RAGE Mediates Podocyte Injury in Adriamycin-induced Glomerulosclerosis
Jiancheng Guo*,
Radha Ananthakrishnan*,
Wu Qu*,
Yan Lu*,
Nina Reiniger*,
Shan Zeng*,
Wanchao Ma,
Rosa Rosario*,
Shi Fang Yan*,
Ravichandran Ramasamy*,
Vivette D'Agati and
Ann Marie Schmidt*
Departments of * Surgery, Ophthalmology, and Pathology, Columbia University Medical Center, New York, New York
Correspondence: Ann Marie Schmidt, Columbia University Medical Center, Department of Surgery, 630 West 168th Street, P&S 17-501, New York, NY 10025. Phone: 212-305-6406; Fax: 212-305-5337; E-mail: ams11{at}columbia.edu
Received for publication October 16, 2007.
Accepted for publication December 18, 2007.
In the kidney, the receptor for advanced glycation end products(RAGE) is principally expressed in the podocyte at low levels,but is upregulated in both human and mouse glomerular diseases.Because podocyte injury is central to proteinuric states, suchas the nephrotic syndrome, the murine adriamycin nephrosis modelwas used to explore the role of RAGE in podocyte damage. Inthis model, administration of the anthracycline antibiotic adriamycinprovokes severe podocyte stress and glomerulosclerosis. In contrastto wild-type animals, adriamycin-treated RAGE-null mice weresignificantly protected from effacement of the podocyte footprocesses, albuminuria, and glomerulosclerosis. Administrationof adriamycin induced rapid generation of RAGE ligands, andtreatment with soluble RAGE protected against podocyte injuryand glomerulosclerosis. In vitro, incubation of RAGE-expressingmurine podocytes with adriamycin stimulated AGE formation, andtreatment with RAGE ligands rapidly activated nicotinamide adeninedinucleotide phosphate (NADPH)-oxidase, via p44/p42 MAP kinasesignaling, and upregulated pro-fibrotic growth factors. Thesedata suggest that RAGE may contribute to the pathogenesis ofpodocyte injury in sclerosing glomerulopathies such as focalsegmental glomerulosclerosis.
The interaction of Receptor for Advanced Glycation End products(RAGE) with its ligands, including Advanced Glycation End products(AGEs), and proinflammatory S-100/calgranulins and high mobilitygroup box-1, mediates cellular stress.1–3 In human andexperimental nephropathy, RAGE is highly expressed in the glomerularvisceral epithelial cell or podocyte.4,5
Treatment of susceptible strains of mice with adriamycin (ADR)evokes rapid podocyte injury, manifested within days as massivefoot process effacement which precedes the development of frankglomerulosclerosis.6–10 Here, we provoked severe podocytestress in mice via administration of ADR to test the hypothesisthat RAGE contributes to glomerular injury.
RAGE Null Mice Are Protected From ADR-induced Podocyte Injury and Glomerulosclerosis
We performed laser-capture microdissection of mouse glomerulito monitor RAGE transcript levels in response to ADR. At 72h, 7 d, and 14 d, compared with normal saline (NS), ADR resultedin increases in RAGE transcripts of approximately 2.1-, 1.7-,and 1.7-fold, respectively (P < 0.001; Figure 1a). On day3, by confocal microscopy, RAGE was expressed in the podocyte,as illustrated by colocalization with synaptopodin epitopes(Figure 1, b through d).
