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 Kestilä||,
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 ||Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland
Correspondence to Dr. Hannu Jalanko, Hospital for Children and Adolescents, University of Helsinki, 00290 Helsinki, Finland. Phone: +358-9-47173708; Fax: +358-9-47173700; E-mail: hannu.jalanko{at}hus.fi
Glomerular epithelial cells (podocytes) play an importantrole in the pathogenesis of proteinuria. Podocyte foot processeffacement is characteristic for proteinuric kidneys, and geneticdefects in podocyte slit diaphragm proteins may cause nephroticsyndrome. In this work, a systematic electron microscopic analysiswas performed of the structural changes of podocytes in twoimportant nephrotic kidney diseases, congenital nephrotic syndromeof the Finnish type and minimal-change nephrotic syndrome (MCNS).The results showed that (1) podocyte foot process effacementwas present not only in proteinuric glomeruli but also in nonproteinuricMCNS kidneys; (2) podocytes in proteinuric glomeruli did notshow detachment from the basement membrane or cell membraneruptures; (3) the number of pinocytic membrane invaginationsin the basal and apical parts of the podocytes was comparablein proteinuric and control kidneys; (4) in proteinuric kidneys,the podocyte slit pore density was decreased by 69 to 80% andup to half of the slits were so "tight" that no visible spacebetween foot processes was seen; thus, the filtration surfacearea between podocytes was dramatically reduced; and (5) inthe narrow MCNS slit pores, nephrin was located in the apicalpart of the podocyte foot process, indicating vertical transferof the slit diaphragm complex in proteinuria. In conclusion,these results suggest that protein leakage in the two nephroticsyndromes studied occurs through defective podocyte slits, andthe other structural alterations commonly seen in electron microscopyare secondary to, not a prerequisite for, the development ofproteinuria.
The striking morphologic change in proteinuric kidneys is thereplacement of discrete glomerular podocytic foot processesby large expanses of flattened epithelial cytoplasm. This abnormalitywas recognized in the earliest electron microscopic studiesin the 1950s (1–3) and has constantly been described inhuman glomerular diseases as well as in animal models of proteinuria.The effacement of the foot processes has been regarded as anabnormal response of the epithelium either to direct injuryor to alterations elsewhere in the glomerulus. The process isprobably associated with changes in the actin cytoskeleton ofthe podocyte foot processes, but its molecular basis is stillnot known (4,5).
The loss of the complex interdigitation of podocytes does notexplain protein leakage in nephrotic kidneys. On the contrary,the wide expanses of epithelial cytoplasm often cover the glomerularbasement membrane (GBM) of the capillary wall so that only occasionalinterruptions are present. These residual sites of previousslit pores may also show close apposition of adjacent podocytecell membranes, forming "tight" or "close" junctions (6–8).Thus, the total area available for the passage of the urinaryultrafiltrate in nephrotic kidney is probably much less thannormal, and it is difficult to see how massive proteinuria occursin such a situation.
Two explanations have been offered to connect the ultrastructuralfindings and proteinuria. The first hypothesis suggests thatproteinuria actually results from the detachment of the epithelialcells from the GBM and formation of focal gaps in the epithelialcovering. In these areas, increased water flux would cause plasmaproteins to be dragged across the GBM filter to the urinaryspace. In animal models, the onset of proteinuria coincideswell with the development of areas of denuded GBM (9,10), andsuch areas have also been described in two reports on humannephrotic kidneys (11,12). The second theory is that, in nephrotickidneys, large amounts of protein would pass through the epithelialcells into urine via active endocytosis. This is supported bythe fact that electron microscopic studies have revealed increasedamounts of pinocytic vesicles, phagosomes, and lysosomes inepithelial cells in human proteinuric kidneys as well as inacute aminonucleoside nephrosis in rats (7,13,14).
