CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice

Abstract

Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function.

Trial registration: ClinicalTrials.gov NCT01440257 NCT01447147.

Keywords: albuminuria; chemokine; chemokine (C-C motif) receptor 2; hyperglycemia; macrophage.

Figures

Fig. 1.

CCX140-B inhibits chemokine (C-C motif) receptor 2 (CCR2) in human primary monocytes. A and B: CCX140-B inhibited chemokine (C-C motif) ligand (CCL)2 (0.1 nM)-induced chemotaxis of human monocytes in buffer (A) and 100% human serum (B). C and D: CCX140-B inhibited CCL2 (5 nM)-induced Ca2+ mobilization in human monocytes (C) and 125I-labeled CCL2 (125I-CCL2; 50 pM) binding to human monocytes (D). E: saturation binding of [3H]CCX140-B to human primary monocytes.

Fig. 2.

CCX140-B inhibits multiple CCR2 ligands. CCX140-B inhibited chemotaxis of human monocytes in 100% human serum toward CCL2/monocyte chemotactic protein (MCP)-1 (A), CCL8/MCP-2 (B), CCL7/MCP-3 (C), and CCL17/MCP-4 (D).

Fig. 3.

Leukocytes from human (h)CCR2 knockin (KI) mice respond to multiple mouse CCR2 ligands and can be inhibited by CCX140-B. Thioglycollate-elicited peritoneal leukocytes from wild-type mice (A) and hCCR2 KI mice (B) exhibited similar chemotaxis profiles to mouse (m)CCL2/MCP-1, mCCL7/MCP-3, and mCCL12/MCP-5. C and D: CCX140-B potently inhibited mCCL2/MCP-1-induced chemotaxis of leukocytes isolated from hCCR2 KI mice (C) but not from wild-type mice (D).

Fig. 4.

CCX140-B inhibits CCR2 in hCCR2 KI mice. A: hCCR2 KI mice and wild-type C57BL/6 mice were equally responsive to thioglycollate challenge, whereas CCR2−/− [knockout (KO)] mice did not accumulate peritoneal leukocytes after thioglycollate challenge. B: CCX140-B inhibited the thioglycollate response in hCCR2 KI mice in a dose-dependent manner. C: thioglycollate-elicited peritoneal leukocytes isolated from wild-type mice and hCCR2 KI mice mobilized similar amounts of intracellular Ca2+ in response to mCCL2/MCP-1. The addition of CCX140-B blocked mCCL2-mediated Ca2+ mobilization in hCCR2 KI cells (C) but not in wild-type cells (D). E and F: CCX140-B did not alter the frequency of peripheral blood monocytes (E) or the levels of plasma CCL2 (F) in thioglycollate-challenged hCCR2 KI mice. *P < 0.05 relative to vehicle-treated mice.

Fig. 5.

CCX140-B improves albuminuria in diabetic hCCR2 KI mice. Diet-induced obese (DIO) hCCR2 KI mice and hCCR2 KI db/db mice were treated with vehicle or 100 mg/kg CCX140-B for 6–8 wk. A and B: in DIO hCCR2 mice, CCX140-B initially decreased the urinary albumin excretion rate (UAER) and urinary albumin-to-creatinine ratio (ACR) and thereafter slowed the progressive increase in the these values. C and D: in hCCR2 db/db mice, CCX140-B reduced both values over the entire treatment period. *P < 0.05 and **P < 0.005 relative to vehicle-treated mice.

Fig. 6.

CCX140-B reverses glomerular hypertrophy and podocyte density diminution in DIO hCCR2 KI mice. DIO hCCR2 KI mice were treated with vehicle or 100 mg/kg CCX140-B for 8 wk. A and B: CCX140-B reduced glomerulus size (A) and increased the density of podocytes (B). C: representative images of podocyte nucleus staining in vehicle-treated (left) and CCX140-B-treated (right) mice. Note the smaller glomerulus size and increased number of podocyte nuclei in the kidney section from the CCX140-B-treated mouse. Original magnification: ×400. *P < 0.05 and **P < 0.005 relative to vehicle-treated mice.

Fig. 7.

CCX140-B rapidly improves hyperglycemia and insulin sensitivity in DIO hCCR2 KI mice. DIO hCCR2 KI mice were treated with vehicle or 100 mg/kg CCX140-B for 14 days. A–C: CCX140-B reduced fasting plasma glucose (A) and fasting plasma insulin (B) relative to vehicle-treated mice and also improved insulin sensitivity as determined by homeostatic model assessment of insulin resistance (C). D and E: CCX140-B reduced adipose tissue inflammatory macrophage numbers (D) but did not affect plasma CCL2 levels (E). F: CCX140-B did not affect mouse body weight. *P < 0.05 and **P < 0.005 relative to vehicle-treated mice.