Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury.

BACKGROUND AND PURPOSE: Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH: Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. KEY RESULTS: NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor alpha-glycyrrhetinic acid (alpha-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of alpha-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by alpha-GA. CONCLUSION AND IMPLICATIONS: Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.

Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector.

Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors.

A role for the non-phosphorylated form of yeast Snf1: tolerance to toxic cations and activation of potassium transport.

The Snf1/AMP-activated protein kinases play a key role in stress responses of eukaryotic cells. In the yeast Saccharomyces cerevisiae Snf1 is regulated by glucose depletion, which triggers its phosphorylation at Thr210 and concomitant increase in activity. Activated yeast Snf1 is required for the metabolic changes allowing starvation tolerance and utilization of alternative carbon sources. We now report a function for the non-activated form of Snf1: the regulation of the Trk high-affinity potassium transporter, encoded by the TRK1 and TRK2 genes. A snf1Delta strain is hypersensitive in high-glucose medium to different toxic cations, suggesting a hyperpolarization of the plasma membrane driving increased cation uptake. This phenotype is suppressed by the TRK1 and HAL5 genes in high-copy number consistent with a defect in K(+) uptake mediated by the Trk system. Accordingly, Rb(+) uptake and intracellular K(+) measurements indicate that snf1Delta is unable to fully activate K(+) import. Genetic analysis suggests that the weak kinase activity of the non-phosphorylated form of Snf1 activates Trk in glucose-metabolizing yeast cells. The effect of Snf1 on Trk is probably indirect and could be mediated by the Sip4 transcriptional activator.

Elevation of sister chromatid exchange frequency in transformed human fibroblasts following exposure to widely used aminoglycosides.

Aminoglycosides are a class of antibiotics that interfere with protein translation. Geneticin and hygromycin are two such agents, which have been shown to exhibit highly toxic effects in mammalian cells. Cloned bacterial genes, which inactivate these antibiotics, have facilitated the establishment of dominant selection systems, which are widely used in eukaryotic molecular genetics. We have examined the effect of aminoglycosides on the sister chromatid exchange (SCE) frequency in transformed human fibroblast cell lines. Geneticin and hygromycin were both found to increase SCE frequency in all cell lines examined, including a cell line derived from a patient with Bloom syndrome, a disorder exhibiting an elevated spontaneous SCE frequency. Induction was seen to occur in a dose-responsive manner and was also observed in cells expressing the resistance genes that inactivate the cellular toxicity of these antibiotics. The implications of these findings for somatic cell genetics and for human gene therapy protocols are discussed.

Hygromycin B therapy of a murine coronaviral hepatitis.

Hepatitis caused by mouse hepatitis virus (MHV-A59), a murine coronavirus, is accompanied by direct infection and replication of virus within the liver. We demonstrate here that the aminoglycoside hygromycin B is able to eliminate MHV-A59 infection from mouse peritoneal macrophages and cultured liver cells in vitro and is also able to reduce levels of virus replication and necrotic liver foci in vivo.