J Am Soc Nephrol 15: 2611–2618, 2004 Podocytes Are Firmly Attached to Glomerular Basement Membrane in Kidneys with Heavy Proteinuria ANNE-TIINA LAHDENKARI,* KARI LOUNATMAA,† JAAKKO PATRAKKA,*‡ CHRISTER HOLMBERG,* JORMA WARTIOVAARA,§ MARJO KESTILA¨ ,ʈ OLLI KOSKIMIES,* and HANNU JALANKO* *Hospital for Children and Adolescents and Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; †Helsinki University of Technology, Laboratory of Electronics Production Technology, Espoo, Finland; ‡Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institut, Stockholm, Sweden; §Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, Helsinki, Finland; and ʈDepartment of Molecular Medicine, National Public Health Institute, Helsinki, Finland Abstract. Glomerular epithelial cells (podocytes) play an im- in the basal and apical parts of the podocytes was comparable portant role in the pathogenesis of proteinuria. Podocyte foot in proteinuric and control kidneys; (4) in proteinuric kidneys, process effacement is characteristic for proteinuric kidneys, the podocyte slit pore density was decreased by 69 to 80% and and genetic defects in podocyte slit diaphragm proteins may up to half of the slits were so “tight” that no visible space cause nephrotic syndrome. In this work, a systematic electron between foot processes was seen; thus, the filtration surface microscopic analysis was performed of the structural changes area between podocytes was dramatically reduced; and (5)in of podocytes in two important nephrotic kidney diseases, con- the narrow MCNS slit pores, nephrin was located in the apical genital nephrotic syndrome of the Finnish type and minimal- part of the podocyte foot process, indicating vertical transfer of change nephrotic syndrome (MCNS). The results showed that the slit diaphragm complex in proteinuria. In conclusion, these (1) podocyte foot process effacement was present not only in results suggest that protein leakage in the two nephrotic syn- proteinuric glomeruli but also in nonproteinuric MCNS kid- dromes studied occurs through defective podocyte slits, and the neys; (2) podocytes in proteinuric glomeruli did not show other structural alterations commonly seen in electron micros- detachment from the basement membrane or cell membrane copy are secondary to, not a prerequisite for, the development ruptures; (3) the number of pinocytic membrane invaginations of proteinuria. The striking morphologic change in proteinuric kidneys is the so that only occasional interruptions are present. These residual replacement of discrete glomerular podocytic foot processes by sites of previous slit pores may also show close apposition of large expanses of flattened epithelial cytoplasm. This abnor- adjacent podocyte cell membranes, forming “tight” or “close” mality was recognized in the earliest electron microscopic junctions (6–8). Thus, the total area available for the passage studies in the 1950s (1–3) and has constantly been described in of the urinary ultrafiltrate in nephrotic kidney is probably much human glomerular diseases as well as in animal models of less than normal, and it is difficult to see how massive pro- proteinuria. The effacement of the foot processes has been teinuria occurs in such a situation. regarded as an abnormal response of the epithelium either to Two explanations have been offered to connect the ultra- direct injury or to alterations elsewhere in the glomerulus. The structural findings and proteinuria. The first hypothesis sug- process is probably associated with changes in the actin cy- gests that proteinuria actually results from the detachment of toskeleton of the podocyte foot processes, but its molecular the epithelial cells from the GBM and formation of focal gaps basis is still not known (4,5). in the epithelial covering. In these areas, increased water flux The loss of the complex interdigitation of podocytes does would cause plasma proteins to be dragged across the GBM not explain protein leakage in nephrotic kidneys. On the con- filter to the urinary space. In animal models, the onset of trary, the wide expanses of epithelial cytoplasm often cover the proteinuria coincides well with the development of areas of glomerular basement membrane (GBM) of the capillary wall denuded GBM (9,10), and such areas have also been described in two reports on human nephrotic kidneys (11,12). The second theory is that, in nephrotic kidneys, large amounts of protein Received March 10, 2004. Accepted June 24, 2004. would pass through the epithelial cells into urine via active Correspondence to Dr. Hannu Jalanko, Hospital for Children and Adolescents, University of Helsinki, 00290 Helsinki, Finland. Phone: ϩ358-9-47173708; endocytosis. This is supported by the fact that