Proc. Nati. Acad. Sci. USAVol. 82, pp. 8508-8512, December 1985Cell Biology

Localization of sialic acid in kidney glomeruli: Regionalization inthe podocyte plasma membrane and loss in experimental nephrosis

(domain fonnation/puromycin-induced nephrosis/lectin-gold technique)

PIERRE M. CHAREST AND JURGEN ROTH*Interdepartmental Electron Microscopy, Biocenter, University of Basle, CH4056 Basle, Switzerland

Communicated by A. Frey-Wyssling, August 9, 1985

ABSTRACT Sialic acid residues were localized by electronmicroscopy in renal glomeruli of normal and puromycin-treated rats with a cytochemical technique that utilized theLimax flavus lectin. In Lowicryl K4M thin sections fromnormal rats, sialic acid residues were found along the plasmamembrane of the various glomerular cell types and in theglomerular basement membrane as well as the mesangialmatrix. In NaDodSO4/PAGE, sialic acid residues of normalglomeruli were mainly confined to a 140-kDa protein previ-ously identified as podocalyxin. The distribution of sialic acidresidues in the podocyte plasma membrane was found to beremarkably regionalized. Based on the differential labelingintensity, three plasma membrane domains could be dermed:the foot process base, the foot process region above the slitdiaphragm, and the body of podocytes. Cytochemical andbiochemical analysis of glomeruli from puromycin-treated ratsshowed a loss of sialic acid residues from glomerularsialoglycocoajugates indicating a perturbated glycosylation.

The renal glomerulus is a complex structure composed ofthree cell types and a highly organized basement membrane.The visceral epithelial cells or podocytes exhibit character-istic features such as a special shape and an unusually thick,negatively charged glycocalyx, the so-called epithelialpolyanion. Based on the shape of the podocytes, two differ-ent plasma membrane regions can be distinguished: (i) thefree surface, which includes the cell body and the footprocess above the slit diaphragm, and (it) the base of the footprocess below the slit diaphragm and in close contact with thelamina rara externa of the basement membrane. This distinc-tion is also reflected by differences in the membrane com-position as revealed by freeze-fracture investigations (1, 2)and staining with various cationic probes (3-8) and differentlectins (9-11). The latter also provided evidence for the sialicacid-rich nature of the epithelial polyanion. Among thefactors responsible for the maintenance of the characteristicshape of podocytes, the presence of the epithelial polyanionseems to be essential since its absence during certain stagesof glomerular differentiation (12) or in certain diseased states(13, 14) as well as experimentally induced neutralization bypolycationic compounds (15) is paralleled by the loss of thetypical shape. In their recent investigations Kerjaschki et al.(16) have been able to identify and characterize the majorsialoglycoprotein of the glomerulus that apparently consti-tutes the main component of the epithelial polyanion. Theso-called podocalyxin is a 140-kDa protein which bindscationic stains and lectins with a certain specificity for sialicacid. By immunoelectron microscopy podocalyxin was foundto be present not only on the surface ofpodocytes but also onthe surface of endothelial cells.

In the present study we have applied a new lectin-goldtechnique to detect sialic acid residues (17) in kidneyglomeruli. This cytochemical technique has two distinctadvantages. First, the Limax flavus lectin possesses a highspecificity for N-acetyl and N-glycolylneuraminic acid whichis in contrast to wheat germ agglutinin (18) and Limuluspolyphemus lectin (19, 20). Second, the postembeddinglabeling technique on Lowicryl K4M thin sections eliminatesproblems of accessibility and allows precise localization oflectin binding sites with the use of gold particles (21). Wewere able to define three distinct plasma membrane regionsin podocytes based on a difference in labeling intensity forsialic acid residues: the foot process base, the foot processregion above the slit diaphragm, and the body of podocytes.Under puromycin-aminonucleoside nephrosis a loss of sialicacid residues in all glomerular structures occurred indicatinga defective glycosylation.

