PDGF-C Expression in the Developing and Normal Adult Human Kidney and in Glomerular Diseases
Frank Eitner*,
Tammo Ostendorf*,
Matthias Kretzler,
Clemens D. Cohen for the ERCB-Consortium,
Ulf Eriksson,
Hermann-Josef Gröne and
Jürgen Floege*
*Division of Nephrology and Immunology, Aachen University, Aachen, Germany; Medical Policlinic, Ludwig Maximilians University of Munich, Munich, Germany; Ludwig Institute for Cancer Research, Stockholm, Sweden; Department of Cellular and Molecular Pathology, German Cancer Research Center Heidelberg, Germany.
Correspondence to Dr. Frank Eitner, Universitätsklinikum Aachen, Medizinische Klinik II (Nephrologie und Immunologie), Pauwelsstr 30, 52074 Aachen, Germany. Phone: 49-241-8089 670; Fax: 49-241-8082 446;
ABSTRACT. PDGF-C is a new member of the PDGF-family and hasrecently been identified as a rat mesangial cell mitogen. Itsexpression and function in human kidneys is unknown. Localizationof PDGF-C protein was analyzed by immunohistochemistry usinga rabbit polyclonal antibody directed against the core-domainof PDGF-C in human fetal kidneys (n = 8), normal adult humankidneys (n = 9), and in renal biopsies of patients with IgAnephropathy (IgAN, n = 31), membranous nephropathy (MGN, n =8), minimal change disease (MC, n = 7), and transplant glomerulopathy(TxG, n = 12). Additionally, PDGF-C mRNA was detected in microdissectedglomeruli by real-time RT-PCR in cases of normal adult kidneys(n = 7), IgAN (n = 27), MGN (n = 11), and MC (n = 13). In thefetal kidney, PDGF-C localized to the developing mesangium,ureteric bud epithelium, and the undifferentiated mesenchyme.In the adult kidney, PDGF-C was constitutively expressed inparietal epithelial cells of Bowman’s capsule, tubularepithelial cells (loops of Henle, distal tubules, collectingducts), and in arterial endothelial cells. A marked upregulationof glomerular PDGF-C protein was seen in MGN and TxG with aprominent positivity of virtually all podocytes. In MC, PDGF-Clocalized to podocytes in a more focal distribution. In MGN,increased glomerular PDGF-C protein expression was due to increasedmRNA synthesis as a 4.3-fold increase in PDGF-C mRNA was detectedin microdissected glomeruli from MGN compared with normal. PDGF-Cprotein was additionally expressed in individual mesangial cellsin TxG. Finally, upregulated PDGF-C protein expression was detectedwithin sclerosing glomerular and fibrosing tubulointerstitiallesions in individual cases from all analyzed groups. We concludethat PDGF-C is constitutively expressed in the human kidneyand is upregulated in podocytes and interstitial cells afterinjury/activation of these cells. E-mail: feitner@ukaachen.de
Members of the platelet-derived growth factor (PDGF) familyof cytokines are involved in different aspects of renal disease,particularly the mediation of glomerular mesangial cell proliferationand the induction of renal interstitial fibrosis (1,2). Untilrecently, the PDGF family comprised three dimers composed ofa PDGF A-chain and B-chain, i.e., PDGF-AA, PDGR-AB, and PDGF-BB,that act through the -subunit and -subunit of the PDGF receptor(PDGFR) (3–5). In 2000, a new member of the PDGF familywas identified and subsequently termed PDGF-C (6). PDGF-C, likePDGF-A and PDGF-B, forms a disulphide-bonded dimer, PDGF-CC.Li et al. (6) identified PDGF-CC as a PDGFR-–specificligand. Whereas the -subunit of the PDGF-receptor is presentin vascular smooth muscle cells and the renal interstitium,the -subunit is constitutively expressed in mesangial and parietalglomerular epithelial cells, in vascular smooth muscle cells,as well as in renal interstitial cells (5). Increased expressionof PDGF receptors at sites of renal injury has been documentedin a large variety of diseases (5).
