Background During kidney fibrosis, a hallmark and promoter of CKD (regardless of the underlying renal disorder leading to CKD), the extracellular-regulated kinase 1/2 (ERK1/2) pathway, is activated and has been implicated in the detrimental differentiation and expansion of kidney fibroblasts. An ERK1/2 pathway inhibitor, trametinib, is currently used in the treatment of melanoma, but its efficacy in the setting of CKD and renal fibrosis has not been explored.Methods We investigated whether trametinib has antifibrotic effects in two mouse models of renal fibrosis -mice subjected to unilateral ureteral obstruction (UUO) or fed an adenine-rich diet-as well as in cultured primary human fibroblasts. We also used immunoblot analysis, immunohistochemical staining, and other tools to study underlying molecular mechanisms for antifibrotic effects.Results Trametinib significantly attenuated collagen deposition and myofibroblast differentiation and expansion in UUO and adenine-fed mice. We also discovered that in injured kidneys, inhibition of the ERK1/2 pathway by trametinib ameliorated mammalian target of rapamycin complex 1 (mTORC1) activation, another key profibrotic signaling pathway. Trametinib also inhibited the ERK1/2 pathway in cultured primary human renal fibroblasts stimulated by application of TGF-b1, the major profibrotic cytokine, thereby suppressing downstream mTORC1 pathway activation. Additionally, trametinib reduced the expression of myofibroblast marker a-smooth muscle actin and the proliferation of renal fibroblasts, corroborating our in vivo data. Crucially, trametinib also significantly ameliorated renal fibrosis progression when administered to animals subsequent to myofibroblast activation.Conclusions Further study of trametinib as a potential candidate for the treatment of chronic renal fibrotic diseases of diverse etiologies is warranted.

Background and Purpose There are no medications currently available to treat metabolic inflammation. Bruton's tyrosine kinase (BTK) is highly expressed in monocytes and macrophages and regulates NF‐κB and NLRP3 inflammasome activity; both propagate metabolic inflammation in diet‐induced obesity. Experimental Approach Using an in vivo model of chronic inflammation, high‐fat diet (HFD) feeding, in male C57BL/6J mice and in vitro assays in primary murine and human macrophages, we investigated if ibrutinib, an FDA approved BTK inhibitor, may represent a novel anti‐inflammatory medication to treat metabolic inflammation. Key Results HFD‐feeding was associated with increased BTK expression and activation, which was significantly correlated with monocyte/macrophage accumulation in the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD‐fed mice inhibited the activation of BTK and reduced monocyte/macrophage recruitment to the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD‐fed mice decreased the activation of NF‐κB and the NLRP3 inflammasome. As a result, ibrutinib treated mice fed HFD had improved glycaemic control through restored signalling by the IRS‐1/Akt/GSK‐3β pathway, protecting mice against the development of hepatosteatosis and proteinuria. We show that BTK regulates NF‐κB and the NLRP3 inflammasome specifically in primary murine and human macrophages, the in vivo cellular target of ibrutinib. Conclusion and Implications We provide “proof of concept” evidence that BTK is a novel therapeutic target for the treatment of diet‐induced metabolic inflammation and ibrutinib may be a candidate for drug repurposing as an anti‐inflammatory agent for the treatment of metabolic inflammation in T2D and microvascular disease.

Sepsis is one of the most prevalent diseases in the world. The development of cardiac dysfunction in sepsis results in an increase of mortality. It is known that Bruton's tyrosine kinase (BTK) plays a role in toll-like receptor signaling and NLRP3 inflammasome activation, two key components in the pathophysiology of sepsis and sepsis-associated cardiac dysfunction. In this study we investigated whether pharmacological inhibition of BTK (ibrutinib 30 mg/kg and acalabrutinib 3 mg/kg) attenuates sepsis associated cardiac dysfunction in mice. 10-week old male C57BL/6 mice underwent CLP or sham surgery. One hour after surgery mice received either vehicle (5% DMSO + 30% cyclodextrin i.v.), ibrutinib (30 mg/kg i.v.), or acalabrutinib (3 mg/kg i.v.). Mice also received antibiotics and an analgesic at 6 and 18 h. After 24 h, cardiac function was assessed by echocardiography in vivo. Cardiac tissue underwent western blot analysis to determine the activation of BTK, NLRP3 inflammasome and NF-κB pathway. Serum analysis of 33 cytokines was conducted by a multiplex assay. When compared to sham-operated animals, mice subjected to CLP demonstrated a significant reduction in ejection fraction (EF), fractional shortening (FS), and fractional area change (FAC). The cardiac tissue from CLP mice showed significant increases of BTK, NF-κB, and NLRP3 inflammasome activation. CLP animals resulted in a significant increase of serum cytokines and chemokines (TNF-α, IL-6, IFN-γ, KC, eotaxin-1, eotaxin-2, IL-10, IL-4, CXCL10, and CXCL11). Delayed administration of ibrutinib and acalabrutinib attenuated the decline of EF, FS, and FAC caused by CLP and also reduced the activation of BTK, NF-κB, and NLRP3 inflammasome. Both ibrutinib and acalabrutinib significantly suppressed the release of cytokines and chemokines. Our study revealed that delayed intravenous administration of ibrutinib or acalabrutinib attenuated the cardiac dysfunction associated with sepsis by inhibiting BTK, reducing NF-κB activation and the activation of the inflammasome. Cytokines associated with sepsis were significantly reduced by both BTK inhibitors. Acalabrutinib is found to be more potent than ibrutinib and could potentially prove to be a novel therapeutic in sepsis. Thus, the FDA-approved BTK inhibitors ibrutinib and acalabrutinib may be repurposed for the use in sepsis.