Figure 1. Homozygous RAGE null mice display decreased podocyte stress, albuminuria, and glomerulosclerosis consequent to ADR. (a-d) ADR upregulates RAGE. (a) ADR, 10.5 mg/kg, was administered to male BALB/cJ mice. Mice were treated with sRAGE or vehicle, PBS, immediately after ADR and continued once daily until death. Laser capture microdissection of glomeruli was performed, RNA prepared, and quantitative real-time PCR performed for detection of murine RAGE transcripts and reported as fold change versus sham treatment (normal saline, NS; and PBS); n = at least 8 mice/group. (b-d) Confocal microscopy was performed on kidneys retrieved 3 d after ADR. Sections were stained with anti-RAGE IgG (b) or anti-synaptopodin IgG (c). In d, merged images reveal that the principal site of RAGE expression is the podocyte. Scale bar: 10 µm. (e-h) ADR and the impact of RAGE deletion on albuminuria and podocyte stress. Wild-type and homozygous RAGE null mice were treated with ADR. (e) Albuminuria. 24 h urine was collected from wild-type and RAGE null mice at 2 and 6 wk after ADR; urinary albumin/creatinine ratio is shown. n = at least 10 mice/group. (f-h) Two wk post-ADR, mice were killed and electron microscopy performed on kidney sections. Quantification of foot process effacement was performed and is shown in h. n = at least 5 mice/group. (i-o) Pathologic analysis. Wild-type and homozygous RAGE null mice were treated with ADR. At two and six wk, mice were killed and sections from kidney stained with PAS (i-l) Quantification was performed to establish mesangial/total glomerular area (m), sclerotic/total glomeruli (n), and tubules with casts/total tubules (o). n = at least 10 mice/group. Original magnifications: i, j x400; k, l x200). (p-q) Real-time PCR for detection of TGF-β1 and CTGF transcripts. On day 3, wild-type or RAGE null mice were killed and laser capture microdissection of glomeruli was performed; RNA was prepared and real-time PCR was performed for detection of transcripts for TGF- β1 (p) and CTGF (q). Results are reported as fold change and compared with wild-type sham-treated controls. n = at least 5 mice/group.
To address the role of RAGE in ADR injury using homozygous RAGEnull mice, we first genotyped three informative microsatellitemarkers (D16Mit165, D16Mit34, D16Mit156) spanning the DOXNPHsusceptibility locus on chromosome 16.11,12 We confirmed thatRAGE null mice, backcrossed more than 12 generations into BALB/cJ,were homozygous for BALB/cJ susceptibility alleles at the DOXNPHlocus.
Upon administration of ADR, RAGE null mice displayed significantlyless excretion of urinary albumin/creatinine versus wild-typemice at 2 wk, 276.5 ± 164.6 and 1228.3 ± 440.9µg/mg, and at 6 wk, 170.0 ± 119.3 and 1111.9 ±405.7 µg/mg, respectively (P < 0.01; Figure 1e). Wewere unable to detect any albumin in the urine of RAGE nullmice at baseline, suggesting that the RAGE null mouse did notdisplay a phenotype at baseline, and that it was partially protectedfrom ADR.
Based on the significant reduction in albuminuria observed inRAGE null mice after ADR, we examined pathology and glomerulargene expression. Two weeks after ADR, marked effacement of podocytefoot processes was observed in wild-type but not in RAGE nullmice (Figure 1, f through h). RAGE null mice displayed significantlyless glomerulosclerosis and cast formation versus wild-typemice (Figure 1, i and k versusFigure 1, j and l). Mean mesangialmatrix/total glomerular area and numbers of sclerotic/totalglomeruli and tubular casts area were significantly higher inwild-type versus RAGE null mice (P < 0.01; Figure 1, m througho). Pre-ADR, RAGE null mice displayed no detectable scleroticglomeruli or tubular casts, indicating that the genetic deletionof RAGE resulted in partial protection from ADR.
ADR induces glomerulosclerosis; thus, we performed quantitativereal-time PCR on laser capture-microdissected glomeruli to detectTGF-β1 and connective tissue growth factor (CTGF) transcripts.Both transcripts were significantly higher in wild-type mice3 d post-ADR versus NS (2.1-fold and 2.9-fold, respectively;P < 0.004; Figure 1, p and q) and were significantly lowerin RAGE null mice glomeruli (P < 0.01).
ADR Generates RAGE Ligands
To establish RAGE-dependent mechanisms in ADR injury, we testedwhether ADR bound RAGE. Anti-ADR IgG-immobilized AffiGel 15was incubated with ADR followed by sRAGE; Figure 2b revealsthat ADR was eluted, but not RAGE (RAGE was assessed by Westernblot; Figure 2d, lane 1). When AffiGel 15 beads were coatedwith anti-RAGE IgG and incubated with sRAGE followed by ADR,ADR was not detected in the eluate (Figure 2c), but eluate containedsRAGE (Western blot; Figure 2d, lane 3). Figure 2a is a typicalADR elution profile, and in Figure 2d, lane 2 is standard sRAGEcontrol detected by anti-RAGE IgG.
