Synthesis of sulfated proteoglycans by bovine glomerularendothelial cells in cu - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:32 |只看该作者 |倒序浏览 |打印

作者：JennySörensson, AnnaBjörnson, MariaOhlson, Barbara J.Ballermann,  BörjeHaraldsson作者单位：1 Department of Nephrology, GöteborgUniversity, SE-405 30 Göteborg, Sweden; and Division of Nephrology, Albert Einstein College ofMedicine, Bronx, New York 10461
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      【摘要】. ^5 G9 K1 X2 C6 {$ m
   It has been suggested that proteinuria iscaused by alterations of the charge selectivity of the basementmembrane and/or the epithelial cell layer (podocytes). However, recentfindings suggest that the endothelial luminal surface coat, consisting of proteoglycans with their connected glycosaminoglycan (GAG) branchesand glycoproteins, may contribute to the permselectivity. Therefore, wewanted to investigate the effects on endothelial GAG synthesis duringnormal and pathological conditions. We treated glomerular endothelialcell cultures with puromycin aminonucleoside (PAN, a nephrosis-inducingagent) or interleukin-1 (IL-1 ) for a total of 72 h andcompared the metabolic turnover and incorporation of[ 35 S]sulfate during the last 2 days. In control cultures,the GAG content in the media supernatants increased 66 ± 6%(mean ± SE) between 12 and 42 h of incubation withradioactivity ( P n = 8). Thecontent of 35 S-labeled GAGs in the media was reduced by31 ± 1% by PAN ( P 0.001, n = 8) and increased by 141 ± 15% by 10 U/ml IL-1 ( P 0.01, n = 8). Treatment withenzymes revealed a dominance of heparan, chondroitin, and dermatansulfate GAGs. Thus the glomerular endothelial cell production of GAGswas increased by IL-1 and reduced by PAN. Therefore, it isconceivable that certain nephrotic conditions may be due to endothelialdysfunction, rather than other renal causes.
      【关键词】 endothelium kidney glomerulus interleukin proteoglycan puromycin aminonucleoside' U" N, |3 Z% S. {# \* {: D7 ^' q
   INTRODUCTION
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GLOMERULAR PERMSELECTIVITY is mainly thought to be regulated at the level ofthe glomerular basement membrane and/or the podocyte slit diaphragm.The endothelial cells of the glomerulus are highly fenestrated and,thus, have not been considered to have prominent restrictiveproperties. However, recent findings suggest that the glomerularendothelial fenestrae may indeed be covered by a diaphragm similar tothe diaphragm that covers fenestrae of other capillary beds( 33 ). The protein PV-1, a component of the diaphragms offenestrae and caveolae, is not present in the glomerular endothelium( 40-42 ). Therefore, the glomerular endothelium mayhave properties different from those of other fenestrated capillary beds.0 W5 }# k6 v4 y$ j
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Earlier studies show a thick cell coat (glycocalyx) covering theendothelial cells ( 25, 29, 32, 46 ). The cell coat is amatrix-like gel composed of proteoglycans, with negatively charged aswell as neutral glycosaminoglycans (GAGs), glycoproteins, and plasmaproteins. In particular, the plasma protein orosomucoid seems to bevital for maintaining normal capillary permeability ( 8, 11, 12 ). Previously, our laboratory also demonstrated thatorosomucoid is produced by endothelial cells ( 37 ). The negatively charged components are abundant in this gel-like cell coat,which is suggested to constitute at least part of the charge barrier inthe kidney and other organs ( 1, 38, 39, 46 ).

