Maria Dolores Sanchez-Niño*,
Ana Belen Sanz*,
Pekka Ihalmo,
Markus Lassila,
Harry Holthofer,
Sergio Mezzano||,
Claudio Aros||,
Per-Henrik Groop,
Moin A. Saleem¶,
Peter W. Mathieson¶,
Robert Langham**,
Matthias Kretzler,
Viji Nair,
Kevin V. Lemley,
Robert G. Nelson,
Eero Mervaala||||,
Deborah Mattinzoli¶¶,
Maria Pia Rastaldi¶¶,
Marta Ruiz-Ortega*,
Jose Luis Martin-Ventura*,
Jesus Egido* and
Alberto Ortiz*
* Fundacion Jimenez Diaz, Universidad Autonoma de Madrid, Fundación Renal Iñigo Alvarez de Toledo, Madrid, Spain; Folkhälsan Institute of Genetics, Folkhälsan Research Center, University of Helsinki, and Division of Nephrology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland; Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Finland; Center for BioAnalytical Sciences, Dublin City University, Ireland; || Division of Nephrology, Universidad Austral, Valdivia, Chile; ¶ Academic and Children's Renal Unit, University of Bristol, Bristol, United Kingdom; **Department of Medicine, St. Vincent's Hospital, University of Melbourne, Victoria, Australia; Division of Nephrology, University of Michigan, Ann Arbor, Michigan; Division of Nephrology, Childrens Hospital Los Angeles, Los Angeles, California; Phoenix Epidemiology and Clinical Research Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Phoenix, Arizona; |||| Institute of Biomedicine/Pharmacology, University of Helsinki, Finland; and ¶¶ Fondazione D'Amico per la Ricerca sulle Malattie Renali, Milan, Italy
Correspondence: Dr. Alberto Ortiz, Unidad de Diálisis, Fundación Jiménez Díaz, Avda Reyes Católicos 2, 28040 Madrid, Spain. Phone: +34-915-504940; Fax: +34-915-442636; E-mail: aortiz{at}fjd.es
Received for publication February 15, 2008.
Accepted for publication July 16, 2008.
Although metabolic derangement plays a central role in diabeticnephropathy, a better understanding of secondary mediators ofinjury may lead to new therapeutic strategies. Expression ofmacrophage migration inhibitory factor (MIF) is increased inexperimental diabetic nephropathy, and increased tubulointerstitialmRNA expression of its receptor, CD74, has been observed inhuman diabetic nephropathy. Whether CD74 transduces MIF signalsin podocytes, however, is unknown. Here, we found glomerularand tubulointerstitial CD74 mRNA expression to be increasedin Pima Indians with type 2 diabetes and diabetic nephropathy.Immunohistochemistry confirmed the increased glomerular andtubular expression of CD74 in clinical and experimental diabeticnephropathy and localized glomerular CD74 to podocytes. In culturedhuman podocytes, CD74 was expressed at the cell surface, wasupregulated by high concentrations of glucose and TNF-, andwas activated by MIF, leading to phosphorylation of extracellularsignal–regulated kinase 1/2 and p38. High glucose alsoinduced CD74 expression in a human proximal tubule cell line(HK2). In addition, MIF induced the expression of the inflammatorymediators TRAIL and monocyte chemoattractant protein 1 in podocytesand HK2 cells in a p38-dependent manner. These data suggestthat CD74 acts as a receptor for MIF in podocytes and may playa role in the pathogenesis of diabetic nephropathy.
Diabetic nephropathy (DN) is one of the major complicationsof diabetes and the most common cause of ESRD. Hyperglycemiaactivates secondary mediators that lead to DN. In this regard,inflammatory cytokines may be critical in the development ofmicrovascular diabetic complications and nephropathy.1 Monocytechemoattractant protein 1 (MCP-1) is considered a diagnosticmarker and therapeutic target in DN.2 TNF-related apoptosisinducing ligand (TRAIL) expression is increased in diabeticnephropathy and induces tubular cell apoptosis.3 A better understandingof secondary mediators of injury may lead to the design of newtherapeutic strategies.