MATERIALS AND METHODSReagents. Antipain, fetuin, puromycin, N-acetylneuramin-

ic acid, and neuraminidase (type X, affinity-purified) wereobtained from Sigma. Limax flavus lectin was from E-YLaboratories (San Mateo, CA) or Calbiochem-Behring.Benzamidin, diisopropyl fluorophosphate, and pepstatin Awere purchased from Fluka.

Experimental Nephrosis. Male rats (Wistar strain, about200-g body weight) were made nephrotic by daily subcuta-neous injection of aminonucleoside of puromycin (1.5mg/100 g of body weight) dissolved in isotonic saline.Kidneys were removed after 7 or 14 days of treatment andprocessed for electron microscopy or for isolation ofglomeruli as described below.

Tissue Processing. Kidneys from control or puromycin-treated rats were fixed by vascular perfusion with 1%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 10 min,followed by immersion of small pieces from kidney cortex inthe same fixative for 50 min. Afterwards, the tissue pieceswere washed three times (10 min each) in buffer and thentreated with 50mM NH4Cl in cacodylate buffer for 30-60 minto block free aldehyde groups.For light microscopy, routine embedding in Epon 812 was

performed, and semithin sections (0.5-1.5 ,um) were pre-pared and mounted on glass slides. For electron microscopy,dehydration at progressively lowered temperature down to-350C and embedding in Lowicryl K4M (22) at -350C wasdone as described (23). Thin sections were mounted onparlodion-carbon-coated nickel grids (150 mesh).

Cytochemical Procedure for Sialic Acid Localization. Sialicacid residues were detected with a cytochemical-affinitytechnique using the lectin from the slug Limaxflavus and thefetuin-gold complex. The details of the technique were givenelsewhere (17, 24).

*To whom reprint requests should be addressed.

8508

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Proc. Natl. Acad. Sci. USA 82 (1985) 8509

Light microscopy. Epon semithin sections were treated toremove the resin (25) and rehydrated. Sections were coveredwith TBS (10 mM TrisHCl, pH 7.4/0.5 M NaCl/2 mMMnCl2/2 mM MgCl2/2 mM CaCl2) for 5 min, then incubatedwith Limaxflavus lectin (150 ,ug/ml in TBS) for 1 hr at roomtemperature, washed twice with TBS (5 min each), exposedto the fetuin-gold complex (diluted to 80 ug fetuin-gold/mlexpressed in terms of fetuin concentration) for 1 hr andwashed twice in TBS (5 min each). Finally, sections wererapidly dehydrated, mounted in Entellan (Merck, Darmstadt,F.R.G.), and examined with a Zeiss Photomicroscope III.Electron microscopy. Ultrathin sections from Lowicryl

K4M embedded kidney cortex were floated on a droplet ofTBS for 5 min then transferred to a droplet of Limaxflavuslectin (100 Ag/ml) for 1 hr at room temperature, rinsed twicewith TBS (2 min each) and placed on a droplet of fetuin-goldcomplex (diluted to give an absorption of 0.35 at 525 nm) for30 min. After two rinses with TBS (2 min each) and a briefrinse with distilled water sections were stained with 2%(wt/vol) aqueous uranyl acetate for 5 min and lead acetate for45 sec and examined with a Zeiss EM 10 electron microscope.

Cytochemical Controls. Sections were exposed to Limaxflavus lectin to which N-acetylneuraminic acid had beenadded to a concentration of 6 mM, 30 min prior to incubation.In other experiments nonlabeled fetuin (30 ,ug/ml, 30 min)was applied after the lectin incubation step followed byfetuin-gold complex. Nonspecific binding of the fetuin-goldcomplex to thin sections was checked by omission of theLimaxflavus lectin incubation step. Other thin sections weretreated with 1 unit/ml or 4 units/ml of neuraminidase (in 0.1M acetate buffer, pH 5.0, with 0.04 M CaCl2 for 1-2 hr at37°C) before incubation with the lectin and fetuin-goldcomplex.