We have previously identified PDGF-C as a potent mitogenic stimulusfor cultured rat mesangial cells in vitro (7). Immunohistochemicalstudies in rat tissues detected a constitutive expression ofPDGF-C within arterial smooth muscle cells and collecting ductepithelial cells. However, a marked upregulation of PDGF-C wasidentified within the mesangium in rat experimental mesangioproliferativeglomerulonephritis (7). Other rat glomerular disease modelswith predominant sclerotic lesions or predominant injury topodocytes were almost completely negative for glomerular PDGF-C.In a single rat model of spontaneous glomerulosclerosis (Milannormotensive glomerulosclerosis), PDGF-C localized to podocytesat sites of focal glomerular injury (7). The expression andpotential role of PDGF-C in human kidneys and in human renaldiseases is completely unknown. In the current study, we analyzedthe tissue localization of PDGF-C by immunohistochemistry indeveloping fetal kidneys, in normal adult kidneys, and in renalbiopsy tissues with different glomerular diseases. Furthermore,we analyzed PDGF-C mRNA expression by real-time RT-PCR in microdissectedglomeruli from patients with different glomerular diseases.The present study is the first to identify a significant increaseof podocytic PDGF-C in human glomerular diseases primarily associatedwith injury to podocytes, i.e., membranous nephropathy (MGN),transplant glomerulopathy (TxG), and to a lesser degree minimalchange disease (MC). Furthermore, mesangial PDGF-C expressionwas detected in TxG, a glomerular disease characteristicallypreceded by mesangiolysis. However, in human mesangioproliferativeIgA nephropathy (IgAN), no upregulation of either glomerularPDGF-C protein or mRNA was detectable.
Analyzed Kidney Tissues
Formalin-fixed, paraffin-embedded renal tissue specimens obtainedbetween 1999 and 2002 at the German Cancer Research Center,DKFZ Heidelberg, Germany, were included in this study. Humantissue was used after approval by, and following the guidelinesof, the local Ethics Committee. Renal biopsy cases with sufficienttissue for immunohistochemical evaluation after completion ofdiagnostic workup were included. Normal adult human kidney tissuewas obtained from kidneys surgically excised because of thepresence of a localized neoplasm. Tissues utilized for thisstudy were obtained from macroscopically normal portions ofkidney located at some distance from the neoplastic process.Eight human fetal kidneys (estimated gestational age rangingfrom 14 to 22 wk) were obtained fresh from tissue examined aftertherapeutic abortion. Both adult and fetal kidney tissues werefixed in formalin and embedded in paraffin. Table 1 summarizesthe details of the analyzed materials.
Antibodies
Rabbit antiserum directed against PDGF-C was generated as describedpreviously in detail (6). An antiserum generated against humanPDGF-CC, affinity-purified against the core domain of PDGF-CC,was utilized in the present study. Specificity of this antiserumfor the detection of PDGF-C has been demonstrated previously(6,7).
Immunohistochemistry
Immunohistochemistry was performed according to previously publishedprotocols (7,8). Briefly, formalin-fixed, paraffin-embeddedtissues were sectioned at 4 µm. Sections were deparaffinizedin xylene and rehydrated in graded ethanols. Endogenous peroxidasewas blocked by incubation in 3% hydrogen peroxide. The sectionswere then incubated for 1 h with the primary antibody dilutedin phosphate-buffered saline (PBS) containing 1% bovine serumalbumin (BSA; Sigma). After washes in PBS, the sections wereincubated with biotinylated goat anti-rabbit antibody (Vector,Burlingame, CA). A Tyramide Signal Amplification (TSA-Indirect;NEN Life Science Products, Boston, MA) was performed accordingto the instructions of the manufacturer. Finally 3,3'-diaminobenzidine(DAB; Sigma) with nickel chloride enhancement was used as thechromogen. Sections were counterstained with methyl green, dehydrated,mounted, and coverslipped. To further confirm the specificityof the immunohistochemical staining, another protocol was performedas previously described (9,10). After microwave treatment of4-µm sections of formaldehyde-fixed paraffin-embeddedbiopsies in citrate buffer and after blockade of endogenousperoxidase activity by incubation of the sections in 3% H2O2at 22°C for 10 min, nonspecific binding sites were saturatedwith 4% skim milk in PBS (pH 7.6) at 22°C for 20 min. Theprimary antibody was added to the sections for 18 h at 4°C.Then, with the application of avidin followed by biotin (avidin/biotinblocking kit; Vector), endogenous biotin, biotin receptors,or avidin binding was labeled. This was followed by applicationof a biotinylated pan-specific secondary antibody (BA-1300;Vector) in 1% BSA/PBS at 22°C for 1 h. A horseradish peroxidase-streptavidincomplex (SA 5004, diluted 1:200; Vector) was then applied at22°C for 1 h. 3-Amino-9-ethylcarbazole or 3,3'-diaminobenzidinesubstrate kits (SK-4200 or SK 4100, respectively; Vector) wereused for specific staining. Counterstaining was performed withhematoxylin at 22°C for 4 min. Negative controls consistedof replacement of the primary antiserum with non-immune rabbitserum.