Macrophage migration inhibitory factor (MIF) is a widely expressedpleiotropic cytokine, exhibiting a broad range of immune andinflammatory activities.4 MIF has been implicated in renal injury.A marked increase in MIF was noted in glomeruli and tubulesin proliferative forms of human glomerulonephritis, where denovo MIF expression was localized to glomerular endothelialand mesangial cells.5 Cultured tubular and mesangial cells produceMIF in response to inflammatory stimuli.6,7 Enhanced podocyteexpression of a MIF transgene induces podocyte injury in mice,independent of leukocyte infiltration, suggesting autocrineeffects of MIF on podocytes.8 Urine MIF concentration is significantlyincreased in proliferative forms of glomerulonephritis and correlateswith the degree of renal injury.9
Evidence has also linked MIF to DN. MIF expression is increasedin experimental DN.10 Serum MIF concentration are elevated inindividuals with type 2 diabetes11 and also locally at sitesinjured by diabetes in patients with proliferative diabeticretinopathy.12
Although MIF was identified as a soluble, T cell–derivedfactor in 1966,13 the nature of its membrane receptor was unknownuntil 2003, when CD74 was shown to be a receptor for MIF.14CD74 antigen (invariant polypeptide of MHC, HLA-DR ) is a typeII transmembrane protein that plays a critical role in classII MHC antigen processing.15 In addition, CD74 may transduceMIF-induced activation of the extracellular signal–regulatedkinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK)cascade and promotes cell proliferation and prostaglandin E2production in leukocytes and fibroblasts.14 On the basis ofthe observation that CD74 expression was increased in transcriptomestudies of human DN, we investigated the role of CD74 in transducingMIF signals in human podocytes. CD74 was found to be increasedin podocytes and tubular cells from humans and animals withDN, and cultured podocytes expressed CD74 that mediated MIFactivation of MAPK as shown by knockdown experiments. Furthermore,MIF promoted MCP-1 and TRAIL expression in podocytes and tubularcells.
Identification of CD74 as an Overexpressed Gene in Human DN
Increased renal interstitial CD74 mRNA expression (a 1.92-foldincrease over living kidney donor controls; P = 0.0015) hadbeen noted by transcriptomic analysis of human DN biopsy tubulointerstitium.3,16We now report similar findings in the renal tubulointerstitiumof a different cohort of DN patients: Pima Indians with DN,on whom CD74 mRNA was increased 1.95-fold over living kidneydonor control subjects (P < 0.0001; Figure 1A). In addition,a 1.64-fold increase in CD74 mRNA was noted in the glomerulifrom Pima Indians with diabetes (P < 0.0001; Figure 1A).By contrast, changes in minimal-change disease (MCD) were <1.5-foldin both compartments (Figure 1A).
Figure 1. Increased CD74 expression in human DN biopsies. (A) Transcriptomic analysis of CD74 mRNA in glomerular and tubulointerstitial compartments of living kidney donors (control, n = 6) and Pima Indians with diabetes (DN, n = 20) and MCD (n = 4). Data are means ± SD; #P < 0.0001 versus control. (B) Semiquantification of CD74 immunoreactivity within the glomerular and tubular compartments in the kidney biopsies from control subjects (n = 5) and patients with DN (n = 5) and MCD (n = 4). **P = 0.01 versus control. (C) Representative image of a control and a DN sample. A marked increase in CD74 staining in DN is visible in both glomerular and tubular cells. (D) Co-localization of CD74 and synaptopodin in podocytes in a glomerulus from DN biopsy.
In an independent second group of patients with DN, immunohistochemistrydemonstrated increased expression of CD74 protein in both theglomerular and tubular compartments (Figure 1, B and C). Bycontrast, no significant changes in protein expression wereobserved in MCD (Figure 1B). Confocal microscopy co-localizedCD74 and synaptopodin, indicating that podocytes contributeto glomerular CD74 expression in DN (Figure 1D).
