Reactivation of M. tuberculosis Infection in Trans-Membrane Tumour Necrosis Factor Mice

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established

Specific Targeting of the Nrf2 Transcription Factor to Prevent Diabetic Kidney Disease Progression by Stimulating the Oxidative Stress Response: A Next-Generation Strategy

This research proposal outlines preclinical pharmacological approaches to elucidate the potential efficacy of Nrf2 activation to ameliorate diabetic kidney disease (DKD). A key vulnerability of the kidney in diabetes stems from its functional reliance on mitochondrial energetics and its susceptibility to oxidative stress. The focus on Nrf2 is linked to this transcription factor’s induction of several antioxidant genes upon activation. The goals of this proposal are thus twofold. First, I seek to demonstrate target engagement of the novel small molecule Nrf2 activator, leading to improvement in functional and histological endpoints in an animal model of DKD. Next, I will evaluate whether selective Nrf2 agonism avoids adverse effects on the endothelin signaling pathway in a preclinical model of DOCA-salt nephropathy. This adverse effect, which can lead to heart failure, was seen in a subset of patients in clinical trials with the nonspecific Nrf2 activator Bardoxolone methyl. The ultimate purpose of this initiative is the development of effective strategies for prevention and management of human diabetic nephropathy, a renal disease demonstrating insufficient activation of the antioxidant stress response. The potential translational impact of this approach is high, as the Nrf2 activator Bardoxolone methyl has already demonstrated efficacy in mild to moderate DKD in clinical trials, though it has demonstrated toxic side effects in advanced DKD. To accomplish these goals, I propose three projects to demonstrate: (1) Nrf2 Dysregulation in a Preclinical Model of DKD; (2) Target Engagement and Functional/Histological Improvement in a Preclinical Model of DKD with an Orally Dosed Small Molecule Nrf2 Activator; and (3) Lack of Toxic Liabilities of an Orally Dosed Specific Small Molecule Nrf2 Activator that are Otherwise Associated with Bardoxolone Methyl Treatment, a Nonspecific Nrf2 Activator

Specific Targeting of the Nrf2 Transcription Factor to Prevent Diabetic Kidney Disease Progression by Stimulating the Oxidative Stress Response: A Next-Generation Strategy

This research proposal outlines preclinical pharmacological approaches to elucidate the potential efficacy of Nrf2 activation to ameliorate diabetic kidney disease (DKD). A key vulnerability of the kidney in diabetes stems from its functional reliance on mitochondrial energetics and its susceptibility to oxidative stress. The focus on Nrf2 is linked to this transcription factor’s induction of several antioxidant genes upon activation. The goals of this proposal are thus twofold. First, I seek to demonstrate target engagement of the novel small molecule Nrf2 activator, leading to improvement in functional and histological endpoints in an animal model of DKD. Next, I will evaluate whether selective Nrf2 agonism avoids adverse effects on the endothelin signaling pathway in a preclinical model of DOCA-salt nephropathy. This adverse effect, which can lead to heart failure, was seen in a subset of patients in clinical trials with the nonspecific Nrf2 activator Bardoxolone methyl. The ultimate purpose of this initiative is the development of effective strategies for prevention and management of human diabetic nephropathy, a renal disease demonstrating insufficient activation of the antioxidant stress response. The potential translational impact of this approach is high, as the Nrf2 activator Bardoxolone methyl has already demonstrated efficacy in mild to moderate DKD in clinical trials, though it has demonstrated toxic side effects in advanced DKD. To accomplish these goals, I propose three projects to demonstrate: (1) Nrf2 Dysregulation in a Preclinical Model of DKD; (2) Target Engagement and Functional/Histological Improvement in a Preclinical Model of DKD with an Orally Dosed Small Molecule Nrf2 Activator; and (3) Lack of Toxic Liabilities of an Orally Dosed Specific Small Molecule Nrf2 Activator that are Otherwise Associated with Bardoxolone Methyl Treatment, a Nonspecific Nrf2 Activator

Dicer1 activity in the stromal compartment regulates nephron differentiation and vascular patterning during mammalian kidney organogenesis

MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle’s loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal while peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation and morphogenic functions including α-smooth muscle actin, integrin-α8, -β1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis

TWEAK-Fn14 Signaling Activates Myofibroblasts to Drive Progression of Fibrotic Kidney Disease

The identification of the cellular origins of myofibroblasts has led to the discovery of novel pathways that potentially drive myofibroblast perpetuation in disease. Here, we further investigated the role of innate immune signaling pathways in this process. In mice, renal injury-induced activation of pericytes, which are myofibroblast precursors attached to endothelial cells, led to upregulated expression of TNF receptor superfamily member 12a, also known as fibroblast growth factor-inducible 14 (Fn14), by these cells. In live rat kidney slices, administration of the Fn14 ligand, TNF-related weak inducer of apoptosis (TWEAK), promoted pericyte-dependent vasoconstriction followed by pericyte detachment from capillaries. In vitro, administration of TWEAK activated and differentiated pericytes into cytokine-producing myofibroblasts, and further activated established myofibroblasts in a manner requiring canonical and noncanonical NF-?B signaling pathways. Deficiency of Fn14 protected mouse kidneys from fibrogenesis, inflammation, and associated vascular instability after in vivo injury, and was associated with loss of NF-?B signaling. In a genetic model of spontaneous CKD, therapeutic delivery of anti-TWEAK blocking antibodies attenuated disease progression, preserved organ function, and increased survival. These results identify the TWEAK-Fn14 signaling pathway as an important factor in myofibroblast perpetuation, fibrogenesis, and chronic disease progression