CD44 Disruption Prevents Degeneration of the Capillary Network in Obstructive Nephropathy via Reduction of TGF-1–Induced Apoptosis
Kasper M.A. Rouschop,
Nike Claessen,
Steven T. Pals,
Jan J. Weening and
Sandrine Florquin
Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands
Address correspondence to: Dr. Sandrine Florquin, Academic Medical Center, Room M2-108, PO Box 22668, 1100 DD Amsterdam, The Netherlands. Phone: +31-20-5664240; Fax: +31-20-6960389; E-mail: s.florquin{at}amc.uva.nl
Received for publication August 3, 2005.
Accepted for publication December 15, 2005.
CD44 is a glycoprotein that is involved in inflammation andcell–cell/cell–matrix interactions, is upregulatedin the kidney upon injury, and leads to fibrosis through enhancementof TGF-1 signaling. Absence of CD44 prevents development ofrenal fibrosis in unilateral ureteral obstruction (UUO). A hallmarkof development of renal fibrosis is the degeneration of thecapillary network. This study shows that CD44 is upregulatedon capillary endothelial cells during UUO. For elucidation ofthe role of CD44 on peritubular endothelial cells in UUO, capillarynetwork degeneration was compared in CD44+/+ and CD44–/–mice. As expected, degeneration of the capillary network wasobserved in CD44+/+ mice during UUO, associated with increasedendothelial apoptosis. However, in the absence of CD44, degenerationof the network is prevented as a result of a decrease in therate of apoptosis in endothelial cells. The divergence in endothelialapoptosis is not correlated to differential vascular endothelialgrowth factor or thrombospondin-1 expression. For further investigationof capillary regression, CD44+/+ and CD44–/– peritubularcapillary endothelial cell lines were established. With theuse of these cells, it is shown that interaction between CD44and its ligand hyaluronic acid enhances the proapoptotic effectof TGF-1 but not thrombospondin-1 on endothelial cells, contributingto the degeneration of the capillary network. Blocking interactionbetween hyaluronic acid and CD44 therefore may be a potentialtherapeutic opportunity to preserve the capillary network andprevent the development of fibrosis in chronic renal disease.
An important determinant of progression of renal disease istubulointerstitial damage, histologically characterized by tubulardilation, atrophy, and accumulation of extracellular matrix(ECM) proteins that lead to the development of fibrosis. CD44is a family of glycoproteins that are encoded by a single genethat consists of 19 exons. Alternative splicing generates differentisoforms and confers specific functions to the CD44 protein(1–4). These isoforms have been implicated in many importantphysiologic and pathologic processes, such as cell–celland cell–matrix interaction, lymphocyte extravasation,wound healing/scarring, cell migration, lymphocyte activation,and binding/presentation of growth factors (5–8).
The shortest (standard) isoform of CD44 does not contain anyof the alternative domains and binds hyaluronic acid (HA) andosteopontin, which are two major components of the extracellularmatrix. The interaction between CD44 and HA is involved in extravasationof inflammatory cells (9,10) and facilitates TGF-1 signaling(11,12). CD44 isoforms that contain the domain encoded by variableexon 3 (CD44v3) contain a heparan sulfate–binding siteand therefore are able to bind several growth factors and facilitatetheir signaling (8).
CD44 is not expressed in the normal kidney, but it is rapidlyupregulated after injury in a number of animal models for kidneydiseases (10,12–15) and human nephropathies (16,17). Previously,we showed that CD44 is crucial in the development of renal fibrosisthrough enhancement of TGF-1 signaling (12). CD44 is also involvedin the maintenance of tubular architecture and reduces tubulardamage in unilateral ureteral obstruction (UUO) by enhancementof beneficial growth factor signaling, such as hepatocyte growthfactor (12,18).
An important contributor to persistent renal injury and progressiveinterstitial fibrosis is the loss of peritubular capillaries(19). Endothelial cell loss is associated with both increasedcell death and decreased cell proliferation. Accordingly, preservationof the capillary network prevented development of fibrosis inthe remnant kidney (20–22).
Various studies have underlined the opposing functions of vascularendothelial growth factor (VEGF) and thrombospondin-1 (TSP-1)in the process of capillary degeneration. In normal kidney,the angiogenic, survival, and trophic factor VEGF is constitutivelyexpressed mainly on tubular epithelial cells. In progressiverenal disease, the expression of VEGF is altered (20–22).In contrast, TSP-1 exerts powerful antiangiogenic actions bycounteracting VEGF-mediated endothelial cell proliferation (23),and it induces endothelial cell apoptosis (24–26). Expressionof TSP-1 is restricted mainly to the interstitium of damagedkidneys. Because both VEGF and TSP-1, the two major mediatorsimplicated in capillary network preservation/regression, areheparan sulfate–binding proteins (24,27) and thus theoreticallyable to bind CD44v3, leading to enhanced signaling, we examinedthe role of CD44 in the degeneration of the peritubular capillaryendothelial network during UUO.
