Bacterial CpG-DNA accelerates Alport glomerulosclerosis by inducing an M1 macrophage phenotype and tumor necrosis factor-α-mediated podocyte loss.

Loss of function mutations in the α3 or α4 chain of type IV collagen cause Alport nephropathy, characterized by progressive glomerulosclerosis. While studying the mechanisms that determine disease progression, we found that the evolution of kidney disease in Col4a3-deficient mice was associated with an influx of immune cell subsets including nonactivated macrophages. This suggested that intrarenal inflammation might accelerate Alport nephropathy. A possible mechanism might be the well-known enhancement of immune recognition by bacterial products. We found that exposure to bacterial endotoxin from 4 to 6 weeks of age did not affect disease progression, whereas an equipotent dose of cytosine-guanine (CpG)-DNA, a synthetic mimic of bacterial DNA, accelerated all aspects of Alport nephropathy and reduced the overall lifespan of Col4a3-deficient mice. This effect was associated with a significant increase of renal CD11b+/Ly6C(hi) macrophages, intrarenal production of inducible nitric oxide synthase, tumor necrosis factor (TNF)-α, interleukin-12, and CXCL10, and loss of podocytes. TNF-α was essential for acceleration of Alport nephropathy, as etanercept (a soluble TNF-α receptor) entirely abrogated the CpG-DNA effect. Thus, systemic exposure to CpG-DNA induces classically activated (M1) macrophages that enhance intrarenal inflammation and disease progression. Hence, factors that modulate the phenotype of renal macrophages can affect the progression of Alport nephropathy and, potentially, other types of chronic kidney diseases.

Ccl2/Mcp-1 blockade reduces glomerular and interstitial macrophages but does not ameliorate renal pathology in collagen4A3-deficient mice with autosomal recessive Alport nephropathy.

Lack of the alpha3 or alpha4 chain of type IV collagen (COL4) causes autosomal recessive Alport nephropathy in humans and mice that is characterized by progressive glomerulosclerosis and tubulointerstitial disease. Renal pathology is associated with chemokine-mediated macrophage infiltrates but their contribution to the progression of Alport nephropathy is unclear. We found Ccl2 to be expressed in increasing amounts during the progression of nephropathy in Col4a3-deficient mice; hence, we blocked Ccl2 with anti-Ccl2 Spiegelmers, biostable L-enantiomeric RNA aptamers suitable for in vivo applications. Ccl2 blockade reduced the recruitment of ex vivo-labelled macrophages into kidneys of Col4a3-deficient mice. We therefore hypothesized that a prolonged course of Ccl2 blockade would reduce renal macrophage counts and prevent renal pathology in Col4a3-deficient mice. Groups of Col4a3-deficient mice received subcutaneous injections of either an anti-mCcl2 Spiegelmer or non-functional control Spiegelmer on alternate days, starting from day 21 or 42 of age. Glomerular and interstitial macrophage counts were found to be reduced with Ccl2 blockade by 50% and 30%, respectively. However, this was not associated with an improvement of glomerular pathology, interstitial pathology, or of overall survival of Col4a3-deficient mice. We conclude that Ccl2 mediates the recruitment of glomerular and interstitial macrophages but this mechanism does not contribute to the progression of Alport nephropathy in Col4a3-deficient mice.

Bacterial lipopeptide triggers massive albuminuria in murine lupus nephritis by activating Toll-like receptor 2 at the glomerular filtration barrier.