Increased CD74 Expression in Experimental DN
In a chronic rat model of DN induced by a single injection ofstreptozotocin (STZ) and characterized by the development ofalbuminuria at 7 mo of follow-up (1071 ± 247 versus 390± 70 µg/24 h; P < 0.02; Supplemental Table 1),CD74 was increased in whole kidney by Western blot (Figure 2,A and B). Immunohistochemistry localized the increased CD74expression to both glomeruli and tubules of DN rats (Figure 2C).In glomeruli, CD74 was localized to podocytes (Figure 2C). Quantificationof immunohistochemistry preparations showed a significant increasein glomerular CD74 staining (Figure 2D).
Figure 2. Expression of CD74 protein in experimental DN. (A) Western blot of whole kidney. Representative image. (B) Western blot quantification expressed as percentage increase over control. Data are means ± SD of 10 rats per group. *P < 0.05 versus control. (C) Immunohistochemistry, representative images of control kidney, DN, and a detail of DN in which podocyte staining for CD74 is observed (arrows). (D) Quantification of glomerular CD74 expression by immunohistochemistry. **P < 0.01 versus control. Magnification, x200.
Expression of CD74 in Cultured Human Podocytes
We found differentiated cultured human podocytes to expressCD74 mRNA and protein (Figure 3). Either a high-glucose mediumor the inflammatory cytokine TNF- increased CD74 mRNA (Figure 3,A and B) and protein expression (Figure 3, C and D). We observedno changes (<7% variation at 24 or 48 h) in CD74 expressionwith mannitol, used as an osmolarity control. The delayed increasein protein is consistent with findings in other cell types stimulatedwith IFN-.17 Confocal microscopy showed that the bulk of CD74localizes to the perinuclear region (Figure 3E). This is consistentwith findings in other cell types.18 We also found CD74 in thenucleus. This is also consistent with the fact that CD74 mayundergo regulated intramembrane proteolysis and migrate to thenucleus.18 Of more relevance to the present research, we alsoobserved CD74 at the cell membrane. Staining for nephrin confirmedthe differentiated state of podocytes (Figure 3E). Flow cytometryconfirmed the presence of CD74 at the cell surface (Figure 3F),supporting its possible role as a receptor.
Figure 3. Expression of CD74 by human cultured podocytes. (A) High glucose increases CD74 mRNA. Real-time reverse transcription–PCR (RT-PCR). Data are means ± SD of three independent experiments. **P < 0.01 versus control 48 h. (B) TNF increases CD74 mRNA. Real-time RT-PCR. Data are means ± SD of three independent experiments. **P < 0.01 versus control. (C) High glucose increases CD74 protein expression. Representative Western blot and quantification. Data are means ± SD of three independent experiments. **P < 0.01 versus control 48 h. (D) TNF increases CD74 protein. Data are means ± SD of three independent experiments. **P < 0.01 versus control 48 h. (E) Confocal microscopy localized CD74 to the perinuclear region and the cell membrane. Nephrin staining used the same secondary antibody and confirmed the differentiated status of podocytes. (F) Flow cytometry of nonpermeabilized cells showed that CD74 is expressed in the cell surface.
Actions of MIF in Cultured Podocytes Are Mediated by Engagement of CD74
We investigated the sensitivity of podocytes to the CD74 ligandMIF. MIF was previously linked to glomerular injury and thecomplications of diabetes,10–12 but the MIF receptor(s)in the kidney had not been characterized. Phosphorylation ofERK1/2 was recently found to be a CD74-mediated MIF effect inextrarenal cells.14 In podocytes, MIF induced phosphorylationof ERK1/2 and p38 MAPK in a time- and dosage-dependent manner(Figure 4). As previously reported for CD74-mediated responsesin extrarenal cells, ERK1/2 phosphorylation in response to MIFpersisted for up to 24 h (Figure 4A). We observed similar resultsfor p38 MAPK (Figure 4, C and D).
Figure 4. MIF activates MAPK in cultured podocytes. (A through D) MIF stimulation results in ERK1/2 (A and B) and p38 (C and D) phosphorylation. Time course (A, **P < 0.01 versus control *P < 0.05 versus control; C, **P < 0.01 versus control *P < 0.05 versus control) and dose-response at 30 min (B, *P < 0.05 versus control; D, **P < 0.01 versus control). Representative Western blots and quantification (means ± SD) of three independent experiments.