Microdissection, RNA Isolation, Real-Time RT-PCR
Human kidney biopsies were obtained in a multicenter study forgene expression analysis in renal biopsies (see Appendix forparticipating centers). Clinical data including gender, age,serum-creatinine, and quantitative proteinuria were obtainedfrom each patient at the time of biopsy. Informed consent ofthe patients and acknowledgment of the respective local ethicalcommittees were obtained. For a detailed description of theprotocol used see reference 11. In brief, directly after biopsya cortical tissue segment was transferred into a commerciallyavailable RNase inhibitor (RNAlater; Ambion), stored at -20°C,and then manually microdissected in glomeruli and tubulointerstitialcompartments in RNAlater. Glomerular microdissection was confirmedby detection of the glomerulus-specific cDNA for Wilms tumorantigen 1. Total RNA was isolated using a commercially availablesilica-gel–based isolation protocol (RNeasy-Mini, Qiagen,Germany). Reverse transcription was performed in a 45-µlvolume, containing 9 µl of buffer, 2 µl of DTT (bothLife Technologies), 0.9 µl of 25 mM dNTP (Amersham Pharmacia,Freiburg, Germany), 1 µl of RNase inhibitor (Rnasin; Promega,Mannheim, Germany), and 0.5 µl of Microcarrier (MolecularResearch Center, Cincinnati, OH), 1 µg of random hexamers(2 mg/ml stock; Roche, Mannheim, Germany), and 200 U of reversetranscriptase (Superscript; Life Technologies) for 1 h at 42°C.Real time RT-PCR was performed on a TaqMan ABI 7700 SequenceDetection System (PE Biosystems, Weiterstadt, Germany) usingheat-activated TaqDNA polymerase (Amplitaq Gold; PE Biosystems,Weiterstadt, Germany). After an initial hold of 2 min at 50°Cand 10 min at 95°C, the samples were cycled 40 times at95°C for 15 s and 60°C for 60 s. PDGF-C and housekeepercDNA templates were quantified by standard curves of dilutedstandard cDNA. Here, the threshold cycle of each sample correspondswith a dilution step of the standard cDNA (arbitrary units).For quantitative analysis, cDNA content of each sample was comparedwith another sample by PDGF-C/housekeeping gene. GAPDH and 18Sribosomal RNA served as housekeeping genes to assess the overallcDNA content, yielding comparable results. Data are given forratios to 18S rRNA.
The following oligonucleotide primers (300 nM) and probes (100nM) were used: Human PDGF-C (gb AF244813; bp 193 to 323): senseprimer 5'-GCCTCTTCGGGCTTCTCC-3', antisense primer 5'-TGAGGATCTTGTACTCCGTTCTGTT-3',fluorescence-labeled probe (FAM) 5'-CCGGCCAGAGACGAGGGACTCA-3';Human Wilms Tumor WT1 (gb X51630; bp1155 to 1221): sense primer5'-AAATGGACAGAAGGGCAGAGC-3', antisense primer 5'-GGATGGGCGTTGTGTGGT-3',fluorescence-labeled probe (FAM) 5'-ACCACAGCACAGGGTACGAGAGCGA-3';commercially available predeveloped TaqMan reagents were usedfor human GAPDH and 18S rRNA. Similar amplification efficienciesfor all targets were demonstrated by analyzing serial cDNA dilutionsshowing a slope value of log input cDNA amount versus (Ct tagetA - Ct target B) of < 0.1. The primers for PDGF-C and WT-1were cDNA-specific, not amplifying genomic DNA. All primersand probes were obtained from Applied Biosystems, Weiterstadt,Germany.
