Protection of Endothelial Cells by Dextran Sulfate in Rats with Thrombotic Microangiopathy
Nobuaki Eto*,
Ichiro Kojima*,
Noriko Uesugi,
Reiko Inagi,
Toshio Miyata,
Toshiro Fujita*,
Richard J. Johnson,
Stuart J. Shankland|| and
Masaomi Nangaku*
* Division of Nephrology and Endocrinology, University of Tokyo, School of Medicine, Tokyo, Japan; Department of Pathology, Fukuoka University, School of Medicine, Fukuoka, Japan; Molecular and Cellular Nephrology, Institute of Medical Sciences, Tokai University, Kanagawa, Japan; Division of Nephrology, Hypertension, and Transplantation, University of Florida, Gainesville, Florida; and || Division of Nephrology, University of Washington, Seattle, Washington
Address correspondence to: Dr. Masaomi Nangaku, Division of Nephrology and Endocrinology, University of Tokyo, School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan. Phone: 81-3-5800-8648; Fax: 81-3-5800-8806; E-mail: mnangaku-tky{at}umin.ac.jp
Received for publication February 4, 2005.
Accepted for publication July 4, 2005.
The characteristic features of thrombotic microangiopathy (TMA)include glomerular and peritubular capillary endothelial cellinjury in association with loss of heparan sulfate proteoglycanson the cell surface and thrombus formation, followed by subsequentischemic tubulointerstitial damage. It therefore was hypothesizedthat dextran sulfate (DXS) may protect the kidney against endothelialdamage in a model of TMA. TMA was induced in rats by renal arteryperfusion of an antiglomerular endothelial antibody, followedby the administration of DXS or vehicle. Renal damage was assessedby histologic analysis and measurements of blood urea nitrogenand creatinine. Whereas control rats developed severe renalfailure with extensive glomerular and tubular injury, administrationof DXS significantly protected renal function and preservedthe glomerular endothelium and peritubular capillaries. Thebeneficial effect of DXS could be attributed to the abilityof DXS to protect endothelial cells from coagulation and complementactivation, as demonstrated by the histologic analysis. In addition,binding of the administered DXS to the surface of the glomerularendothelium was confirmed in TMA rats, suggesting that DXS actsas a "repair coat" of injured glomerular endothelium. In conclusion,DXS protects the kidney from experimental TMA. This protectionmay be mediated by DXS’s binding directly to the surfaceof glomerular endothelium and amelioration of coagulation, complementactivation, and cellular matrix loss.
Injury to the vascular endothelium is critical in inflammationand promotion of a procoagulant state and is likely to be ofmajor importance in the pathogenesis of various kidney disorders,such as glomerulonephritis, vasculitis, allograft rejection,ischemia-reperfusion injury, and thrombotic microangiopathy(TMA) (1–4). Endothelial injury is also involved in theinitiation and propagation of glomerulosclerosis and ESRD (5–11).
The hemolytic uremic syndrome (HUS) and related TMA are clinicalsyndromes characterized by thrombocytopenia, nonimmune hemolyticanemia, and variable degrees of renal insufficiency (10–13).Most cases of HUS are secondary to enteric infection with certainverotoxin-producing strains of Escherichia coli (particularlyO157:H7) (14), but some cases are associated with genetic defectsin complement inhibition on endothelium (15). Regardless, thehallmark of HUS is injury to the glomerular and other renalmicrovascular endothelium, characterized by arteriolar and capillaryswelling, apoptosis, and/or detachment of endothelial cells(16,17). Although most patients have complete recovery of renalfunction, a significant number of patients develop eventualend-stage renal failure. To understand better the pathophysiologyand potential therapies, we recently developed a model of TMAin rats by selective renal artery perfusion with anti-endothelialcell antibody (1). This model is characterized by severe glomerularand peritubular capillary endothelial injury, showing the characteristicfeatures of clinical TMA. Similar to severe forms of HUS, therenal injury does not resolve, and progressive glomerular andtubulointerstitial damage ensues (1,4). To date, therapy forthe renal involvement in TMA is limited.