Quantification of Labeling. The data are based on theevaluation of three different glomeruli each from threecontrol rats. Photographs from glomerular loops were takenon 70-mm film and projected on a graphic tablet connected toan Apple Ile computer. The intensity oflabeling expressed asnumber of gold particles per ,um length of plasma membranewas estimated at the foot process base, the free surface ofthefoot process above the slit diaphragm, and the main body ofthe podocytes.

Isolation of Glomeruli. Kidneys from control andpuromycin-treated (7 days and 14 days) rats were perfusedwith oxygenated Hanks' balanced salt solution containingprotease inhibitors (1 ug of pepstatin A/ml, 1 ug ofantipain/ml, 1 mM benzamidin, and 1 mM diisopropylfluorophosphate). Cortical slices were finely minced at 4°Cwith a razor blade. The cortical slush was dispersed in Hanks'balanced salt solution containing the protease inhibitors in 15ml glass tubes, and the larger fragments were allowed tosediment. Glomeruli were enriched in the supernatant andwere isolated under dissecting microscopes.NaDodSO4. Freshly isolated glomeruli were pelleted in a

Microfuge and resuspended in NaDodSO4 sample buffer asdescribed by Kerjaschki et al. (16). Aliquots (%2 mg ofprotein/ml) were heated for 5 min in a boiling water bath.Aliquots (100 ,l) were loaded on a 5-10%6 gradient polyacryl-amide gel containing 10% (wt/vol) sucrose, 1.5 M Tris'HCl(pH 8.8), 8 mM EDTA, and 0.4% NaDodSO4. Gels wereelectrophoresed at about 7 mA overnight. Afterwards theywere processed for staining with (i) Coomassie blue [0.25% in7.5% (vol/vol) acetic acid/20% (vol/vol) methanol], (ii) silverstaining according to Oakley et al. (26), and (iii)Stains Allaccording to Green and Pastenka (27). Samples of isolatedglomeruli were incubated before NaDodSO4/PAGE withneuraminidase [20 units/ml in 0.1 M acetate buffer (pH 5),with 0.04 M CaCl2 for 16 hr at 370C].

Transfer to Nitrocellulose and Lectin Overlay. Proteins inNaDodSO4 gels were transferred to nitrocellulose sheets for

45 min at 40C according to Towbin et al. (28). The gels werethen stained with Coomassie blue to assess the completenessof the transfer. Paper strips were excised and incubated withradioiodinated Limax flavus lectin in TBS containing 0.5%bovine serum albumin for 30 min, rinsed several times withTBS, dried, and exposed for autoradiography. Some paperstrips were incubated with lectin to which 10 mM N-acetylneuraminic acid had been added.

RESULTS

Light Microscopy. In semithin sections of kidneys fromnormal rats incubated with Limax flavus lectin followed byfetuin-gold complex, intense red coloration appeared overthe various glomerular structures such as at the surface ofthepodocytes, along the capillary lumen, and the basementmembrane region (Fig. 1). The staining was completelyabolished under the different control conditions.

Electron Microscopy. Incubation of Lowicryl K4M thinsections from kidney cortex of normal animals resulted in adense staining with gold particles, which indicated the pres-ence of sialic acid residues since this labeling could becompletely abolished in the different cytochemical controlincubations. Gold particle labeling was observed along theentire plasma membrane ofpodocytes and endothelial cells aswell as throughout the glomerular basement membrane (Fig.2A). The results in Table 1 show that the plasma membraneof the podocyte main body exhibited the highest gold particledensity when compared to the foot process region above theslit diaphragm and the foot process base below the slitdiaphragm, which had 60%o and 25% of the labeling intensity,respectively.Examination of thin sections from glomeruli ofpuromycin-

treated rats revealed the characteristic changes ofpodocytesshape as described (29-32). After 7 days of puromycin treat-ment most podocytes displayed this altered shape which was

FIG. 1. Rat kidney; Epon embedding; semithin section stainedwith Limax flavus lectin followed by fetuin-gold complex. Intenselabeling is present at the surface of podocytes, along the luminalaspect of capillaries and parietal Bowman's capsule. Proximaltubular cells are also stained. (Bar = 20 lum.)

Cell Biology: Charest and Roth

8510 Cell Biology: Charest and Roth

I*

F, ..

