Multipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the alpha3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to 10 of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, proteinuria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice.

The monocyte chemoattractant protein CCL2 is crucial for monocyte and T cell recruitment from the vascular to the extravascular compartment at sites of inflammation. CCL2 is expressed in human lupus nephritis and was shown to mediate experimental lupus; therefore, CCL2 antagonists may be beneficial for therapy. This study describes the L-enantiomeric RNA oligonucleotide mNOX-E36, a so-called Spiegelmer that binds murine CCL2 with high affinity and neutralizes its action in vitro and in vivo. The mirror image configuration of the Spiegelmer confers nuclease resistance and thus excellent biostability. mNOX-E36 does not induce type I IFN via Toll-like receptor-7 or cytosolic RNA receptors, as recently shown for certain synthetic D-RNA. Autoimmune-prone MRL lpr/lpr mice that were treated with a polyethylene glycol form of mNOX-E36 from weeks 14 to 24 of age showed prolonged survival associated with a robust improvement of lupus nephritis, peribronchial inflammation, and lupus-like inflammatory skin lesions. Thus, mNOX-E36 -based inhibition of CCL2 represents a novel strategy for the treatment of autoimmune tissue injury, such as lupus nephritis.

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 -3PEG, 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 -3PEG 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 -3PEG reduced the number of glomerular macrophages by 40% compared with nonfunctional (control) Spiegelmertreated 1K db/db mice. This result was associated with protection from diffuse glomerulosclerosis and significantly improved the glomerular filtration rate. mNOX-E36 -3PEG 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.

Diabetic nephropathy is associated with interstitial macrophage infiltrates, but their contribution to disease progression is unclear. We addressed this question by blockade of chemokine receptor (CCR)1 because CCR1 mediates the macrophage recruitment to the renal interstitium. In fact, when CCR1 was blocked with BL5923, a novel orally available CCR1 antagonist, the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with advanced diabetic nephropathy. Likewise, BL5923 (60 mg/kg, twice a day) orally administered from months 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, transforming growth factor-beta1, and collagen I-alpha1 when compared with untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for interstitial macrophages for tubulointerstitial injury, loss of peritubular microvasculature, interstitial inflammation, and fibrosis in type 2 diabetic db/db mice. These data identify oral treatment with the CCR1 antagonist BL5923 as a potential therapy for late-stage diabetic nephropathy.

Human Alport disease is caused by a lack of the ␣3-, 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.