Mice and Experimental Protocol
CD44 knockout on C57Bl/6 background (CD44–/–) (28)and C57Bl/6 wild type (CD44+/+) male mice (6 to 8 wk old) werebred in our animal facility. Right kidney UUO or sham surgerywas performed as described previously (12). Mice were killedhumanely (n = 6 for each group) at days 1, 3, 7, and 14 afterUUO or sham surgery. All experimental procedures were approvedby the Animal Care and Use Committee of the University of Amsterdam(Amsterdam, The Netherlands).
Histology and Immunohistochemistry
Renal tissues were fixed in 10% formalin for 12 h and embeddedin paraffin in a routine manner. For immunohistochemistry, antigenretrieval was performed by incubation in a 10-mM sodium citratesolution (pH 6.0) for 10 min at 98°C in a microwave oven,except for VEGF (clone ab-4; Calbiochem, Darmstadt, Germany)and TSP-1 (Calbiochem), which was performed in 10 mM Tris/1mM EDTA (pH 9). After endogenous peroxidase activity was blockedwith 0.3% H2O2 in methanol and free protein binding sites wereblocked with normal goat serum, sections were probed with theantibody. As a negative control, we used species- and isotype-matchedantibodies. After incubation with the secondary (horseradishperoxidase [HRP] labeled) antibodies (DAKO, Glostrup, Denmark),bound antibodies were visualized by developing peroxidase activityusing 3,3-diamino-benzidine tetrachloride (Sigma Chemical Co.,St. Louis, MO). Double immunostaining for endothelial cellsand CD44, proliferation, or apoptosis was identified by immunostainingwith CD34 (Cedarlane, Hornby, ON, Canada) and respectively anti-CD44(clone IM7.8.1; ATCC, Livermore, CA), anti–proliferatingcell nuclear antigen (anti-PCNA; DAKO), or anti-active caspase-3(Cell signaling Technology, Beverly, MA). The anti-CD34 antibodywas probed with a secondary antibody anti-rat IgG2a–alkalinephosphatase (AP) (Zymed, San Francisco, CA), CD44 with anti-ratIgG2b-HRP, PCNA with an anti-mouse IgG2a-HRP, and caspase-3with anti-rabbit IgG-HRP. AP activity was visualized with VectorBlue, and HRP activity was visualized with Novared (Vector Laboratories,Burlingame, CA).
Flow Cytometric Analysis
Before surgery, 3 and 7 d after UUO, kidneys were harvestedand single-cell suspensions were made by straining the tissuethrough a 40-µm mash. Erythrocytes were subjected to hypotoniclysis. The cell suspensions were incubated with anti–CD34-FITCand CD44-PE (both BD Pharmingen, Erembodegem, Belgium) for 60min at 4°C. CD44 expression on endothelial cells was analyzedby flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes,NJ).
Histopathologic Scoring
CD34-positive areas and TSP-1 expression in the renal cortexwere analyzed using a digital image analysis program (Imagepro-plus, Mediacybernetics, Gleichen, Germany); values are expressedas a percentage of the total cortex. Glomeruli and arterieswere excluded from quantification. To evaluate the number ofproliferating and apoptotic peritubular capillary endothelialcells (CD34-positive cells), we analyzed 10 randomly chosen,nonoverlapping fields (x200). Values are expressed as cells/mm2.VEGF expression was analyzed as the percentage VEGF-expressingtubuli.
Isolation of Peritubular Capillary Endothelial Cells
Immortomice (CBA/ca x C57Bl/10 hybrid; Charles River Laboratories,Maastricht, The Netherlands) were crossed with CD44+/+ and CD44–/–for more than four generations. Kidneys of CD44+/+ and CD44–/–mice homozygous for the temperature-sensitive SV40 large T antigen(H-2kb-tsA58) were collected. Removal of the glomeruli was performedas described by Takemoto et al. (29). In short, kidneys wereperfused with 10 x 107 magnetic beads (Ø 4.5 µm;Sigma) in PBS. Kidneys were cut into small pieces (approximately1 mm3) and digested in 1 mg/ml collagenase A (Roche Diagnostics,Mannheim, Germany) in Hanks balanced salt solution, for 15 minat 37°C. The digested kidneys were pressed through a 100-µmcell strainer. Glomeruli were removed using a magnetic particleconcentrator. In addition, the remaining cell suspension wasfiltered through a 40-µm cell strainer to remove any residualglomeruli.