;.vAEnd

>?/ ,^} , .*fK t;$

t.s .= w r4 $

C.} 'a i;. i I,, atWe ' t oh . 11a;W and as < And r

'4zz ah' "8*.St'+ I+ .s

FIG. 2. Lowicryl K4M thinsections; postembedding labelingwith Limax flavus lectin andfetuin-gold complex. (A) Controlrat. Labeling for sialic acid resi-dues as indicated by the goldparticles occurs along the plasmamembrane of podocytes (Pod)and capillary endothelial cells(End) and is present throughoutthe glomerular basement mem-brane (GBM). The differential la-beling for sialic acid along theplasma membrane of a podocyteis shown in the inset toA and thequantitative data are given in Ta-ble 1. (B) After 7 days ofpuromy-cin treatment, only sparse label-ing for sialic acid can be detectedat the plasma membrane ofpodocytes which display signifi-cant changes from their normalshape. Similarly, a strong reduc-tion in labeling can be seen at theendothelial cell surface and theglomerular basement membrane.(C) Specific label for sialic acid isno longer detectable in the abovementioned structures after 14days of puromycin treatmentwhen the specialized shape ofthepodocytes is lost. (Bars = 0.5EAMs)

accompanied by an almost complete loss in labeling for sialicacid residues (Fig. 2B). Few podocytes still exhibited footprocess-like structures that were very sparsely labeled withgold particles. There was no detectable labeling after 14 daysofpuromycin treatment on any region ofthe podocyte plasmamembrane (Fig. 2C). No labeling was found at the plasmamembrane of endothelial cells and throughout the glomerularbasement membrane.The protein analysis by NaDodSO4/PAGE of freshly

isolated glomeruli from control rats confirmed data obtainedby Kerjaschki et al. (16). Multiple bands were seen afterCoomassie blue or silver staining (Fig. 3). After staining withStains All to detect mainly sialoproteins and other anioniccomponents (27) only a few bands were seen (Fig. 3): a majorblue stained band with an apparent Mr of 140,000 (also seenin silver-stained gels) and two minor bands of about 125,000and 110,000. In addition, a red-stained major band with anapparent Mr of about 46,000 and some minor bands of lowermobility were present. Protein samples treated withneuraminidase before NaDodSO4/PAGE showed afterStains All procedure only a very faint blue staining of the

140,000 bands. In autoradiograms of nitrocellulose strips thathad been incubated with 251I-labeled Limaxflavus lectin, aMr140,000 band and further bands with higher and lowermobility were detected (Fig. 3). Directly extracted isolatedglomeruli from puromycin-treated rats showed a similarstaining pattern with Coomassie blue when compared tocontrol animals (Fig. 3), although the staining intensity ofseveral bands with high mobility was decreased after 14 daysof treatment. With Stains All the pattern of bands appearedsimilar to control animals; however, none of the bandsexhibited a blue staining (Fig. 3). Most of the red-stainedbands were less intense after 14 days of nephrosis. Inautoradiograms no bands were detectable after 7 and 14 daysof puromycin treatment.

DISCUSSION

We have used a cytochemical approach for high-resolutionlocalization of sialic acid residues in renal glomeruli ofnormaland nephrotic rats. The specificity of the Limaxflavus lectinfor N-acetylneuraminic acid a2,6- or a2,3-linked to galactose

Proc. Natl. Acad. Sci. USA 82 (1985)

Proc. Natl. Acad. Sci. USA 82 (1985) 8511

Table 1. Quantitative evaluation of the distribution of sialic acid residues on the plasma membrane of different regions of podocytes

Main body Foot process Foot process baseGlomerulum Particles/,um Length, Am Particles/Mm Length, Mm Particles/Mzm Length, Mm

Rat A1 12.0 ± 4.2 38.4 8.0 ± 2.5 56.0 3.4 ± 2.1 21.22 12.3 ± 3.0 40.1 9.2 ± 2.3 48.6 5.3 ± 2.4 19.03 13.6 ± 3.1 21.4 9.6 ± 2.4 28.4 4.0 ± 1.9 15.8X* 12.4 ± 3.8 99.9 8.9 ± 2.6 133.0 4.3 ± 2.4 56.0