The production of GAGs by the glomerular cells was first reported inthe early 1980s ( 18 ). Most studies have dealt with themesangial and epithelial cells; only a few studies have focused on theglomerular endothelial cells (GECs). Kasinath ( 21 ) studied sulfated GAGs produced by GECs under control conditions and in thepresence of transforming growth factor- 1 (TGF- 1). TGF- 1 causedan almost twofold increase in the synthesis of GAGs. Moreover, it wasshown that the endothelial cells synthesize not only heparan sulfatebut also chondroitin and dermatan sulfate ( 21, 35 ). Otherstudies measured the GAG synthesis and turnover in vitro and in vivoand in response to puromycin aminonucleoside (PAN). Akuffo et al.( 2 ) found that glomerular GAGs mainly in the glomerularbasement membrane and mesangial matrix have a more rapid turnover ratethan GAGs in other tissues. They also observed a decrease in GAGturnover rate in rats in response to PAN at day 5 oftreatment, when proteinuria was significant. A similar finding wasreported earlier for the glomerular basement membrane by Garin andShirey ( 10 ). In another study, it was shown that theglycocalyx covering the endothelial cells contains the GAG hyaluronan,which is needed to maintain a selective barrier to macromolecules( 14 ).. M  {; T9 W4 U( r1 Z
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Our concern was that because so few reports deal with the endothelialcell coat as a part of the permselective barrier in the glomeruli,its involvement in certain pathological conditions of the kidney couldhave been overlooked. To address this concern, we treated cultures ofbovine GECs with PAN or interleukin (IL)-1 to assess whether thesynthesis of sulfated GAGs was altered in any way. PAN was chosenbecause it has previously been extensively used to induce anephrosis-like condition in vivo in the rat ( 30, 34 ).IL-1 is a cytokine with a wide variety of proinflammatory actions,such as endothelial cell activation and induction of leukocyte adhesionmolecules and other cytokines, as well as inducible nitric oxidesynthase ( 9 ). Several studies have shown that IL-1increases the amount of GAG synthesis by glomerular cells ( 20, 26, 44 ). In addition, it has been shown thatincreased biosynthesis of GAGs enhances glomerular injury( 6, 45 ).

Our primary goal was to analyze the hydrodynamic size and chemicalcomposition of the molecules produced by the endothelial cells. Therate of production over time was also studied and compared with that inthe presence of PAN or IL-1.

METHODS3 v, I  e$ c7 g" q+ `  q

Medium and reagents. GECs were cultured in culture flasks (Costar, Cambridge, MA) in ahumidified, 5% CO 2 atmosphere at 37°C. The cultureflasks were coated with attachment factor (gelatin; Cascade Biologics, Portland, OR). RPMI 1640 medium was supplemented as follows:penicillin, streptomycin, and amphotericin B (Cascade Biologics), 20⺶al calf serum (FCS), bovine fibroblast growth factor(acidic, 8 ng/ml for cloning medium and 4 ng/ml for growth medium; R & D Systems), bovine brain extract (700 µl from stock solution;BioWhittaker, Västra Frölunda, Sweden), and heparin (2 µg/ml; Lövens, Malmö, Sweden). Trypsin (0.125%; LifeTechnologies, Täby, Sweden) was used for passaging of the cells,and collagenase (type IV, Sigma, St. Louis, MO) was used to digest thewhole glomeruli. Stainless steel sieves (180, 106, and 75 µm;Endecotts, London, UK) were used to extract the glomeruli./ {1 A/ i# s! K; b" |

Isolation of glomeruli. Kidneys from calves younger than 1 yr were obtained from aslaughterhouse; within minutes after harvest, they were placed inmedium containing antibiotics and stored on ice. Within 1 h afterharvesting of the kidney, glomeruli were isolated and treated withcollagenase to obtain single cells and cell clusters according to theprotocol described by Ballermann ( 5 ). The pelletcontaining single cells and cell clusters was resuspended in cloningmedium and plated onto gelatin-coated petri dishes. When theendothelial cell colonies had reached ~300 cells, they weretransferred to 24-well plates by the use of cloning cylinders. Thecells were subcloned in the event that pure cultures were not obtainedthe first time. When the cells were confluent, they were passaged andmoved to 25-cm 2 flasks, and the medium was changed togrowth medium.) U& T* R: S8 k0 r3 f4 H