Figure 2. ADR generates RAGE ligands. (a-d) ADR does not bind RAGE. In panel b, anti-ADR IgG-immobilized AffiGel 15 was incubated first with ADR followed by sRAGE, ADR was eluted, but not RAGE (Western blot) (d, lane 1). When AffiGel 15 beads were coated with anti-RAGE IgG and incubated with sRAGE followed by ADR, ADR was not detected in the eluate (c), but eluate contained sRAGE (Western blot) (d, lane 3). In panel d, lane 2 is standard sRAGE control detected by anti-RAGE IgG. Panel a is a typical ADR elution profile. (e-p) ADR induces generation of AGE and ROS. (e-h) ADR generates AGEs. On day 3 post-ADR, kidney cortices were retrieved and ELISA for detection of AGE epitopes performed (e). In f through h, confocal microscopy using anti-AGE (f) or anti-synaptopodin IgG (g) was performed and localized AGE epitopes to the podocyte as indicated by merged studies in panel h (original magnification x200). (i-p) Wild-type and RAGE null mice were injected with ADR. (i) NADPH oxidase activity at 3 d post-ADR was determined in kidney cortex. (j) Renal cortex malondialdehyde levels were determined on days 3 and 7 after ADR. (k-l) Three days post-ADR, glomeruli were laser capture microdissected and real-time PCR was performed to determine iNOS transcripts (fold change compared with wild-type non-ADR controls) (k). In l, total nitrite and nitrate levels were determined in kidney cortex. (m-p) Nitrotyrosine epitopes in kidney cortex were studied by ELISA on days 3 and 7 after ADR; in panels n through p, confocal microscopy using either anti-nitrotyrosine (n) or anti-synaptopodin IgG (o) was performed and localized sites of nitrotyrosine epitopes to the podocyte as indicated by merged studies in (p) (original magnification x200). In e, i, j, k, l, and m, n = at least 7 mice/group. (q-v) ADR stimulates S-100/calgranulin generation. Confocal microscopy. Sections from NS (Con) or ADR-treated mice on day 3 were subjected to confocal microscopy for detection of S-100 (r, u) and synaptopodin (q, t) epitopes. Merged images are illustrated in panels s and v. Scale bar (r-w) = 10 µm.
Because ADR did not bind RAGE, we then hypothesized that ADRgenerated RAGE ligands. On day 3, approximately 1.8-fold higherAGE epitopes were identified in the cortex of wild-type miceversus NS and were significantly lower in RAGE null cortex (P< 0.05). The primary site of AGE after ADR was the glomerulus,and especially the podocyte, based on colocalization with anti-synaptopodinIgG (Figure 2, f through h).
Previous work linked ADR to reactive oxygen species (ROS) generationas a mediator of injury, but did not identify the specific mechanismsby which ROS were generated.8,9Compared with wild-type micereceiving NS, mice receiving ADR displayed an approximately3.1-fold increase in renal cortex nicotinamide adenine dinucleotidephosphate (NADPH)-oxidase activity on day 3 (P < 0.000001);this increase was significantly less in RAGE null mice (P <0.05; Figure 2i). Malondialdehyde, a lipid peroxidation product,was significantly higher in the cortex of wild-type mice treatedwith ADR versus RAGE null mice at 3 and 7 d (P < 0.01; Figure 2j).
Additional sources of ROS were assessed; compared with NS, animalstreated with ADR revealed approximately 2.5-fold increase iniNOS glomerular transcripts on day 3, whereas no significantincrease in iNOS mRNA was observed in RAGE null mice (Figure 2k).Levels of total nitrite and nitrate and nitrotyrosine epitopeswere significantly higher in wild-type versus RAGE null micecortex (Figure 2, l and m). Confocal microscopy localized nitrotyrosineepitopes primarily to the glomerulus, and at least in part tothe podocyte, based on colocalization with anti-synaptopodinIgG (Figure 2, n through p).
ADR increased expression of another RAGE ligand in the glomerulus,S-100/calgranulins. As illustrated in Figure 2t through v, S-100/calgranulinepitopes were evident at low levels in NS-treated wild-typemice. Upon ADR, S-100/calgranulin epitopes were increased inthe glomerulus (Figure 2r), in part in podocytes, based on colocalizationwith anti-synaptopodin IgG (Figure 2, q through s).
