No abstract

Background. Autophagy is an intracellular bulk degradation process induced by cell starvation. Autophagy was recently reported to be induced by various stresses such as hypoxia, ischemia/reperfusion, toxins, and denatured proteins, and to affect cell survival and death. Light chain 3-II (LC3-II) is specifically located on double membrane-bound autophagosomes that envelop disused proteins or organelles. Method. Transgenic mice in which green fluorescent protein (GFP) was fused to LC3(LC3-GFP) were administered cisplatin (20 mg/kg). After euthanasia at times between 0-72 hours, kidneys were excised for immunohistochemical analyses.Microscopic examinations of the generated NRK-52E cell lines stably transfected with LC3-GFP, and western blot analyses of NRK-52E cells were undertaken after cisplatin treatment with or without autophagy inhibitors and beclin 1 siRNA.Results. Autophagosomes increased in the proximal tubular cells of transgenic mice from 12 hours after cisplatin injection (20 mg/kg). The time course for this was faster than those for tubular necrosis and apoptosis. Autophagosomes also increased in NRK-52E cells after cisplatin treatment, with the time course for this faster than that for apoptosis. When autophagy was suppressed by autophagy inhibitors or beclin 1 siRNA, the level of apoptosis was also suppressed. Conclusion.Autophagy occurs in proximal tubular cells after cisplatin treatment and is involved in cell death in renal tubular injury. Our data suggested that autophagy is a kind of cell damage index and cells with activated autophagy will be scavenged by apoptosis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 and in other cases induces apoptosis or type-2 programmed cell death [6][7][8]. Chronic myocardial ischemia/reperfusion injury induces autophagy increment and apoptosis decrement [9]. Inhibition of autophagy prevents neuron death after hypoxic-ischemic injury in neonatal mice [10]. However, in acute kidney injury (AKI), the association between autophagy and cell death is not obvious.Cisplatin is a major chemotherapeutic drug against solid tumors. One of the most important side effects of cisplatin is nephrotoxicity. Cisplatin is freely filtered at the Subjects and Methods MaterialsAnti-light chain 3 antibody was purchased from MBL (Nagoya, Japan).Anti-aquaporin-1 antibody was purchased from Abcam (Cambridge, MA).Anti-cleaved caspase 3 and rapamycin were purchased from Cell Signaling (Danvers, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Cell CultureThe NRK-52E cell line was obtained from ATCC (Manassas, VA), and cultured in DMEM medium containing 10% fetal bovine serum, 100 u...

The therapeutic effects of bacteriophage (phage) KPP12 in Pseudomonas aeruginosa keratitis were investigated in mice. Morphological analysis showed that phage KPP12 is a member of the family Myoviridae, morphotype A1, and DNA sequence analysis revealed that phage KPP12 is similar to PB1-like viruses. Analysis of the phage KPP12 genome did not identify any genes related to drug resistance, pathogenicity or lysogenicity, and so phage KPP12 may be a good candidate for therapeutic. KPP12 showed a broad host range for P. aeruginosa strains isolated from clinical ophthalmic infections. Inoculation of the scarified cornea with P. aeruginosa caused severe keratitis and eventual corneal perforation. Subsequent single-dose administration of KPP12 eye-drops significantly improved disease outcome, and preserved the structural integrity and transparency of the infected cornea. KPP12 treatment resulted in the suppression of neutrophil infiltration and greatly enhanced bacterial clearance in the infected cornea. These results indicate that bacteriophage eye-drops may be a novel adjunctive or alternative therapeutic agent for the treatment of infectious keratitis secondary to antibiotic-resistant bacteria.

The insertion/deletion (I/D) polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene is involved in the development of cardiovascular diseases. We compared the ACE mRNA expression originating from the allele with a deletion (D allele) and that from the allele with an insertion (I allele) in human white blood cells from ID heterozygotes. We identified the mRNA from the I allele by using the G2215A polymorphism that lies in exon 15 and that was linked to the I/D polymorphism. RNA samples were obtained from 12 healthy heterozygotes of both I/D and G2215A, and every insertion was shown to be linked to 2215G. ACE mRNA was amplified by the reverse transcription/polymerase chain reaction (RT-PCR) method with an end-labeled antisense primer. The PCR products were digested with HaeII and separated by electrophoresis, and the relative radioactivities of the 2215A and 2215G bands were measured on an auto-image analyzer. The results showed that, in every cases, the intensity of the 2215A product (D allele origin) was higher than that of the 2215G product (I allele origin). The mean ratio of 2215A to 2215G was 1.79 (1.11-2.62). Thus, the D allele leads to higher expression of the ACE mRNA and may affect the renin-angiotensin system in local regions.

The present study demonstrates that TNF-alpha induces renal tubular cell damage in RPTEC and AT1/AT2 receptor blockers showed cytoprotective effects probably via at least partly different mechanism.