Physiologically, endothelial cells (EC) are covered with a layerof heparan sulfate proteoglycans (HSPG), which are crucial forthe anticoagulant and anti-inflammatory properties of the endothelium.HSPG is shed from the cell membrane on activation of EC, exposinga procoagulant and proinflammatory cell surface (18). Moreover,HSPG regulate multiple functions such as leukocyte–endothelialinteractions and extravasation (19) and are released rapidlyunder conditions of inflammation and tissue damage (20).
Dextran sulfate (DXS) is a semisynthetic analog of the glycosaminoglycanfamily, which includes heparin, heparan sulfate, dermatan sulfate,and chondroitin sulfate. Several studies reported that certainglycosaminoglycans prevent the complement cascade by activatingC1 inhibitor-mediated inactivation of C1s (21–23). Wuilleminet al. (24) indicated that DXS has the most potent C1s inactivationability among various glycosaminoglycans. Taken together, wehypothesized that the DXS treatment in experimental TMA couldreduce microvascular injury and protect renal functions.
Animal Model and Experimental Study Design
Male Wistar rats were purchased from Nippon Seibutsu ZairyoCenter Co. (Saitama, Japan). All rats were housed in cages ina temperature- and light-controlled environment in an accreditedanimal care facility, with free access to water. All studiesconformed to the principles of the Guide for Animal Experimentationat the University of Tokyo.
Chemical Reagent
DXS was purchased from Kowa Pharmaceutical Company (Osaka, Japan).Injection was performed via superior mesenteric artery at afinal concentration of 50 mg/kg in saline. Biotinylated DXSwas prepared by linking hydrazide biotin to the DXS, by a commercialmethod, which is available from Dojin-Chemical (Kumamoto, Japan).
Purification of Goat Antiglomerular EC IgG
Goat anti-EC IgG for renal artery perfusion was purified byusing a caprylic acid precipitation method, as described previously(1). The purity of antiglomerular EC IgG was checked by SDS-PAGE.
Induction of the Renal TMA Model
The left kidney was removed 1 d before the induction of disease.The renal TMA model was induced by selective renal artery perfusionof the right kidney, through the superior mesenteric artery,with the purified IgG fraction of goat anti-glomerular endothelium(GEN) antibody, as reported previously (1). Kidneys were perfusedwith 0.2 ml of PBS (pH 7.2), followed by 50 mg/kg body wt anti-GENEC IgG or PBS (control). Ischemia time was <6 min.
Experimental Protocol
For investigating the effect of DXS in this model, experimentalTMA was induced as described above in 41 male rats that weighed200 to 230 g. Rats were divided into two groups: An experimentalgroup received 50 mg/kg DXS (n = 22), and a control group receivedPBS (n = 19) administered via the right renal artery 10 minafter the anti-GEN antibody perfusion. Twenty-four hours afterrenal perfusion, a blood sample was obtained via tail vein forthe measurement of serum blood urea nitrogen (BUN) and creatininelevels. Two days after induction of the disease, a blood samplewas obtained by cardiac puncture. The rats were killed, anda biopsy was taken for histologic analysis. For demonstratingfunctioning endothelium, 250 µg of biotinylated lectin(Lycopersicin esculentum lectin; Vector Laboratories, Burlingame,CA) was injected via the tail vein exactly 4 min before therats were killed.
Serum BUN and Creatinine Measurements
Serum BUN levels were determined colorimetrically with a commercialkit that used the urease-indophenol method to measure urea nitrogen(Wako Pure Chemical Industries, Tokyo, Japan). Serum creatininelevels were determined colorimetrically with a commercial kitthat used the Jaffe’s method (Wako Pure Chemical Industries).
