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http://www.kidney-international.org & 2006 International Society of Nephrology
Progression of kidney disease: Blocking leukocyte recruitment with chemokine receptor CCR1 antagonists 1 + H-J Anders1, V Ninichuk1 and D Schlondorff 1
Nephrological Center, Medical Policlinic, Ludwig Maximilians-University Munich, Munich, Germany
Chronic kidney disease (CKD) is usually associated with interstitial leukocytic cell infiltrates, which may contribute to disease progression by production of proinflammatory, proapoptotic, and profibrotic mediators. Recruiting leukocytes into the kidney involves local expression of chemotactic cytokines, that is, chemokines, that interact with respective chemokine receptors on the leukocyte’s outer surface. Thus, specific chemokine receptor antagonists may represent an attractive therapeutic concept to interfere with renal leukocyte recruitment. Among the proinflammatory chemokine receptors, chemokine receptor (CCR)-1 has nonredundant roles for leukocyte adhesion to activated vascular endothelium and for transendothelial migration. In fact, blocking CCR-1 with specific small-molecule antagonists was shown to retard progression in various types of rodent CKD models. Here we discuss the perspective of CCR-1 as a new potential target for the treatment of CKD.
Inflammatory cell infiltrates are a hallmark of chronic kidney diseases (CKDs). Infiltrating leukocytes produce proinflammatory and profibrotic cytokines that contribute to fibroblast proliferation, myofibroblast transdifferentiation, matrix production, and tubular atrophy.1 Recent advances in the understanding of the molecular mechanisms that regulate renal leukocyte recruitment suggest chemokines and chemokine receptors as new targets for specific pharmacological intervention. Chemokine receptors are a family of seventransmembrane-spanning G-protein-coupled receptors expressed on the surface of leukocytes.2 As chemokine receptors can bind multiple proinflammatory chemokines, therapeutic blockade of chemokine receptors rather than individual chemokine ligands may be the preferred strategy in order to interfere with leukocyte recruitment to sites of tissue injury. Here we discuss the emerging perspective for therapeutic blockade of chemokine receptor (CCR)-1 in CKDs.
Kidney International (2006) 69, 29–32. doi:10.1038/sj.ki.5000053 KEYWORDS: chronic kidney disease; chemokines; leukocytes; inflammation; progression
Correspondence: H-J Anders, Medizinische Poliklinik der LMU, Pettenkoferstr. 8a, 80336 Munich, Germany. E-mail:
[email protected] Received 20 September 2005; accepted 26 September 2005 Kidney International (2006) 69, 29–32
WHY FOCUS ON CHEMOKINE RECEPTORS?
Upon injury, parenchymal tissues produce inflammatory mediators that enhance the expression of adhesion molecules such as selectins and integrins and the presentation of chemokines at the luminal endothelial cell surface of capillaries adjacent to the tissue lesion. Selectins mediate the rolling process of leukocytes along the endothelial surface by interaction with their respective carbohydrate ligands. The rolling facilitates the subsequent endothelial–leukocyte interactions through chemokine activation of leukocyte integrins, resulting in firm binding to their endothelial immunoglobulin–like integrin ligands and adhesion (Figure 1). The chemokines may either be generated by injured endothelial cells or by the surrounding tissue and then be transcytosed across the endothelium by chemokine receptor-like proteins such as the Duffy antigen-related chemokine receptor (DARC). The chemokines are then exposed at the luminal endothelial cell surface on specific glycosaminoglycans such as heparan sulfate proteoglycans. The interaction of a chemokine with its corresponding chemokine receptor can cause conformational changes of the leukocyte integrins, leading to firm adhesion of the leukocyte to endothelial cells. The concerted interaction of adhesion molecules, endothelial 29
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Rolling
H-J Anders et al.: Progression of kidney disease
Adhesion
Diapedesis
Renal vascular compartments
Monocyte
Chemokine receptors
Glomerulus
-integrins
CD31
W
PSGL-1
ICAM
HSPG Selectin
CK
CD99
P-selectin
Peritubular vessel VCAM
GAG VCAM-1 Chemokine
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differ in: Vascular endothelial cell
Shear stress Basement membrane
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Interstitial space Chemotaxis
Chemokine and Chemokine receptor expression
Adhesion molecules Endothelial selectins Ig-like integrin ligands
Chemokine presentation Chemokine transporters Interceptors (e.g. DARC) Heparansulfate-Glycosaminoglycans
Figure 2 | Schematic view of differences in adhesion molecules, heparin sulfate proteoglycans, and other glycosaminoglycans, transcytosis and presentation of chemokines (e.g. DARC) on glomerular and peritubular vascular endothelia.
