Recent findings indicate that elderly patients with acute kidney injury (AKI) have an increased incidence of progression to chronic kidney disease (CKD) due to incomplete recovery from an acute insult. In the current study, a co-morbid model of AKI was developed to better mimic the patient population and to investigate whether age exacerbates the fibrosis and inflammation that develop in the sequelae of progressive kidney disease following acute injury. Young (8–10 weeks) and aged (46–49 weeks) C57BL/6 mice were subjected to 30 min bilateral renal ischemia-reperfusion (I/R) to induce AKI. The aged animals have greater mortality and prolonged elevation of plasma creatinine correlating with less tubular epithelial cell proliferation compared to the young. Six weeks post-reperfusion, interstitial fibrosis is greater in aged kidneys based on picrosirius red staining and immunolocalization of cellular fibronectin, collagen III and collagen IV. Aged kidneys 6 weeks post-reperfusion also express higher levels of p53 and p21 compared to the young, correlating with greater increases in senescence associated (SA) β-galactosidase, a known marker of cellular senescence. A higher influx of F4/80+ macrophages and CD4+ T lymphocytes is measured and is accompanied by increases in mRNA of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α). Importantly, microvascular density is significantly less, correlating with an increase in nitro-tyrosine, a marker of oxidative stress. Collectively, these data demonstrate that prolonged acute injury in the aged animals results in an accelerated progression of kidney disease in a chronic state.

Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear. In this study, we used a renal bilateral ischemiareperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b + cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies. Macrophage populations were isolated from kidney homogenates by fluorescence-activated cell sorting for whole genome microarray analysis.

A key goal of molecular/cell biology/biotechnology is to identify essential genes in virtually every physiological process to uncover basic mechanisms of cell function and to establish potential targets of drug therapy combating human disease. The current article describes a semester-long, project-oriented molecular/cellular/biotechnology laboratory providing students, within a framework of bone cell biology, with a modern approach to gene discovery. Students are introduced to the topics of bone cells, bone synthesis, bone resorption, and osteoporosis. They then review the theory of microchip gene arrays, and study microchip array data generated during the differentiation of bone-resorbing osteoclasts in vitro. The class selects genes whose expression increases during osteoclastogenesis, and researches them in small groups using web-based bioinformatics tools. Students then go to a biotechnology company website to find and order siRNAs designed to “knockdown” expression of the gene of interest. Students then learn to transfect these siRNAs into osteoclasts, stimulate the cells to differentiate, assay osteoclast differentiation in vitro, and measure specific gene expression using real-time PCR and immunoblotting. Specific siRNA knockdown resulting in a decrease in osteoclastogenesis is indicative of a gene's physiological relevance. The results are analyzed statistically, and presented to the class in groups. In the past two years, students identified several genes essential for optimal osteoclast differentiation, including Myo1d. The students hypothesize that the myo1d protein functions in osteoclasts to deliver important proteins to the cell surface via vesicular transport along microfilaments. Student response to the new course was overwhelmingly positive.

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo , in a dose-dependent manner. Finally, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids.

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.