PDGF-C Expression in Normal Adult Human Kidney
The immunohistochemical staining pattern was very consistentin the nine analyzed adult human kidneys (summarized in Table 2).Within the glomerular compartment, parietal epithelial cellswere consistently positive for PDGF-C in all cases (Figure 1A).The glomerular tuft was however negative for PDGF-C. Very fewindividual positive cells were rarely identified within theglomerular tuft (data not shown). In view of the infrequency,we were unable to identify the phenotype of these cells by morphologiccriteria. Tubular epithelial cells frequently expressed PDGF-C(Figure 1B). PDGF-C expression localized to the loop of Henle,distal tubules, and collecting ducts, whereas proximal tubules(as identified by the presence of a brush border) did not showdetectable PDGF-C expression. Few individual PDGF-C–expressingcells were localized throughout the interstitium in a scattereddistribution. There was a strong expression of PDGF-C withinendothelial cells of renal arteries and arterioles (Figure 1C).Endothelial cells in the glomerular or peritubular microvasculatureor in veins did not demonstrate detectable PDGF-C expression.In addition to the prominent endothelial PDGF-C expression,there was a weaker and more scattered positivity within thesmooth muscle cell–rich media of arterial vessels. Identicalresults were obtained with two different staining methods usedin this study. Negative control tissues that were incubatedwith equal amounts of non-immune rabbit serum did not demonstrateany staining signal in all analyzed normal and diseased cases.
Figure 1. PDGF-C protein expression in normal adult human kidney (A through C) and in human fetal development (D and E). Immunohistochemistry using a PDGF-C–specific polyclonal antibody results in a black/brown color product. (A) In the normal adult human kidney, PDGF-C expression is absent within the glomerular tuft. Parietal epithelial cells are consistently positive for PDGF-C in all cases. (B) Tubular epithelial cells frequently express PDGF-C. By morphologic criteria, PDGF-C localizes to collecting ducts. (C) Prominent expression of PDGF-C is detectable within arterial endothelial cells of renal arteries and arterioles. (D) In the developing human kidney, strong PDGF-C expression is frequently detected within the ureteric buds. (E) Metanephric blastema (arrows) demonstrates strong PDGF-C expression. Early glomerular stages, like the illustrated s-shape body, do not express PDGF-C. (F) In more differentiated glomeruli, PDGF-C expression is restricted to the developing mesangium while all other glomerular cell types remain negative. Additionally, parietal epithelial cells express PDGF-C. Original magnifications: x600 in A through D; x1000 in E through F.
PDGF-C Expression in the Developing Fetal Kidney
In the developing human kidney, PDGF-C showed a distinct localizationpattern (summarized in Table 3). Strong PDGF-C expression wasfrequently detected within ureteric buds (Figure 1D) and withinthe metanephric blastema (Figure 1E). Early glomerular stages,i.e., glomerular vesicles, comma-shape, or s-shape glomeruli,were almost completely negative for PDGF-C (Figure 1E). However,few s-shape glomeruli weakly expressed PDGF-C in the mesangialstalk region (data not shown). In more differentiated glomeruli,PDGF-C was restricted to the developing mesangium while allother glomerular cell types, including developing endothelialcells as well as podocytes, remained uniformly negative (Figure 1F).Additionally, parietal epithelial cells expressed detectablePDGF-C in more differentiated glomeruli (Figure 1F). In thevasculature, PDGF-C regularly localized to smooth muscle cellsin smaller arteries and arterioles. In these arterial vessels,PDGF-C was additionally expressed by endothelial cells.