To confirm that MIF actions on podocytes were mediated by CD74,we knocked down CD74 by specific small interfering RNA (siRNA;Figure 5A). As a result, podocyte ERK1/2 and p38 phosphorylationresponses to MIF were lost (Figure 5, B and C).
Figure 5. Downregulation of CD74 prevents MIF actions on podocytes. siRNA downregulation of CD74 protein (Western blot; A) prevents ERK1/2 (B) and p38 (C) phosphorylation induced by 50 ng/ml MIF for 30 min. Data are means ± SD of three independent experiments. (B) *P < 0.05 versus control; **P < 0.01 versus MIF alone. (C) *P < 0.05 versus control; **P < 0.01 versus MIF alone.
MIF Increases TRAIL and MCP-1 Expression in Podocytes and Tubular Cells
MCP-1 and TRAIL are mediators of renal injury in diabetes.2,3MIF increased TRAIL mRNA in podocytes in a time-dependent manner(Figure 6A). Inhibitors of ERK1/2 and p38 prevented the upregulationof TRAIL and MCP-1 expression (Figure 6, B and C), suggestingthe need for the combined participation of both pathways.
Figure 6. MIF increases TRAIL and MCP-1 expression in podocytes. (A) Time course of TRAIL mRNA expression in podocytes treated with 10 ng/ml MIF. Real-time RT-PCR. Data are means ± SD of three independent experiments. *P < 0.05 versus control. (B and C) In cells stimulated with 10 ng/ml MIF for 24 h, pretreatment with inhibitors of ERK1/2 (20 µM PD98059) and p38 MAPK (5 µM SB203580) for 1 h prevented the induction of TRAIL mRNA (B, *P < 0.05 versus control, **P < 0.01 versus MIF alone) and MCP-1 mRNA (C, *P < 0.05 versus control, **P < 0.01 versus MIF alone). Data are means ± SD of three independent experiments.
High glucose also upregulated CD74 expression in cultured HK2human proximal tubular cells (Figure 7, A and B), and MIF alsoincreased TRAIL and MCP-1 expression in these cells (Figure 7,C through E).
Figure 7. CD74 expression and MIF actions in cultured tubular cells. (A) High glucose increases CD74 mRNA expression at 24 h. Real-time RT-PCR. Data are means ± SD of three independent experiments. #P < 0.0001 versus control. (B) High glucose increases CD74 protein expression at 24 h. Representative Western blot and quantification. Data are means ± SD of three independent experiments. #P < 0.0001 versus low-glucose control. (C) Exposure to 10 ng/ml MIF for 24 h increases TRAIL expression in tubular cells. Representative Western blot and quantification. Data are means ± SD of three independent experiments. *P < 0.05 versus control. (D and E) Cells were pretreated with inhibitors of ERK1/2 (20 µM PD98059) and p38 MAPK (5 µM SB203580) for 1 h and stimulated with 10 ng/ml MIF for 24 h: Effect on TRAIL mRNA (D, *P < 0.05 versus control, **P < 0.01 versus MIF alone) and MCP-1 mRNA expression (E, *P < 0.05 versus control, **P < 0.01 versus MIF alone). Data are means ± SD of three independent experiments.
We previously observed de novo TRAIL expression in diabeticglomeruli.3 We now report that podocytes are sites of TRAILexpression in human DN (Figure 8A) and that, in addition totubular cells,3 TRAIL kills podocytes cultured in a diabeticmilieu (Figure 8B).
Figure 8. TRAIL is expressed by podocytes in diabetic kidney injury and is lethal for cultured podocytes. (A) Representative TRAIL immunohistochemistry of a human glomerulus from a patient with DN. The arrow points a stained podocyte. De novo TRAIL expression in glomeruli was found in 13 of 17 biopsies from patients with DN by our group, whereas no glomerular staining for TRAIL was observed in control glomeruli.3 (B) Culture for 24 h in the presence of 100 ng/ml TRAIL increases the apoptosis rate in podocytes cultured in a high-glucose milieu. *P < 0.05 versus low glucose; **P < 0.01 versus high glucose. Data are means ± SD of three independent experiments.
