Imatinib ameliorates renal disease and survival in murine lupus autoimmune disease
2006; Elsevier BV; Volume: 70; Issue: 1 Linguagem: Inglês
10.1038/sj.ki.5001528
ISSN1523-1755
AutoresCarla Zoja, Daniela Corna, Daniela Rottoli, Cristina Zanchi, Mauro Abbate, Giuseppe Remuzzi,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoPlatelet-derived growth factor (PDGF) has been proved to play an important role in progressive glomerular disease of systemic lupus. The present study investigated the tyrosine kinase inhibitor of PDGF receptor, imatinib, as a novel therapeutic approach for the cure of lupus nephritis in New Zealand Black/White (NZB/W)F1 hybrid mice with established disease. Two groups of NZB/W mice (N=30 each group), starting at 5 months of age, were given by gavage vehicle or imatinib (50 mg/kg b.i.d). Fifteen mice for each group were used for the survival study. The remaining were killed at 8 months. Imatinib significantly (P=0.0022) ameliorated animal survival with respect to vehicle. The drug significantly delayed the onset of proteinuria (% proteinuric mice, 7 and 8 months: 33 and 47 vs vehicle, 80 and 87, P<0.05) and limited the impairment of renal function. Imatinib protected the kidney against glomerular hypercellularity and deposits, tubulointerstitial damage, and accumulation of F4/80-positive mononuclear cells and α-smooth actin-positive myofibroblasts. The abnormal transforming growth factor-β mRNA expression in kidneys of lupus mice was reduced by imatinib. In conclusion, findings of amelioration of animal survival and renal manifestations in NZB/W lupus mice with established disease by imatinib suggests the possibility to explore whether imatinib may function as steroid-sparing drug in human lupus nephritis. Platelet-derived growth factor (PDGF) has been proved to play an important role in progressive glomerular disease of systemic lupus. The present study investigated the tyrosine kinase inhibitor of PDGF receptor, imatinib, as a novel therapeutic approach for the cure of lupus nephritis in New Zealand Black/White (NZB/W)F1 hybrid mice with established disease. Two groups of NZB/W mice (N=30 each group), starting at 5 months of age, were given by gavage vehicle or imatinib (50 mg/kg b.i.d). Fifteen mice for each group were used for the survival study. The remaining were killed at 8 months. Imatinib significantly (P=0.0022) ameliorated animal survival with respect to vehicle. The drug significantly delayed the onset of proteinuria (% proteinuric mice, 7 and 8 months: 33 and 47 vs vehicle, 80 and 87, P 4 mg/day) was evaluated at different stages of the disease in the two experimental groups. As shown in Figure 2, imatinib significantly delayed the onset of proteinuria as compared to vehicle. Thus, at 7 and 8 months of age, the percentage of proteinuric mice was 33 and 47% in the treated group vs 80 and 87% in the vehicle-group (P<0.05). At 9 months, the percentage of proteinuric mice in the two groups increased to 86 and 100%, respectively. Basal levels of blood urea nitrogen (BUN) as determined in all mice at 2 months of age were 30 mg/dl, during the experimental period. In the vehicle group, renal function progressively deteriorated so that at 7, 8, and 9 months of age, 67, 88, and 100% of lupus mice, respectively, had abnormal BUN values. By contrast, in the imatinib group, percentages reached only 27% (7 months) and 47% (8 and 9 months).Table 1Cumulative % mice with BUN> 30 mg/dlMonths of age2678910Vehicle (%)0276788100100Imatinib (%)0027474753BUN, blood urea nitrogen.BUN levels>30 mg/dl were considered abnormal (normal range: 14–29 mg/dl). Open table in a new tab BUN, blood urea nitrogen. BUN levels>30 mg/dl were considered abnormal (normal range: 14–29 mg/dl). Serum levels of circulating anti-DNA antibodies measured in 2-month-old NZB/W mice averaged 10±1.4 U/ml. Anti-DNA antibodies significantly increased during time averaging 144±61 U/ml at 5 months of age, before treatment, and reaching 270±125 U/ml at 8 months in NZB/W mice given vehicle (P<0.01 and P<0.001 vs 2 months, respectively). In mice treated with imatinib, anti-DNA formation was slightly retarded (203±92 U/ml). By light microscopy, focal glomerular hypercellularity was found in the kidneys of mice killed at 5 months of age, time at which the treatment was started (score 0.6±0.2). By immunofluorescence, immunoglobulin G (IgG) and complement 3 (C3) deposits