Peritubular capillary endothelial cells were isolated as describedby Langley et al. (30). In short, the remaining cell suspensionwas grown in DMEM supplemented with 10% FCS and 2 mM l-glutamine(all Life Technologies, Rockville, MD) and 25 ng/ml IFN- (R&DSystems, Minneapolis, MN) at 33°C until confluence. Confluentcells then were placed for 8 d at 37°C without additionof IFN-. These cells then were stimulated using 10 ng/ml murineTNF- (R&D Systems) for 4 h. Cells were labeled with vascularcellular adhesion molecule-1 (VCAM-1)–FITC and E-selectin–PE(both BD Pharmingen). Peritubular capillary endothelial cellswere sorted using a FACSaria (BD Pharmingen). Purified endothelialcells were cultured in medium as described above with additionof IFN- in permissive conditions. Cells were cultured for 8d without IFN-; in this period, expression of SV40 is lost (datanot shown) and cells differentiate into their original phenotype.
Differentiated endothelial cells were detached from the culturesystem using 1 mM EDTA. Endothelial cells were characterizedusing antibodies directed against CD31 (Abcam, Cambridge, UK),von Willebrand factor (vWF, F8; DAKO), and CD34-PE and CD44-PE(both from BD Pharmingen). Secondary antibodies goat anti-rat–FITC(CD31-staining) and goat anti-rabbit–FITC (vWF staining)were obtained from DAKO.
Peritubular Capillary Endothelial Cell Apoptosis
Fully differentiated CD44+/+ and CD44–/– peritubularcapillary endothelial cells were incubated at low serum (0.5%FCS) for 16 h followed by a 24-h incubation with TGF-1 (R&DSystems) or TSP-1 (Sigma) with or without addition of 50 µg/mlHA (derived from rooster comb; Sigma). Apoptosis of endothelialcells was determined by flow cytometry, using Annexin-V–FITC(BD Pharmingen) and 7-amino-actinomycin D (7-AAD) (Via-Probe;BD Pharmingen). Annexin-V–positive cells but negativefor 7-AAD were considered apoptotic.
Statistical Analyses
Multiple comparisons were performed using a repeated measuresANOVA corrected by a Bonferroni post hoc test.
De Novo CD44 Expression on Peritubular Endothelial Cells during UUO
Under normal conditions, CD44 is not expressed in the kidneyexcept on passenger leukocytes. Already 3 d after UUO, we observedde novo expression of CD44 on the capillary network of CD44+/+mice, as determined by double immunostaining for CD34 and CD44(Figure 1A). Flow cytometric analysis of a single cell suspensionprepared from renal tissue 3 and 7 d after UUO confirmed CD44expression on CD34-positive capillary endothelial cells (Figure 1B).In sham-operated kidneys, the percentage of CD44-positiveendothelial cells (CD34-labeled cells) was approximately 5%.The number of CD44-positive cells increased after UUO to reachapproximately 30% after 3 d (data not shown) and 40% after 7d.
Figure 1. Capillary endothelial cells express CD44 after injury. Double immunostaining for CD44 (red) and CD34 (blue) reveals coexpression as soon as 3 d after unilateral ureteral obstruction (UUO; A). Flow cytometric analysis of endothelial cells (CD34 positive) in total kidney cell suspensions revealed CD44 expression after UUO (B). Only 5% double labeled (CD34 and CD44), of the total CD34-positive cells, expressed CD44 after sham operation. Seven days after UUO, 40% of the CD34-positive cells expressed CD44. Magnification, x600 in A.
CD44 Deficiency Decreases Capillary Network Degeneration
In progressive renal diseases, development of fibrosis is associatedwith capillary network regression. To unravel the role of CD44on peritubular capillary endothelial cells during UUO, we comparedthe capillary network in CD44+/+ and CD44–/– mice.
Seven days after UUO, regression of the capillary network inthe CD44+/+ mice was apparent, whereas the capillary networkof the CD44–/– mice was still preserved (Figure 2).Degeneration of the capillary network correlated to thedevelopment of fibrosis (r = –0.52, P < 0.001) as determinedby CD34 staining and hydroxyproline contents (12).
Figure 2. CD44 deficiency prevents capillary regression. Immunostaining for CD34 revealed degeneration of the capillary network in CD44+/+ (left) mice () but not in CD44–/– (right) mice (); staining is quantified by digital analysis. Data are presented as mean ± SEM; n = 6. Magnification, x100.
Degeneration of the capillary network could be due to decreasedproliferation and/or increased apoptosis of the capillary endothelialcells in CD44+/+ as compared with CD44–/–. We observeda sharp rise in the number of proliferating endothelial cellsat day 3 after UUO, which, however, was similar in both phenotypes(Figure 3A). Apoptosis of endothelial cells increased afterUUO in both phenotypes. However, 7 and 14 d after UUO, we observeda two-fold increase in the number of apoptotic peritubular capillaryendothelial cells in CD44+/+ compared with CD44–/–kidneys (Figure 3B).