Table 3. PDGF-C expression in the developing human fetal kidney
PDGF-C Expression in Human Glomerular Diseases
Given the marked mesangial PDGF-C upregulation in rat mesangioproliferativeglomerulonephritis and human renal development and its roleas a mesangial cell mitogen in vitro, we next analyzed the expressionof PDGF-C in human mesangioproliferative IgAN (summarized inTable 2). Thirty-one cases of IgAN were selected to representa variety of pathologic lesions of IgAN. Cases included minimallesions, classical cases with prominent mesangial hypercellularityand segmentally increased matrix deposition/sclerosis, and caseswith severe focal necrosis and crescents. Within the analyzedglomeruli, there was a consistent positivity of all parietalepithelial cells (Figure 2A). The glomerular tuft of the majorityof all cases was completely negative for PDGF-C. There was noimmunohistochemical signal localized to mesangial cells. Caseswith prominent mesangial hypercellularity, as illustrated inFigures 2A and 2B, were regularly negative for PDGF-C in themesangium. A weak granular staining signal for PDGF-C localizedto occasional podocytes and glomerular endothelial cells. However,the weak and infrequent character of this staining pattern wasconsidered with some caution as it was just above the levelof nonspecific background signal. In contrast, IgAN biopsieswith segmental necrosis/crescentic lesions typically demonstratedan increase in glomerular PDGF-C expression. Extracapillaryproliferates and podocytes adjacent to segmental necrosis stronglyexpressed PDGF-C (Figure 2, C and D). In all IgAN biopsies,PDGF-C was expressed by the loop of Henle, distal tubules, andcollecting ducts. Identical with the findings in normal adultkidneys, a strong expression of PDGF-C was identified in arterialendothelial cells. Additionally, individual arterial smoothmuscle cells as well as individual cells within the adventitiaof arteries labeled positively for PDGF-C.
Figure 2. PDGF-C expression in human IgA nephropathy (IgAN). Immunohistochemistry using a PDGF-C specific polyclonal antibody results in a black/brown color product. (A) In cases of IgAN, glomerular PDGF-C remains largely negative despite the presence of prominent focal mesangial hypercellularity. PDGF-C expression of parietal epithelial cells is unchanged compared with normal adult human tissues (compare with Figure 1A). (B) Higher power magnification illustration of Figure 2A indicating the absent mesangial PDGF-C. (C) In IgAN biopsies with segmental necrosis, strong PDGF-C expression is detected in podocytes adjacent to segmental necrosis. (D) Higher power magnification illustration of Figure 2C, indicating strong PDGF-C expression within podocytes (arrows). Original magnifications: x400 in A and C; x1000 in B and D.
In cases with a marked injury of podocytes, we identified strongexpression of podocytic PDGF-C expression (Table 2). All casesof MGN expressed PDGF-C within the glomeruli. PDGF-C was localizedto the cytoplasm of podocytes (Figure 3, A and B). Podocyteswere identified by morphologic criteria as adjacent cells locatedat the outer aspect of the glomerular basement membrane. A verysimilar finding was obtained in the cases of TxG. Podocytes,both in a focal and segmental or a global distribution, labeledpositively for PDGF-C (Figure 3C). Individual biopsies thathad features of a transplant glomerulitis frequently had detectablePDGF-C in glomerular segments. In these cases, PDGF-C was locatedto focal glomerular endothelial cells as well as mesangial cells(Figure 3D). Within the tubulointerstitium and the vasculature,PDGF-C expression was unchanged compared with the normal adultand the IgAN cases. Immunohistochemical analysis of cases ofMC detected PDGF-C expression in the cytoplasm of podocytes(data not shown). Compared with the prominent positivity inMGN, the staining pattern in MC was more focal.
Figure 3. PDGF-C expression in human membranous nephropathy and transplant glomerulopathy. Immunohistochemistry using a PDGF-C–specific polyclonal antibody results in a red color product. (A) All cases of membranous nephropathy express PDGF-C within the glomeruli. (B) Higher power magnification illustration of Figure 3A showing localization of PDGF-C within the cytoplasm of podocytes (arrows). (C) In the cases of transplant glomerulopathy, glomerular PDGF-C was detected in all cases. (D) Higher power magnification illustration of Figure 3C indicating strong expression of PDGF-C in individual podocytes (arrows). Original magnifications: x400 in A and C; x1000 in B and D.