The electron microscopic studies mainly were performed at atime when little was known of the molecular basis of the glomerularfiltration. Traditionally, the podocyte alterations were believedto be associated with increased leakiness of the GBM (4,15),although the possible role of the slit diaphragm connectingthe podocyte foot processes was also suggested (16). Duringthe past few years, the work on genetic diseases has indicatedthat the podocyte slit diaphragm is probably more importantthan the GBM in the protein sieving. Defects in many podocyteproteins, such as nephrin, Neph1, podocin, Fat, and CD2AP, resultin nephrotic syndrome (17–21). How the classic electronmicroscopic observations on nephrotic kidneys and the new molecularfindings fit together have not been studied.
In this study, we analyzed the ultrastructure of the podocytesand their attachment to the GBM in kidney samples from patientswith congenital nephrotic syndrome of the Finnish type (NPHS1)and minimal-change nephrotic syndrome (MCNS). The NPHS1 kidneysare especially suitable for these studies because the moleculardefect in this disorder is known, and the kidneys show massiveproteinuria with little histologic lesions or renal failure.NPHS1 is caused by mutations in NPHS1 gene that encodes a majorslit diaphragm protein, nephrin (17). Patients with severe mutationsin NPHS1 do not express nephrin in the glomerulus and have adefective podocyte slit diaphragm (22). Conversely, MCNS isthe most common nephrotic disease in children with unknown causeand a prototype of "acquired" nephrotic syndromes. Proteinuriais fluctuating in MCNS, which offers an opportunity to studykidneys in different phases of the disease.
Samples
Renal tissue blocks for electron microscopy (EM) were obtainedby core needle biopsies taken on clinical indications from patientswho were treated between 1969 and 2003 at the Hospital for Childrenand Adolescents, University of Helsinki. Some of the biopsiesfrom NPHS1 children were taken at nephrectomy, when the kidneysstill had normal blood flow. In total, 10 renal biopsies frompatients with MCNS and eight biopsies from patients with NPHS1were included. The diagnosis of MCNS was based on clinical dataas well as renal histology. Five MCNS patients had proteinuriavarying from 6 to 10.4 g/L, and five were in remission at thetime of biopsy. The diagnosis of NPHS1 was verified by analysisof the NPHS1 gene in every case. As controls, we used 11 renalbiopsy samples from patients with no proteinuria. These sampleswere obtained from patients who underwent control renal biopsybecause of having a renal transplant (three cases), a livertransplant (three cases), or tubulointerstitial nephritis (TIN)(five cases). Cryo-EM and immunogold labeling were performedon four of the MCNS and three control samples. Two of the MCNSpatients were in remission at the time of sampling.
Scanning EM
The glomeruli for scanning EM (SEM) were isolated under thedissection microscope immediately after the biopsies. The sampleswere fixed with 2.5% (wt/vol) phosphate-buffered glutaraldehydefor 2 h. Some samples were also treated with 1% (wt/vol) osmiumtetroxide. After washing and dehydration in ethanol, critical-pointdrying was performed using the Bal-Tec CDP 030 critical-pointdrying unit. The samples were mounted on aluminum stubs withdouble-sided tape and silver glue and then sputter coated withplatinum or chromium. The specimens were observed using bothZeiss scanning electron microscope DMS 962 and a Jeol fieldemission scanning electron microscope JSM 6335F.
Transmission EM
The preparation of the renal biopsies for transmission EM (TEM)was performed according to standard procedures. The tissue blockswere fixed by 2.5% glutaraldehyde and embedded in Epon. Eachsample was sectioned in series so that 10 grids were collectedfrom levels that were 1.2 µm apart from each other. Fiveto 10 different capillaries were studied from each glomeruluswith an FEI Tecnai F12 electron microscope at 80 kV. The analysisof the podocyte pores was performed at a magnification of 8200.All areas that were suspected to show denuded GBM were photographedfor further inspection.