Rat B1 15.4 ± 2.9 27.0 9.8 ± 1.9 45.1 3.9 ± 2.0 16.02 16.5 ± 3.1 25.9 10.3 ± 1.6 37.1 4.2 ± 2.5 12.93 14.9 ± 2.9 8.0 9.8 ± 2.2 30.4 3.9 ± 1.8 17.3X* 15.7 ± 3.0 60.9 10.1 ± 1.8 112.6 4.0 ± 2.1 46.2

Rat C1 15.5 ± 3.5 24.8 9.2 ± 1.8 36.1 3.2 ± 1.5 18.52 17.2 ± 3.9 16.0 9.6 ± 1.6 26.5 2.7 ± 1.5 22.63 18.9 ± 3.3 20.3 10.2 ± 1.5 67.3 2.8 ± 1.9 31.1X* 17.0 ± 3.8 61.2 9.7 ± 1.6 129.9 2.8 ± 1.6 72.2

*The weighted mean ± SEM number of particles per Aum for the three glomeruli examined and the total length of podocyte plasma membraneevaluated from the three glomeruli listed.

and a2,6-linked to N-acetyl-D-galactosamine as well as forN-glycolylneuraminic acid a2,6-linked to galactose and 9-0-acetylated N-acetylneuraminic acid a2,6-linked to galactose(33, 34) makes it a superior probe when compared to cationichistochemical stains, or lectins such as wheat germ agglutininor limulin. The sialic acid linkages recognized by the Limax

1 2 3 4 5 6 7 8

HI200 kDa

140kDa-

116 kDa-92 kDa

66kDa-

45kDa-

_, ;fIFIG. 3. Extracts of isolated glomeruli solubilized in NaDodSO4

sample buffer and separated on a 5-10%6 gradient polyacrylamide gel.Lanes: 1, silver staining; 2, overlay with III-labeled Limax flavuslectin of glomerular extracts separated on a 5-10%o gradient poly-acrylamide gel after transfer to nitrocellulose. Lanes 3-5, Coomassieblue staining, and lanes 6-8, Stains All staining of glomerularextracts from control animal (lanes 3 and 6), 7 days (lanes 4 and 7)and 14 days (lanes 5 and 8) of puromycin treatment of rats. Multiplebands are present after silver staining and Coomassie blue staining inthe control animals. A 140-kDa band which can be seen in silver-stained specimen is labeled with 125I-labeled Limaxflavus lectin andstains blue with Stains All in control animals. 125I-labeled Limaxflavus lectin binds in addition to bands with higher and lowermobility, and Stains All reveals further bands with higher mobility.In puromycin-treated animals, the Coomassie blue staining pattern issimilar to control animals but after 14 days of treatment the intensityof staining of several low molecular weight bands is decreased. WithStains All only red, but no more blue, stained bands can be detected.