We further addressed whether PDGF-C was locally produced withinthe diseased glomeruli in the course of the glomerulonephritis.We therefore microdissected individual glomeruli of normal adultkidneys and of biopsies with IgAN, MGN, or MC and analyzed PDGF-CmRNA expression by real-time RT-PCR. Consistent with our resultsobtained by immunohistochemistry at the protein level, we foundno significant upregulation of PDGF-C mRNA expression in IgANcompared with normal adult kidneys (Figure 4). However, caseswith the diagnosis of MGN demonstrated a significant increasein glomerular PDGF-C mRNA (Figure 4). Cases of MC reached anintermediate result. While some cases of MC demonstrated increasedglomerular PDGF-C mRNA expression, the whole group did not differsignificantly from the normal controls. These results indicatethat the upregulation of podocytic PDGF-C protein that was detectedby immunohistochemistry in MGN was related to an increase inlocal PDGF-C mRNA synthesis. Further analysis of clinical parametersrevealed no significant correlation between glomerular PDGF-CmRNA expression and serum-creatinine, amount of proteinuria,age, or gender in any of the groups (data not shown).
Figure 4. PDGF-C mRNA expression in microdissected glomeruli. No significant difference of glomerular PDGF-C mRNA is observed in IgA nephropathy (IgAN) and minimal change disease (MC) as compared with normal adult kidney (normal). Glomerular PDGF-C mRNA is significantly increased in membranous nephropathy (MGN) as compared with normal adult kidneys or IgA nephropathy. Graphical points represent data obtained from individual patients.
PDGF-C Expression is Increased at Sites of Fibrosing Tubulointerstitial Injury
Individual cases of all analyzed groups had developed variabledegrees of tubulointerstitial disease, including tubular atrophy,tubulointerstitial leukocyte infiltration, and tubulointerstitialfibrosis. The severity of the tubulointerstitial lesions correlatedwith the severity of the glomerular lesions in general. At sitesof fibrosing lesions, tubulointerstitial PDGF-C expression wasmarkedly upregulated in all cases analyzed (Figure 5, A and B).There was typically a finely granular immunohistochemicalsignal in fibrosing areas. Even at high magnifications (Figure 5B),we were unable to clearly identify whether PDGF-C was localizedintracellularly within interstitial cells or within the extracellularmatrix.
Figure 5. PDGF-C expression in fibrosing tubulointerstitial lesions. Immunohistochemistry using a PDGF-C–specific polyclonal antibody results in a black/brown color product. (A) In a case of IgA nephropathy that was associated with severe tubulointerstitial injury, tubulointerstitial PDGF-C expression is markedly upregulated. (B) Higher power magnification illustration of Figure 5A indicating the typical fine granular immunohistochemical signal in fibrosing areas. Original magnifications: x400 in A; x1000 in B.
PDGF cytokines have been identified as important mediators inthe pathogenesis of glomerular diseases (1,2). The present studyis the first to identify PDGF-C in human kidneys. PDGF-C isconstitutively expressed in the adult human kidney and localizesto parietal epithelial cells, distal tubular epithelial cells,and arterial endothelial cells. After predominant injury topodocytes, which is typically seen in membranous nephropathyand transplant glomerulopathy, there is a significant upregulationof glomerular PDGF-C. PDGF-C localizes immunohistochemicallyto podocytes. A significant upregulation of PDGF-C is additionallydetected within the tubulointerstitium localizing to fibrosinglesions independent of the primary diagnosis of the differentcases.
