Microscopic Imaging Nephron Physiol 2006;103:p75–p81 DOI: 10.1159/000090623
Published online: March 10, 2006
Imaging Glomeruli in Renal Biopsy Specimens Carrie L. Phillips a, b Vincent H. Gattone, II c Stephen M. Bonsib b a
Department of Medicine, Division of Nephrology and Indiana Center for Biological Microscopy, and Departments of b Pathology and c Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Ind., USA
Key Words Glomeruli Two-photon fluorescence microscopy Three dimensions Imaging
Abstract Glomerular capillary loops are complex vascular filters composed of interdigitating podocytes and fenestrated endothelial cells with an intervening proteoglycan-rich extracellular matrix. This arrangement is crucial to maintaining the filtration barrier but renders the glomerulus difficult to analyze by conventional two-dimensional histochemical techniques. When pathologic lesions distort glomerular architecture, its complex morphology is even more challenging to interpret. Fortunately, recent advances in microscopes and computer software now enable glomerular enthusiasts to dissect this complex structure with finer detail. In this review we explore the application of new methodologies such as two-photon microscopy that optimize three-dimensional, multicolor imaging and single-cell segmentation of glomerular components. Copyright © 2006 S. Karger AG, Basel
Introduction
The unique and complex structure of the nephron and its associated glomerular filtration were initially demonstrated by Bowman in 1842. A century ago, Huber [1905]
© 2006 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail
[email protected] www.karger.com
Accessible online at: www.karger.com/nep
was able to elucidate glomerular morphology by serial reconstruction of the intricate glomerular architecture and its complex development from a hollow vesicle to structurally and functionally segmented mature nephrons. Through the efforts of these pioneers and other morphologists, we recognize that glomerular tufts are threedimensional (3-D) vascular filters composed of a complex network of anastomosing capillary loops. The success of each sieve is dependent upon an intricate scaffolding of extracellular matrix (ECM) that supports specialized cells. Podocytes, or visceral epithelial cells, extend foot processes over the urinary aspect of glomerular basement membranes (GBM) while fenestrated endothelial cells carpet the vascular surface [Pavenstadt et al., 2003]. Mesangial cells and their supporting matrices serve to anchor the capillary tufts while providing contractile properties that regulate capillary blood flow and secretory properties that mediate inflammation and immunity [Stockand and Sansom, 1998]. The architectural complexity of glomeruli is difficult to appreciate by traditional two-dimensional (2-D) histologic techniques and can be daunting if pathologic lesions are present. Here we review 2-D and 3-D imaging techniques that facilitate exploration of glomeruli using microscopes that appeal to both the basic science researcher as well as hospital-based renal pathologists. Emphasis will be placed on two-photon fluorescence microscopes, relatively new instruments that permit deep optical imaging of thick biological samples labeled with fluorescently tagged markers (fig. 1, 2).
Carrie L. Phillips Indiana University School of Medicine, Department of Medicine Division of Nephrology and Indiana Center for Biological Microscopy 950 West Walnut Street, Room R2-202, Indianapolis, IN 46202 (USA) Tel. +1 317 274 1266, Fax +1 317 274 8575, E-Mail
[email protected]
1
2
p76
Nephron Physiol 2006;103:p75–p81
Phillips /Gattone /Bonsib
Brightfield Microscopy
Brightfield microscopy of glass-mounted tissue sections has been the mainstay of morphologists for centuries [Bellis, 2005]. Preparation of tissue sections from formalin-fixed, paraffin-embedded specimens is relatively inexpensive, the glass slides can be stored for decades at room temperature and hundreds of histological and immunohistochemical stains can be routinely applied with consistent results. Unfortunately brightfield microscopy, a type of light microscopy, offers less contrast and resolution in comparison to confocal or two-photon fluorescence microscopy. For brightfield microscopy, contrast is provided by staining dehydrated 2- to 5-m-thick tissue sections with dyes (e.g., hematoxylin and eosin to distinguish nuclei from cell cytoplasm) or chromogens (e.g., diaminobenzidine that provides the brown stain for immunoperoxidase reactions). The observer sees contrast when visible light, usually originating from a halogen bulb, passes through the stained tissue and on through a series of magnifying lenses that focus a 2-D image on the retina. For renal pathologists, brightfield micros-
