Renal ischemia reperfusion injury (IRI) is associated with significant morbidity and mortality. Given the importance of microRNAs (miRNAs) in regulating gene expression, we examined expression profiles of miRNAs following renal IRI. Global miRNA expression profiling on samples prepared from the kidneys of C57BL/6 mice that underwent unilateral warm ischemia revealed nine miRNAs (miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805, and miR-194) that are differentially expressed following IRI when compared with sham controls. These miRNAs were also differently expressed following IRI in immunodeficient RAG-2/ common γ-chain double-knockout mice, suggesting that the changes in expression observed are not significantly influenced by lymphocyte infiltration and therefore define a lymphocyte-independent signature of renal IRI. In vitro studies revealed that miR-21 is expressed in proliferating tubular epithelial cells (TEC) and up-regulated by both cell-intrinsic and -extrinsic mechanisms resulting from ischemia and TGF-β signaling, respectively. In vitro, knockdown of miR-21 in TEC resulted in increased cell death, whereas overexpression prevented cell death. However, overexpression of miR-21 alone was not sufficient to prevent TEC death following ischemia. Our findings therefore define a molecular fingerprint of renal injury and suggest miR-21 may play a role in protecting TEC from death.kidney | miR-21 | tubular epithelial cells | programmed cell death protein 4 I schemic injury followed by reperfusion results in the clinical syndrome of acute kidney injury, a common clinical problem (1-4). The initial nonimmune hypoxic injury and subsequent reperfusion leads to activation of innate and adaptive immune responses, resulting in tissue damage (5). Following injury, a repair process involving cellular proliferation must take place to regain renal function (6). Ischemia reperfusion injury (IRI) is also inevitable in kidney transplantation and contributes to delayed graft function and long-term changes in kidney transplants that affect outcome (7-9). Although IRI is clearly a major clinical problem in native kidneys and in the setting of renal transplantation, the pathogenesis of renal IRI is not fully understood.MicroRNAs (miRNAs) are a class of small, noncoding RNAs ≈21-22 nucleotides in length that regulate gene expression and many disease processes (10, 11). A role for miRNAs in kidney disease is rapidly emerging (12). Numerous hallmarks of IRI (9), such as apoptosis (13), fibrosis (14), epithelial-mesenchymal transition (15), and TLR signaling (16), are regulated by miRNAs in other settings. Recent work has also revealed a role for miRNAs in the regulation of cardiac (13) and hepatic IRI (17).We hypothesized that miRNA expression patterns may serve as a biomarker of kidney injury. To test this hypothesis we reasoned that it would be necessary to examine miRNA expression patterns in an unbiased manner and examine whether differences in miRNA expression patterns were the result of lymphocyte in...

Despite significant progress in understanding the origin of soluble CD14 (sCD14), its physiological function remains largely unknown. Recent research has produced contradictory observations suggesting that sCD14 may have either beneficial or detrimental properties in protection against LPS-induced endotoxin shock. To resolve this controversy and to establish a mouse model suitable for elucidation of the functions of human CD14 (hCD14) in vivo, we generated several lines of transgenic mice bearing different copy numbers of the hCd14 transgene on a murine Cd14 -/-background. The hCD14 was entirely capable of complementing loss of mouse CD14 to mediate cellular responses to LPS. Serum levels of sCD14 in a founder with multiple copies of the transgene were several times higher than in transgenic animals with a single copy of Cd14. Furthermore, mice with high levels of hCD14 were hypo-responsive to LPS and survived a lethal dose of LPS. Further inquiry into the mechanism of the hypo-response to LPS revealed that protection is associated with the higher amounts of circulating LPS. Most of this circulating LPS can be immunoprecipitated with anti-CD14 antibodies. These results suggest that sCD14 blocks circulating LPS by limiting the amount of monocyte-bound LPS and thus reduces inflammatory responses.