Administration of Soluble RAGE Suppresses ADR-mediated Injury in BALB/cJ Mice
As ADR generated RAGE ligands in vivo, it was logical to testthe effect of the ligand decoy. Soluble RAGE (sRAGE), administeredintraperitoneally immediately after ADR, was continued dailyuntil death. Administration of sRAGE blocked upregulation ofRAGE transcripts in glomeruli versus ADR/phosphate-bufferedsaline (PBS) at 72 h, 7 and 14 d (P < 0.01; Figure 1a).
Compared with ADR/PBS-treated mice, where significant podocytefoot process effacement was noted (Figure 3, a, c, and e), sRAGE-treatedanimals significantly decreased effacement (Figure 3, b, d,f, and g) at 2 and 6 wk (P < 0.01; Figure 3g). Compared withmice treated with ADR/PBS, sRAGE-treated mice displayed significantlydecreased excretion of urinary albumin/creatinine, 809.6 ±365.9 versus 191.1 ± 49.9 µg/mg at 2 wk and 1363.0± 403.0 versus 249.8 ± 107.0 µg/mg at 6wk, respectively; P < 0.05 (Figure 3h).
Figure 3. Administration of soluble RAGE suppresses ADR-mediated podocyte stress, albuminuria, and glomerulosclerosis. Wild-type mice were treated with ADR and sRAGE, 100 µg/d, or PBS. (a-g) Effect of ADR on foot process effacement and podocyte loss; 2 and 6 wk post-ADR, mice were killed and electron microscopy performed on kidney sections. Quantification of foot process effacement is shown in panel g. (h) Effect of ADR on albuminuria; 24-h urine was collected from wild-type mice treated with sRAGE or PBS at 2 and 6 wk after ADR; urinary albumin/creatinine ratio is shown. n = at least 10 mice/group. (i-l) Impact of ADR on glomerulosclerosis: effect of sRAGE. At 6 wk after ADR, kidneys were retrieved and subjected to PAS staining (i-j). Quantification was performed to establish mesangial/total glomerular area (k) and sclerotic/total glomeruli (l). n = at least 10 mice/group. Scale bar: i-j = 50 µm. (m-p) Real-time PCR for detection of TGF-β1 and CTGF transcripts. On days 3 and 7 after ADR, mice were killed, and laser capture microdissection of glomeruli was performed; RNA was prepared and real-time PCR performed for detection of transcripts for TGF- β1 (m, o) and CTGF (n, p). Results are reported as fold change compared with wild-type sham-treated controls. n = at least 10 mice/group.
Post-ADR, mesangial area/total glomerular area, and numbersof sclerotic to total glomeruli were significantly higher inPBS- versus sRAGE-treated mice (P < 0.05; Figure 3, i throughl). Laser capture microdissection of glomeruli 3 d after ADRrevealed an approximately 2.3-fold and approximately 3.2-foldincrease in transcripts for TGF-β1 and CTGF in ADR/PBS-treatedmice versus NS/PBS-treated mice, respectively (P < 0.0003;Figure 3, m and n). Significantly reduced transcripts for bothmolecules were noted in sRAGE-treated mice at 3 d (P < 0.0005;Figure 3, m and n). However, at 7 d, there were no differencesin TGF-β1 transcripts (Figure 3o). At 7 d, although anapproximately 2.2-fold increase in CTGF transcripts was notedin ADR/PBS versus NS-treated mice, significantly reduced transcriptswere seen in ADR/sRAGE-treated mice (P < 0.01; Figure 3p).
Administration of sRAGE Suppresses Oxidative Stress
AGE epitopes were higher in ADR- versus NS-treated mice, andmice receiving ADR and sRAGE demonstrated significantly lessAGEs in the cortex (P < 0.05; Figure 4a). On days 3 and 7,administration of ADR/PBS resulted in a significant increasein NADPH oxidase activity in kidney cortex versus NS/PBS (2.8-foldand 2.7-fold, respectively; P < 0.01; Figure 4b), which wassignificantly decreased by sRAGE (P < 0.01; Figure 4b). Levelsof malondialdehyde in cortex of sRAGE-treated mice were significantlylower at 6 and 72 h after ADR (P < 0.01; Figure 4c). Comparedwith ADR/PBS-treated mice, glomerular iNOS transcripts in sRAGE-treatedmice were lower at 24 h, 3 d, and 7 d by approximately 1.4-,2.1-, and 2.4-fold versus PBS, respectively (P < 0.01; Figure 4d).Levels of total nitrite and nitrate were significantly higherin ADR/PBS-treated mice versus sRAGE-treated animals at 3 hand 3 d post-ADR (P < 0.05; Figure 4e). Three days afterADR, sRAGE-treated mice displayed significantly lower nitrotyrosineepitopes in cortex (P < 0.01; Figure 4f).