MIF is a pleiotropic cytokine that has been implicated in thedevelopment of renal injury5–8,10; however, to date, thereceptor mediating MIF actions on kidney cells had not beencharacterized. We now show (1) that the expression of CD74,a receptor for MIF, is increased in human DN as well as in experimentalDN; (2) that podocytes and tubular epithelial cells expressCD74 both in culture and in vivo; and (3) that CD74 mediatessignal transduction initiated by MIF in these cells, increasingthe expression of TRAIL and MCP-1. These results identify CD74as a potential therapeutic target in DN.
We confirmed increased CD74 expression in human DN at the mRNAand protein levels in two different cohorts of patients. Findingsin the rat model corroborated those in humans. Although it isconceivable that CD74 plays a role in other forms of renal diseasein which MIF has been found to be increased, data from patientswith MCD suggest that it is not upregulated in all forms ofglomerular injury. In this regard, high glucose levels couldcontribute to the increased podocyte and tubular cell CD74 expressionin patients with diabetes; however, increased glomerular CD74may not be specific from DN. In fact, preliminary studies showedthat CD74 mRNA expression is increased in hypertensive nephropathy(P.I. et al., unpublished observation). In this regard, podocyteCD74 was also upregulated by inflammatory cytokines, such asTNF-, that are present in a number of glomerular pathologies,including DN.19
MIF was previously implicated in renal injury. Increased glomerularand tubular MIF expression as well as urinary excretion wasnoted in human and experimental glomerular injury, includingDN.5,9,10 Despite this, the cellular targets of MIF in glomerularinjury had not been studied in detail and the receptor mediatingMIF actions had not been identified. We now show that the expressionof the MIF receptor CD74 is increased in epithelial tubularand glomerular cells in DN, thereby placing cytokine and receptorin the same compartments. The contribution of MIF to renal injurycould involve promoting inflammatory mediator expression andmodulation of apoptosis and cell proliferation.4 In this regard,there is in vivo evidence of a direct injurious role for MIFon glomerular cells: Transgenic expression of MIF in podocytesled to proteinuria, azotemia, and podocyte injury in the absenceof glomerular hypercellularity, suggesting that podocytes aredirect targets for MIF.8
MIF activated ERK1/2 and p38 MAPK in podocytes. ERK1/2 and p38MAPK have been implicated in the progression of various glomerulopathies.20They are phosphorylated in podocytes from patients with DN.21MAPK pathways mediate different signaling events either aloneor in concert with other pathways.22 p38 MAPK play a criticalrole in the regulation of proinflammatory cytokine production,cell survival and apoptosis, and the stability of the cytoskeleton.23–26Phosphorylation of p38 MAPK is increased in injured podocytes,and p38 MAPK inhibition suppressed podocyte injury in experimentalnephrotic syndrome.20 Increased glomerular ERK1/2 phosphorylationwas also observed in these models.20 The ERK cascade transmitssignals from many extracellular agents to regulate cellularprocesses through actions on more than 160 substrates.20 ERK1/2and p38 activation were required for MIF-induced induction ofTRAIL and MCP-1 in podocytes and tubular cells. TRAIL is a lethalcytokine whose expression increases in tubular cells and appearsde novo in podocytes in DN.3 TRAIL induces tubular cell apoptosisin a diabetic milieu and is also lethal for podocytes.
In summary, we have identified a novel role of CD74 in glomerularinjury. As a receptor for MIF, CD74 could contribute to podocyteinjury in susceptible patients with diabetes by activating MAPKcascades and increasing TRAIL and MCP-1 expression.
Transcriptomic Analysis
Human renal biopsies were collected in a multicenter study,the European Renal cDNA Bank (ERCB).3,16 The protocol was approvedby the local ethical committees. We obtained informed consentfrom patients according to local guidelines, and we processedsamples according to the ERCB protocol. For oligonucleotidearray–based gene expression profiling, we included a totalof 30 kidney biopsies from individual patients: Pretransplantationkidney biopsies from living donors used as control (n = 6) andprotocol biopsies from Pima Indians with histologic diagnosisof DN (n = 20) and from patients with MCD as a proteinuric nephropathycontrol (n = 4; Supplemental Table 2).
