesion of mesangial cells to the base- because podocytes develop in “cloche” these genes have reduced numbers and ment membrane and subsequent loop- mutants that have no endothelia.43 complexity of capillary loops, defects ing of glomerular capillaries.18

Figure 6. Glomeruli from Pod1 and Foxc2 null mice. Glomeruli from wild-type (left) or null mice (right). Note the dilated capillary loop(s) and abnormal clump of mesangial cells (me) in the glomeruli from null mice. FoxC2 photomicrographs courtesy of Minoru Takemoto, MD, PhD, Department of Clinical Cell Biology and Medicine, Chiba University Graduate School of Medicine, Chiba, Japan.

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DEVELOPMENTAL INFLUENCE CONCLUSIONS Podocyte differentiation, mesangial Pod1/lmx1b/Foxc2 cell recruitment and capillary looping Podocyte Glomerulogenesis provides important clues into glomerular function in health and disease. At the core of the glomerulus is a complex structure known as the capillary tuft that consists of invaginations in the basement Alpha3 integrin Same as Pod1/lmx1b/Foxc2 membrane surrounded by podocytes and VEGF-A Endothelial migration, survival and differentiation endothelial and mesangial cells. Gene-targeting studies in mice and human genetic stud- Flk-1 (VEGFR-2) ies provide molecular footholds into some of thekeypathwaysinvolvedincapillarytuftde- Endothelial cell velopment; these include VEGF signaling,

Mesangial cell ingrowth and PDGF-B signaling, and enzymes such as PDGF-B capillary looping PLCe1. Importantly, these studies provide a new understanding about cross-talk among PDGFR-β theresidentcellsoftheglomerulus(Figure7). However, it is clear that many genes and pathways that will help to define the unique properties of the glomerular microvascula- Mesangial cell ture, which in turn is required for formation of the urinary filtrate, remain to be uncov- ered. As additional pathways are described, they will provide potential avenues for thera- peutic intervention. Figure 7. Signaling pathways of capillary tuft development. The resident cells of the capillary tuft (podocyte, endothelial, and mesangial cells) each play critical roles in capillary tuft development. The development of the tuft is influenced by cellular cross-talk ACKNOWLEDGMENTS (solid arrows) and through various genes, transcription factors, and growth factors. These factors affect different aspects of capillary tuft development as noted by the dashed This work was funded by a Kidney Foundation arrows. of Canada grant and Canadian Institutes of Health Research (CIHR) grants MOP-62931 Human Genetic Mutations that (WT1), another transcription factor and MOP-77756 to S.E.Q. and National Insti- Disrupt the Capillary Tuft expressed by podocytes; the second is tutes of Health grants F32 DK070434 and Diffuse mesangial sclerosis is a serious found in Pierson syndrome, which is K08DK076970 to M.V. glomerular lesion usually resulting in caused by mutations in the GBM pro- We thank Dragana Vukasovic for expert ESRD by age 5 in humans. Pathologic tein LAMB254; and, more recently, the secretarial assistance. S.E.Q. is the recipient of analysis of glomeruli from patients third is found in truncating mutations a Canada Research Chair, Tier II, and a Pre- with this form of sclerosis suggests a in the phospholipase epsilon C1 en- mier of Ontario Research Excellence Award. major defect in glomerular develop- zyme, which result in a heritable auto- ment. Capillary loops are reduced in somal recessive form of diffuse mesan- 55 number, and persistence or re-expres- gial sclerosis. Mice that overexpress DISCLOSURES ␣ sion of -SMA, a marker of early mes- the Denys-Drash mutant WT1 allele None. angial cell differentiation, is seen. Fail- exhibit defects in glomerular develop- ure of isoform switching of the mental with fewer capillary loops, and VEGF-A gene—from the 165 to the a knockdown of Plce1 in zebrafish re- REFERENCES 165␤ isoform—has also been noted in sults in developmental arrest of the sin- glomeruli from these patients.53 Three gle midline glomerulus, fusion of the 1. Saxen L: Organogenesis of the Kidney, 55,56 genetic mutations have been identified foot processes, and edema. How Cambridge, Cambridge University Press, in sporadic or inherited forms of this mesangial sclerosis results from these 1987 disease. The first is found in patients genetic defects is not entirely clear, but 2. Abrahamson DR: Glomerulogenesis in the with Denys-Drash syndrome, a disor- given that within the glomerulus, both developing kidney. Semin Nephrol 11: 375– der characterized by gonadal and renal of these genes are most highly ex- 389, 1991 3. Hatini V, Huh SO, Herzlinger D, Soares VC, dysgenesis that results from a mutation pressed by podocytes, it is likely that Lai E: Essential role of stromal mesenchyme in the Wilms’ tumor suppressor gene altered cross-talk plays a role. in kidney morphogenesis revealed by tar-

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