Cryo-EM and Immunogold Labeling for Nephrin
For cryosectioning, biopsy samples from renal cortex were fixedin phosphate-buffered 3.5% paraformaldehyde and 0.02%glutaraldehyde.After fixation and washing, the samples were embedded in 10%gelatin, infiltrated with 2.3 M sucrose in PBS, and frozen inliquid nitrogen. Immunolabeling was done by the Tokuyasu method(23,24). Approximately 70-nm-thick sections were labeled for60 min with antinephrin and then incubated with fresh proteinA coupled to 10 nm of gold for 30 min. Polyclonal rabbit antinephrinantibody against the intracellular domain of nephrin was used(25). The second antibody (protein A–gold conjugate) waspurchased from University of Utrecht, School of Medicine, Departmentof Cell Biology. Sections were observed on an electron microscope(Tecnai F12; FEI, Eindhoven, Holland) and photographed for detailedanalysis.
Podocyte Structure in SEM
Glomeruli were prepared for SEM analysis from nine control kidneysand from six proteinuric kidneys, including four patients withNPHS1 and two patients with MCNS. The control kidneys showeda well-preserved organization of the glomerular capillary wallwith podocyte nuclear areas and primary, secondary, and tertiaryprocesses (Figure 1A). With high magnification, one could seethe slender foot processes with a rough surface and occasionalfilaments connecting them (Figure 1B).
Figure 1. Scanning electron microscopy (SEM) micrographs of control and proteinuric minimal-change nephrotic syndrome (MCNS) kidneys. (A) A normal capillary loop in a control patient tubulointerstitial nephritis (TIN) showing a podocyte cell body (arrow) and organized branching of the epithelial processes. (B) Micrograph of a control kidney at high magnification showing foot processes (arrowheads) and thin strands in between (arrows). (C) A capillary loop in proteinuric MCNS patient showing less organized epithelial processes. Podocyte cell body is marked with an arrow. (D) A closer view of a capillary loop of proteinuric MCNS glomerulus showing thin primary processes (arrows) extending from the cell body (arrowhead). (E and F) The surface of a capillary wall at high magnification showing poorly developed terminal processes. No gaps between the epithelial cells are seen.
In NPHS1 glomeruli, the podocyte cell bodies were prominentand had a balloon-like appearance (Figure 2, A and C). Oftenonly one primary process connected the cell body to a flat cytoplasmicsheet with no branching (Figure 2, B and C). Thus, epithelialcytoplasmic plates with narrow cell borders usually coveredthe major part of the capillary wall (Figure 2, B and D). Variationin the appearance, however, was evident, and in some areas,primitive ramification (Figure 2E) or slender processes withfilamentous connections were observed (Figure 2F). Especiallyin NPHS1, there were also occasional lumpy protrusions of thecytoplasm, which were aggregated on the surface of the podocytefoot processes. Most important, no areas of denuded GBM or holesin the epithelial cell covering were observed in the NPHS1 glomeruli.
Figure 2. SEM micrographs of NPHS1 kidneys. (A) A capillary loop at low magnification showing numerous cell bodies. The primary process (arrow) ends as an epithelial sheet. Area marked with a square is shown in B. (B) A high-magnification image of the capillary wall showing narrow and winding cell borders where epithelial sheets contact with each other (arrows). (C) A portion of a capillary wall depicting nuclear area of a podocyte (arrow) with a thin and slender primary process (arrowhead). (D) A portion of a NPHS1 glomerulus showing swollen podocytes with smooth appearance. No cytoplasmic holes are seen on the surface. (E) A high-magnification image shows disorganized processes on a capillary wall. (F) Parallel slender processes with filamentous grooves in NPHS1 glomerulus.
The organization of podocyte processes in MCNS glomeruli wasnot, in general, as severely affected as in NPHS1. Branchingof the primary processes was present in some areas (Figure 1, C and D).Also, foot processes could be recognized, but theywere disorganized and had a thin and a flattened appearance(Figure 1, E and F). No areas of bare GBM were detected as wasthe case in NPHS1 kidneys.