were found in glomeruli with predominant mesangial distribution (Figure 3). The presence of immune complex disease was confirmed by electron microscopy analysis showing both mesangial and subendothelial electron dense deposits (Figure 3). No or very sparse glomerular deposits of IgG and C3 nor glomerular electron dense deposits were detected in 2-month-old NZB/W mice (Figure 3). As shown in Table 2, at 8 months, NZB/W mice given vehicle revealed glomerular changes consisting of intracapillary hypercellularity associated with focal extracapillary proliferation. Immune-type deposits were detected in the mesangium and in the glomerular capillary walls with subendothelial distribution. Tubular damage, interstitial inflammation, and fibrosis were present. Treatment with imatinib limited glomerular hypercellularity, deposits, and tubulointerstitial damage (P<0.05 vs vehicle) (Table 2).Table 2Effect of imatinib treatment on renal histology in NZB/W miceGlomeruliTubulo-interstitial damageIntracapillary hypercellularity (score)Extracapillary hypercellularity (score)Deposits (score)(score)Vehicle2.13±0.132.5±0.32.1±0.21.30±0.1Imatinib1.33±0.22*1.5±0.3*1.2±0.3*0.75±0.1*NZB/W, New Zealand Black/White.Data are mean±s.e. *P<0.05 vs vehicle. Open table in a new tab NZB/W, New Zealand Black/White. Data are mean±s.e. *P<0.05 vs vehicle. At 8 months, in vehicle-treated mice, there were diffuse granular deposits of IgG and C3 in the mesangium and in the glomerular capillary wall. In mice treated with imatinib, less IgG and C3 deposits were detected than that of mice treated with vehicle, but a statistical significance was not reached (score, IgG: 1.80±0.16 vs 2.27±0.13; C3: 1.60±0.19 vs 2.21±0.21). A marked accumulation of F4/80-positive monocytes/macrophages was present in the renal interstitium of NZB/W mice given vehicle (85±6 vs CD-1 control mice: 4±1 cells/high-power field (HPF), P<0.01). Treatment with imatinib significantly (P<0.01) reduced peritubular F4/80-positive cell accumulation (45±8 cells/HPF) (Figure 4). Infiltrates of monocytes/macrophages were also observed at periglomerular level in vehicle-mice (13±2 cells/HPF vs controls, 0.9±0.2 cells/HPF, P<0.01), and were limited by the drug (6±2 cells/HPF, P<0.05 vs vehicle) (Figure 4). In control kidneys, interstitial immunostaining for α-smooth muscle actin (α-SMA) was present only in sparse cells (score, 0.35±0.12). In the kidneys of lupus mice given vehicle, the interstitial expression of α-SMA was markedly increased (score, 2.06±0.05). This increase was significantly (P<0.05) prevented by imatinib (score, 1.37±0.23) (Figure 5). As to the mechanism underlying imatinib's effect on the accumulation of α-SMA-positive cells, we have analyzed the expression of transforming growth factor-β (TGF-β), a strong stimulus for the differentiation of fibroblasts and other cell types to α-SMA-positive myofibroblasts. Real-time polymerase chain reaction (PCR) analysis showed that TGF-β mRNA levels were increased in the kidneys of NZB/W mice given vehicle (fourfold increase over CD-1 control mice). Cytokine upregulation was significantly (P<0.05) reduced by imatinib, to the extent that in treated mice, renal TGF-β expression increased by only twofold over controls. In the present study, we have investigated the tyrosine kinase inhibitor imatinib as a novel therapeutic option for the cure of lupus nephritis. The drug retarded the evolution of renal disease of NZB/W lupus mice and ameliorated survival as compared to untreated animals. Specifically, imatinib orally administered after the onset of immune complex deposition delayed the development of proteinuria to a significant extent, prevented renal function impairment, and limited glomerular hypercellularity, immune type deposits, tubular damage, and interstitial inflammation and fibrosis. These effects were associated with lower levels of circulating anti-DNA antibodies with respect to NZB/W mice given vehicle. Imatinib has been originally developed as a therapeutic agent for chronic myeloid leukemia.25.Druker B.J. Tamura S. Buchdunger E. et al.Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.Nat Med. 1996; 2: 561-566Crossref PubMed Scopus (3039) Google Scholar, 26.Deininger M. Buchdunger E. Druker B.J. The development of imatinib as a therapeutic agent for chronic myeloid leukemia.Blood. 