flavus lectin are found in both N- and O-linked oligosac-charides of glycoproteins and in gangliosides (35). Thepresence of anionic sites most probably representing sialicacid has already been shown (3-8, 36). What is particularlystriking in the present work is the demonstration of aregionalized distribution of sialic acid residues in thepodocyte plasma membrane in which three domains can bedistinguished. Until now clearcut compositional differenceshave been found between the plasma membrane of the footprocess base and the rest of the podocyte plasma membraneabove the slit diaphragm with respect to the number and sizeof intramembrane particles (1, 2), of filipin binding (1, 2), orlectin labeling (11). On the basis of the cytochemicallydetectable distribution of sialic acid residues, however, wecould disclose the existence of three membrane domainswhich are (i) the foot process base below the slit diaphragm,(it) the free part of the foot process above the slit diaphragm,and (iii) the main cell body. It is not clear how this andpreviously described regionalization of other lectin bindingsites (11) are established and maintained, or how these sitesfunction. Compositional and functional epithelial cell polarityexpressed in the existence ofan apical and basolateral plasmamembrane domain is thought to be associated with thepresence of tight junctions (37-41). However, recent obser-vations on polarized virus budding indicate that the presenceof complete tight junctions is not essential for the expressionof epithelial polarity (42). Interestingly, it was noted that cellinteraction with a substrate or another cell may be sufficientto establish plasma membrane component segregation need-ed for polarized virus budding. There is also evidence thatcytoskeletal elements may be involved in the control andmaintenance of specialized distribution of plasma membranecomponents (38, 43, 44). The presence of histochemicallydemonstrable epithelial polyanion seems to be essential forthe normal arrangement of the foot processes and slits inpodocytes. This has been most convincingly shown by Seileret al. (15) in experiments where they perfused kidneys fromnormal rats with polycations to neutralize cell surface anionicsites. Glomerular epithelial changes similar to those found inpuromycin-induced nephrosis were observed together with amarkedly suppressed staining by colloidal iron suggesting theabsence of cell surface anionic sites was an important factorin the morphological changes. Reperfusion with polyanionsresulted in an almost complete restoration of the glomerularstructures especially of the foot process and slit pore ar-rangement. A loss ofanionic sites from the plasma membraneof podocytes has been shown to occur in puromycin-induced

Cell Biology: Charest and Roth

8512 Cell Biology: Charest and Roth

nephrosis (45, 46) or human nephrotic syndrome (14) con-comitantly with the 'development of foot process abnormal-ities and proteinuria. Staining by colloidal iron was absentfrom the foot process base whereas the free surface of thepodocytes showed a normal staining. Furthermore, a loss ofanionic sites (46) and of heparan sulfate proteoglycan (47) inthe glomerular basement membrane was observed inpuromycin-nephrosis. In this study we have shown by in situcytochemistry the gradual loss of sialic acid residues asvisualized with a specific lectin from all regions of thepodocyte plasma membrane in puromycin aminonucleosidenephrosis. Sialic acid residues were also not more detectableover the other glomerular structures. These findings could befurther confirmed on NaDodSO4/PAGE gels of isolatedglomeruli from nephrotic rats when processed with Stains Allor in the lectin overlay where we were not able to demon-strate sialic acid. Of great interest are the data obtained withthe Stains All procedure since they show that a 140-kDaprotein [which is according to Kerjaschki et al. (16) thepodocalyxin] is still detectable but not more positive for sialicacid. This seems to indicate that there is a defective sialyla-tion of podocalyxin under conditions of puromycin-nephrosis. During the preparation of our manuscript anabstract has been published by Kerjaschki et al. (48) withdata in accordance with our findings. They observed bio-chemically a reduced content of sialic acid and galactose ofpodocalyxin in puromycin-induced nephrosis. By immuno-electron microscopy no difference in labeling intensity forpodocalyxin was found between control and puromycin-treated rats. At present it is unclear how puromycin-treat-ment may affect the sialylation of podocalyxin and of othersialoglycoconjugates of the glomerulum. In this contextpuromycin was shown to affect the assembly of oligosaccha-ride chains in the thyroid gland (49). An effect on sugartransport in isolated rat adipocytes was reported (50). Inisolated Golgi fractions from rat liver, puromycin directlyinhibited galactosyltransferase activity upon binding to Golgimembranes (51). These data and observations by freeze-fracture electron microscopy and lectin labeling (39) provideevidence that the normal degree of plasma membrane spe-cialization in podocytes is either greatly perturbed or lost inpuromycin aminucleoside nephrosis.

Note Added In Proof. Full data on puromycin-induced podocalyxinchanges have now been published (52).

We thank Daniel Wey for preparing the photographs and EricaOesch for word processing and editing. This study was supported bythe Swiss National Science Foundation Grant 3.443-0.83 and theKanton Basel-Stadt. P.M.C. was a recipient of a postdoctoralscholarship from Fonds pour la formation de Chercheurs et Aide Ala Recherche Fund, Quebec City, Canada.

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Proc. Natl. Acad. Sci. USA 82 (1985)