Podocyte Foot Processes in Transmission EM
A systematic transmission EM (TEM) analysis of podocyte-GBMattachment, podocyte pinocytic invaginations, and the frequencyand the quality of the podocyte slit pores was performed onseven MCNS, four control, and four NPHS1 kidneys. Four of theseven MCNS samples were obtained in relapse and three in theremission phase of the disease. A total of 1544 visual fieldscorresponding to 5250 µm of the GBM could be analyzedin serial sections cut at 1.2-µm intervals (Table 1).
Table 1. Podocyte slit pores, sites of detachment, and pinocytic invaginations in NPHS1, MCNS and control kidneysa
Podocyte Foot Process Effacement
In contrast to controls, the effacement of the podocyte footprocesses was evident in all proteinuric kidneys and also insamples from the three MCNS patients who had been in remissionfrom 5 d to 4 mo (Figure 3). The podocyte pore density (numberof slits per underlying GBM length) was decreased from 1542pores/mm GBM in controls to 308 pores/mm in NPHS1 and to 478pores/mm in proteinuric MCNS kidneys (Table 1). Pore densitywas reduced also in nonproteinuric patients with MCNS. One patient,who had been in remission for 4 mo, still had slit pores halfof the normal (Table 1).
Figure 3. Transmission EM (TEM) micrographs of the glomerular capillary wall. (A) Normal podocyte architecture with filtration slits and slit diaphragms (arrowheads) are seen in a control kidney (TIN). (B) In NPHS1, foot processes show effacement with slit pores located far apart. The pores are narrow (arrows) and lack detectable slit diaphragms. Note the invaginations on the basal surface facing the glomerular basement membrane (GBM; arrowheads). (C) Capillary wall of a proteinuric MCNS kidney shows partial effacement and close junctions (arrows) between the foot processes. (D) In the remission phase of MCNS, more podocyte slit pores are open, but broad epithelial sheets are still present. Also, normal-width pores with slit diaphragm are seen (arrowheads).
Attachment of the Podocytes to the GBM
Epithelial cells were seen to cover the GBM in all samples,and no areas of detachment were noticed even in NPHS1 glomeruliwith massive proteinuria. NPHS1 glomeruli contained occasionalballoon-like spaces, which, however, were located intracellularlyand not between the podocyte and the GBM (Figure 4, A and B).Two MCNS glomeruli had a small area of denuded GBM at the edgeof the section, where capillaries were easily subjected to distension(Figure 4, D and E). The first patient was in remission, andthe other had a relapse at the time of biopsy. The areas inthe two samples presented 0.03 to 0.3% of the whole length ofthe GBM screened. The epithelial covering was seen to vary inthickness, which partly resulted from a different plane of across-section (Figure 4C).
Figure 4. TEM micrographs of podocytes. (A) Three intracytoplasmic vacuoles (arrows) in podocytes of an NPHS1 kidney. (B) A closer view of a podocyte with a vacuole shows that the cell is attached to the underlying GBM. (C) Micrograph of a control kidney showing a short area with a thin podocyte layer. Such areas were seen in all samples. (D) A portion of a capillary wall from an MCNS patient in remission showing a short area where GBM was not covered by epithelial cells. The length of the denuded area is 2.1 µm. (E) A short area of bare GBM in a kidney from an MCNS patient with proteinuria. The length of the area is 0.55 µm. The areas of bare GBM in micrographs D and E were the only ones detected in an extensive survey.
Podocyte Invaginations
To asses the endocytic activity in the podocytes, we countedthe number of membranous invaginations (coated pits) at thebasal and apical surface. As shown in Table 1, no clear differencein their frequency was found between proteinuric and nonproteinurickidneys. These invaginations were seen in all samples and bothin areas with effacement and in the areas where podocytes showednormal architecture (Figure 5, A and B).
