Assessment Of Housekeeping Genes In Inflammatory Mouse Models Of Lung Disease

Abstract Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used “housekeeping” genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10–12 weeks old (Pkd1flox/flox:Nestincre/Pkd1flox/-:Nestincre, n = 10) and non-cystic (NC) controls (Pkd1flox/flox/Pkd1flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1+/-, n = 6) and wild-type (WT) controls (Pkd1+/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.

Download Full-text

Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models

Scientific Reports ◽

10.1038/s41598-021-99366-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Juan J. Muñoz ◽

Ana C. Anauate ◽

Andressa G. Amaral ◽

Frederico M. Ferreira ◽

Elieser H. Watanabe ◽

...

Keyword(s):

Mouse Models ◽

Expression Profiles ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Renal Disorder ◽

Ideal Number ◽

Autosomal Dominant Polycystic Kidney ◽

Suitable Reference ◽

Stage Renal Disease ◽

End Stage

AbstractAutosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used “housekeeping” genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10–12 weeks old (Pkd1flox/flox:Nestincre/Pkd1flox/−:Nestincre, n = 10) and non-cystic (NC) controls (Pkd1flox/flox/Pkd1flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1+/−, n = 6) and wild-type (WT) controls (Pkd1+/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.

Download Full-text

Chemokine receptor 2-targeted molecular imaging in pulmonary fibrosis

10.1101/2020.03.04.960179 ◽

2020 ◽

Author(s):

Steven L. Brody ◽

Sean P. Gunsten ◽

Hannah P. Luehmann ◽

Debbie H. Sultan ◽

Michelle Hoelscher ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Lung Disease ◽

Mouse Models ◽

Chemokine Receptor ◽

Pet Imaging ◽

Lung Fibrosis ◽

Ex Vivo ◽

Inflammatory Monocytes ◽

Fibrotic Process ◽

Fibrotic Lung Disease

AbstractIdiopathic pulmonary fibrosis (IPF) is a progressive, inflammatory lung disease that is monitored clinically by measures of lung function, without effective molecular markers of disease activity or therapeutic efficacy. Lung immune cells active in the pro-fibrotic process include inflammatory monocyte and interstitial macrophages that express the C-C motif chemokine receptor 2 (CCR2). CCR2+ monocyte lung influx is essential for disease phenotypes in models of fibrosis and identified in lungs from subjects with IPF. Here, we show that our peptide-based radiotracer 64Cu-DOTA-ECL1i identifies CCR2+ inflammatory monocytes and interstitial macrophages in multiple preclinical mouse models of lung fibrosis, using positron emission tomography (PET) imaging. Mice with bleomycin-induced fibrosis treated with blocking antibodies to interleukin-1β, a mediator of fibrosis associated with CCR2+ cell inflammation, or with pirfenidone, an approved anti-fibrotic agent, demonstrated decreased CCR2-dependent interstitial macrophage accumulation and reduced 64Cu-DOTA-ECL1i PET uptake, compared to controls. Lung tissues from patients with fibrotic lung disease demonstrated abundant CCR2+ cells surrounding regions of fibrosis, and an ex vivo tissue-binding assay showed correlation between radiotracer localization and CCR2+ cells. In a phase 0/1 clinical study of 64Cu-DOTA-ECL1i PET, healthy volunteers showed little lung uptake, while subjects with pulmonary fibrosis exhibited increased uptake, notably in zones of subpleural fibrosis, reflecting the distribution of CCR2+ cells in the profibrotic niche. These findings support a pathologic role of inflammatory lung monocytes/macrophages in fibrotic lung disease and the translational use of 64Cu-DOTA-ECL1i PET to track CCR2-specific inflammation for image-guided therapy.One Sentence SummaryPET imaging of CCR2+ cells in lung fibrosis identifies a therapeutic response in mouse models and displays a perifibrotic signal in subjects with IPF.

Download Full-text

Characterization of Mouse Models of Mycobacterium avium Complex Infection and Evaluation of Drug Combinations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04841-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2129-2135 ◽

Cited By ~ 10

Author(s):

Claire Andréjak ◽

Deepak V. Almeida ◽

Sandeep Tyagi ◽

Paul J. Converse ◽

Nicole C. Ammerman ◽

...

Keyword(s):

Lung Disease ◽

Mouse Models ◽

Mycobacterium Avium ◽

Treatment Efficacy ◽

Model Systems ◽

Mouse Strains ◽

Combination Drug ◽

Content Type ◽

The Impact

ABSTRACTTheMycobacterium aviumcomplex is the most common cause of nontuberculous mycobacterial lung disease worldwide; yet, an optimal treatment regimen forM. aviumcomplex infection has not been established. Clarithromycin is accepted as the cornerstone drug for treatment ofM. aviumlung disease; however, good model systems, especially animal models, are needed to evaluate the most effective companion drugs. We performed a series of experiments to evaluate and use different mouse models (comparing BALB/c, C57BL/6, nude, and beige mice) ofM. aviuminfection and to assess the anti-M. aviumactivity of single and combination drug regimens,in vitro,ex vivo, and in mice.In vitro, clarithromycin and moxifloxacin were most active againstM. avium, and no antagonism was observed between these two drugs. Nude mice were more susceptible toM. aviuminfection than the other mouse strains tested, but the impact of treatment was most clearly seen inM. avium-infected BALB/c mice. The combination of clarithromycin-ethambutol-rifampin was more effective in all infected mice than moxifloxacin-ethambutol-rifampin; the addition of moxifloxacin to the clarithromycin-containing regimen did not increase treatment efficacy. Clarithromycin-containing regimens are the most effective forM. aviuminfection; substitution of moxifloxacin for clarithromycin had a negative impact on treatment efficacy.

Download Full-text

Pulmonary Emphysema: When More is Less

Physiology ◽

10.1152/physiol.00027.2006 ◽

2006 ◽

Vol 21 (6) ◽

pp. 396-403 ◽

Cited By ~ 11

Author(s):

David G. Morris ◽

Dean Sheppard

Keyword(s):

Quality Of Life ◽

Lung Disease ◽

Mouse Models ◽

Pulmonary Circulation ◽

Pulmonary Emphysema ◽

Blood Oxygen ◽

Molecular Events ◽

Capillary Bed ◽

Genetically Manipulated

Pulmonary emphysema results from the loss of intricate alveolar architecture and progressive simplification of small and highly effective gas-exchanging units into large, inefficient cyst-like spaces. Because of the loss of alveolar gas-exchanging units and the capillary bed within them, blood oxygen levels eventually fall and pressures within the pulmonary circulation rise. Recent insights from genetically manipulated mouse models have refined our understanding of the molecular events that prevent or promote the development of pulmonary emphysema. Capitalizing on an improved molecular understanding of emphysema with improved therapeutics has the potential to enhance both the survival and quality of life of patients with this common lung disease.

Download Full-text