Chemokine gradient
Tissue macrophage
subsets of interest include monocytes/macrophages and T cells.1,4 Site of tissue injury
Figure 1 | Leukocyte migration from the vascular to the extravascular compartment. In the vascular compartment, the rolling leukocyte interacts through P-selectin glycoprotein ligand with P-selectin on the endothelial surface. Chemokines are produced by activated vascular endothelial cells and presented at the vascular lumen by binding to glycosaminoglycans. Chemokines ligate chemokine receptors on the surface of leukocytes, which activates integrins (e.g. intercellular adhesion molecule-1) to interact with adhesion molecules on the endothelial cell surface (e.g. ICAM or vascular cell adhesion molecule-1).
heparan sulfate proteoglycans, and the chemokine system provides a versatile system that allows for a high degree of variability, depending on, for example, the specific microvascular bed. In the kidney this is illustrated by the difference in, for example, glycosaminoglycans, integrins, and selectins present in the high shear-stress, high-permeability glomerular microcirculation versus the low shear-stress peritubular microcirculation (Figure 2). These differences will also influence chemokine transcytosis (absence of DARC on glomerular endothelial cells versus presence on peritubular capillaris) and presentation by the different glycocalyx of these vascular beds (Figure 2). Different types of chemokine receptors are expressed in a restricted manner on specific leukocyte subsets. The picture is further complicated by the fact that, for example, monocytes and T-cells will change the type of chemokine receptor that they express on their cell surface depending on their state of activation and maturation.3 As multiple chemokine receptors are involved in adhesion and chemotaxis of the different leukocyte subsets, it would be desirable to identify those that have nonredundant functions for recruitment of the immune cell subsets of interest. In the context of CKD, the leukocyte 30
RATIONALE FOR FOCUSSING ON THE CHEMOKINE RECEPTOR CCR1
T cells, neutrophils, monocyte/macrophages, and dendritic cells can express CCR1, which binds multiple proinflammatory chemokine ligands, that are, chemokine ligand (CCL)-3 (macrophage inflammatory protein (MIP)-1a), CCL5 (RANTES), CCL7 (membrane cofactor protein-3), CCL14 hemofiltrate CC chemokine (HCC)-1, CCL15 (HCC-2), CCL16 (HCC-4), and CCL23 (MIP-3).2 For example, CCL5 immobilized on vascular endothelium induces leukocyte arrest via CCR1 but not CCR5, while transendothelial migration is supported by both receptors.5 Thus, CCR1-dependent adhesion represents a proximal and crucial event in leukocyte adhesion, suggesting that lack of CCR1 or CCR1 blockade may interfere with leukocyte recruitment in vivo. In fact, intravital microscopy of the microcirculation of the cremaster muscle in mice revealed that leukocyte adhesion and transendothelial migration in response to the chemokine CCL3 were markedly reduced in CCR1-deficient mice or in wild-type mice given a CCR1 antagonist.6 These data indicate an important role for CCR1 in monocyte/macrophage and T-cell adhesion to activated vascular endothelium, the critical initial step for leukocyte transmigration, and hence recruitment to extravascular sites of injury. The relevance of CCR1 for leukocyte extravasation was also demonstrated in various inflammatory disease models such as Schistosoma-induced granuloma formation, cardiac allograft rejection, or renal unilateral ureteral obstruction.7–9 Furthermore, CCR1 appears to have an additional nonredundant role in activating macrophages in a proinflammatory environment.6 Thus, CCR1 has important functions in leukcoyte adhesion, transendothelial migration, and activation, which provides a rationale for validating CCR1 as a therapeutic target in renal disease models. Kidney International (2006) 69, 29–32
H-J Anders et al.: Progression of kidney disease
CCR1- MEDIATES INTERSTITIAL BUT NOT GLOMERULAR LEUKOCYTE ACCUMULATION