Figure 5. TEM micrographs of podocyte invaginations and slit pores. (A) Two basal invaginations are seen in foot processes of a control kidney (arrows). (B) Two basal invaginations (arrows) and one apical invagination (arrowhead) in a podocyte effacement area. Sample from an MCNS patient in relapse. (C) A widened slit pore (80 nm) occasionally seen in NPHS1 kidneys. (D) A narrow slit pore with filamentous material but no slit diaphragm in an NPHS1 glomerulus. (E) Cell contact area seen in an NPHS1 kidney (arrow). (F) A widened slit pore (60 nm) with no slit diaphragm in a proteinuric MCNS glomerulus. (G) A tight pore in proteinuric MCNS glomerulus. (H) Partially closed pore in MCNS in remission where filamentous material is seen in apical part of the contacting foot processes.
Podocyte Slit Pores in TEM and Immuno-EM
The glomeruli studied by TEM contained a total of 3445 podocyteslit pores, and the average width was 27 to 28 nm in control,NPHS1, and MCNS kidneys as measured from high-magnificationmicrographs. In proteinuric kidneys, however, the width of aslit pore showed a large variation and was occasionally increasedup to 50 to 80 nm (Figure 5, C and F). However, more often,the slit pores looked narrow, and in highly proteinuric NPHS1glomeruli, almost half (47%) of the pores were so "tight" thatno visible slit between adjacent foot processes was seen (Figure 5G).Pores were classified as "tight" or "closed" when therewas only a narrow space (5 to 10 nm) or no visible space betweenthe membranes of the neighboring foot processes. This observationis partly dependent on the angle of the cross-section, because20% of the pores looked tight also in control kidneys. In MCNSglomeruli, 19 to 66% of the slit pores gave a similar image(Table 1).
In glutaraldehyde-fixed samples, normal slit diaphragms givea filamentous image on TEM (Figure 3A). We have previously foundthat this filament is totally missing in the open slit poresin NPHS1 kidneys (with no nephrin expression) and greatly reduced(by 39%) in proteinuric MCNS kidneys (26). The analysis in thiswork revealed that 13.7% of the pores in NPHS1 glomeruli haddiffuse filamentous material (Figure 5, D and E, Table 1). Thesepores represented 3.4% (relapse) and 5.6% (remission) of theslits in MCNS (Figure 5H), which is clearly more than in controls(0.9%). However, as a result of the dispersion, no statisticaldifference could be verified. Immuno-EM of the MCNS glomerulishowed that nephrin, which is a major component of a normalslit diaphragm, was located to the apical part of this filamentousmaterial in narrow slit pores (Figure 6B). In wider pores, nephrinwas seen at the level of the filamentous material either inthe apical region of the foot process (Figure 6, C and D) orat the "right" level above the GBM (Figure 6A).
Figure 6. Immuno-EM for nephrin. (A) Nephrin labeling in the remission phase of MCNS showing normal slit diaphragms, which are located in basal areas of foot processes. Gold particles corresponding to nephrin are seen in the close vicinity of adjacent membranes at the level of slit diaphragm. One pore is tight, and nephrin is located in the apical part of the podocyte (arrows). (B) A closer view of a narrow slit pore with filamentous material in MCNS glomerulus. (C) An apical slit diaphragm seen in the relapse phase of MCNS. (D) In relapse, there also are some open slit pores that contain a normal-looking slit diaphragm, but the location of the slit diaphragm is still high.
In this study, we analyzed the structural changes in glomerularepithelial cells (podocytes) in two important nephrotic kidneydiseases, NPHS1 and MCNS. The loss of the normal podocyte footprocess organization (effacement, fusion) was evident in proteinuricNPHS1 and MCNS kidneys, as reported previously in nephrotichuman and animal kidneys (3,9,27). On the average, the lesionswere more severe in NPHS1 kidneys with constant massive proteinuriathan in MCNS kidneys. Foot process broadening, however, waspresent also in nonproteinuric MCNS kidneys from patients whohad been in remission from 5 d to 4 mo, indicating that proteinleakage is not directly associated with the epithelial lesions.