We manually microdissected tubulointerstitial and glomerularcompartments from cortical tissue segments. We isolated totalRNA using a commercially available isolation protocol. For probelabeling, we used a modification of the Eberwein protocol. Affymetrixmicroarray analysis following the manufacturer's protocol hasbeen described. We initially obtained image files through AffymetrixGeneChip software (MAS5).16 Subsequently, we performed robustmultichip analysis using RMAexpress (http://statwww.berkeley.edu/users/bolstad/RMAExpress).Starting from the normalized robust multichip analysis, theSignificance Analysis of Microarrays (SAM; version 1.21, http://www-stat.stanford.edu/tibs/SAM/)software was applied using a false discovery rate of 1% to identifygenes that were significantly differentially regulated betweenthe analyzed groups.3,16 We evaluated changes in expressionof 1.5-fold for significance. On the basis of this analysis,we found CD74 to be significantly upregulated in the glomerularand interstitial compartments of patients with DN.
Cell Culture and Reagents
Human podocytes are an immortalized cell line, as describedpreviously,27 transfected with a temperature-sensitive SV40gene construct and a gene encoding the catalytic domain of humantelomerase.28 At a permissive temperature of 33°C, the cellsremain in an undifferentiated proliferative state, whereas raisingthe temperature to 37°C results in growth arrest and differentiationto the parental podocyte phenotype. Undifferentiated podocytecultures were maintained at 33°C in RPMI 1640 medium withpenicillin; streptomycin; insulin, transferrin, and selenite;and 10% FCS. Once cells had reached 70 to 80% confluence, theywere cultured at 37°C for at least 14 d before use, whenfull differentiation had taken place. For experiments, cellswere cultured in serum-free medium 24 h before the additionof the stimuli and throughout the experiment. For high-glucoseexperiment, glucose was added in the medium to reach a finalconcentration of 700 mg/dl versus control medium with 200 mg/dlglucose. The same amount of mannitol was added as an osmolaritycontrol. TNF- (30 ng/ml) was from Immugenex (Los Angeles, CA),and TRAIL (100 ng/ml) was from Alexis (Läufelfingen, Switzerland).3Culture of HK2 human proximal tubular epithelial cells (ATCC,Rockville, MD) and the assessment of apoptosis as hypodiploidcells by flow cytometry have been described.3,29 PD98059 andSB203580 (Stressgen Bioreagent, Ann Arbor, MI) were dissolvedin DMSO.
Animal Model
We studied two groups of ten 10-wk-old Wistar Kyoto rats (Criffa,Barcelona, Spain). We induced diabetes by a single intraperitonealinjection of STZ (Sigma, St. Louis, MO), 50 mg/kg in 0.01 Mcitrate buffer (pH 4.5).30 Control rats received the STZ vehicle.We killed rats at 7 mo after induction of diabetes.31 All studieswere performed in accordance with the European Union normative.We administered insulin (1 to 4 IU subcutaneously; InsulatardNPH; Novo Nordisk, Bagsvaerd, Denmark) weekly so as to preventdeath but not with the aim of totally correcting hyperglycemia.We initiated insulin administration 7 d after STZ, having checkedthat the animals had glycemia >400 mg/dl (Glucocard; Menarini,Barcelona, Spain). We measured systolic BP monthly in conscious,restrained rats by the tail-cuff sphygmomanometer (NARCO, Biosystems,Austin, TX). Albuminuria was measured by ELISA (Celltrend, Luckenwalde,Germany; Supplemental Table 1).