Our current understanding of the biology of the cytokine PDGF-Cis still very limited. A first study by Li et al. (6) identifiedPDGF-CC as a PDGFR-specific ligand, while Gilbertson et al.(12) additionally demonstrated that PDGF-CC could activate thebeta-chain in the heterodimeric receptor complex but not ina homodimeric PDGFR receptor. Little is known about the potentialfunctions of PDGF-C. Both, Li et al. (6) and Gilbertson et al.(12) have identified PDGF-C as a potent mitogenic stimulus forseveral mesenchymal cells in vitro. Furthermore, transgenicoverexpression of PDGF-C in the mouse heart induced strong proliferationof cardiac fibroblasts and resulted in subsequent interstitialexpansion with features of interstitial fibrosis (6). Furtherstudies located PDGF-C expression in vascular smooth musclecells as well as in endothelial cells (13). Additionally, PDGF-Cwas capable of inducing proliferation of human coronary arteryand aortic smooth muscle cells in vitro, suggesting that PDGF-Cparticipates in vascular development and pathology (13). Expressionanalysis of PDGF-C in adult and developing mouse tissues revealeda widespread and dynamic expression of PDGF-C in different organs(14). A strong expression was particularly prominent at sitesof developing epidermal openings, indicating a function of PDGF-Cin this developmental step (14).
The expression of the main PDGF-C receptor PDGFR has previouslybeen analyzed in human developing, normal adult, and in diseasedkidneys. In the developing kidney, PDGFR is present in interstitialcells, in vascular arcades, in the invaginating mesangium andthe stalk region of early glomerular differentiation stagesand strongly in the adventitia of the fetal arterial architecture(15). In normal adult kidneys, PDGFR was extensively expressedby interstitial cells and only occasionally by mesangial cells(15). In cases of renal arteriosclerosis and/or renal vascularrejection, a widespread expression of PDGFR was noted in renalcell types involved in fibrotic and sclerosing processes (16).Gesualdo et al. (17) detected only a slight increase of PDGFRexpression in diseased kidneys, mainly at the interstitial level,while a few cases of lupus nephritis also showed a moderateincrease of PDGFR at the glomerular level. In another studyin biopsies from patients with glomerular diseases, Stein-Oakleyet al. (18) identified increased PDGFR in patients with IgANand focal glomerulosclerosis, particularly in areas of mesangialhypercellularity. Taken together, these data show that PDGFRis expressed in the normal renal interstitium and is upregulatedat sites of renal fibrosis. One PDGFR ligand, PDGF-A, is constitutivelyexpressed in normal renal interstitium (19). PDGF-A was additionallydetected in glomerular visceral epithelial cells, vascular arterialsmooth muscle cells, and vascular endothelial cells in rejectingrenal allograft (19,20).
Correlation of the expression analyses of PDGF-C and its receptormight offer new insights into potential functional mechanismsof this cytokine. In the developing human kidney, increasedPDGF-C expression occurs in close proximity to sites of previouslyreported PDGFR expression. PDGF-C localizes to the metanephricblastema (PDGFR to interstitial cells) and to arterial endothelialand smooth muscle cells (PDGFR to adventitial cells), indicatinga potentially paracrine action of PDGF-C in vascular and interstitialdevelopment. In the fetal glomeruli, both the ligand PDGF-Cas well as the receptor PDGFR localize to the developing mesangium,indicating a potentially paracrine or even autocrine role forPDGF-C in developing mesangial cells.
The identification of increased PDGF-C at sites of fibrosing/sclerosinginjury in diseased kidneys might indicate a functional rolefor PDGF-C in this process. The other known PDGFR ligand, PDGF-A,is constitutively expressed in normal interstitium; therefore,a role of increased PDGFR in fibrosing renal interstitium hasremained speculative. Our current data suggest that upregulatedexpression and signaling of PDGF-C via upregulated PDGFR mighthave a unique role in mediating renal fibrosis.