Assessing Renal Pathology
Tissue fixed in methyl Carnoy’s solution was processedand paraffin-embedded. Three-micrometer sections were stainedwith the periodic acid-Schiff (PAS) reagent and counterstainedwith hematoxylin. An indirect immunoperoxidase method was usedto identify the following antigens: Proliferating cell nuclearantigen (PCNA) with anti-PCNA (PC10, mouse IgG2a; DAKO, Glostrup,Denmark), vascular endothelial growth factor (VEGF) with anti-VEGF(rabbit IgG; Santa Cruz Biotechnology, Santa Cruz, CA). To identifythe renal microvascular EC, we chose two methods. One is anindirect immunoperoxidase method, which used mAb JG-12 (BenderMedSystems, Vienna, Austria) (6). The other is the lectin perfusionmethod, which used biotinylated lectin (25). Single-labelingstudies used biotinylated species-specific secondary antibodies,followed by streptavidin–horseradish peroxidase (HRP)complex (DAKO). Chromogenic color was developed with 3,3'-diaminobenzidinetetrahydrochloride (DAB). Double-labeling studies were performedas described previously (25), using biotinylated species-specificsecondary antibodies, followed by streptavidin-HRP complex (DAKO),with labeling being revealed using DAB as a chromogen (brown).After staining the first antigen, the remaining peroxidase activitywas extinguished with 3% H2O2 in methanol for 10 min. The remainingbiotin was blocked by incubation with avidin solution (VectorLaboratories), after which the first antibodies for the secondantigen were applied, followed by suitable secondary biotinylatedantibodies. Color was developed with DAB plus 0.08% nickel (black).Paraffin sections were also applied to the terminal dUTP nick-endlabeling (TUNEL) staining method using a commercially availablekit (Trevigen, Gaithersburg, MD).
Tissues for immunofluorescent staining were embedded in OCTcompound (Miles, Inc., Elkhart, IN) and snap-frozen in liquidN2. Four-micrometer frozen sections were cut on a rotary microtomeand mounted onto silanated microscope slides. Fibrin was detectedby staining with FITC-conjugated goat anti-fibrinogen IgG (Cappel,Durham, NC). Deposition of the pathogenic goat anti-EC antibodywas detected with a biotinylated anti-goat IgG (secondary antibody;DAKO), followed by incubation with Oregon green/Neutralite avidin(Molecular Probes, Eugene, OR). C3 deposition was detected bystaining with FITC-conjugated goat anti-rat C3 (Cappel). Frozensections were also used to an indirect immunoperoxidase stainingmethod. To identify heparan sulfate, anti–heparan sulfatemouse IgM (JM403, Seikagaku Kogyo, Tokyo, Japan) was used, followedby anti-mouse IgM-HRP complex (Cappel).
Quantification of Renal Histology
Quantification was performed in a blinded manner on 25 randomlyselected glomeruli/perfused kidney on all experimental and controlanimals, for each of the following variables. For glomerularthrombi, a semiquantitative scoring system was used as follows:0, normal; 1, 0 to 25% of glomerular area involved; 2, 25 to50% of glomerular tuft area involved; 3, 50 to 75% of tuft areainvolved; 4, >75% of tuft area involved. For glomerular ECanalysis, a semiquantitative scoring system was also used asfollows: 0, no positive glomerular tuft staining for endothelium;1, 1 to 25% of glomerular tuft positive for endothelium; 2,25 to 50% positive; 3, 50 to 75% positive; 4, 75 to 100% positive.PAS stain quantification was performed separately in two differentparts. For glomerular scoring, 25 randomly selected glomeruliwere quantified as follows: 0, normal; 1, 0 to 25% of glomerulararea involved; 2, 25 to 50% of glomerular tuft area involved;3, 50 to 75% of tuft area involved; 4, >75% of tuft areainvolved. For tubular scoring, 25 randomly selected x400 pictureswere quantified as follows: 0, normal; 1, 0 to 25% of tubulearea involved; 2, 25 to 50% of tubule area involved; 3, 50 to75% of tubule involved; 4, >75% of tubule are involved.