Figure 3. Increased endothelial apoptosis in CD44+/+ compared with CD44–/– obstructed kidneys. (A) Double immunostaining for proliferating cell nuclear antigen (PCNA; red) and CD34 (blue; as indicated by arrows) revealed no differences in endothelium proliferation in CD44+/+ kidneys () and CD44–/– kidneys () after UUO; data are presented as double-positive cells/mm2. (B) Double staining for active caspase-3 (red) and CD34 (blue; as indicated by arrow) revealed increased apoptosis in CD44+/+ () compared with CD44–/– () of peritubular endothelial cells; data are presented as double-positive cells/mm2. All data are presented as mean ± SEM; n = 6. Magnification, x400.
Similar Expression Levels of VEGF and TSP-1 in CD44+/+ and CD44–/– Obstructed Kidneys
Because VEGF has mitogenic and protective functions on endothelialcells, we determined VEGF expression during UUO. VEGF was constitutivelyexpressed on tubular epithelial cells. During UUO, VEGF expressionwas still observed on intact tubuli (Figure 4), but dilatedtubuli failed to express VEGF. No differences were detectedbetween CD44+/+ and CD44–/– obstructed kidneys.
Figure 4. Vascular endothelial growth factor (VEGF) expression after UUO. Immunostaining for VEGF revealed comparable expression in CD44+/+ (top) kidneys () and CD44–/– (bottom) kidneys (). Data are presented as mean ± SEM; n = 6. Magnification, x200.
TSP-1 is a very potent proapoptotic molecule for capillary endothelialcells. TSP-1 expression was confined to the tubulointerstitialarea in UUO. Despite significant differences in apoptotic rateof peritubular capillary endothelial cells, digital image analysisrevealed no differences in expression of TSP-1 between CD44+/+and CD44–/– mice (Figure 5). To unravel the pathogenicmechanism of endothelial CD44 expression, we established CD44+/+and CD44–/– peritubular capillary endothelial celllines.
Figure 5. Thrombospondin-1 (TSP-1) expression after UUO. Immunostaining for TSP-1 revealed interstitial expression of TSP-1. Comparable expression was observed in CD44+/+ (top) kidneys () and CD44–/– (bottom) kidneys (). Data are presented as mean ± SEM; n = 6. Magnification, x300.
Characterization of Peritubular Capillary Endothelial Cells
After 8 d of differentiation at 37°C without IFN-, cellsno longer expressed SV40 or PCNA (data not shown). Exposureto 10 ng/ml TNF- for 4 h induced high levels of VCAM-1 (Figure 6A)and E-selectin (Figure 6B). The endothelial marker CD34(Figure 6C) was not expressed by in vitro cultured capillaryendothelial cells. vWF (Figure 6D) and CD31 (Figure 6E) bothwere expressed constitutively as determined by flow cytometry.Expression patterns of VCAM-1, E-selectin, CD34, vWF, and CD31were comparable in CD44+/+ and CD44–/– cell lines.As expected, CD44–/– endothelial cells expressedno CD44, whereas CD44+/+ cells express high levels of CD44 (Figure 6F).
Figure 6. Peritubular capillary endothelial cell characterization. Flow cytometric analysis on fully differentiated peritubular capillary endothelial cells, 4 h after TNF- stimulation (gray line), revealed increased expression of vascular cellular adhesion molecule-1 (VCAM-1; A) and E-selectin (B), compared with nonstimulated cells (filled area). Endothelial cells in vitro do not express CD34 (C); gray line represents CD34 labeled cells, and filled area is the control isotype antibody. Endothelial cells express von Willebrand factor (vWF; D) and CD31 (E); gray line represents specific antibody labeled cells, and filled are is the control isotype antibody. CD44+/+ endothelial cells (gray line) express CD44; CD44–/– cells (filled area) do not express CD44 (F).
CD44-HA Interaction Enhances TGF-1–but not TSP-1–Induced Apoptosis in Capillary Endothelial Cells
The viability and apoptotic rate of fully differentiated, nondividingCD44+/+ and CD44–/– endothelial cells were similarunder differentiation conditions. Addition of HA had no effecton the percentage of apoptosis of CD44+/+ and CD44–/–endothelial cells. Cells were stimulated for 24 h using differentconcentrations of TGF-1 with or without addition of 50 µg/mlHA (Figure 7A). Various concentrations of TGF-1 failed to induceapoptosis. However, when HA was added simultaneously to thecell cultures, the rate of apoptosis of endothelial cells thatexpressed CD44 increased but not in CD44-deficient endothelialcells. This observation suggests that CD44-HA ligation is requiredfor TGF-1 induction of apoptosis in endothelial cells.