Another novel and important aspect of the present study is theidentification of podocytic PDGF-C in human membranous nephropathy,transplant glomerulopathy, and minimal change disease. Our previousstudies in rat experimental glomerulonephritis had revealeda focal expression of PDGF-C in some podocytes in a model ofglomerulosclerosis (Milan glomerulosclerosis) (7). GlomerularPDGF-C was undetectable in a model of membranous nephropathy(passive Heymann nephritis) (7). In human membranous nephropathy,however, there was a prominent PDGF-C expression in all analyzedcases. Immunohistochemistry localized PDGF-C to the podocytecytoplasm. Furthermore, our data indicate a local productionof PDGF-C because glomerular PDGF-C mRNA was equally upregulatedas PDGF-C protein in these cases. Transplant glomerulopathyis another human disease that is thought to be associated withprominent injury to the podocytes. A strong podocytic PDGF-Cexpression mimicked the results of the membranous nephropathycases. Interestingly, PDGF-C remained undetectable in podocytesduring human renal development. From a more general perspective,these data might indicate that the activating injury (dedifferentiation)of podocytes in membranous and transplant nephropathy differsfrom the activation (differentiation) of podocytes in fetalglomerular development and results in distinct PDGF-C expression.
Finally, our data indicate a role for PDGF-C in modulating mesangialcell behavior in vivo. One central finding in our previous reportanalyzing PDGF-C expression in rat models of glomerular diseaseswas a marked and apparent de novo upregulation of mesangialPDGF-C in mesangioproliferative glomerulonephritis (7). Furthermore,PDGF-C acted as a potent rat mesangial cell mitogen in vitro(7). The present data obtained in the human organism are lessconsistent. Proliferating developing mesangial cells overexpressedPDGF-C in the human fetal glomerulus, indicating a role forPDGF-C in human mesangial cell proliferation in vivo. In adulthuman mesangioproliferative glomerulonephritis that was analyzedin IgA nephropathy and mesangioproliferative lupus nephritis(our own unpublished results), mesangial PDGF-C overexpressionremained absent. Additionally, RT-PCR did not detect any significantupregulation of PDGF-C mRNA in IgAN. In contrast, in human transplantglomerulopathy, a disease typically preceded by mesangiolysis,PDGF-C expression was seen in individual mesangial cells. Thus,a different mesangial stimulus, i.e., mesangiolysis as it occursin rat anti-Thy 1.1 nephritis or human TxG, apparently is neededfor mesangial PDGF-C expression.
In conclusion, the present study is the first to identify distinctexpression patterns of the novel cytokine PDGF-C in human adultand developing kidneys. An upregulated expression was seen inpodocytes or interstitial cells and was associated with injuryor activation of these specific cell types. Specific inhibitionof PDGF-C in vivo will help to further define the roles of PDGF-Cin renal development and disease.
Acknowledgments
The technical help of Kerstin Schenk, Gabi Dietzel, Gerti Minartz,Andrea Cosler, and Claudia Schmidt is gratefully acknowledged.This work was supported in part by grants SFB 542 C7 (FE, TO,JF) and SFB 405 B10 (HJG) from the Deutsche Forschungsgemeinschaft(DFG), by a grant from the Swedish Research Council (grant K2001–03P-12070–05B)and the Novo Nordisk Foundation (UE), and by grant DHGP01KW9922/2and the Else-Kröner Fresenius Foundation (MK). FE is arecipient of a stipend of the German Kidney Foundation (DeutscheNierenstiftung).
C Cohen, M Kretzler, and D Schlöndorff, Munich; F Delarueand JD Sraer, Paris; MP Rastaldi and G D’Amico, Milano;P Doran and HR Brady, Dublin; D Mönks and C Wanner, Würzburg;AJ Rees, Aberdeen; P Brown, Aberdeen; F Strutz and G Müller,Göttingen; P Mertens and J Floege, Aachen; N Braun andT Risler, Tübingen; L Gesualdo and FP Schena, Bari; J Gerthand G Stein, Jena; R Oberbauer and D Kerjaschki, Vienna; M Fischerederand B Krämer, Regensburg; W Samtleben and W Land, Munich;H Peters and HH Neumayer, Berlin; K Ivens and B Grabensee, Düsseldorf.
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Received for publication October 2, 2002.
Accepted for publication January 23, 2003.
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Top
Abstract
Introduction
-subunit and 
-subunit of the PDGF receptor (PDGFR) (3–5). In 2000, a new member of the PDGF family was identified and subsequently termed PDGF-C (6). PDGF-C, like PDGF-A and PDGF-B, forms a disulphide-bonded dimer, PDGF-CC. Li et al. (6) identified PDGF-CC as a PDGFR-