Figure 4. Administration of soluble RAGE suppresses AGE generation and ROS in ADR-treated mice. Wild-type mice were treated with ADR and sRAGE or PBS. (a) ADR generates AGEs: effect of sRAGE. On day 3 post-ADR, kidney cortices were retrieved, and ELISA for detection of AGE epitopes was performed. n = at least 7 mice/group. (b-f) ADR generates oxidative stress: effect of sRAGE. At the indicated times after ADR, kidneys were retrieved, and cortices were prepared for examination of NADPH oxidase activity (b), malondialdehyde epitopes (c), iNOS (laser capture-microdissected glomeruli), and total nitrate and nitrite (d-e), and nitrotyrosine ELISA (f). n = at least 7 mice/group. *P < 0.05, **P < 0.01.
Effects of ADR and RAGE Ligands on Murine Podocytes: AGE Generation and Upregulation of Growth Factors
Our findings suggested that ADR stimulated RAGE ligand generationin podocytes in vivo. To further probe this concept, we incubatedcultured murine podocytes with ADR and found increased productionof AGEs (Figure 5a). These findings led us to test the roleof RAGE on podocyte stress; we used RAGE ligand S-100b, as S-100/calgranulinwas increased by ADR in vivo.2 Compared with sham, incubationof podocytes with S-100b (10 µg/ml) resulted in significantlyincreased transcripts for TGF-β1 (P < 0.05; Figure 5b).Pretreatment with anti-RAGE IgG, but not nonimmune IgG, significantlyattenuated S-100b-stimulated TGF-β1 transcripts (P <0.0002; Figure 5c).
Figure 5. ADR generates AGEs and pro-fibrotic factors in cultured podocytes. (a) ADR. Cultured murine podocytes were differentiated and incubated with ADR, 3 µg/ml for 3 d. Control cells were subjected to no (baseline) or NS treatment. Cell supernatants were retrieved on day 3 and subjected to AGE ELISA. (b-g) S-100b-RAGE interaction in podocytes upregulates TGF-β1 and CTGF. (b) Murine podocytes were incubated with S-100b, 10 µg/ml, or sham, for the indicated times; glomerular RNA was prepared and real-time PCR was performed for detection of TGF- β1 transcripts. (c) Pretreatment of podocytes with anti-RAGE IgG or nonimmune IgG at the indicated concentration for 30 min was performed before incubation with S-100b for 30 min. (d) Cells were treated with sham, Ad vector alone, or Ad DN RAGE followed by stimulation with S-100b for 30 min. RNA was prepared and real-time PCR was performed for detection of TGB-β1 transcripts. (e-g) CTGF. Podocytes were stimulated with S-100b (10 µg/ml) for the time course (e) alone or in the presence of pretreatment with anti-RAGE or nonimmune IgG for 30 min (f) and in the presence of Ad vector alone or Ad DN RAGE (g). In panels f and g, S-100 incubation was performed over 4 h. In panels e through g, RNA was prepared and real-time PCR was performed for detection of CTGF transcripts. In all cases, experiments were performed in at least triplicates and the mean ± standard error is reported. *P < 0.05, **P < 0.01.
To probe the requirement for RAGE-mediated signal transduction,we prepared adenoviral vectors expressing dominant negative(DN) RAGE, cytoplasmic domain-deleted RAGE.1–3 WhereasS-100b/Ad vector revealed significant increases in TGF-β1transcripts versus sham, a marked suppression of S-100b-stimulatedupregulation of TGF-β1 transcripts was noted in the presenceof Ad DN RAGE (Figure 5d). Incubation of podocytes with S-100bresulted in a significant approximately 1.9-, 2.4-, 1.8-, and1.8-fold increase in CTGF transcripts at 2, 4, 6, and 8 h, respectively,versus sham (P < 0.05; Figure 5e). Pretreatment of the podocyteswith anti-RAGE IgG significantly attenuated CTGF transcriptsby S-100b (P < 0.0002; Figure 5f), as did introduction ofAd DN RAGE (P < 0.01; Figure 5g).