Fig. 1. The glomerulus and its cellular components are virtually dissected in 3-D. A and E are SEM photomicrographs of immersion-fixed glomeruli and B–D, F–L are volumes from perfusionfixed rat kidneys collected by two-photon microscopy. A Acellular capillary loops from a human kidney biopsy specimen after extraction of cells reveals the smooth outer surface of normal GBM. B Two-photon microscopy permits exploration of the interior of a glomerulus, as shown in a single 0.4-m optical slice through GBM labeled with Lens culinaris-rhodamine that was one in a series of focal planes used to build the 3-D volume shown in C (110.5 m (x-axis) ! 110.5 m (y-axis) ! 38.4 m (z-axis)). D Luminal space (red) within these anastomosing capillary loops was virtually selected or ‘segmented’ with computer software that permits free rotation and quantification, unlike the static loops imaged by SEM in A. E SEM provides remarkable surface resolution of podocytes including the tight arrangement of their foot processes. When these epithelial cells are labeled in situ with anti-vimentin antibody and imaged by two-photon microscopy, their cell bodies and major processes can be explored in 3-D (F) because their foot processes which lack vimentin are no longer visualized. Podocytes were segmented with computer software (G) for individualized study (H). Glomerular components can be further dissected with anti-WT for podocyte nuclei (green, I), anti-podocalyxin or GLEPP1 for podocyte foot processes (green in J and K, respectively) or anti-factor VIII for endothelial cells (green, L). GBM is labeled with L. culinaris-rhodamine (tan, I–K) and podocytes are labeled with anti-vimentin (tan, L). B, C, F and I–L were rendered with Voxx software and D, G and H were segmented with Amira software.
Microscopy of Glomeruli
copy with histological stains allows relatively rapid identification of tissue perturbations due to cell proliferation or matrix alterations resulting from immune complex deposition (fig. 2A). However, when analyzing typical immunoperoxidase reactions, discrimination is often limited to determining whether a cell shows ‘positive’ or ‘negative’ antibody labeling without much intracellular detail. Another disadvantage of brightfield microscopy is that 3-D data cannot be easily obtained from 2-D images. Generating 3-D data from glass-mounted tissues requires laborious examination of serial sections, assembled by an artist’s rendering or sequential scanning and digital alignment with computer software. Justification for 3-D analysis of glomerular pathology in human disease has been provided by the brightfield microscopy studies of Fogo et al. [1995], who elegantly demonstrated by serial histologic sectioning a twofold increase in the detection of segmental glomerular scars, as compared to single 2-D sections, in renal biopsies of children with nephrotic syndrome and focal segmental glomerulosclerosis. In a subsequent study by Fuiano et al. [1996], serial
Fig. 2. Pathologic diagnosis of human biopsy specimens. A–C are taken from patients with membranous glomerulopathy and D–F are
from a single patient with lupus nephritis showing both proliferative and membranous lesions. A Jones’ silver stain outlines in black the GBM alterations of membranous glomerulopathy, i.e., vacuoles (arrow) and spikes resulting from unseen deposits of IgG and complement C3. B 3-D craters along the urinary aspect of the GBM are revealed by SEM after podocytes and a subepithelial blanket of immune deposits are chemically removed. C SEM of lupus membranous glomerulopathy following selective removal of podocytes reveals actual subepithelial immune deposits on the outer aspect of the GBM. D When visualized with 2-D widefield epifluorescence, the subepithelial granular immune deposits of membranous glomerulopathy can be seen along peripheral capillary loops (arrowhead) but are nearly impossible to resolve from subendothelial and mesangial deposits in this biopsy from a patient with lupus nephritis. E The corresponding frozen tissue remaining after cryosectioning, archived at –80 ° C since 1998, was thawed and labeled with anti-IgG (green) and Lens culinaris lectin (GBM, brown). When imaged in situ with two-photon microscopy and rendered in 3-D with Voxx software, deposits of IgG can be seen blanketing the entire subepithelial surface of the GBM (arrowheads, comparable to image C) but also discerned are small patches of subendothelial immunoglobulin (straight arrows) that appear as green islands floating on a brown sea of matrix (capillary loop tunnels receding into the volume are indicated with curved arrows). These focal patches of subendothelial deposits can be easily missed in 70-nm ultrasections examined in 2-D with TEM (F, podo = podocyte, en = endothelial cell, subepithelial deposits indicated with arrowheads, mesangial deposits marked with an asterisk).