Figure 7. TGF-1–but not TSP-1–induced apoptosis is enhanced by CD44–hyaluronic acid (HA) interaction. Fully differentiated capillary endothelial cells were stimulated for 24 h with TGF-1 or TSP-1 with or without addition of 50 µg/ml HA. Apoptosis was determined by flow cytometry. TGF-1 was capable of inducing apoptosis only in CD44+/+ cells after CD44-HA ligation (A). TSP-1 induced apoptosis in endothelial cells independent of the receptor-ligand complex, CD44-HA (B). , CD44+/+; , CD44–/–; , CD44+/+ with HA; and , CD44–/– with HA; data are presented as mean ± SEM; n = 3. *P < 0.05 (CD44+/+ with HA versus CD44+/+); #P < 0.05 (CD44+/+ with HA versus CD44–/– with HA).
As expected, TSP-1 induced apoptosis in a dose-dependent manner(Figure 7B). However, in contrast to TGF-1, CD44 or interactionof CD44 with HA had an effect only on TSP-1–induced apoptosisat 20 ng/ml. At higher concentrations, this effect was no longerobserved.
A cascade of events is initiated during the progression of tubulointerstitiallesions, including release of cytokines/chemokines and growthfactors, expression of adhesion molecules, inflammatory infiltrate,renal epithelial cell damage, accumulation of myofibroblasts,capillary network degeneration, and finally fibrosis. CD44 isupregulated in various diseases, including experimental nephropathiesand human renal diseases (13–17). CD44 therefore may beconsidered as a regulatory molecule in the cascade that leadsto tubulointerstitial damage. Using a mouse model of chronicobstructive nephropathy, we showed that CD44 protects tubulifrom injury but also induces the development of fibrosis (12).
In this study, we determined the role of CD44 in the degenerationof the capillary network. Loss of peritubular and glomerularcapillaries is correlated with the severity of renal disease(19,20). Ohashi et al. (22) documented that capillary networkregression resulted from increased endothelial cell loss andimpaired regeneration. As documented in other diseases (10,13),we show that the capillary endothelial cells express CD44 afterrenal injury. As expected, the capillary network degeneratedin CD44+/+ after UUO. Remarkably, the capillary network waswell preserved in CD44–/– obstructed kidneys. Thedegree of proliferation of peritubular capillary endothelialcells was comparable in mice of both phenotypes. However, thenumber of apoptotic endothelial cells was less in CD44–/–compared with CD44+/+, which may contribute to the preservationof the capillary network in CD44–/– obstructed kidneys.Many reports emphasize an important role for VEGF in proliferationof endothelial cells and preservation of the capillary network(20–22). Activation of CD44 by HA or activating antibodiesstimulates VEGF production in vitro in human vascular endothelialcells (31). However, no differences in VEGF expression couldbe detected in CD44+/+ and CD44–/– obstructed kidneys.This is in accordance with the comparable number of proliferatingendothelial cells in CD44+/+ and CD44–/– mice. Apotent inducer of apoptosis for endothelial cells is TSP-1 (24,25).Therefore, we analyzed expression of TSP-1. No difference inexpression of TSP-1 in mice of both genotypes that may explainthe difference observed in endothelial cell apoptosis was found,yet CD44 may enhance signaling of TSP-1 because TSP-1, likehepatocyte growth factor, is a heparan sulfate–bindinggrowth factor (24), the signaling of which can be enhanced inthe presence of CD44v3 (8,12). However, using conditionallyimmortalized capillary endothelial cell lines derived from CD44+/+or CD44–/– kidneys, we were unable to show a clearfacilitating role for CD44 in TSP-1–induced apoptosisof capillary endothelial cells. Interaction of HA with CD44was only at 20 ng/ml TSP-1 able to increase apoptosis in CD44+/+endothelial cells, suggesting a possible role for CD44 in TSP-1signaling. Lowering TSP-1 concentrations below 20 ng/ml TSP-1resulted in background apoptosis levels.
Besides TSP-1, TGF-1 can induce apoptosis in endothelial cells(32,33). Previously, we showed that in our model, TGF-1 is highlyexpressed (12). Addition of high concentrations of TGF-1 wasonly moderately capable of inducing apoptosis in capillary endothelialcells, but a synergetic effect of HA was observed on TGF-1–inducedendothelial cell apoptosis. The potentiation of TGF-1 proapoptoticactivity was observed only in capillary endothelial cells thatexpressed CD44. This suggests that ligation of CD44-HA facilitatesTGF-1–induced apoptosis in peritubular capillary endothelialcells. Effects of CD44-HA interactions in TGF-1 signaling weredescribed previously in a tumor cell line (11) and also in tubularepithelial cells (12). Bourguignon et al. (11) showed that CD44forms a complex with TGF-1 receptor I and to a lesser extentTGF-1 receptor II. Stimulation of CD44 with HA results in anincreased threonine/serine kinase activity, resulting in increasedsmad2/3 phosphorylation. In agreement, we showed in vivo inCD44+/+ and CD44–/– mice (12) that the absence ofCD44 resulted in decreased activation of the smad2/3 proteins.This decreased TGF-1 signaling in turn prevented the developmentof fibrosis in CD44–/– mice. It is generally consideredthat TGF-1 is the major growth factor involved in transdifferentiationof tubular epithelial cell (TEC) into myofibroblasts (34). Whereasmyofibroblasts generally are thought to derive from transdifferentiationof TEC and from resident fibroblasts, increasing evidence hasbeen gathered that myofibroblasts can also derive from hematopoieticcells (35). We observed no transdifferentiation of endothelialcells into myofibroblasts (data not shown). Although it is wellestablished that myofibroblasts play a major role in the developmentof renal fibrosis, the relative contribution of the different"types" of myofibroblasts is still a matter of debate.