Immunohistochemistry. The cells were grown in chamber slides (Becton Dickinson) precoatedwith gelatin (Cascade Biologics) until they reached 70% confluence.The cells were rinsed briefly with 0.01 M PBS-0.01% Triton X-100, pH7.4, and fixed with 4% paraformaldehyde-PBS, pH 7.4, for 15 min atroom temperature. After fixation, the cells were rinsed with PBS-TritonX-100 for 20 min. The cells were incubated in 50 mM NH 4 Clin 0.01 M PBS-0.15 M NaCl, pH 7.4, to block aldehyde groups. After theywere rinsed, the cells were treated with 0.1% Triton X-100-PBS for 30 min and incubated with 5% low-fat milk in PBS for 1 h at roomtemperature. Primary human von Willebrand factor antibody (rabbitmonoclonal; catalog no. F-3520, Sigma), diluted 1:500 in the blockingsolution, was applied, and the cells were incubated for 1 h atroom temperature. As a control for contaminating cells in the culture,smooth muscle -actin antibody (mouse monoclonal; catalog no. A-2547,Sigma), diluted 1:400, was used under the conditions described above.As a negative control, we used cells in which incubation with theprimary antibody was replaced by incubation with rabbit or mousenonspecific IgG (Sigma), and a different cell type (rat cardiomyocytes)was incubated as described above for the GECs. The cells were incubatedwith secondary antibodies conjugated with horseradish peroxidase (HRP;Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h and thendeveloped with diaminobenzidine-H 2 O 2.( ~8 m1 c' K# p6 K+ K" ~  \; L
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Further characterization of the cells using acetylatedlow-density lipoprotein. The cells were treated with trypsin and seeded onto chamber slides(Falcon, Becton Dickinson, Meylan Cedex, France) precoated with gelatin(Cascade Biologics). The cells were allowed to grow under normalconditions until they reached 70% confluence. The medium was removed,and 0.3 ml of acetylated low-density lipoprotein (Dil-Ac-LDL;Biomedical Technologies, Stroughton, MA), diluted to 10 µg/ml in RPMImedium, was added to each well. After incubation for 3 h at37°C, the cells were washed briefly three times with 0.01 M PBS, pH7.4. The cells were fixed with 3% formaldehyde for 20 min on a rotaryshaker at room temperature and then washed once with PBS. The PBS andthe chamber wells were removed, and the cells were mounted. The cellswere examined by fluorescence microscopy at 570 nm.

Biosynthesis of GAGs. GAG biosynthesis by GECs was measured by a modification of the methodof Arisaka et al. ( 3 ). The cells (P 6 ) wereallowed to grow to 70% confluence in 75-cm 2 culture flasks(Costar, Cambridge, MA) before they were stimulated with 1 nM PAN(Sigma) or 10 or 20 U/ml IL-1 (Sigma). Eight 75-cm 2 culture flasks were used for each concentration. As control, we usedeight flasks of nonstimulated cells grown under normal conditions.Stimulation was performed for 24 h at 37°C in a humidified, 5%CO 2 atmosphere. The cells were "starved" in standardmedium with 2% FCS and half of the amount of bovine brainextract during the study. For metabolic labeling of GAGs, 35 S-labeled sodium sulfate (10 µCi/ml; NEX041, NEN,Boston, MA) was added to each flask after initial treatment with thetest substances for 24 h. Conditioned media from the cell flaskswere collected 12, 24, 36, and 48 h after the 35 S-labeled sodium sulfate was added. At 72 h, afterthe cells were washed extensively with Hanks' balanced salt solution,cell layers were scraped off and the resultant cell suspension wascentrifuged. The protein content of the cell fraction was measured bythe method of Lowry et al. ( 24 ).$ C) Z5 l$ t3 J7 C6 P: g4 A6 Q
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HPLC analysis of the cell culture media. The samples from the cell culture media at different hours ofstimulation were fractionated (BioSep SEC-S3000 column, Phenomenex, Torrance, CA) according to molecular size as follows: 41-50,50-59, 102Å. Then each sample was placed in a vial with fresh quenching fluidand analyzed for 10 min in a beta counter (model L6 6500, Beckman).Calibrated standards were analyzed after every 16th sample to allowaccurate estimations of Stokes-Einstein (SE) radii.