Effects of RAGE Ligands on Murine Podocytes: Activation of NADPH Oxidase via p44/42 MAP Kinase Signaling
Our in vivo findings implicated RAGE and generation of ROS inADR nephropathy; thus, we tested the impact of RAGE ligandson regulation of NADPH oxidase in cultured podocytes. Incubationwith S-100b rapidly activated NADPH oxidase; at 5 and 15 min,approximately 2.1-fold and approximately 1.7-fold increasesin activity were noted versus sham, respectively (P < 0.05;Figure 6a). The effects of S-100b were dose- and RAGE-dependent(Figure 6b), as reduction was observed with anti-RAGE IgG (Figure 6c)and Ad DN RAGE (P < 0.01; Figure 6d).
Figure 6. RAGE ligand S-100b activates NADPH oxidase in cultured podocytes (a-d). S-100b-RAGE activates NADPH oxidase. Podocytes were incubated with sham versus S-100b for the indicated times (a) and dose (b) followed by assessment of NADPH oxidase activity. Pretreatment with anti-RAGE or nonimmune IgG at the indicated concentrations was performed for 30 min followed by incubation with S-100b (10 µg/ml) for 10 min (c). (d) The impact of Ad vector or Ad DN RAGE was assessed in podocytes stimulated with S-100b, 5 µg/ml, for 10 min. (e-h) S-100b-RAGE activates signal transduction. (e-g) Effect of RAGE on MAP kinase activation. Podocytes were stimulated with S-100b, 10 µg/ml, for the indicated time or sham. Where indicated, cells were pretreated 10 min with the p44/p42 MAP kinase inhibitor (PD98059, 10 µM) or the p38 MAP kinase inhibitor (SB203580, 10 µM) or anti-RAGE IgG (12.5 µg/ml). Cell lysates were prepared and subjected to Western blotting for phospho- followed by total anti-p44/42 MAP kinase IgG (e-f) or anti-p38 MAP kinase IgG (g). Density of phospho/total kinase bands were normalized and ratios reported. (h) Effect of RAGE signaling on activation of NADPH oxidase. Podocytes were pretreated with PD98059 or SB203580 followed by S-100b, 10 µg/ml, for 10 min. NADPH oxidase activity was determined. In all cases, experiments were performed in at least triplicates and the mean ± standard error is reported. *P < 0.05, **P < 0.01.
Activation of p44/p42 and p38 MAP kinases is suggested to playkey roles in ADR-mediated nephropathy.13 Incubation of culturedpodocytes with S-100b enhanced phosphorylation of p44/p42 MAPkinase at 5 and 15 min (11.0-fold and 15.0-fold versus sham;P < 0.01; Figure 6e), in a manner blocked by anti-RAGE IgG(P < 0.01; Figure 6f). Incubation with S-100b resulted insignificant, time-dependent increases in p38 MAP kinase phosphorylationwith peak at 15 min (5.8-fold; P < 0.05; Figure 6g).
To address by what mechanism RAGE ligands activated NADPH oxidase,podocytes were treated with inhibitors of either p44/42 MAPkinase or p38 MAP kinase, and then stimulated with S-100b. Theseexperiments identified roles for p44/p42 MAP kinase, as PD98059resulted in nearly complete suppression of activation of NADPHoxidase by S-100b (P < 0.01), whereas SB203580 had no effect(Figure 6h).
Ligand-RAGE interaction promotes generation of ROS, at leastin part via NADPH oxidase, here shown for the first time inthe podocyte.14 As ROS may trigger AGE generation,15,16 we proposethat ADR initiates events, which, via RAGE, contribute to podocytestress. Our studies do not exclude that ADR-RAGE directly initiatesthe burst of ROS. Immediate roles for RAGE are less likely,however, as ADR is not a RAGE ligand. Furthermore, althoughoxidative stress is suppressed in RAGE null and sRAGE-treatedmice, it is not abolished. ADR also stimulates increases inS-100/calgranulins, ligands linked to cell stress,17–20at least in part in podocytes.
Signal transduction via RAGE is essential for modulation ofcellular properties by ADR. In murine podocytes, S-100b increasesphosphorylation of both p44/p42 and p38 MAP kinases, implicatedin mitogenic, inflammatory, and death- or survival-provokingpathways.21–24 We examined these two MAP kinase familymembers, as recent studies elucidated specific roles for p44/p42and p38 MAP kinase in podocyte stress.25–27In vivo, administrationof inhibitors of these pathways to rodents receiving ADR attenuatedproteinuria.25 Our data suggest that RAGE ligands signal throughp44/p42 MAP kinase in the podocyte, and not p38 MAP kinase,to stimulate activation of NADPH oxidase.