2005; 105: 2640-2653Crossref PubMed Scopus (1018) Google Scholar By inhibiting BCR-ABL tyrosine kinase, it targets the molecular event implicated as the direct cause of the disease. Although highly specific, imatinib occupies the adenosine triphosphate-binding site of several tyrosine kinase molecules such as ABL, ABL-related gene product, the PDGF receptor, and the stem cell factor receptor c-kit, impeding the ensuing signal transduction.26.Deininger M. Buchdunger E. Druker B.J. The development of imatinib as a therapeutic agent for chronic myeloid leukemia.Blood. 2005; 105: 2640-2653Crossref PubMed Scopus (1018) Google Scholar Clinical trials in chronic myeloid leukemia showed that the drug produced an unprecedented rate of hematologic and cytogenetic remission.26.Deininger M. Buchdunger E. Druker B.J. The development of imatinib as a therapeutic agent for chronic myeloid leukemia.Blood. 2005; 105: 2640-2653Crossref PubMed Scopus (1018) Google Scholar Moreover, imatinib yielded encouraging results in patients with gastrointestinal stroma tumors caused by c-kit mutations and myeloproliferative disorders with rearrangements of PDGF receptor, diseases where the activation of an imatinib-sensitive kinase is central to the pathogenesis.29.Hochhaus A. Imatinib mesylate (Glivec, Gleevec) in the treatment of chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GIST).Ann Hematol. 2004; 83: S65-S66PubMed Google Scholar, 30.Pardanani A. Tefferi A. Imatinib targets other than bcr/abl and their clinical relevance in myeloid disorders.Blood. 2004; 104: 1931-1939Crossref PubMed Scopus (105) Google Scholar, 31.George D. Targeting PDGF receptors in cancer – rationales and proof of concept clinical trials.Adv Exp Med Biol. 2003; 32: 141-151Crossref Scopus (63) Google Scholar The inhibitory effect of imatinib on PDGF receptor activation suggested its potential application to nonmalignant diseases in which PDGF has been strongly implicated, such as glomerulonephritis. Imatinib ameliorated experimental mesangial proliferative glomerulonephritis causing significant reductions in mesangial cell proliferation, numbers of activated/α-SMA-positive mesangial cells and extracellular matrix accumulation.27.Gilbert R.E. Kelly D.J. McKay T. et al.PDGF signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis.Kidney Int. 2001; 59: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar Renoprotective effects of imatinib have been recently reported against the diabetic nephropathy of apolipoprotein E-knockout mice, a model of accelerated renal disease associated with increased renal expression of PDGF-B.28.Lassila M. Jandeleit-Dahm K. Seah K.K. et al.Imatinib attenuates diabetic nephropathy in apolipoprotein E-knockout mice.J Am Soc Nephrol. 2005; 16: 363-373Crossref PubMed Scopus (107) Google Scholar The drug administered starting immediately after diabetes induction for 20 weeks reduced albuminuria and limited glomerular changes. Tubulointerstitial inflammation and fibrosis were also attenuated by the treatment. Protective effects achieved in NZB/W lupus mice by interrupting PDGF signal transduction with imatinib, while emphasizing the role of PDGF in the manifestation of the disease, suggest the PDGF pathway as a therapeutic target in lupus nephritis. Experimental studies showed progressively increasing PDGF mRNA expression in the renal cortex of lupus mice during the evolution of the disease.19.Nakamura T. Ebihara I. Nagaoka I. et al.Renal platelet-derived growth factor gene expression in NZB/W F1 mice with lupus and ddY mice with IgA nephropathy.Clin Immunol Immunopathol. 1992; 63: 173-181Crossref PubMed Scopus (31) Google Scholar, 21.Entani C. Izumino K. Takata M. et al.Expression of platelet-derived growth factor in lupus nephritis in MRL/MpJ-1pr/1pr mice.Nephron. 1997; 77: 100-104Crossref PubMed Scopus (6) Google Scholar Moreover, in renal tissue from patients with diffuse proliferative lupus nephritis, gene and protein expression of both PDGF-B chain and PDGF-β receptor increased in correlation with the histologic grade. Sites of localization were the mesangium, cells of the capillary walls, crescents, and mononuclear cells infiltrating the interstitium.22.Matsuda M. Shikata K. Makino H. et al.Gene expression of PDGF and PDGF receptor in various forms of glomerulonephritis.Am J Nephrol. 1997; 17: 25-31Crossref PubMed Scopus (33) Google Scholar In the current study, PDGF-receptor tyrosine kinase inhibition was associated with reduced accumulation of monocytes/macrophages in the kidneys, suggesting that PDGF may contribute, together with other cytokines,32.Zoja C. Liu X.