Immunofluorescence and Immunohistochemistry Staining
We performed immunohistochemistry for CD74 in human kidney biopsieson formalin-fixed paraffin sections (4 µm) using HistostainSP Kit (Broad Spectrum, AEC, 95–9943; Zymed Labs, SanFrancisco, CA). Antigen was retrieved by microwave heating (10min in citric acid, 10 mM [pH 6.0]). Endogenous peroxidase activitywas quenched with 3%. Sections were blocked with serum, incubatedwith anti-CD74 (rabbit polyclonal, sc-20082, 1:30; Santa CruzBiotechnology, Santa Cruz, CA) overnight at 4°C, and stainedwith biotinylated secondary antibody. For the semiquantificationof staining intensity, we visually assessed 10 random views(x200 magnification) in each slide as 0 to 3 (0, no staining;1, moderate staining; 2, strong staining; 3, very strong staining)in a blinded manner. We calculated the mean value for all ofthe views in one slide. CD74 expression was expressed as percentagechange over control. The nonaffected parts of tumor nephrectomiesserved as a control group (n = 5), and we studied patients withDN (n = 5) and MCD (n = 4; Supplemental Table 2). TRAIL immunohistochemistryhas been described.3
For immunofluorescence, the unfixed renal tissue was embeddedin Tissue-Tek (Società Italiana Chimici, Rome, Italy),snap-frozen in a mixture of isopentane and dry-ice, and storedat –80°C. Subsequently, 5-µm sections were fixedin cold acetone and stained with rabbit anti-CD74 (Santa CruzBiotechnology) followed by AlexaFluor 488 goat anti-rabbit IgG(Molecular Probes, Invitrogen, Milan, Italy). After washing,the procedure was repeated with the second primary antibody(mouse anti-synaptopodin; Progen, Frankfurt, Germany), followedby AlexaFluor 546 goat anti-mouse (Molecular Probes). Sectionswere mounted with antifading mounting medium (Vectashield; VectorLaboratories, Burlingame, CA). Specificity of labeling was demonstratedby the lack of staining after substituting proper control IgG(rabbit primary antibody isotype control and mouse primary antibodyisotype control, both from Zymed, Invitrogen) for the primaryantibody.
In rats immunohistochemistry was carried out in paraffin-embeddedtissue sections 5 µm thick.31 The primary antibody wasgoat polyclonal anti-CD74 (1:60, Santa Cruz Biotechnology).Sections were counterstained with Carazzi's hematoxylin. Negativecontrols included incubation with a nonspecific Ig of the sameisotype as the primary antibody. The percentage of positiveglomerular surface area was quantified in 20 glomeruli per rat.
Western Blot
Tissue and cell samples were homogenized in lysis buffer31;then separated by 10 or 12% SDS-PAGE under reducing conditionsand transferred to polyvinylidene difluoride membranes (Millipore,Bedford, MA); blocked with 5% skim milk in PBS/0.5% vol/volTween 20 for 1 h; washed with PBS/Tween; and incubated withgoat polyclonal anti-CD74 (1:500), mouse monoclonal anti–p-ERK(1:500), rabbit polyclonal anti-ERK1/2 (1:2000), mouse monoclonalanti-p-p38 (1:500), or goat polyclonal anti-p38 (1:2500; allfrom Santa Cruz Biotechnology), and mouse monoclonal anti-TRAIL(1:1000, Pharmingen). Antibodies were diluted in 5% milk PBS/Tween.Blots were washed with PBS/Tween and subsequently incubatedwith appropriate horseradish peroxidase–conjugated secondaryantibody (1:2000; Amersham, Aylesbury, UK). After washing, theblots were developed with the chemiluminescence method (ECL;Amersham). Blots were then probed with mouse monoclonal anti–-tubulinantibody (1:2000; Sigma) and levels of expression were correctedfor minor differences in loading.
Real-Time Reverse Transcription–PCR
RNA was isolated by Trizol (Invitrogen, Paisley, UK).31 Onemicrogram of RNA was reverse-transcribed with High CapacitycDNA Archive Kit (Applied Biosystems, Foster City, CA). Real-timePCR reactions were performed on an ABI Prism 7500 sequence detectionPCR system (Applied Biosystems) according to the manufacturer'sprotocol using the Ct method.31 Expression levels are givenas ratios to glyceraldehyde-3-phosphate dehydrogenase. Predevelopedprimer and probe assays were obtained for human glyceraldehyde-3-phosphatedehydrogenase, MCP-1, TRAIL, and CD74 from Applied Biosystems.