Our studies confirmed that an early effect of ADR was extensivepodocyte foot process effacement. Key podocyte actin-bindingproteins, such as -actinin 4 and synaptopodin, maintain podocyteshape and integrity.28–33 Although podocytes express RAGE,RAGE does not appear essential for maintenance of normal glomerularstructure and function, as RAGE null mice at baseline do notdisplay foot process effacement, glomerular sclerosis, or detectablealbuminuria.
Importantly, RAGE null mice were vulnerable to ADR. First, thesemice were homozygous for BALB/cJ susceptibility alleles at theDOXNPH locus (chromosome 16). The gene encoding RAGE is in theMHC class III region on chromosome 17. Second, our studies revealedthat p44/42 MAP kinase was a central signaling pathway by whichRAGE ligand evoked activation of NADPH oxidase in podocytes.In distinct studies, p44/42 MAP kinase was found not to be impactedby deletion of the RAGE gene in smooth muscle cells.34 Lastly,RAGE null mice demonstrated only partial protection from theeffects of ADR.
Podocyte depletion may follow lethal oxidative stress and thefailure of this terminally differentiated cell to proliferate.27The significant protection afforded by RAGE deletion or antagonismin preserving podocytes is likely linked mechanistically toprotection from glomerulosclerosis, as previous studies establishedthe etiologic link between progressive podocyte loss and glomerulosclerosisby diphtheria toxin-induced podocyte depletion using transgenicrats.35 Importantly, the mice used in this study were globallydevoid of RAGE. As others have suggested contributory rolesfor macrophages and certain T lymphocyte subsets in the pathogenesisof ADR nephropathy,7,36–41 it is possible that RAGE-dependentnonpodocyte roles contributed to ADR injury. Future studies,using tissue-targeted RAGE null mice, will explore this concept.
Taken together, we propose that consequent to ADR, ligand-RAGEinteraction amplifies generation of ROS, at least in part viaNADPH oxidase. Diverse changes in cellular properties ensue,which evoke albuminuria and glomerulosclerosis. The identificationof AGEs and RAGE in glomeruli of human focal segmental glomerulosclerosisand other progressive glomerular diseases4 suggests that theligand-RAGE mechanism may contribute to diverse sclerosing glomerulopathies.
Animal Studies
Animal experiments were performed with the approval of the InstitutionalAnimal Care and Use Committee of Columbia University. HomozygousRAGE null mice were bred into BALB/cJ background to the 12thgeneration. Wild-type BALB/cJ mice (Jackson Laboratories, BarHarbor, ME) were controls. Male mice (age 8 wk), body weight20 to 25 g, were treated with a single dose of ADR (doxorubicinHCl) (Ben Venue Laboratories, Bedford, OH), 10.5 mg/kg, by tailvein injection. Murine sRAGE, was prepared as described.1–3,5
Albuminuria
Urine at 24 h was collected in metabolic cages, and urine albuminand creatinine were determined using kits from Exocell (Philadelphia,PA).
Morphometry
Kidney sections, 3 µm, were stained with periodic acid-Schiff(PAS) (Sigma-Aldrich, St Louis, MO). Images were scanned intoa computer and quantification of mesangial matrix in nuclei-freeregions, and glomerular area was performed using a Zeiss microscope(Thornwood, NY) and an image analysis system. Forty glomeruliper animal were selected randomly. Podocyte foot process effacementwas determined by electron microscopy in at least five glomeruliper mouse.
Confocal Microscopy
Confocal microscopy was performed using a Lasersharp 2000 BioRadscanning confocal microscope (Bio-Rad, Hercules, CA). Polyclonalrabbit anti-human RAGE IgG4 and mouse anti-human synaptopodinIgG (Maine Biotechnology, Portland, ME) were used. Frozen sections(5 µm) were incubated with anti-RAGE IgG followed by biotin-conjugatedanti-rabbit IgG (Sigma-Aldrich) and with streptavidin-conjugatedAlexa Fluor 555 (Invitrogen, Carlsbad, CA). Other antibodiesincluded antisynaptopodin IgG, rabbit antinitrotyrosine IgG(Upstate Group, Lake Placid, NY), and anti-S-100 IgG, Sigma-Aldrich).