Nephron Physiol 2006;103:p75–p81
p77
morphometric analysis of renal biopsy specimens revealed that sclerotic lesions could be missed in single sections because segmental scars averaged less than 15% of the total glomerular volume. In another serial section study of focal segmental glomerulosclerosis, Bonsib [1999] demonstrated that tubular and glomerular injury develop in parallel, since atrophic tubules are derived from glomeruli only partially affected by sclerosis. These manual brightfield analyses of serial histologic sections require extensive time and effort. While scanning electron microscopy (SEM) offers a 3-D perspective, the volumes are opaque and the interior of the tissue cannot be easily explored. Therefore, intraglomerular pathology, like glomerulosclerotic lesions, would be missed.
When combined with cell digestion techniques [Bonsib, 1985], SEM offered the earliest 3-D images of granular immune complexes and dramatic GBM damage in renal biopsies of patients with membranous glomerulopathy (fig. 2B, C). While the subepithelial localization of these membranous deposits may be discerned at high resolution with TEM (fig. 2F), a necessary tool of renal pathologists, this 2-D technique fails to capture the full distribution and character of immunoglobulin and complement deposition. Comparable analysis could be accomplished more easily and quickly by optical sectioning with other types of light microscopy, i.e. confocal laser scanning or two-photon fluorescence microscopy, with the added advantage that critical proteins and other molecules can be identified with fluorescent markers.
Electron Microscopy Widefield Epifluorescence Microscopy
Ultrastructural examination of biological structures began in the 1930s when Max Knott and Ernst Ruska invented the electron microscope and Albert Claude focused this technology on mitochondria in the 1940s [Palade, 1971]. Compared to brightfield microscopes, electron microscopes offered greater resolving power to early biologists seeking a closer look inside cells. Instead of photons of light, the electron microscope utilizes the shorter wavelength of electrons and electromagnetic lenses to provide nanometer resolution to evaluate subcellular and molecular structure. For SEM, an electron beam stimulates the generation of secondary electrons that are ejected from the sample, collected by a detector and converted into a signal that conveys 3-D information to a 2-D viewing screen. Limited aspects of glomerular 3-D structure have been revealed by SEM of kidneys from animal models and human biopsy specimens. For example, SEM has provided insight into the complexity of anastomosing capillary loops (fig. 1A) and podocyte architecture (fig. 1E). Podocytes are remarkably intricate cells with elaborate branching processes that end as interdigitating foot processes or pedicles [Pavenstadt et al., 2003]. Transmission electron microscopy (TEM) and SEM have unraveled the sequential morphologic alterations that develop in proteinuric states where podocyte process simplification occurs with disappearance of their cell pedicles [Arakawa and Tokunaga, 1972; Farquhar et al., 1957] or complete podocyte loss resulting in adherence of naked GBM to Bowman’s capsule [Kriz and Lemley, 1999].
p78
Nephron Physiol 2006;103:p75–p81
Advances in fluorescence microscopy address many of the limitations of brightfield and electron microscopy. Like brightfield microscopy, fluorescence microscopy relies on photons of light to visualize cells or tissue samples. However, fluorescence microscopes do not require conventional histologic stains to impart contrast in samples but instead have the capacity to excite (excitation wavelength) and capture light emitting from (emission wavelength) fluorochromes that decorate cells and tissues [Herman et al., 2000–2005]. By the 1970s, widefield epifluorescence microscopes focused light from mercury bulbs onto immunolabeled renal biopsy specimens and were crucial in differentiating among various glomerulonephritides such as IgA nephropathy [Berger, 1969]. The instruments are relatively inexpensive in comparison to laser scanning confocal microscopes (at least a tenfold difference in price for basic systems) and can be readily outfitted with filters that discriminate among commonly employed fluorochromes (e.g., fluorescein or rhodamine) or equipped with cameras for the collection of video, digital or film-based 2-D images. The major disadvantage of widefield epifluorescence is an inability to exclude out-offocus light which requires the user to manually focus up and down through various focal planes (z-axis) to distinguish morphologic features such as granular deposits of IgG in a biopsy specimen from a patient with lupus nephritis (fig 2D). While 5-m-thick cryosections can be rapidly screened in this manner, the observer is compromised in his or her ability to finely discriminate labeled structures within individual focal planes and lacks the
Phillips /Gattone /Bonsib
capacity to assemble individual focal planes into discernible 3-D compositions. These limitations can be answered by optical sectioning a series of focal planes (Z-stack or Z-series) with laser scanning confocal or two-photon fluorescence microscopy.