In SEM, the most dramatic change was observed in NPHS1 kidneys,where podocytes often gave a "tadpole" image instead of thenormal "octopus"-like structure. The balloon-like cell bodiesoften hung from a filamentous primary process that was attachedto a large cytoplasmic plate. This finding is different fromthat seen in rats (10) and from the two previous reports onthe SEM findings in human nephrotic kidneys (28,29). In thesestudies, the podocyte nuclear portions were found to be attenuated,and spreading of the cell body area obliterated the cytoplasmicbranching. It is interesting that the SEM images of the capillarywall in NPHS1 varied so that also more normal areas were observed.Primitive branching was focally evident, resembling the changesin MCNS kidneys. This favors the idea that the epithelial changesare associated with proteinuria as such and not caused by thedefect in the podocyte slit diaphragm. In SEM, the other majorfinding was that the epithelial sheets in NPHS1 and MCNS kidneysdid not show membrane ruptures, as has been reported in theanimal models (30).
The major observation in our work was that epithelial cell detachmentand formation of areas of denuded GBM were not found in theproteinuric glomeruli. The extensive search for such areas inSEM and TEM revealed only two small patches of bare GBM in twoMCNS kidneys (one in relapse and the other in remission). Thisis clearly opposite to the results reported previously, especiallyin the aminonucleoside nephrosis in rats (9,14,30,31). In additionto the animal models, focal areas of externally denuded GBMhave been observed in the glomeruli from nephrotic patientswith focal glomerulosclerosis (11,31), amyloidosis (32), anddiabetes (33). Also, Yoshikawa et al. (12) found small fociof epithelial detachment in 16 of the 33 patients with MCNSand nephrosis. The findings in human diseases, however, areless clear than in the rats.
Detachment of the epithelium suggests that the mechanism bywhich epithelial cells and the GBM normally adhere to one anotheris impaired. The other possibility is that foot process retractionleads to separation of neighboring podocytes. That we did notsee areas of denuded GBM in NPHS1 is interesting in two ways.First, it shows that even massive proteinuria (up to 100 g/L)is possible without problems in podocyte-GBM adherence. Second,the NPHS1 kidneys lack the slit diaphragm and its major component,nephrin, and large defects in podocyte contacts with separationof the foot processes would seem possible. This was clearlynot the case, however, indicating that the epithelial cell connectionsand the basal anchoring were enough to prevent the formationof epithelial gaps.
The tissue sections studied represented only a small portionof the whole kidney, and it is possible, especially in MCNS,that focal epithelial gaps were missed because they were notincluded in the analyzed material. For obtaining maximal coverage,the sections used in the analyses were taken 1 µm apart(every 20th microtome section), which, of course, did not completelysolve the problem. Two pieces of information, however, speakagainst the "epithelial gap" theory. In animal models, the denudedareas have been numerous (one gap in every fifth section) (10),suggesting that the 200 sections analyzed from the 10 proteinuricMCNS glomeruli in our work should have contained 40 gaps, insteadof one small area in the edge of a section. Also, the kineticsof the foot process alterations seem "wrong." In MCNS, proteinuriatypically resolves within a few days after commencement of prednisonetherapy, whereas the podocyte lesions seem to normalize veryslowly (within months). Thus, the rapid response is more easilyexplained by molecular reorganization of podocyte proteins ratherthan healing of the possible epithelial gaps, representing asevere form of injury.