What are the molecular mechanisms of bacterial infections triggering or modulating lupus nephritis? In nephritic MRL(lpr/lpr) mice, transient exposure to bacterial cell wall components such as lipopeptide or lipopolysaccharide (LPS) increased splenomegaly, the production of DNA autoantibodies, and serum interleukin (IL)-6, IL-12 and tumour necrosis factor (TNF) levels, and aggravated lupus nephritis. Remarkably, bacterial lipopeptide induced massive albuminuria in nephritic but not in non-nephritic mice. This was associated with down-regulation of renal nephrin mRNA and redistribution from its normal localization at foot processes to the perinuclear podocyte area in nephritic MRL(lpr/lpr) mice. Bacterial lipopeptide activates Toll-like receptor 2 (TLR2), which we found to be expressed on cultured podocytes and glomerular endothelial cells. TNF and interferon (IFN)-gamma induced TLR2 mRNA and receptor expression in both cell types. Albumin permeability was significantly increased in cultured podocytes and glomerular endothelial cells upon stimulation by bacterial lipopeptide. LPS also induced moderate albuminuria. In summary, bacterial lipopeptide and LPS can aggravate glomerulonephritis but only lipopeptide potently induces severe albuminuria in MRL(lpr/lpr) mice.

Late onset of Ccl2 blockade with the Spiegelmer mNOX-E36-3'PEG prevents glomerulosclerosis and improves glomerular filtration rate in db/db mice.

Diabetic kidney disease is associated with monocyte chemoattractant CC chemokine ligand 2 (CCL2)-dependent glomerular and interstitial macrophage recruitment. In addition, nephropathy is delayed in Ccl2 mutant diabetic mice. However, whether the late onset of therapeutic Ccl2 blockade modulates the progression of advanced diabetic nephropathy remains unknown. We addressed this question by antagonizing Ccl2 with mNOX-E36-3'PEG, an anti-Ccl2 L-enantiomeric RNA aptamer (ie, a Spiegelmer), which binds murine Ccl2 and blocks the recruitment of ex vivo-labeled macrophages to the kidneys of db/db mice with type 2 diabetes. We injected mNOX-E36-3'PEG subcutaneously at a dose of 50 mg/kg three times per week into uninephrectomized (1K) db/db mice with advanced glomerulopathy from 4 to 6 months of age. mNOX-E36-3'PEG reduced the number of glomerular macrophages by 40% compared with nonfunctional (control) Spiegelmer-treated 1K db/db mice. This result was associated with protection from diffuse glomerulosclerosis and significantly improved the glomerular filtration rate. mNOX-E36-3'PEG also reduced renal Ccl2 mRNA and protein expression compared with control Spiegelmer-treated 1K db/db mice of the same age. Together, the late onset of therapeutic Ccl2 blockade, eg, with specific Spiegelmers, offers protection from diffuse glomerulosclerosis in type 2 diabetic db/db mice and, thus, may represent a novel therapeutic strategy for advanced glomerulosclerosis.

Delayed chemokine receptor 1 blockade prolongs survival in collagen 4A3-deficient mice with Alport disease.

Human Alport disease is caused by a lack of the alpha3-, 4-, or 5-chain of type IV collagen (COL4A). Affected humans and COL4A3-deficient mice develop glomerulosclerosis and progressive renal fibrosis in the presence of interstitial macrophages, but their contribution to disease progression is under debate. This question was addressed by treating COL4A3-deficient mice with BX471, an antagonist of chemokine receptor 1 (CCR1) that is known to block interstitial leukocyte recruitment. Treatment with BX471 from weeks 6 to 10 of life improved survival of COL4A3-deficient mice, associated with less interstitial macrophages, apoptotic tubular epithelial cells, tubular atrophy, interstitial fibrosis, and less globally sclerotic glomeruli. BX471 reduced total renal Cll5 mRNA expression by reducing the number of interstitial CCL5-positive cells in inflammatory cell infiltrates. Intravital microscopy of the cremaster muscle in male mice identified that BX471 or lack of CCR1 impaired leukocyte adhesion to activated vascular endothelium and transendothelial leukocyte migration, whereas leukocyte rolling and interstitial migration were not affected. Furthermore, in activated murine macrophages, BX471 completely blocked CCL3-induced CCL5 production. Thus, CCR1-mediated recruitment and local activation of macrophages contribute to disease progression in COL4A3-deficient mice. These data identify CCR1 as a potential therapeutic target for Alport disease or other progressive nephropathies associated with interstitial macrophage infiltrates.