Confocal and Two-Photon Fluorescence Microscopy
Table 1. Advantages and disadvantages of two-photon fluorescence
microscopy Resolution less than SEM and TEM but comparable to other forms of light microscopy Multiple antibodies can be employed (also available with WEM and CLSM, but challenging with BFM, TEM and SEM) Tissue can be optically sectioned along z-axis and resultant volumes can be explored in 3-D (available with CLSM but not with BFM, WEM, TEM and SEM) 3-D images superior to CLSM due to better depth of imaging and less photobleaching
The first commercial confocal laser scanning fluorescent microscopes were introduced in the 1980s following the essential contributions of scientists such as Ernst Abbe who laid down the principles of optical microscopy in the later part of the 19th century, his young contemporary Paul Nipkow who first converted 2-D optical images into electrical signals and Marvin Minsky who patented the first confocal microscope in 1957 [Inoue, 1995]. Depending on the required excitation wavelength, these instruments rely on a variety of lasers providing visible light (e.g., argon-ion, krypton-ion, mixture of krypton and argon, helium-neon and helium-cadmium) and/or ultraviolet light [Gratton and vandeVen, 1995]. Each system employs a confocal pinhole that rejects out-of-focus light thereby solving the major limitation encountered with widefield epifluorescence. These features, combined with high-quality objectives, motorized stages and computerized raster scanning of individual focal planes, provided an extremely powerful tool to cell biologists who could now collect thin optical slices of fluorescently labeled specimens. Around 15 years ago, another form of fluorescence microscopy that optically sections tissue was launched [Denk et al., 1990]. Because of its ability to image hundreds of micrometers into tissues, the two-photon fluorescence microscope is a morphologic imaging tool that can help us examine glomeruli in ways never before imaged (see table 1, Denk et al. [1995], and the review by K.W. Dunn, this issue). Using excitation wavelengths nearly twice as long as confocal systems (e.g., 800 nm), two-photon microscopes detect a variety of fluorescently labeled probes to delineate the distribution and special relationships of matrix, nuclear and cytoplasmic components within biological samples. With titaniumsapphire lasers, this technology affords deep optical imaging from thick tissues, such as 1-mm diameter needle cores from renal biopsies (fig. 2E) that have been labeled with fluorescent markers. Volume rendering software [Clendenon et al., 2002, and see review by J. Clendenon et al., this issue] enables two-photon microscopy to re-
veal in situ and 3-D relationships between and within cells and associated ECM plus they permit 3-D microscopy volumes to be rotated in near real-time around any axis and viewed from any direction. In figure 1B–L, we have applied this imaging technique to the investigation of rat glomeruli permitting selective exposure of podocyte nuclei, cell body and primary processes, or tertiary processes, and defining their 3-D relationship to capillary loop basement membranes or endothelial cells. For example, podocalyxin (fig. 1J), a sialomucin on the urinary surface of podocytes, links to the actin cytoskeleton via ezrin and maintains the intercellular spaces between the foot processes [Kerjaschki et al., 1984; Orlando et al., 2001; Sawada et al., 1986]. Wilms’ tumor suppressor gene 1 (fig. 1I) regulates expression of podocalyxin [Palmer et al., 2001], influences glomerular capillary loop diameter [Natoli et al., 2002] and has been linked to Deny-Drash syndrome [Pelletier et al., 1991] and formation of glomerular crescents [Guo et al., 2002]. Mice deficient in glomerular epithelial protein-1 (GLEPP1, fig. 1K), a receptor tyrosine phosphatase required for podocyte development [Wang et al., 2000], have altered distribution of the intermediate cytoskeletal protein vimentin and exhibit blunting and widening of podocyte foot processes [Wharram et al., 2000]. Many of these molecules are affected by, or may be responsible for glomerular dysfunction in proteinuric disorders [Barisoni and Kopp, 2002]. On the luminal side
Microscopy of Glomeruli
Nephron Physiol 2006;103:p75–p81
More expensive than BFM and WEM but comparable to CLSM, TEM and SEM systems BFM = Brightfield microscopy; CLSM = confocal laser scanning microscopy; SEM = scanning electron microscopy; TEM = transmission electron microscopy; WEM = widefield epifluorescence microscopy.
p79
of the GBM are endothelial cells that may be altered in nephrosis [Futrakul et al., 2003] but could be easily analyzed in 3-D with antibodies such as anti-factor VIII (fig. 1L).