electron micro- Fax: ϩ358-9-47173700; E-mail: [email protected] scopic studies have revealed increased amounts of pinocytic 1046-6673/1510-2611 vesicles, phagosomes, and lysosomes in epithelial cells in Journal of the American Society of Nephrology human proteinuric kidneys as well as in acute aminonucleoside Copyright © 2004 by the American Society of Nephrology nephrosis in rats (7,13,14). DOI: 10.1097/01.ASN.0000139478.03463.D9 The electron microscopic studies mainly were performed at 2612 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2611–2618, 2004 a time when little was known of the molecular basis of the drying unit. The samples were mounted on aluminum stubs with glomerular filtration. Traditionally, the podocyte alterations double-sided tape and silver glue and then sputter coated with plati- were believed to be associated with increased leakiness of the num or chromium. The specimens were observed using both Zeiss GBM (4,15), although the possible role of the slit diaphragm scanning electron microscope DMS 962 and a Jeol field emission connecting the podocyte foot processes was also suggested scanning electron microscope JSM 6335F. (16). During the past few years, the work on genetic diseases has indicated that the podocyte slit diaphragm is probably more Transmission EM important than the GBM in the protein sieving. Defects in The preparation of the renal biopsies for transmission EM (TEM) was performed according to standard procedures. The tissue blocks many podocyte proteins, such as nephrin, Neph1, podocin, Fat, were fixed by 2.5% glutaraldehyde and embedded in Epon. Each and CD2AP, result in nephrotic syndrome (17–21). How the sample was sectioned in series so that 10 grids were collected from classic electron microscopic observations on nephrotic kidneys levels that were 1.2 ␮m apart from each other. Five to 10 different and the new molecular findings fit together have not been capillaries were studied from each glomerulus with an FEI Tecnai F12 studied. electron microscope at 80 kV. The analysis of the podocyte pores was In this study, we analyzed the ultrastructure of the podocytes performed at a magnification of 8200. All areas that were suspected to and their attachment to the GBM in kidney samples from show denuded GBM were photographed for further inspection. patients with congenital nephrotic syndrome of the Finnish type (NPHS1) and minimal-change nephrotic syndrome Cryo-EM and Immunogold Labeling for Nephrin (MCNS). The NPHS1 kidneys are especially suitable for these For cryosectioning, biopsy samples from renal cortex were fixed in studies because the molecular defect in this disorder is known, phosphate-buffered 3.5% paraformaldehyde and 0.02%glutaralde- and the kidneys show massive proteinuria with little histologic hyde. After fixation and washing, the samples were embedded in 10% lesions or renal failure. NPHS1 is caused by mutations in gelatin, infiltrated with 2.3 M sucrose in PBS, and frozen in liquid NPHS1 gene that encodes a major slit diaphragm protein, nitrogen. Immunolabeling was done by the Tokuyasu method (23,24). Approximately 70-nm-thick sections were labeled for 60 min with nephrin (17). Patients with severe mutations in NPHS1 do not antinephrin and then incubated with fresh protein A coupled to 10 nm express nephrin in the glomerulus and have a defective podo- of gold for 30 min. Polyclonal rabbit antinephrin antibody against the cyte slit diaphragm (22). Conversely, MCNS is the most com- intracellular domain of nephrin was used (25). The second antibody mon nephrotic disease in children with unknown cause and a (protein A–gold conjugate) was purchased from University of Utre- prototype of “acquired” nephrotic syndromes. Proteinuria is cht, School of Medicine, Department of Cell Biology. Sections were fluctuating in MCNS, which offers an opportunity to study observed on an electron microscope (Tecnai F12; FEI, Eindhoven, kidneys in different phases of the disease. Holland) and photographed for detailed analysis. Materials and Methods Results Samples Podocyte Structure in SEM Glomeruli were prepared for SEM analysis from nine con- Renal tissue blocks for electron microscopy (EM) were obtained by core needle biopsies taken on clinical indications from patients who trol kidneys and from six proteinuric kidneys, including four were treated between 1969 and 2003 at the Hospital for Children and patients with NPHS1 and two patients with MCNS. The con- Adolescents, University of Helsinki. Some of the biopsies from trol kidneys showed a well-preserved organization of the glo- NPHS1 children were taken at nephrectomy, when the kidneys still merular capillary wall with podocyte nuclear areas and pri- had normal blood flow. In total,