Previous reports implicated CD44 in Fas-induced apoptosis; however,these reports are contradictory. One study showed that CD44stimulation downregulates Fas expression and Fas-mediated apoptosisof pulmonary cancer cells (36), but another study describedthat CD44 markedly enhanced Fas expression and subsequentlyFas-induced apoptosis of these cells (37). These contradictoryobservations prompted us to investigate Fas expression in ourmodel by immunostaining. No difference in Fas expression wasobserved between CD44+/+ and CD44–/– UUO kidneys(data not shown).
Although CD44 increases the number of apoptotic endothelialcells, we showed previously that in the same model, expressionof CD44 on TEC decreases apoptosis of TEC (12). This observedparadoxic effect of CD44 on TEC and endothelial cells may berelated to the multiple modifications of CD44, e.g., alternativesplicing; glycosylation; addition of keratin, heparan, or chondroitinsulfate side chains. This modifies the properties of CD44 andtherefore may contribute to enhancement of certain growth factorsignaling, whereas it inhibits others. Although the cellularprocesses that lead to alternative splicing still are poorlyunderstood, expression of CD44 isoforms seems to be tissue specific.Therefore, the enhancement or inhibition of growth factor signalingby CD44 may also be tissue specific.
Although we show a clear role for CD44 together with TGF-1 inthe degeneration of the capillary network, this does not excludeinvolvement of other growth factors. Moreover, it remains unclearwhether development of fibrosis is dependent on capillary degenerationor vice versa. This study together with that of Kang et al.(20,21) shows that capillary degeneration and development offibrosis are associated with each other. Blocking interactionbetween HA and CD44 therefore may be a potential therapeuticopportunity to prevent progression of chronic renal diseases.Not only may the degeneration of the capillary network be preventedbut also the development of fibrosis (12).
This report shows that expression of CD44 in the injured kidneycontributes to the degeneration of the capillary network, atleast in part through enhanced TGF-1–induced apoptosisof endothelial cells.
Acknowledgments
This research was funded by The Netherlands Organisation forScientific Research (grants 907-00-004 and 015-001-061) andthe Dutch Kidney Foundation (grant PC 125).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI: Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons.
Proc Natl Acad Sci U S A 89
: 12160
–12164, 1992[Abstract/Free Full Text]
Dougherty GJ, Landorp PM, Cooper DL, Humphries RK: Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte homing receptor expressed by hemopoietic cells.
J Exp Med 174
: 1
–5, 1991[Abstract/Free Full Text]
Gunthert U: CD44: A multitude of isoforms with diverse functions.
Curr Top Microbiol Immunol 184
: 47
–63, 1993[Medline]
Tolg C, Hofmann M, Herrlich P, Ponta H: Splicing choice from ten variant exons establishes CD44 variability.
Nucleic Acids Res 21
: 1225
–1229, 1993[Abstract/Free Full Text]
Weiss L, Slavin S, Reich S, Cohen P, Shuster S, Stern R, Kaganovsky E, Okon E, Rubinstein AM, Naor D: Induction of resistance to diabetes in non-obese diabetic mice by targeting CD44 with a specific monoclonal antibody.
Proc Natl Acad Sci U S A 97
: 285
–290, 2000[Abstract/Free Full Text]
Siegelman MH, DeGrendele HC, Estess P: Activation and interaction of CD44 and hyaluronan in immunological systems.
J Leukoc Biol 66
: 315
–321, 1999[Abstract]
Jones M, Tussey L, Athanasou N, Jackson DG: Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action.
J Biol Chem 275
: 7964
–7974, 2000[Abstract/Free Full Text]
van der Voort R, Taher TE, Wielenga VJ, Spaargaren M, Prevo R, Smit L, David G, Hartmann G, Gherardi E, Pals ST: Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met.
J Biol Chem 274
: 6499
–6506, 1999[Abstract/Free Full Text]
DeGrendele HC, Estess P, Siegelman MH: Requirement for CD44 in activated T cell extravasation into an inflammatory site.
