13 research outputs found
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CCR2-Mediated Uptake of Constitutively Produced CCL2: A Mechanism for Regulating Chemokine Levels in the Blood.
C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gαi, Gαs or Gαq/11)-independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in <30 minutes. We also found that constitutive receptor internalization/recycling and ligand uptake are not universal across monocyte-expressed chemokine receptors. For example, CXCR4 does not internalize constitutively. In summary, we describe a mechanism that explains the numerous preclinical and clinical reports of increased CCL2 plasma levels following in vivo administration of CCR2 antagonists. These findings suggest that constitutive CCL2 secretion by monocytes and other cell types is counteracted by constant uptake and internalization by CCR2-expressing cells. The effectiveness of CCR2 antagonists in disease settings may be dependent upon this critical equilibrium
CCR2-Mediated Uptake of Constitutively Produced CCL2: A Mechanism for Regulating Chemokine Levels in the Blood
CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice
CCR2 antagonism leads to marked reduction in proteinuria and glomerular injury in murine models of focal segmental glomerulosclerosis (FSGS)
<div><p>Focal segmental glomerulosclerosis (FSGS) comprises a group of uncommon disorders that present with marked proteinuria, nephrotic syndrome, progressive renal failure and characteristic glomerular lesions on histopathology. The current standard of care for patients with FSGS include immunosuppressive drugs such as glucocorticoids followed by calcineurin inhibitors, if needed for intolerance or inadequate response to glucocorticoids. Renin-angiotensin-aldosterone (RAAS) blockers are also used to control proteinuria, an important signature of FSGS. Existing treatments, however, achieved only limited success. Despite best care, treatment failure is common and FSGS is causal in a significant proportion of end stage renal disease. Thus, an unmet need exists for novel disease modifying treatments for FSGS. We employed two widely-used murine models of FSGS to test the hypothesis that systemic inhibition of chemokine receptor CCR2 would have therapeutic benefit. Here we report that administration CCX872, a potent and selective small molecule antagonist of CCR2, achieved rapid and sustained attenuation of renal damage as determined by urine albumin excretion and improved histopathological outcome. Therapeutic benefit was present when CCX872 was used as a single therapy, and moreover, the combination of CCX872 and RAAS blockade was statistically more effective than RAAS blockade alone. In addition, the combination of CCR2 and RAAS blockade was equally as effective as endothelin receptor inhibition. We conclude that specific inhibition of CCR2 is effective in the Adriamycin-induced and 5/6 nephrectomy murine models of FSGS, and thus holds promise as a mechanistically distinct therapeutic addition to the treatment of human FSGS.</p></div
CCX872 improves renal function as assessed by UACR, serum creatinine and BUN in adriamycin challenged mice.
<p>Mice were challenged with Adriamycin as described in Methods. Test compound treatment was begun one hour prior to the Adriamycin challenge. Urine was collected for measurement of UAER (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192405#pone.0192405.s003" target="_blank">S3 Table</a>) and creatinine at the indicated time points, and UACR was calculated as described in Methods. Serum creatinine and BUN were measured at time of terminal bleed after two weeks of treatment. Error bars represent standard error of the mean. N = 10/group at week 1, and N = 8 at week 2. <i>p</i> values were calculated on each treated group to vehicle treated animals using student’s t-test (GraphPad Prism).</p
IHC analysis of kidney sections from 5/6 nephrectomy mice for changes in glomerulus size and podocyte density.
<p>The kidneys (8 animals/treated groups) were harvested 5 weeks after initiation of treatment, fixed and stained as described in Methods. All representative images of podocyte nuclear staining in various treatments were at 400X magnification. Red dashed line indicates glomeruli shape. Note the smaller glomerulus size and increased number of podocyte nuclei in the kidney section from the CCX872 and RAAS treated mice.</p
CCX872 improves renal function as assessed by UACR, serum creatinine, BUN and cystatin C in 5/6 nephrectomized mice.
<p>Mice underwent a 5/6 nephrectomy as described in Methods. Three weeks post-surgery the mice were randomized to the groups indicated above for the study period of 4 weeks. Urine was collected for measurement of albumin and creatinine at the indicated time points, and UACR was calculated as described in Methods. Serum creatinine, BUN and Cystatin C were measured at time of terminal bleed after four weeks of treatment. Error bars represent standard error of the mean. N = 10/group, and N = 6 at week 3. <i>p</i> values were calculated between each drug-treated group and vehicle treated animals (asterisk), or between CCX872/RAAS blocker combination and RAAS blocker alone (octothorpe) using student’s t-test (GraphPad Prism).</p
Relative efficacy of CCR2, RAAS, and endothelin receptor blockade in the 5/6 nephrectomy model as assessed by UACR (mg/mg).
<p>Mice underwent a 5/6 nephrectomy as described in Methods. Three weeks post-surgery the mice were randomized to the indicated groups for the study period of 4 weeks. Urine was collected for measurement of UACR at the indicated time points, as described in Methods. Error bars represent standard error of the mean. N = 10/group at week 1, and N = 6 at week 4. <i>p</i> values were calculated between each drug-treated group and vehicle treated animals (asterisk), or between the CCX872/RAAS blocker combination and RAAS blocker alone (octothorpe) using student’s t-test (GraphPad Prism).</p
Potency and Pharmacokinetics of CCX872.
<p>A. Inhibition of Radio-labeled mJE Binding to WEHI cells. CCX872 was added at the indicated concentrations and competed with the binding of murine CCL2 (JE) to the cells, as described in Methods. The IC<sub>50</sub> was determined to be 270nM (n = 8 for each data point on IC<sub>50</sub> curve). Nonlinear regression (curve fit) analysis showed slope coefficient R<sup>2</sup> = 0.78. B. Pharmacokinetic profile. CCX872 was administered by s.c. injection and the concentration in the blood was determined by LC-MS/MS from plasma samples.</p