For analysis of C3 and anti-GEN antibody deposition, image analysiswas used. In brief, the photographs of 20 randomly selectedglomeruli were saved in JPEG files, and the electric densitieswere measured using an image processing and analysis programImage-J software (NIH, MD). The percentage of positive areain glomeruli was calculated under the threshold setting by 130to 255.
Binding of Biotin-Labeled DXS to Vasculature in Rats with TMA
Left kidney was removed 1 d before the experiment. Rats withTMA were induced by selective renal artery perfusion of theright kidney with the goat anti-glomerular EC antibody as describedabove. Rats without TMA were induced by selective renal arteryperfusion of the right kidney with PBS. After 10 min, both groups’kidneys were perfused with 50 mg/kg biotin-labeled DXS. Oneday after the injection, rats were killed and tissues were embeddedin OCT compound and snap-frozen in liquid N2. Deposition ofthe DXS in the kidney was detected with biotinylated DXS, followedby incubation with streptavidin Alex488 (Molecular Probes, Eugene,OR).
Electron Microscopy
Kidney specimens for electron microscopy (EM) were fixed inice-cold 1.4% glutaraldehyde (pH 7.4), postfixed in osmium tetroxide,dehydrated in graded ethanols, and embedded in epoxy resin.Ultrathin sections were stained with uranyl acetate and leadcitrate and examined with a JEM-100CX II (Nihon Denshi, Tokyo,Japan) electron microscope.
Statistical Analyses
Differences between the DXS and vehicle-treated groups wereevaluated with the unpaired t test. Nonparametric data, includingthe histologic scores, were analyzed by Mann-Whitney test. Valueswere considered significant at P < 0.05.
Renal Function Preserved in Animals Treated with DXS
Serum BUN and creatinine levels of normal rats at the same agesof experimental TMA rats were 14.88 ± 1.01 and 0.58 ±0.03 mg/dl, respectively. There was a significant increase inserum BUN levels in control TMA rats that were given vehicletreatment on day 1 and day 2 with measures of 57.10 ±6.11 and 63.53 ± 9.99 mg/dl, respectively (P < 0.001versus normal). In contrast, serum BUN levels in TMA rats thatwere given DXS treatment on day 1 and day 2 were 41.65 ±5.14 and 40.98 ± 6.39 mg/dl, respectively (P < 0.05versus vehicle treatment at each time point).
Similar to BUN, DXS treatment significantly improved serum creatininelevel compared with control TMA rats that were given vehicletreatment on day 1 (0.84 ± 0.08 and 1.07 ± 0.10mg/dl, respectively; P < 0.05) and on day 2 (0.86 ±0.11 and 1.30 ± 0.21 mg/dl, respectively; P < 0.05).Taken together, these results indicated that DXS treatment improvedrenal function in rats with experimental TMA.
DXS Administration Ameliorated Histologic Injury in Rats with TMA
Light microscopic analysis with PAS staining revealed characteristicchanges of TMA in the control vehicle-treated group (Figure 1).Glomeruli demonstrated generalized hypocellularity, withindistinct capillary walls, often accompanied by intraluminalthrombi. Many tubules were dilated, often with sloughing ofepithelial cells into the tubule lumen, cast formation, or patchyareas of necrosis. In contrast, both the glomerular and tubularabnormalities were significantly improved in the DXS-treatedrats (Figure 1, Table 1).
Figure 1. Renal histologic injury in experimental thrombotic microangiopathy (TMA). Periodic acid-Schiff staining showed that the rats with TMA displayed thrombi in glomerular capillary loops, tubular necrosis, sloughing of tubular epithelial cells, and tubular cast formation (A and C). Dextran sulfate (DXS)-treated rats had less injury and showed preservation of glomerular and tubular architecture (B and D). Magnification, x200 in A and B; x400 in C and D.