Enzyme digestion of proteoglycans. Three different enzymes known to degrade proteoglycans were used totreat the final media samples (taken at 72 h). After digestion, the samples were fractionated in an HPLC (see HPLC analysis of the cell culture media ). After fractionation, the samples were analyzed using a scintillation counter for assessment of the amount ofdegraded GAGs compared with the same sample without enzyme treatment.

Chondroitin ABC lyase (EC 4.2.2.4, Sigma; dissolved in 1 MTris · HCl, pH 8.0), which degrades chondroitinand dermatan sulfate, in a final concentration of 0.1 U/ml wasincubated with the samples for 2 h at 37°C.% e2 J. `. w2 F* ?$ m

Heparinase III (EC 4.2.2.8, Sigma; dissolved in 0.1 M sodium acetate,pH 7.0) in a final concentration of 50 mU/ml was incubated with thesamples for 4 h at 37°C.1 C( l" x* w& b4 L4 B  M

Hyaluronidase (EC 3.2.1.35, Sigma; dissolved in sodium phosphatebuffer 0.15 M NaCl, pH 6.0) in a final concentration of 1 mg/mlwas incubated with the samples for 1 h at 37°C. This enzymedegrades hyaluronan and chondroitin sulfate (A and C), but becausehyaluronan does not contain sulfate, the treatment will reveal thechondroitin sulfate component of the GAGs.9 r) w. |5 @1 C$ {" c: W
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All three enzyme reactions were ended by lowering the temperature to 20°C.
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Western blot. The proteins were denatured and then separated on a 10% Bis-Trispolyacrylamide gel (Nupage, Novex, San Diego, CA), which was run at 200 V for 35 min. The proteins were then blotted onto a polyvinylidenedifluoride membrane (Novex) in a standard manner (3 h at 25 V and 0.25 A). After transfer, the membrane was blocked for 1 h at roomtemperature in 5% nonfat dry milk-0.25% gelatin dissolved inTris-buffered saline (10 mM Tris · HCl, pH 7.5, and 150 mM NaCl) with 0.1% Tween. The membrane was incubated for1 h at room temperature with a primary bovine heparan sulfateproteoglycan antibody (mouse monoclonal IgG1, Biogenesis, Poole, UK)diluted 1:100 in the blocking solution. After the membrane was rinsed in TBS-0.1% Tween, it was incubated with a secondary antibody conjugated with HRP (anti-mouse HRP, Amersham). The protein-antibody complexes were visualized by enhanced chemiluminescence (ECL  andHyperfilm ECL, Amersham) according to the manufacturer's instructions.% O- h" H5 r1 M8 ~  l" K0 p! _9 D
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RT-PCR. Synthesis of cDNA was carried out with 1 µg of RNA from GEC; aspositive control, we used RNA from bovine kidney. The RT reaction wascarried out for 50 min at 42°C and for 5 min at 70°C in RT buffer(Promega, Madison, WI) in the presence of 1 µg of random primer(Promega), 15 U of avian myeloblastosis virus RT (Promega), 20 U ofRNasin (Promega), and dNTP mix (1.5 mM of each base) in a total volumeof 20 µl. PCR amplification was performed in a 50-µl reactionbuffer with 5 µl of the cDNA used as a template, PCR buffer [50mmol/l KCl, 10 mmol/l Tris · HCl (pH 8.3), 1.5 mmol/l MgCl 2, and 0.001% (wt/vol) gelatin (Perkin-ElmerCetus, Norwalk, CT)], 0.2 µmol/l of each forward and reverse heparan sulfate primer (oci5), dNTP (10 nM each), and 2.5 U of Taq DNA polymerase (Promega). The following PCR program was used: 94°C for 4 min (94°C for 15 s, 54°C for 15 s, 72°C for30 s) for 30 cycles and 72°C for 10 min (GeneAmp PCR System2400, Perkin-Elmer). The sequences of the heparan sulfate primers wereas follows: 5' AAC TAC CCA AGC CTG ACT CCA C 3' (forward) and 5' ATCTCC ACC ACA CCT GCC ATA C 3' (reverse) with a product size of 479 bp. The primers were located between 541-562 (forward) and998-1,019 (reverse) bp in the human heparan sulfate cDNA sequence.
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RESULTS
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GEC culture. After ~7 days, the glomerular cells started to appear as smallcolonies of different morphology, of which about one of five was ofendothelial origin. After 12 days, we observed clones large enough fortransfer to a 24-well plate (Fig. 1 A ). The cells were ofendothelial origin as judged by light microscopy and by endocytosis ofthe endothelial cell-specific marker Dil-Ac-LDL as observed byfluorescence microscopy (Fig. 1 B ) and expression of factor VIII-related antigen. The cells stained negatively for smooth muscle -actin, and no staining was observed for Dil-Ac-LDL or factorVIII-related antigen in the negative controls.: @5 X. _: o; a: j  b9 ]' J$ L