Science 278
: 672
–675, 1997[Abstract/Free Full Text]
Rouschop KM, Roelofs JJ, Claessen N, da Costa Martins P, Zwaginga JJ, Pals ST, Weening JJ, Florquin S: Protection against renal ischemia reperfusion injury by CD44 disruption.
J Am Soc Nephrol 16
: 2034
–2043, 2005[Abstract/Free Full Text]
Bourguignon LY, Singleton PA, Zhu H, Zhou B: Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells.
J Biol Chem 277
: 39703
–39712, 2002[Abstract/Free Full Text]
Rouschop KM, Sewnath ME, Claessen N, Roelofs JJ, Hoedemaeker I, van der Neut R, Aten J, Pals ST, Weening JJ, Florquin S: CD44 deficiency increases tubular damage but reduces renal fibrosis in obstructive nephropathy.
J Am Soc Nephrol 15
: 674
–686, 2004[Abstract/Free Full Text]
Lewington AJ, Padanilam BJ, Martin DR, Hammerman MR: Expression of CD44 in kidney after acute ischemic injury in rats.
Am J Physiol Regul Integr Comp Physiol 278
: R247
–R254, 2000[Abstract/Free Full Text]
Sibalic V, Fan X, Loffing J, Wuthrich RP: Upregulated renal tubular CD44, hyaluronan, and osteopontin in kdkd mice with interstitial nephritis.
Nephrol Dial Transplant 12
: 1344
–1353, 1997[Abstract/Free Full Text]
Takazoe K, Foti R, Tesch GH, Hurst LA, Lan HY, Atkins RC, Nikolic-Paterson DJ: Up-regulation of the tumour-associated marker CD44V6 in experimental kidney disease.
Clin Exp Immunol 121
: 523
–532, 2000[CrossRef][Medline]
Roy-Chaudhury P, Khong TF, Williams JH, Haites NE, Wu B, Simpson JG, Power DA: CD44 in glomerulonephritis: Expression in human renal biopsies, the Thy 1.1 model, and by cultured mesangial cells.
Kidney Int 50
: 272
–281, 1996[Medline]
Florquin S, Nunziata R, Claessen N, van den Berg FM, Pals ST, Weening JJ: CD44 expression in IgA nephropathy.
Am J Kidney Dis 39
: 407
–414, 2002[CrossRef][Medline]
Florquin S, Rouschop KM: Reciprocal functions of hepatocyte growth factor and transforming growth factor-beta1 in the progression of renal diseases: A role for CD44?
Kidney Int Suppl 64
: 15
–20, 2003[CrossRef]
Bohle A, Mackensen-Haen S, Wehrmann M: Significance of postglomerular capillaries in the pathogenesis of chronic renal failure.
Kidney Blood Press Res 19
: 191
–195, 1996[Medline]
Kang DH, Joly AH, Oh SW, Hugo C, Kerjaschki D, Gordon KL, Mazzali M, Jefferson JA, Hughes J, Madsen KM, Schreiner GF, Johnson RJ: Impaired angiogenesis in the remnant kidney model: I. Potential role of vascular endothelial growth factor and thrombospondin-1.
J Am Soc Nephrol 12
: 1434
–1447, 2001[Abstract/Free Full Text]
Kang DH, Hughes J, Mazzali M, Schreiner GF, Johnson RJ: Impaired angiogenesis in the remnant kidney model: II. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function.
J Am Soc Nephrol 12
: 1448
–1457, 2001[Abstract/Free Full Text]
Ohashi R, Shimizu A, Masuda Y, Kitamura H, Ishizaki M, Sugisaki Y, Yamanaka N: Peritubular capillary regression during the progression of experimental obstructive nephropathy.
J Am Soc Nephrol 13
: 1795
–1805, 2002[Abstract/Free Full Text]
Iruela-Arispe ML, Bornstein P, Sage H: Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro.
Proc Natl Acad Sci U S A 88
: 5026
–5030, 1991[Abstract/Free Full Text]
de Fraipont F, Nicholson AC, Feige JJ, Van Meir EG: Thrombospondins and tumor angiogenesis.
Trends Mol Med 7
: 401
–407, 2001[CrossRef][Medline]
Lawler J: Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth.
J Cell Mol Med 6
: 1
–12, 2002[Medline]
Guo N, Krutzsch HC, Inman JK, Roberts DD: Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells.
Cancer Res 57
: 1735
–1742, 1997[Abstract/Free Full Text]
Forster-Horvath C, Meszaros L, Raso E, Dome B, Ladanyi A, Morini M, Albini A, Timar J: Expression of CD44v3 protein in human endothelial cells in vitro and in tumoral microvessels in vivo.