Table 1. Semiquantitative analysis of histologic and immunochemical studiesa
Thrombus Formation
For evaluating thrombus formation in TMA, glomerular fibrindeposition was assessed by immunofluorescent staining with antifibrinogenantibody. Glomerular thrombi were common and more severe incontrol TMA vehicle-treated rats, compared with DXS-treatedrats with TMA, although the difference did not reach statisticalsignificance (0.80 ± 0.14; DXS-treated group, 1.48 ±0.25; vehicle-treated group; P = 0.063; Figure 2, A and B, Table 1).
Figure 2. Thrombus formation and complement activation in the glomeruli of TMA rats. Marked deposition of fibrin was observed in the glomeruli of TMA rats (A). DXS treatment demonstrated less fibrin deposition (B). Similarly, marked deposition of C3 was observed in the glomeruli of TMA rats (C). DXS treatment resulted in less C3 deposition in glomeruli (D). Magnification, x400.
Complement Activation Inhibited by DXS in TMA Animal
Complement activation in glomeruli, a previously reported featureof this model, was assessed by immunofluorescent staining withanti-C3 antibody. C3 deposition was observed along the capillarywall in both experimental and control animals. However, theintensity of C3 staining was significantly decreased by DXStreatment (0.96 ± 0.24%; DXS-treated group, 26.78 ±5.76%; vehicle-treated group, P < 0.0001; Figure 2, C andD, Table 2).
Table 2. Image analysis of histologic and immunochemical studies
DXS Administration Protected Glomerular Endothelium and Vasculature
Changes in EC morphology and density were studied by stainingtissue sections with the EC-specific mouse mAb JG-12. The majorityof glomeruli and peritubular capillaries demonstrated a decreasein JG-12 staining in control TMA rats (Figure 3A). However,the reduction in JG-12 staining was significantly milder inexperimental TMA rats given DXS (vehicle-treated group 1.85± 0.12; DXS-treated group, 2.97 ± 0.14; P <0.0001; Figure 3B, Table 1). In addition, lectin staining showedthat glomerular endothelium and peritubular capillaries wereseverely damaged in vehicle-treated rats (Figure 3C). Thesedata showing preservation of glomeruli endothelium suggestedthat DXS might be acting as a protectant factor for EC.
Figure 3. Preservation of the glomerular and peritubular capillary network by DXS treatment. JG-12 staining demonstrated preservation of the glomerular and peritubular capillary endothelium in TMA rats with DXS treatment (B) than in TMA rats without DXS treatment (A). Sections were double-stained with biotinylated lectin and anti–proliferating cell nuclear antigen (PCNA). Lectin staining (brown) demonstrated preservation of the glomerular and peritubular capillary endothelium in TMA rats by DXS treatment (D) than in TMA rats without DXS treatment (C). In contrast, PCNA-positive cells (black) in the glomeruli without DXS treatment (D) were markedly increased than those in the glomeruli without DXS treatment (C). Expressions of vascular endothelial growth factor in the glomeruli without DXS treatment (E) were markedly increased than those in the glomeruli without DXS treatment (F). Magnification, x400.
Cellular Apoptosis and Proliferation
TUNEL-positive signal had not been seen in the glomeruli ofboth vehicle- and DXS-treated groups (data not shown). However,a diffuse and significant increase in the number of PCNA-positivecells was noted in vehicle-treated glomeruli (Figure 3C), althoughfew PCNA-positive cells were observed in the glomeruli in DXS-treatedrats (Figure 3D). Nuclear positive signals for PCNA were observedin the glomeruli without lectin staining. It is interestingthat staining of VEGF showed a similar pattern; positive signalswere observed in vehicle-treated glomeruli but not in DXS treatment(Figure 3, E and F).
EM Analysis
In control TMA vehicle-treated rats, glomeruli showed severestasis of red blood cells within dilated capillary lumen (Figure 4A).Fragmented red blood cells and fibrin strands were occasionallynoticed. Glomerular capillaries demonstrated diffuse subendothelialexpansion and swelling or degenerative changes of EC (Figure 4B).In contrast, glomeruli in TMA DXS-treated rats maintainednormal structure, and endothelial fenestrations remained intact(Figure 4, C and D).