Fig. 1. A : glomerular endothelial cells in passage 0,where cells grow out from a small cell cluster in the topright corner in a flowing manner. B :endothelium-specific marker acetylated low-density lipoproteinendocytosed by glomerular endothelial cells and visualized byfluorescence microscopy at 570 nm.
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GAG synthesis. The fractionated cell media showed that most incorporated radioactivityin the control cultures was in the hydrodynamic radius range of70-90 Å. During the control period, there was a gradual increasein the concentration of incorporated radioactivity from 12 to 42 h(mean of 36- and 48-h samples) of 66 ± 6% ( n = 8, P GAGs, namely, controls 3,180 ± 270 cpm/ml, puromycin 3,900 ± 270 cpm/ml, and IL-1 (10 U/ml)3,820 ± 590 cpm/ml, while IL-1B (20 U/ml) was significantlyincreased already at 10 min (5,800 ± 660 cpm/ml). Treatment withPAN reduced the concentration by 31 ± 1%, P n = 8, during the same time period. IL-1, onthe other hand, increased the synthesis of sulfated macromolecules significantly compared with control during the entire time period (Fig. 2 ). At 48 h, the concentration of 35 S-labeled GAGs was twice as high as at 12 h forboth IL-1 treatment groups (10 and 20 U/ml, P n = 8). Moreover, treatment with IL-1 (10 U/ml) gave a value of 9,200 ± 570 cpm/ml at 42 h.  R9 ?) p1 S- D* G$ z1 m& Y

Fig. 2. Distribution of newly synthesized glycosaminoglycans (GAGs). PAN, 1 nM puromycin aminonucleoside; IL-1, 10 and 20, 10 and 20 U/mlinterleukin-1. SE, Stokes-Einstein. Values are means ± SE( n = 8). P P
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The mean of incorporated sulfate in all four groups distributed amongSE radii reveals the same pattern of incorporation as in Fig. 2, andmost incorporated sulfate was found in the fractions of large molecules(SE radius = 70-90 Å; Fig. 3 ).. K" _" o' f- S9 A

Fig. 3. Cell medium activity of incorporated 35 S for the 7 molecular fractions. Bars represent means of 4 collections (from24-48 h). Synthesis was decreased in PAN-treated group andincreased in IL-1 -treated groups, as in Fig. 2.- N  p0 y( V+ v' f+ e) t% d