Conclusion
The glomerulus is a target for many renal diseases. The application of 2-D antibody techniques to human renal biopsies has enabled investigators to detect aberrations in many glomerulopathies. Immunohistochemical identification of specific proteins has been a useful tool to examine cellular dysfunction and matrix alterations in disease, while electron microscopy demonstrated the higher order structural – functional relationships of the podocyte, basement membrane and mesangium. Coupling these techniques has provided valuable insight into the unique cellular and molecular biology of the glomerulus, and their deviations associated with the diseased state. Most glomerular pathophysiologic studies employ methodologies that provide morphologic and/or molecular information but lack a 3-D perspective. Such information may be crucial in an organ where structural relationships and function are highly integrated. Advanced microscopy
techniques, especially those employing molecular recognition, are necessary for 3-D characterization of structural and functional alterations of the diseased glomerulus. The power of two-photon microscopy in optically dissecting a complex structure like the glomerulus is that the 3-D perspective will provide a new and better appreciation of changes associated with glomerulopathies.
Acknowledgements Microscopy images were collected in compliance with Indiana University School of Medicine’s Human Research and Animal Care Committees. The authors are grateful to Roger Wiggins (Ann Arbor, Mich.) for donating antibodies to GLEPP1 and podocalyxin, and to Eric Andreoli, Jeffrey Clendenon, Tameka Conley, Gregory Cook, Michael Goheen, Jens Nürnberger, Elizabeth Ross, Heidi Sterrett, Heather Ward and Moo-Nahm Yum for assistance in collection and rendering of microscopy volumes. C.L.P. acknowledges support from the Indiana Genomics Initiative grant from the Lilly Endowment to Indiana University School of Medicine (INGEN), National Institutes of Health George M. O’Brien Kidney Research Center DK61594 and NIH Young Investigator grant DK02785. V.H.G. acknowledges NIH DK65504. Select images from this work were presented in poster form at the 2002 meetings of the American Society of Nephrology (Philadelphia) and the American Society for Cell Biology (San Francisco).
References Arakawa M, Tokunaga J: A scanning electron microscope study of the glomerulus. Further consideration of the mechanism of the fusion of podocyte terminal processes in nephrotic rats. Lab Invest 1972;27:366–371. Barisoni L, Kopp JB: Modulation of podocyte phenotype in collapsing glomerulopathies. Microsc Res Tech 2002;57:254–262. Bellis M: Light microscopes, electron microscopes, scanning electron microscope; in History of the Microscope. New York, The New York Times Co, 2005 (http://inventors.about.com/library/ inventors/blmicroscope.htm). Berger J: IgA glomerular deposits in renal disease. Transplant Proc 1969;1:939–944. Bonsib SM: Scanning electron microscopy of acellular glomeruli in nephrotic syndrome. Kidney Int 1985;27:678–684. Bonsib SM: Focal-segmental glomerulosclerosis. The relationship between tubular atrophy and segmental sclerosis. Am J Clin Pathol 1999; 111:343–348. Bowman W: On the structure and use of the malpighian bodies of the kidney, with observations on the circulation through the gland. Philos Trans R Soc Lond Biol 1842;132:57–80.
p80
Clendenon JL, Phillips CL, Sandoval RM, Fang S, Dunn KW: Voxx: a PC-based, near realtime volume rendering system for biological microscopy. Am J Physiol 2002; 282:C213– C218. Denk W, Piston DW, Webb WW: Two-photon molecular excitation in laser-scanning microscopy; in Pawley JB (ed): Handbook of Biological Confocal Microscopy. New York, Plenum Press, 1995, pp 445–458. Denk W, Strickler JH, Webb WW: Two-photon laser scanning fluorescence microscopy. Science 1990;248:73–76. Farquhar MG, Vernier RL, Good RA: An electron microscope study of the glomerulus in nephrosis, glomerulonephritis, and lupus erythematosus. J Exp Med 1957;106:649–660. Fogo A, Glick AD, Horn SL, Horn RG: Is focal segmental glomerulosclerosis really focal? Distribution of lesions in adults and children. Kidney Int 1995;47:1690–1696. Fuiano G, Comi N, Magri P, Sepe V, Balletta MM, Esposito C, Uccello F, Dal Canton A, Conte G: Serial morphometric analysis of sclerotic lesions in primary ‘focal’ segmental glomerulosclerosis. J Am Soc Nephrol 1996;7: 49–55.