Figure 4. Electron microscopic analysis of the glomeruli in control TMA vehicle-treated rats (A and B) and TMA DXS-treated rats (C and D). (A) Marked reduction or occlusion of the capillary lumina by red blood cells, fibrin strands (F), expanded subendothelial space, and swelling of endothelial cells (EC). Electron lucent fluffy materials (arrow), fragmented red blood cells (*), and fibrin strands occupied the subendothelial spaces. Endothelial cells (E) had enlarged cytoplasm with an increased number of organelle. (B) In the most severely affected area, capillary lumina revealed irregular dilation with marked fibrinous exudate (F), amorphous material, cellular debris (arrowhead), and fragmented blood cells. Denudation and fragmentation of EC (arrow) was noticed. (C) Almost normal configuration of a glomerulus. A few red blood cells could be detected in the loops. (D) Glomerular EC showed no remarkable change and preserved fenestrations (arrow). Magnification, x2000 in A, B, and C; x12,000 in D.
Binding of Biotin-Labeled DXS to Vasculature in Rats with TMA
To confirm the binding of DXS to the glomerular endothelium,rats with TMA or without TMA received biotin-labeled DXS injection10 min after injection of anti-GEN antibody or PBS, respectively.One day after injection, kidney sections were analyzed by fluorescencemicroscopy. Biotin-DXS staining was detected on the surfaceof the glomerular endothelium in the kidney with TMA, whereasno staining was detected in the kidney without TMA (Figure 5).These results indicated that DXS could bind only to the surfaceof injured endothelium.
Figure 5. Binding of biotin-labeled DXS to rat glomerular endothelium. Marked binding of DXS was observed in the glomeruli of TMA-induced rats (A). In contrast, no binding of DXS was observed in the glomeruli of non-TMA rats (B). Magnification, x200.
DXS Does Not Affect Anti-GEN Antibody Binding
Finally, to ensure that the results of this study were not duesimply to differences in the binding of the disease-inducingantibody, we examined anti-GEN antibody deposition by stainingtissue sections with the anti-goat IgG antibody. The densityof goat anti-GEN was observed without any differences betweenvehicle-treated and DXS-treated rats (Table 2).
TMA is a significant cause of acute renal failure, and in someinstances, patients progress to ESRD. The experimental modelof TMA has provided a unique opportunity to study disease mechanismsand potential therapies. For example, VEGF protects the kidneyin this model (26). The beneficial effect of VEGF may comprisenot only a survival factor but also an angiogenic factor forglomerular capillary endothelium. Neutralization studies ofa specific complement regulatory protein (27) and studies withC6-deficient animals have also shown a crucial role of complementactivation in glomerular endothelium injury in this model (28).In this study, we show that DXS injection after the injectionof antiglomerular EC antibody protected the glomerular endotheliumand preserved renal functions.
Although TMA can be caused by several diseases, they all sharecommon histologic features within the kidney. These includesignificant renal microvascular EC injury, complement activation,platelet infiltration, fibrin deposition, and renal failure(1,4). These changes are due to injury primarily to the glomerularEC. This provides the rationale for our study to test the hypothesisthat DXS, a member of the glycosaminoglycan family, can protectanimals with experimental TMA. The major findings in our studywere that DXS treatment significantly improved renal function,and this was accompanied by marked preservation of tissue injury.Staining with JG-12 antibody and estimation of glomerular endotheliumby perfusion of biotinylated lectin demonstrated that DXS treatmentprotected the glomerular endothelium from the damage in ratswith experimental TMA. We observed more PCNA-positive cellsas well as upregulation of VEGF in vehicle-treated glomeruli.It is likely that these results reflect compensatory responsesagainst glomerular endothelial damage, as we reported previously(1).