Podocytes can endocytose proteins, and it has been proposedthat during nephrosis, large amounts of protein may pass throughthe epithelial cells into urinary space via cytoplasmic vesiclesand vacuoles. Increased endocytic activity in podocytes wasdescribed already in the early EM studies of human proteinurickidneys (1,2). Pinocytic invaginations (coated pits) have alsobeen described repeatedly in aminonucleoside nephrosis in rats(6,7,14,30,34,35). The epithelial vacuoles have been suggestedto communicate with the extracellular space overlying the GBMon one side and with urinary space on the other side of thecell. The flattening of foot processes in nephrosis may facilitatethe uptake and transport of proteins by pinocytosis (36). Thereis also evidence that the epithelium functions as a monitorto recover proteins that leak through the basement membraneeven in normal filtration (37,38). Recently, Kim et al. (39)found that mice that had defects in the podocytic endocytosis(CD2AP deficiency) had increased susceptibility to glomerularinjury, suggesting that protein clearance by podocytes may bean important modulator of glomerular diseases. On the basisof these data, we tried to evaluate the endocytic activity ofthe podocytes in NPHS1 and MCNS kidneys by counting the pinocyticinvaginations in the basal membrane as well as in the apicalareas of the cell membrane. The analysis, however, showed nodifference in the invagination frequency between proteinuricand nonproteinuric glomeruli, which suggests that endocytosisdoes not play a significant role in protein transport throughthe capillary wall in NPHS1 or MCNS.
The number of podocyte slit pores was dramatically reduced inproteinuric kidneys. They also showed qualitative changes inSEM and TEM, so that up to 50% of the residual slits were closedor tight. This phenomenon has previously been described in humannephrotic kidneys (6,7) as well as in aminonucleoside nephrosisin rats (8,35,40). It is probable that many of the tight slitsrestrict normal ultrafiltration (and protein leakage), and,on the basis of our survey, the area of "open" slit pores wasreduced to 10% of normal in NPHS1 kidneys and to 23% in nephroticMCNS kidneys. Because neither of these diseases is associatedwith impaired glomerular clearance of water and small solutes,it seems that the filtration surface of the capillary wall innormal kidneys is extensive.
Our knowledge of the normal podocyte slit pore and the slitdiaphragm has greatly increased during the past few years (4,41,42).The slit diaphragm area has been reported to contain at leastnephrin, Neph1, FAT, and P-cadherin, which interact with eachother and also with the podocyte proteins, such as podocin,CD2AP, and ZO-1. The precise molecular architecture of slitdiaphragm, however, needs further clarification. It is clearthat EM is a crude method for studying such a complex proteinstructure as the slit diaphragm. However, previously, we observedthat the normal slit diaphragm image in the open slit poresis completely lacking in NPHS1 kidneys (22) and reduced in MCNSkidneys (26). A new finding in this report was that one couldsee filaments between the neighboring foot processes in high-powerSEM of normal glomerulus. In TEM, numerous filaments and a fuzzymaterial with no clear slit diaphragm layer was detected inproteinuric kidneys. Also, the immunogold labeling of the cryosectionsrevealed nephrin only in the apical parts of the narrow slitsin MCNS kidneys. This is in accordance with previous findingsin rats indicating that the slit diaphragm may move upward duringnephrosis and also fits the idea that the reverse changes inMCNS patients in remission resemble the normal differentiationprocess that occurs in the fetal period (9,35,43).
In conclusion, the results in the present work favor the ideathat proteinuria in NPHS1 and MCNS is caused by molecular changesin the slit diaphragm structure, and the loss of the complexinterdigitation of podocyte foot processes is a secondary lesionthat is not critical for the development of protein leakage.
Acknowledgments
This work was supported by grants from the Finnish Academy,the Sigrid Juselius Foundation, Pediatric Research Foundation,and Helsinki University Central Hospital Research Fund.
We thank Mervi Lindman, Tuike Helmiö, Arja Strandel, PirkkoLeikas-Lazanyi, and Mikko Jalanko for excellent technical assistance.We are also grateful to Eija Jokitalo for advice regarding immunogoldlabeling.
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Received for publication March 10, 2004.
Accepted for publication June 24, 2004.
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Top
Abstract
Introduction
40 gaps, instead of one small area in the edge of a section. Also, the kinetics of the foot process alterations seem "wrong." In MCNS, proteinuria typically resolves within a few days after commencement of prednisone therapy, whereas the podocyte lesions seem to normalize very slowly (within months). Thus, the rapid response is more easily explained by molecular reorganization of podocyte proteins rather than healing of the possible epithelial gaps, representing a severe form of injury. 