-H. Donadelli R. et al.Renal expression of monocyte chemoattractant protein-1 in lupus autoimmune mice.J Am Soc Nephrol. 1997; 8: 720-729PubMed Google Scholar to inflammatory cell influx into the kidney in lupus nephritis. A chemotactic activity of PDGF on monocytes as well as granulocytes and fibroblasts has been documented in vitro.33.Siegbahn A. Hammacher A. Westermark B. Heldin C.H. Differential effects of the various isoforms of platelet-derived growth factor on chemotaxis of fibroblasts, monocytes, and granulocytes.J Clin Invest. 1990; 85: 916-920Crossref PubMed Scopus (214) Google Scholar Another significant finding here was the reduction after imatinib of α-SMA-positive myofibroblasts in the renal interstitium. This could be either the result of a direct effect on PDGF, known to induce transdifferentiation of renal fibroblasts into α-SMA-positive myofibroblasts,34.Tang W.W. Ulich T.R. Lacey D.L. et al.Platelet-derived growth factor-BB induces renal tubulointerstitial myofibroblast formation and tubulointerstitial fibrosis.Am J Pathol. 1996; 148: 1169-1180PubMed Google Scholar or involve other profibrotic factors such as TGF-β, activated through PDGF receptor-dependent pathways.28.Lassila M. Jandeleit-Dahm K. Seah K.K. et al.Imatinib attenuates diabetic nephropathy in apolipoprotein E-knockout mice.J Am Soc Nephrol. 2005; 16: 363-373Crossref PubMed Scopus (107) Google Scholar, 35.Fraser D. Wakefield L. Phillips A. Independent regulation of transforming growth factor-beta1 transcription and translation by glucose and platelet-derived growth factor.Am J Pathol. 2002; 161: 1039-1049Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Our data showing that the overexpression of TGF-β mRNA in the kidneys of lupus mice was decreased by imatinib are in line with findings of reduced expression of TGF-β in the glomeruli and the tubulointerstitium after treatment with the drug in diabetic mice.28.Lassila M. Jandeleit-Dahm K. Seah K.K. et al.Imatinib attenuates diabetic nephropathy in apolipoprotein E-knockout mice.J Am Soc Nephrol. 2005; 16: 363-373Crossref PubMed Scopus (107) Google Scholar On the other hand, the antifibrotic effect afforded by imatinib could be related to the fact that TGF-β may directly signal through the c-ABL tyrosine kinase, a target of imatinib. Thus, in cultured fibroblasts, TGF-β directly stimulated c-ABL activity, independently of Smad 2/3 phosphorylation or PDGF receptor activation, and the inhibition of c-ABL by imatinib prevented TGF-β-induced morphologic transformation and extracellular matrix gene expression.36.Daniels C.E. Wilkes M.C. Edens M. et al.Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis.J Clin Invest. 2004; 114: 1308-1316Crossref PubMed Scopus (495) Google Scholar Finally, in a mouse model of bleomycin-induced pulmonary fibrosis, treatment with imatinib was found to prevent cytokine-induced lung fibrosis.36.Daniels C.E. Wilkes M.C. Edens M. et al.Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis.J Clin Invest. 2004; 114: 1308-1316Crossref PubMed Scopus (495) Google Scholar The finding that imatinib treatment slightly retarded anti-DNA antibody formation as compared to vehicle reflects the immunoregulatory potential of the drug in NZB/W mice. Consistent with this possibility, imatinib delayed to some extent immune complex deposition as suggested by less glomerular IgG and C3 staining, thereby resulting in the attenuation of the downstream disease. In vitro studies have shown that imatinib, at concentrations achieved in vivo, inhibited the proliferation37.Dietz A.B. Souan L. Knutson G.J. et al.Imatinib mesylate inhibits T-cell proliferation in vitro and delayed-type hypersensitivity in vivo.Blood. 2004; 104: 1094-1099Crossref PubMed Scopus (156) Google Scholar and activation38.Cwynarski K. Laylor R. Macchiarulo E. et al.Imatinib inhibits the activation and proliferation of normal T lymphocytes in vitro.Leukemia. 2004; 18: 1332-1339Crossref PubMed Scopus (116) Google Scholar of T cells via inhibition of early signaling mediated by the T-cell receptor.39.Seggewiss R. Lore K. Greiner E. et al.Imatinib inhibits T-cell receptor-mediated T-cell proliferation and activation in a dose-dependent manner.Blood. 2005; 105: 2473-24
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