DXS can protect the endothelium via several potential mechanisms.DXS has an anticoagulation effect, although the affinity ofDXS to antithrombin is three orders smaller than the affinityof heparin (29). It has also been known for a decade that lowmolecular weight DXS is an efficient inhibitor of complementactivation (24). Indeed, our results show that glomerular C3staining was reduced in the DXS group compared with control.Studies have shown that DXS inhibits all three pathways of complementactivation and dose-dependently protected pig cells from depositionof human complement, and the EC-protective effect of DXS correlatedwith binding of the substance to the cells (30).
Complement-mediated EC activation and damage have also beendemonstrated in the pathophysiology of acute vascular rejectionin xenotransplantation (31). DXS inhibits complement in vitroexperiments using human serum and porcine cells (32). Similarly,DXS inhibited complement in vivo by preventing hamster cardiacxenografts from undergoing acute vascular rejection, and DXSin combination with cyclosporin A significantly prolonged xenograftsurvival rate (33). Taken together, we postulate that DXS amelioratedrenal injury in our study by inhibiting the coagulation cascadeand by inhibiting complement activation.
Another important mechanism of endothelium protection was proposedby Laumonier et al. (30), who suggested that DXS acted locallyand might functionally replace HSPG that are known to be shedfrom the EC surface upon activation. HSPG modulate the actionsof a large number of extracellular ligands (34,35) and are involvedin the preservation of the critical anticoagulant surface ofvascular EC (36). They called these effects of DXS a "repaircoat" by re-establishing an anticoagulant and anti-inflammatorysurface (30). In this study, by using a specific antibody forglomerular heparan sulfate, we showed that most parts of glomerularloops lost HSPG in TMA-induced rats, whereas HSPG covered allparts of glomerular loops in normal rats (Figure 6). In addition,by using biotin-labeled DXS, we showed that DXS binds to theglomerular endothelium in TMA-induced rats. The binding of DXSto the endothelium requires EC damage, because DXS did not bindto the endothelium of uninjured normal rats.
Figure 6. Immunodetection of heparan sulfate proteoglycan (HSPG) molecules in the glomeruli. HSPG covered the glomerular capillary loops in normal rats (A). However, marked decrease of HSPG was observed in the glomerular capillary loops of TMA-induced rats (B). Magnification, x400.
Recent analysis has revealed that HSPG exists on the surfaceof the glomerular basement membrane (GBM) and endothelium (37).HSPG on the GBM is considered to play the key role for chargeselective moiety of glomerular protein filtration, because quantitativechanges in HSPG have been observed in a number of proteinuricnephropathies (38). HSPG on the glomerular endothelium is consideredto play a role in leukocyte infiltration, because lack of HSPGpromotes the increase in vascular permeability and leukocyteextravasation under inflammatory conditions (19). However, proteinuriaor leukocyte infiltration into the glomeruli could not be observedin this model of TMA (1). This result also suggests that replacedDXS bears functions, such as "repair coat," other than havingbeen previously reported. Another possible mechanism of renoprotectionby DXS is prevention of the deposition of anti-GEN IgG on thesurface of glomerular endothelium, but this is unlikely becauseour immunofluorescence studies demonstrated that the depositionof anti-GEN IgG was not different between the two groups.
Although DXS injection via right renal artery was effectivein this study, intravenous injection of DXS at the same dosagehad no beneficial effects on renal functions, EC injury, orcomplement activation (data not shown). A possible explanationfor this difference by injection route might be dose-dependenceof DXS, i.e., a much higher dose of DXS might be needed. Itshould be noted that no signs of acute toxicity of DXS administrationwere observed by either way of injection.
In conclusion, DXS has an important protective effect in experimentalTMA. This is likely mediated by complement inhibition, anticoagulation,and endothelial protection by re-establishing the intact surfaceof injured endothelium. Supplementation of glycosaminoglycansmay present a new approach for treatment of glomerular endothelialinjury.
Acknowledgments
We thank Jeff Pippin (University of Washington, Seattle, WA)and Yuko Izuhara (Tokai University School of Medicine, Kanagawa,Japan) for technical assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication February 4, 2005.
Accepted for publication July 4, 2005.
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Abstract
Introduction