ELISA for Detection of AGE and Nitrotyrosine Epitopes
Kidney cortex was homogenized and protein concentrations weremeasured (Bio-Rad). AGE ELISA was performed using T-gel (PierceChemical, Rockford, IL)-affinity-purified chicken anti-AGE followedby anti-chicken IgY (Sigma-Aldrich). Ribose glycated albuminwas the standard. ELISA for the detection of nitrotyrosine epitopeswas performed using a kit from Oxis Research (Oxis Research,Portland, OR).
Assay for Detection of Malondialdehyde (MDA) and NOS Activity
Assay for detection of MDA was performed using the MDA-586 Assaykit, and levels of total nitrite and nitrate were measured usinga kit from Oxis Research Products.
NADPH Oxidase Activity
Kidney protein (25 µg) and lucigenin (250 µM) (Alexis,San Diego, CA) were added to HEPES buffer (Sigma-Aldrich), followedby NADPH (10 mM; Sigma-Aldrich). The samples were immediatelyexamined in a Lumat LB9501 luminometer (PE Wallac, Gaithersburg,MD) for 10 s.
Laser Capture and Real-time Quantitative PCR
Laser capture of 150 glomeruli from three sections per mousewas performed with PixCell II (Arcturus, CA) and pooled forRNA extraction. cDNA was synthesized with TaqMan Reverse TranscriptionReagents Kit (PE Applied Biosystems, Foster City, CA). The primersand probes for RAGE, TGF-β1, CTGF, and β-actin werepurchased from PE Applied Biosystems. Real-time PCR was performedin an ABI Prism 7900 Sequence Detection System (PE Applied Biosystems)with TaqMan PCR Master Mix. The relative target mRNA level wascalculated by the comparative Ct method as instructed by themanufacturer.
Cell Culture
Murine podocytes (provided by Dr. Paul Klotman, Mount SinaiSchool of Medicine, NY) were propagated and differentiated asdescribed. Cells were stimulated with ADR, 3 µg/ml (5µM) for 3 d.42,43 Supernatants were retrieved for AGEELISA. In other studies, podocytes were stimulated with S-100B.Certain cells were preincubated with PD98059 and SB203580 Calbiochem(San Diego, CA). Ad-DN-RAGE was made by insertion of DN RAGEexpressing cDNA fragment into AdenoEasy Adenovirus expressionsystem (Invitrogen), as driven by the CMV promoter.
RAGE and ADR Binding Assay
Rabbit anti-human RAGE IgG (100 µg) or rabbit anti-ADRIgG (100 µg) (Alexis) was immobilized onto AffiGel 15(Bio-Rad). Anti-RAGE IgG-coupled AffiGel 15 beads were saturatedwith sRAGE and incubated with 0.2 mg/ml ADR in normal saline.The beads were washed and eluted with low pH buffer (pH 2.5).The eluate was applied on spectrum photodensitometer to detectADR or subjected to Western blotting to detect sRAGE. A similarprocedure using anti-ADR coupled AffiGel 15 beads to test ifsRAGE would bind ADR was performed.
Statistical Analysis
Mean ± SE is reported. A one-way ANOVA was performed;if the F test resulted in a P value less than 0.05, multiplecomparisons were made by Tukey's pair-wise testing, which conservesthe overall type I error of 0.05. All data were analyzed bythe SAS system software (SAS Institute, Cary, NC).
A.M.S. receives research support from TransTech Pharma, Inc.and is a member of their Scientific Advisory Board.
Acknowledgments
This work was supported by grants from the United States PublicHealth Service.
The authors thank Dr. Ali Gharavi for his work in determiningthat the homozygous RAGE null mice used in this study were homozygousfor BALB/cJ susceptibility alleles at the DOXNPH locus. Theauthors also thank Ms. Latoya Woods for expert assistance inthe preparation of this manuscript.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
2.1-fold and 
-actinin 4 and synaptopodin, maintain podocyte shape and integrity.28–33 Although podocytes express RAGE, RAGE does not appear essential for maintenance of normal glomerular structure and function, as RAGE null mice at baseline do not display foot process effacement, glomerular sclerosis, or detectable albuminuria. 