Compartment-specific recruitment of different leukocyte populations may involve different glycosaminoglycans and adhesion molecules (Figure 2), but also different chemokines and chemokine receptors. For example, in mice with renal interstitial fibrosis injection of fluorescently labeled monocytes and T cells results in their localization to the renal interstitium. When CCR1-deficient leukocytes or leukocytes pretreated with a CCR1 antagonist were used, almost no renal localization occurred after injection,9 demonstrating the importance of CCR1 for the recruitment of monocytes and T cells into the renal interstitial compartment. By contrast, similar cell transfer studies in mice with immune complex glomerulonephritis revealed that CCR1 does not participate in monocyte recruitment into the inflamed glomeruli,10 illustrating that CCR1 mediates the recruitment of leukocytes to the interstitial but not to the glomerular compartment. Glomerular leukocyte recruitment may be mediated by other chemokine receptors such as CCR2 and CCR5.11,12 CCR1 BLOCKADE IMPROVES THE OUTCOME OF VARIOUS TYPES OF EXPERIMENTAL KIDNEY DISEASE
Human renal biopsy studies have confirmed renal CCR1 expression on macrophages and T cells of predominantly interstitial inflammatory cell infiltrates in various forms of kidney diseases.13 Interstitial inflammation is considered to contribute significantly to the progression of renal diseases. Thus, to evaluate the contribution of CCR1 to this infiltrate requires interventional studies with CCR1 antagonists or with mice made genetically deficient for CCR1 in experimental models of kidney disease. Unilateral ureteral obstruction in C57BL/6 mice
Unilateral ureteral obstruction is used as a model for interstitial fibrosis commonly associated with chronic nephropathies. Within a few days after surgery the inflammatory infiltrate of obstructed kidneys express CCR1 messenger ribonucleic acid (mRNA) at high levels compared to nonobstructed kidneys.14 Injections with BX471, a smallmolecule nonpeptide CCR1 antagonist, markedly reduced interstitial macrophages and T-cell infiltrates. This was associated with less tubular injury and interstitial fibrosis, as determined by interstitial fibroblasts, fibrotic interstitial volume, and mRNA and protein expression for collagen I.15 Interestingly, the CCR1 antagonist was still effective in slowing down the process when started at a time point when substantial interstitial fibrosis was already present. These data suggest that interfering with interstitial leukocyte recruitment can prevent or slow down tubular cell injury and interstitial fibrosis, two major factors for progressive loss of renal function in CKD. Lupus-like glomerulonephritis of MRLlpr/lpr mice
MRLlpr/lpr mice are a valuable model of spontaneous immune complex glomerulonephritis that shares several similarities Kidney International (2006) 69, 29–32
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with human lupus nephritis. Administration of the CCR1 antagonist to MRLlpr/lpr mice with established kidney disease reduced the numbers of interstitial macrophages, T cells, and smooth muscle actin-positive myofibroblasts and diminished renal tumor growth factor (TGF)-b mRNA expression and interstitial collagen I deposits.10 The improvement of histopathological parameters was associated with reduced blood urea nitrogen levels. Surprisingly, the glomerular pathology and the number of glomerular macrophages were not reduced by the CCR1 antagonist, so that proteinuria also remained unaffected. These data indicate that CCR1dependent leukocyte recruitment into the interstitial compartment contributes significantly to the progression of renal failure in MRLlpr/lpr mice. They also show again (see above) that CCR1 does not appear to be involved in leukocyte infiltration into the glomerulus and therefore blocking CCR1 does not ameliorate glomerular pathology. Adriamyin-induced nephrotic syndrome in Balb/c mice
Proteinuria is a prognostic factor for the progression of CKD. While CCR1 blockade did not have an effect on glomerular proteinuria in lupus mice, the beneficial effects of CCR1 antagonism might still apply to the tubulointerstitial pathology associated with nephrotic syndrome. This issue was addressed by testing the CCR1 antagonist BX471 in the model of adriamycin-induced focal segmental glomerulosclerosis in Balb/c mice. In this model, glomerulosclerosis is associated with nephrotic syndrome and progressive interstitial fibrosis. BX471 injections from week 2 to 6 after induction of the model reduced interstitial macrophage and T-cell infiltrates as well as interstitial volume and the numbers of fibroblast-specific protein-1-positive interstitial fibroblasts.16 Again, glomerular macrophage infiltrates and proteinuria were not affected by BX471. These data suggest that CCR1 antagonism can mitigate the progressive interstitial fibrosis associated with nephrotic range proteinuria. Collagen4 A3-deficient mice as a model for autosomal recessive Alport syndrome