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We previously reported the Bruton's tyrosine kinase (BTK) inhibitors ibrutinib and acalabrutinib improve outcomes in a mouse model of polymicrobial sepsis. Now we show that genetic deficiency of the BTK gene alone in Xid mice confers protection against cardiac, renal, and liver injury in polymicrobial sepsis and reduces hyperimmune stimulation (“cytokine storm”) induced by an overwhelming bacterial infection. Protection is due in part to enhanced bacterial phagocytosis in vivo , changes in lipid metabolism and decreased activation of NF-κB and the NLRP3 inflammasome. The inactivation of BTK leads to reduced innate immune cell recruitment and a phenotypic switch from M1 to M2 macrophages, aiding in the resolution of sepsis. We have also found that BTK expression in humans is increased in the blood of septic non-survivors, while lower expression is associated with survival from sepsis. Importantly no further reduction in organ damage, cytokine production, or changes in plasma metabolites is seen in Xid mice treated with the BTK inhibitor ibrutinib, demonstrating that the protective effects of BTK inhibitors in polymicrobial sepsis are mediated solely by inhibition of BTK and not by off-target effects of this class of drugs.

Mounting evidence has linked the metabolic disease to neurovascular disorders and cognitive decline. Using a murine model of a high‐fat high‐sugar diet mimicking obesity‐induced type 2 diabetes mellitus (T2DM) in humans, we show that pro‐inflammatory mediators and altered immune responses damage the blood‐brain barrier (BBB) structure, triggering a proinflammatory metabolic phenotype. We find that disruption to tight junctions and basal lamina due to loss of control in the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) causes BBB impairment. Together the disruption to the structural and functional integrity of the BBB results in enhanced transmigration of leukocytes across the BBB that could contribute to an initiation of a neuroinflammatory response through activation of microglia. Using a humanized in vitro model of the BBB and T2DM patient post‐mortem brains, we show the translatable applicability of our results. We find a leaky BBB phenotype in T2DM patients can be attributed to a loss of junctional proteins through changes in inflammatory mediators and MMP/TIMP levels, resulting in increased leukocyte extravasation into the brain parenchyma. We further investigated therapeutic avenues to reduce and restore the BBB damage caused by HFHS‐feeding. Pharmacological treatment with recombinant annexin A1 (hrANXA1) or reversion from a high‐fat high‐sugar diet to a control chow diet (dietary intervention), attenuated T2DM development, reduced inflammation, and restored BBB integrity in the animals. Given the rising incidence of diabetes worldwide, understanding metabolic‐disease‐associated brain microvessel damage is vital and the proposed therapeutic avenues could help alleviate the burden of these diseases.

Using a global formyl-peptide receptor (Fpr)2 knockout mouse colony, we have reported the modulatory properties of this pro-resolving receptor in polymicrobial sepsis. Herein, we have used a humanized FPR2 (hFPR2) mouse colony bearing an intact or a selective receptor deficiency in myeloid cells to dwell on the cellular mechanisms. hFPR2 mice and myeloid cell-specific hFPR2 KO (abbreviated to KO) mice were subjected to cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Compared with hFPR2 mice, CLP caused exacerbated cardiac dysfunction (assessed by echocardiography), worsened clinical outcome and impaired bacteria clearance in KO mice. This pathological scenario was paralleled by increased recruitment of pro-inflammatory monocytes and reduced M2-like macrophages within the KO hearts. In peritoneal exudates of KO mice, we quantified increased neutrophil and MHC II+ macrophage numbers, but decreased monocyte/macrophage and MHC II- macrophage recruitment. hFPR2 up-regulation was absent in myeloid cells and local production of lipoxin A4 was reduced in septic KO mice. Administration of the FPR2 agonist Annexin A1 (AnxA1) improved cardiac function in hFPR2 septic mice, but had limited beneficial effects in KO mice, in which the FPR2 ligand failed to polarize macrophages towards an MHC II- phenotype. In conclusion, FPR2 deficiency in myeloid cells exacerbates cardiac dysfunction and worsens clinical outcome in polymicrobial sepsis. The improvement of cardiac function and the host immune response by AnxA1 is more effective in hFPR2 competent septic mice.