Microvasc Res 68
: 110
–118, 2004[CrossRef][Medline]
Schmits RFJ, Gerwin N, Senaldi G, Kiefer F, Kundig T, Wakeham A, Shahinian A, Catzavelos C, Rak J, Furlonger C, Zakarian A, Simard JJ, Ohashi PS, Paige CJ, Gutierrez-Ramos JC, Mak TW: CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity.
Blood 90
: 2217
–2233, 1997[Abstract/Free Full Text]
Takemoto M, Asker N, Gerhardt H, Lundkvist A, Johansson BR, Saito Y, Betsholtz C: A new method for large scale isolation of kidney glomeruli from mice.
Am J Pathol 161
: 799
–805, 2002[Abstract/Free Full Text]
Langley RR, Ramirez KM, Tsan RZ, Van Arsdall M, Nilsson MB, Fidler IJ: Tissue-specific microvascular endothelial cell lines from H-2K(b)-tsA58 mice for studies of angiogenesis and metastasis.
Cancer Res 63
: 2971
–2976, 2003[Abstract/Free Full Text]
Murphy JF, Lennon F, Steele C, Kelleher D, Fitzgerald D, Long AC: Engagement of CD44 modulates cyclooxygenase induction, VEGF generation, and proliferation in human vascular endothelial cells.
FASEB J 19
: 446
–448, 2005[Abstract/Free Full Text]
Choi ME, Ballermann BJ: Inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth factor-beta receptors.
J Biol Chem 270
: 21144
–21150, 1995[Abstract/Free Full Text]
Fierlbeck W, Liu A, Coyle R, Ballermann BJ: Endothelial cell apoptosis during glomerular capillary lumen formation in vivo.
J Am Soc Nephrol 14
: 1349
–1354, 2003[Abstract/Free Full Text]
Fan JM, Ng YY, Hill PA, Nikolic-Paterson DJ, Mu W, Atkins RC, Lan HY: Transforming growth factor-beta regulates tubular epithelial-myofibroblast transdifferentiation in vitro.
Kidney Int 56
: 1455
–1467, 1999[CrossRef][Medline]
Direkze NC, Forbes SJ, Brittan M, Hunt T, Jeffery R, Preston SL, Poulsom R, Hodivala-Dilke K, Alison MR, Wright NA: Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice.
Stem Cells 21
: 514
–520, 2003[Abstract/Free Full Text]
Yasuda M, Tanaka Y, Fujii K, Yasumoto K: CD44 stimulation down-regulates Fas expression and Fas-mediated apoptosis of lung cancer cells.
Int Immunol 13
: 1309
–1319, 2001[Abstract/Free Full Text]
Fujii K, Fujii Y, Hubscher S, Tanaka Y: CD44 is the physiological trigger of Fas up-regulation on rheumatoid synovial cells.
J Immunol 167
: 1198
–1203, 2001[Abstract/Free Full Text]
1–Induced Apoptosis
Top
Abstract
Introduction
1 signaling. Absence of CD44 prevents development of renal fibrosis in unilateral ureteral obstruction (UUO). A hallmark of development of renal fibrosis is the degeneration of the capillary network. This study shows that CD44 is upregulated on capillary endothelial cells during UUO. For elucidation of the role of CD44 on peritubular endothelial cells in UUO, capillary network degeneration was compared in CD44+/+ and CD44–/– mice. As expected, degeneration of the capillary network was observed in CD44+/+ mice during UUO, associated with increased endothelial apoptosis. However, in the absence of CD44, degeneration of the network is prevented as a result of a decrease in the rate of apoptosis in endothelial cells. The divergence in endothelial apoptosis is not correlated to differential vascular endothelial growth factor or thrombospondin-1 expression. For further investigation of capillary regression, CD44+/+ and CD44–/– peritubular capillary endothelial cell lines were established. With the use of these cells, it is shown that interaction between CD44 and its ligand hyaluronic acid enhances the proapoptotic effect of TGF-
(R&D Systems, Minneapolis, MN) at 33°C until confluence. Confluent cells then were placed for 8 d at 37°C without addition of IFN-
(R&D Systems) for 4 h. Cells were labeled with vascular cellular adhesion molecule-1 (VCAM-1)–FITC and E-selectin–PE (both BD Pharmingen). Peritubular capillary endothelial cells were sorted using a FACSaria (BD Pharmingen). Purified endothelial cells were cultured in medium as described above with addition of IFN-
) but not in CD44–/– (right) mice (
); staining is quantified by digital analysis. Data are presented as mean ± SEM; n = 6. Magnification, x100.
, CD44+/+ with HA; and 
, CD44–/– with HA; data are presented as mean ± SEM; n = 3. *P < 0.05 (CD44+/+ with HA versus CD44+/+); #P < 0.05 (CD44+/+ with HA versus CD44–/– with HA).