Flow Cytometry Analysis of Cell Surface CD74 Expression
Cells were detached with 2 mM EDTA and 5 x 105 cells were incubatedfor 30 min at 4°C with 8 µg/ml rabbit anti-CD74 antibody(Santa Cruz Biotechnology) or control IgG followed by a 30-min4°C incubation with 1:100 FITC secondary antibody (Pharmingen,San Diego, CA).32 Mean cell fluorescence was calculated usingCell Quest Software (Becton Dickinson, Franklin Lakes, NJ).
Confocal Microscopy
Cells plated onto Labtek slides were fixed in 4% paraformaldehydeand permeabilized in 0.2% Triton X-100 in PBS for 10 min each.After washing in PBS, cells were incubated overnight at 4°Cwith rabbit polyclonal anti-CD74 antibody (1:50; Santa CruzBiotechnology) or rabbit polyclonal anti-nephrin (1:100),33followed by incubation with FITC secondary antibody (1:200;Sigma). Cell nuclei were counterstained with propidium iodide.After washing, cells were mounted in 70% glycerol in PBS andanalyzed with a DM-IRB confocal microscope (Leica DM, Bannockburn,IL).31
Transfection of siRNA
Cells were grown in six-well plates (Costar, Cambridge, MA)and transfected with a mixture of 25 nmol/ml siRNA (Santa Cruz),Opti-MEM I Reduced Serum Medium and Lipofectamine 2000 (Invitrogen).After 18 h, cells were washed and cultured for 48 h in completemedium and serum-depleted for 24 h before addition of MIF. Thistime point was selected from a time course of decreasing CD74protein expression in response to siRNA. A negative controlscrambled siRNA provided by the manufacturer did not reduceCD74 protein.
Statistical Analysis
Data are given as means ± SD. Mann-Whitney, two-sidedt test or one-way ANOVA was applied to indicate significantlydifferent mean values in comparison with the control group.P < 0.05 was considered statistically significant.
This study was supported by grant FIS 06/0046, SAF03/884, LSHB-CT-2004-6(ADDNET), LSHB-CT-2007-036644 (DIALOK), Sociedad Españolade Nefrología, ISCIII-RETIC REDinREN/RD06/0016, and Comunidadde Madrid/FRACM/S-BIO0283/2006. Salary support was providedby FIS to A.B.S., MEC to M.D.S.N., Programa IntensificaciónActividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM)to A.O., and the Finnish Kidney Foundation and the Kyllikkiand Uolevi Lehikoinen Foundation for P.I. Fondecyt 1080083 toS.M. This research was supported, in part, by the IntramuralResearch Program of the National Institute of Diabetes and Digestiveand Kidney Diseases.
Anneli von Behr is acknowledged for technical assistance andMar Gonzalez García-Parreño and Alberto Puimefor technical help.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Supplemental information for this article is available onlineat http://www.jasn.org/.
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Abstract
Introduction
, and was activated by MIF, leading to phosphorylation of extracellular signal–regulated kinase 1/2 and p38. High glucose also induced CD74 expression in a human proximal tubule cell line (HK2). In addition, MIF induced the expression of the inflammatory mediators TRAIL and monocyte chemoattractant protein 1 in podocytes and HK2 cells in a p38-dependent manner. These data suggest that CD74 acts as a receptor for MIF in podocytes and may play a role in the pathogenesis of diabetic nephropathy. 
) is a type II transmembrane protein that plays a critical role in class II MHC antigen processing.15 In addition, CD74 may transduce MIF-induced activation of the extracellular signal–regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) cascade and promotes cell proliferation and prostaglandin E2 production in leukocytes and fibroblasts.14 On the basis of the observation that CD74 expression was increased in transcriptome studies of human DN, we investigated the role of CD74 in transducing MIF signals in human podocytes. CD74 was found to be increased in podocytes and tubular cells from humans and animals with DN, and cultured podocytes expressed CD74 that mediated MIF activation of MAPK as shown by knockdown experiments. Furthermore, MIF promoted MCP-1 and TRAIL expression in podocytes and tubular cells. 
tibs/SAM/) software was applied using a false discovery rate of 1% to identify genes that were significantly differentially regulated between the analyzed groups.3,16 We evaluated changes in expression of 
1.5-fold for significance. On the basis of this analysis, we found CD74 to be significantly upregulated in the glomerular and interstitial compartments of patients with DN. 