Enzyme treatments. After enzyme treatment, we found most of the cell medium activity inthe fractions containing small molecules, confirming that the largeproteoglycans had been degraded (Fig. 4 ).6 Y: U5 b0 T  y4 ?4 M; R- ]
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Fig. 4. Cell medium activity of incorporated 35 S for the 7 molecular fractions after enzyme treatment. Values represent means ofIL-1 -treated (10 and 20 U/ml) and control groups.- L$ y* t/ k4 L2 ]( F# _+ n1 }
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Compared with the enzyme-free solutions, we noted a reduction of 35 S activity in the fractions containing larger molecules(SE radii 69 Å). Similar patterns were seen in the controls andIL-1 groups, and the data were pooled. Because PAN treatment loweredthe rate of incorporation of sulfate, the activity was not high enough to allow us to determine the effects of the enzymes in this group. Cellmedium treated with chondroitinase ABC contained 54 ± 3% ofcontrol ( P 69 ± 2% of control ( P contained 76 ± 6% ofcontrol ( P hyaluronan, which is nonsulfated, and chondroitin sulfate;chondroitinase ABC degrades chondroitin and dermatan sulfate. Thismeans that heparan sulfate accounts for 30.6%, chondroitin sulfate for23.8%, and dermatan sulfate for 22.6% of the total amount of GAGs(Fig. 5 ).
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Fig. 5. Relative proportion of heparan sulfate (30.6%),chondroitin sulfate (23.8%), and dermatan sulfate (22.6%) of GAGs asdetermined by cell medium activity of incorporated 35 69 Å) from IL-1 -treated (10 and 20 U/ml)and control groups.' i* r# L/ p; T" N
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Protein synthesis. The protein concentrations were similar for the different groups( n = 8 in each group), indicating similar numbers ofcells and no apparent general toxicity of the drugs (Fig. 6 )." B2 A# v( z6 U$ m

Fig. 6. Cellular protein concentrations measured by Lowry assay.There were no significant differences between groups.

PCR. The expression of heparan sulfate proteoglycan mRNA from the differentgroups of GEC and cortex from whole kidney was examined by RT-PCR.Transcripts of the expected size (479 bp) were found in all samples(data not shown).

Western blot. Western blot analysis of heparan sulfate proteoglycan was performed,and 55-kDa bands in the protein extracts from the GECs indicate thatheparan sulfate core protein was present in all treatment groups (Fig. 7 ).% p6 Q% ^# M8 b6 v

Fig. 7. Densitometry [arbitrary units (AU)] of Western blot ofheparan sulfate core protein. PAN concentration is 1 nM. Values aremeans ± SE ( n = 2).

DISCUSSION
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The production of GAGs by GECs has been far less studied than GAGsynthesis by epithelial and mesangial cells. In this study, wedemonstrate that PAN and IL-1 affect GAG synthesis in the GECs. Thusthe amount of metabolic labeling was decreased in the PAN-treated cellcultures. When the cells were stimulated with IL-1, an increase inmetabolic labeling was observed. Because the protein concentrationswere similar in the different treatment groups (Fig. 6 ), it was mainlythe degree of sulfation that was affected. The 35 S-labeledGAG content of the culture medium was fractionated according to size byHPLC. When examining the size distribution of the samples, we found apeak at 50-90 Å. In all molecular size fractions, we found adecreased synthesis of GAGs after treatment with PAN and an increasedsynthesis after IL-1 treatment. The pattern was 70 Å). This fits well withthe notion that proteoglycans are large molecules, consisting of a coreprotein with GAG chains, such as heparan sulfate, attached ( 13, 23 ). The sulfation is carried out by several isoenzymes, and thereactions are often incomplete, leading to a large variation insulfation of the heparan sulfate GAGs ( 22, 31 ).7 b1 A- \9 n. \, L