Collagen4 A3-deficient mice develop CKD similar to human autosomal recessive Alport syndrome. Interstitial macrophages rather than TGF-b-induced fibroblast accumulation and interstitial matrix deposition cause tubular atrophy and renal dysfunction in that model.17 A role of interstitial macrophages for disease progression is also supported by our study in collagen4 A3-deficient mice treated with BX471 from 6 to 10 weeks of age.6 BX471 increased the mean survival from 69 days in vehicle-treated collagen4 A3deficient mice to 86 days (P ¼ 0.0002). This survival benefit was associated with a reduction of interstitial macrophages, apoptotic tubular cells, and in morphometric indices of tubular atrophy, interstitial fibrosis, and peritubular capillary cross sections. Again proteinuria remained unaltered. Thus, blocking CCR1-dependent interstitial macrophage recruitment delayed progression of interstitial disease and death from renal failure in this model of Alport nephropathy. 31
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H-J Anders et al.: Progression of kidney disease
Nephropathy of db/db mice with type II diabetes
ACKNOWLEDGMENTS
Recent data provide evidence for a role of inflammatory cells also in the progression of diabetic nephropathy. Blocking CCR1 in diabetic nephropathy could address this issue. Male db/db mice with hyperinsulinemic diabetes develop nephropathy associated with interstitial macrophage infiltrates at 6 month of age.18 Interstitial macrophage recruitment in db/db mice appears to be CCR1-dependent, as injections with BX471 from 5 months of age for 10 days reduced the number of macrophages in the renal interstitium compared to vehicle-treated db/db mice (unpublished observation). Whether this will eventually delay the progression of diabetic nephropathy in this model remains to be examined.
REFERENCES
Kidney allograft dysfunction in rabbits and rats
Cardiac transplant studies in CCR1-deficient mice first suggested a role for CCR1 in allograft rejection.8 Horuk et al.19 studied the effects of CCR1 blockade with BX471 on allograft dysfunction in a model of kidney transplantation in rabbits. BX471 improved serum creatinine level and renal survival as compared to placebo-treated rabbits. Furthermore, BX471 was as effective as cyclosporin in preventing extensive infarction of transplanted kidneys. Another study addressed the role of CCR1 in chronic allograft nephropathy. BX471 was given from day 21 to 42 after transplantation of kidneys from Fischer 344 rats into Lewis rats.20 BX471 reduced the number of ED-1-positive macrophages and the interstitial fibrosis in the allografts. These data suggest that CCR1-dependent leukocyte recruitment and activation contributes to acute and chronic allograft nephropathy, and that blocking CCR1 may have beneficial effects on renal survival after kidney transplantation.
This work was supported by grants from the EU Network of Excellence ‘MAIN’ (FP6-502935) to HJA and DS and the Else-KroenerFresenius Foundation, the Deutsche Forschungsgemeinschaft (LU 612/4-1), the German Renal Foundation, and the Wilhelm SanderFoundation to HJA. DS was supported by the EU consortium FP5: QLG1-CT-2002-01215.
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SUMMARY
CCR1 antagonism with specific small-molecule antagonists can effectively prevent recruitment of monocytes and T cells into the renal interstitial compartment. CCR1 blockade proved to be effective in various types of experimental kidney disease in rodents even when treatment is initiated after the disease process is established. It remains to be evaluated whether CCR1 blockade has additive effects to current treatments of CKD, for example, blockade of the renin–angiotensin system. Clearly, however, glomerular pathology and proteinuria remain unaffected by CCR1 blockade, so that chemokine receptors other than CCR1 (e.g. CCR2) may contribute to glomerular leukocyte infiltration. Based on the experimental data available, we propose that the therapeutic effects of CCR1 antagonists – in combination with established therapies – should be evaluated in an attempt to slow down the tubulointerstitial component of progressive human kidney diseases.
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