Nephron Physiol 2006;103:p75–p81
Futrakul N, Kittikowit W, Yenrudi S: Reduced endothelial factor VIII staining in renal microcirculation correlates with hemodynamic alteration in nephrosis. Ren Fail 2003; 25: 759–764. Gratton E, vandeVen MJ: Laser sources for confocal microscopy; in Pawley JB (ed): Handbook of Biological Confocal Microscopy. New York, Plenum Press, 1995, pp 69–97. Guo JK, Menke AL, Gubler MC, Clarke AR, Harrison D, Hammes A, Hastie ND, Schedl A: WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis. Hum Mol Genet 2002;11:651–659. Herman B, Centonze Frohlich VE, Lakowicz JR, Murphy DB, Spring KR, Davidson MW: Fluorescence microscopy, introduction to fluorescence. Olympus Microscopy Resource Center, Melville/NY, Olympus America Inc, 2000– 2005 (http://www.olympusmicro.com/primer/ techniques/fluorescence/fluorhome.html). Huber C: On the development and shape of uriniferous tubules of certain higher mammals. Am J Anat Suppl 1905;4:1–98.
Phillips /Gattone /Bonsib
Inoue S: Foundations of confocal scanned imaging in light microscopy; in Pawley JB (ed): Handbook of Biological Confocal Microscopy. New York, Plenum Press, 1995, pp 1–17. Kerjaschki D, Sharkey DJ, Farquhar MG: Identification and characterization of podocalyxin – the major sialoprotein of the renal glomerular epithelial cell. J Cell Biol 1984;98:1591–1596. Kriz W, Lemley KV: The role of the podocyte in glomerulosclerosis. Curr Opin Nephrol Hypertens 1999;8:489–497. Natoli TA, Liu J, Eremina V, Hodgens K, Li C, Hamano Y, Mundel P, Kalluri R, Miner JH, Quaggin SE, et al: A mutant form of the Wilms’ tumor suppressor gene WT1 observed in Denys-Drash syndrome interferes with glomerular capillary development. J Am Soc Nephrol 2002;13:2058–2067. Orlando RA, Takeda T, Zak B, Schmieder S, Benoit VM, McQuistan T, Furthmayr H, Farquhar MG: The glomerular epithelial cell antiadhesin podocalyxin associates with the actin cytoskeleton through interactions with ezrin. J Am Soc Nephrol 2001;12:1589–1598.
Microscopy of Glomeruli
Palade GE: Albert Claude and the beginnings of biological electron microscopy. J Cell Biol 1971;50:5d–19d. Palmer RE, Kotsianti A, Cadman B, Boyd T, Gerald W, Haber DA: WT1 regulates the expression of the major glomerular podocyte membrane protein podocalyxin. Curr Biol 2001;11: 1805–1809. Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev 2003;83: 253–307. Pelletier J, Bruening W, Kashtan CE, Mauer SM, Manivel JC, Striegel JE, Houghton DC, Junien C, Habib R, Fouser L, et al: Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell 1991;67: 437–447.
Sawada H, Stukenbrok H, Kerjaschki D, Farquhar MG: Epithelial polyanion (podocalyxin) is found on the sides but not the soles of the foot processes of the glomerular epithelium. Am J Pathol 1986;125:309–318. Stockand JD, Sansom SC: Glomerular mesangial cells: electrophysiology and regulation of contraction. Physiol Rev 1998;78:723–744. Wang R, St John PL, Kretzler M, Wiggins RC, Abrahamson DR: Molecular cloning, expression, and distribution of glomerular epithelial protein 1 in developing mouse kidney. Kidney Int 2000;57:1847–1859. Wharram BL, Goyal M, Gillespie PJ, Wiggins JE, Kershaw DB, Holzman LB, Dysko RC, Saunders TL, Samuelson LC, Wiggins RC: Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low glomerular filtration rate. J Clin Invest 2000;106:1281–1290.
Nephron Physiol 2006;103:p75–p81
p81