The time-course study showed that the PAN effect had a slower onsetthan the IL-1 effect. The significant reduction induced by PAN after36 h remained at this low level at 48 h. The high dose ofIL-1 induced a significant increase at 12 h ( P and the concentration of sulfated GAGs continued to riseduring the time course of the study ( P (10 U/ml) resulted in no significant increase at12 h, but thereafter the pattern was similar to the higher dose(20 U/ml). For the control cells, the concentration of sulfated GAGsincreased slightly during the experiment (Fig. 2 ). In addition, weexamined the presence of mRNA for heparan sulfate in the RNA obtainedfrom the cells. All groups showed expression of heparan sulfate mRNA.We also obtained evidence for heparan sulfate core protein synthesis by the cells.
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It is known that human umbilical vein endothelial cells produce mainlyheparan sulfate ( 29 ). However, in a study investigating GECs, it was shown that mainly chondroitin sulfate chains were attachedto the core proteins of the proteoglycans ( 35 ). This difference in expression of GAGs could be interpreted as macro- andmicrovascular endothelium reflecting different characteristics, aspreviously noted by Zetter ( 48 ). We found that three main GAGs were present in bovine GECs: heparan, chondroitin, and dermatan sulfate (Fig. 5 ).
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PAN causes nephrotic syndrome in rats when administeredintraperitoneally ( 19, 27, 30, 34 ). It is thereforeinteresting to note that the drug also downregulates the incorporationand synthesis of GAGs by the endothelial cells. The endothelial cell coat ( 27, 30, 34 ) has been suggested to contribute to the permselective properties of the capillaries ( 1, 17, 38, 39, 47 ). A decreased synthesis of GAGs could increase permeability of the layer covering the fenestrae and, thus, increase the sieving ofmacromolecules ( 14 ). It has previously been shown that PAN has effects on the GEC fenestrations, which were decreased in size andamount during PAN-induced nephrosis ( 4 ). Thus, during certain pathological conditions, the charge barrier may be reduced as aresult of a decreased amount of negatively charged components of theendothelial cell coat. This is in line with previous observations fromour group where we showed that orosomucoid, a glycoprotein known to beimportant for the glomerular permselectivity [probably because of itshigh negative charge ( 1, 11, 12 )], was synthesized by theendothelial cells ( 37 ). Thus it seems that the GECs can modify their expression and turnover of cell coat components in response to different stimuli. The importance of the GAGs isillustrated in a study where the gene for the enzyme responsible forsulfation (heparan sulfate 2- O -sulfotransferase) of heparansulfate was knocked out. The mice did not develop kidneys as a resultof failure of ureteric bud branching and mesencymal condensation, andthey died shortly after birth ( 7 ).- I- G$ D8 ~5 z5 c7 \& D

In this study, we found marked changes in the degree of sulfation withPAN and IL-1 treatment but no significant changes in proteinconcentrations. This is in accordance with the observations ofHolthöfer et al. ( 16 ), who found similar effects ofPAN on the sulfation of podocalyxin.
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IL-1 is a proinflammatory cytokine that is involved in theacute-phase reaction during inflammation and is known to activate endothelial cells. In this study, we used one of the two available molecular forms of IL-1, IL-1. The two forms (IL-1 and IL-1 ) have similar effects and bind to the same receptors ( 9 ).Our choice of IL-1 was based on a previous study on endothelial cell activation ( 44 ). In that study, it was also shown thatGECs synthesize IL-1 constitutively, but the major effects fromIL-1 are usually thought to come from infiltrating macrophages.IL-1 has been shown to strongly and rapidly upregulate inflammatory mediators such as intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 ( 15, 43 ). It has also been shown that IL-1 upregulates the expression of the neutral GAG hyaluronan in microvascular endothelium ( 28 ). In addition toupregulating inflammatory mediators, we found that IL-1 also inducedan increased synthesis of sulfated GAGs by the GECs, an effect that canbe a part of the cell defense to maintain the permselective properties of the glomerular barrier ( 20 ). There may, however, bedifferences between micro- and macrovascular endothelium, because thesynthesis of GAGs was downregulated when porcine aortic endothelialcells were stimulated with IL-1 ( 36 ).

In summary, the GECs produced sulfated GAGs with a hydrodynamic size of50-90 Å. Heparan, chondroitin, and dermatan sulfate were thethree main GAG components synthesized by bovine GECs. The rate ofproduction of GAGs decreased during PAN treatment and increased duringIL-1 treatment. We propose that the GAGs produced by the GECs may bea vital part of the glomerular charge barrier. Indeed, certainnephrotic conditions may actually be due to endothelial dysfunction,rather than pure renal disorders.

ACKNOWLEDGEMENTS

We appreciate the gift of calf kidneys from M. Larsson(Dalsjöfors Slaughterhouse).: v" Q$ [4 t5 }; P; q) n) M
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