The vertebrate immune system has evolved to recognize nucleic acids of bacterial and viral origin. Microbial DNA, as well as synthetic oligonucleotides based on these motifs, activates innate immune pathways mediated by the family of Toll‐like receptors (TLR) initiating a cascade of signals in immune cells necessary for responses to pathogens. However, not all of the proteins that participate in TLR‐mediated responses have been identified. In studies described herein, we observed significant variation in innate immune responses among selected wild‐derived strains of mice. Specifically, we show that mice of MOLF/Ei, Czech/Ei, and MSM/Ms strains are hypo‐responsive to polyinosinic‐polycytidylic acid (poly(I:C)) because of a mutation in Tlr3. In addition, we discovered a hypo‐response to cytosine guanine dinuleotide in MOLF/Ei mice and established that it is not linked to Tlr9, but to another locus. Further inquiry revealed that this hypo‐response is transmitted as a monogenic dominant trait that can be mapped and cloned through positional cloning methods. These results suggest the existence of a novel molecule that can alter pro‐inflammatory signals or activate additional signal transduction pathways. In addition, they support the wild‐derived mouse strain as a forward genetic tool for the identification of novel immunological phenotypes.

Drug–drug interaction ( DDI ) studies are described for tezacaftor/ivacaftor, a new cystic fibrosis transmembrane conductance regulator modulator therapy for the treatment of cystic fibrosis. Three phase I DDI studies were conducted in healthy subjects to characterize the DDI profile of tezacaftor/ivacaftor with cytochrome P450 ( CYP )3A substrates, CYP 3A inhibitors, and a permeability glycoprotein (P‐gp) substrate. The effects of steady‐state tezacaftor/ivacaftor on the pharmacokinetics ( PK s) of digoxin (a P‐gp substrate), midazolam, and ethinyl estradiol/norethindrone ( CYP 3A substrates) were evaluated. Effects of strong (itraconazole) and moderate (ciprofloxacin) CYP 3A inhibitors on tezacaftor/ivacaftor PK s were also determined. Tezacaftor/ivacaftor increased digoxin area under the curve ( AUC ) by 30% but did not affect midazolam, ethinyl estradiol, or norethindrone exposures. Itraconazole increased the AUC of tezacaftor 4‐fold and ivacaftor 15.6‐fold. Ciprofloxacin had no significant effect on tezacaftor or ivacaftor exposure. Coadministration of tezacaftor/ivacaftor may increase exposure of sensitive P‐gp substrates. Tezacaftor/ivacaftor is unlikely to impact exposure of drugs metabolized by CYP 3A, including hormonal contraceptives. Strong CYP 3A inhibitors significantly increase the exposures of tezacaftor and ivacaftor.

Mouse-derived macrophages have the unique ability to restrict or permit Legionella pneumophila intracellular growth. The common inbred mouse strain C57BL/6J (B6) restricts L. pneumophila growth, whereas macrophages derived from A/J mice allow >10 3 -fold bacterial growth within three days. This phenotypic difference was mapped to the mouse Naip5 allele. The B6 restrictive Naip5 allele is dominant, and six amino acid changes in its product were predicted to control permissiveness. By using the wild-derived mouse strain MOLF/Ei, we found that MOLF/Ei-derived macrophages also restrict L. pneumophila growth, yet the Naip5 protein is identical to the A/J Naip5 at the six-amino-acid signature. The MOLF/Ei restrictive trait, unlike that of B6-derived macrophages, was not dominant over the A/J trait. In spite of this phenotypic difference, the L. pneumophila growth restriction in MOLF/Ei macrophages was mapped to the Naip5 region as well, indicating that the originally predicted change in the A/J Naip5 allele may not be critical for restriction. In the product of the A/J Naip5 permissive allele, there are four unique amino acid changes that map to a NACHT-like domain. Similar misregulating mutations have been identified in the NACHT domains of Nod-like receptor (NLR) proteins. Therefore, one of these mutations may be critical for restriction of L. pneumophila intracellular growth, and this parallels results found with human NLR variants with defects in the innate immune response.