A model of hemodialysis after acute kidney injury in rats.

BACKGROUND: Acute kidney injury (AKI) is frequent among critically ill patients. Renal replacement therapy (RRT) is often required to deal with severe complications of AKI. This technique is however associated with side effects such as hemodynamic instability and delayed renal recovery. In this study, we aimed to describe a novel model of hemodialysis in rats with AKI and depict a dialysis membrane performance. METHODS: Eighteen Sprague-Dawley rats received 0.75% adenine-rich diet to induce AKI. After 2 weeks, nine underwent an arterio-venous extracorporeal circulation (ECC) (ECC group) for 2 h without a dialysis membrane on the circuit and nine received a hemodialysis session (HD group) for 2 h with an ECC circuit. All rats were hemodynamically monitored, and glomerular filtration rate (GFR) was measured by transcutaneous fluorescence after the injection of FITC-Sinistrin. Blood samples were collected at different time points to assess serum creatinine and serum urea concentrations and to determine the Kt/V. Sinistrin concentration was also quantified in both plasma and dialysis effluent. RESULTS: After 2 weeks of adenine-rich diet, rats exhibited a decrease in GFR. Both serum urea and serum creatinine concentrations increased in the ECC group but remained stable in the HD group. We found no significant difference in serum creatinine and serum urea concentrations between groups. At the end of experiments, mean serum urea was 36.7 mmol/l (95%CI 19.7-46.9 mmol/l) and 23.6 mmol/l (95%CI 15.2-33.5 mmol/l) in the ECC and HD groups, respectively (p = 0.15), and mean serum creatinine concentration was 158.0 µmol/l (95%CI 108.1-191.9 µmol/l) and 114.0 µmol/l (95%CI 90.2-140.9 µmol/l) in the ECC and HD groups, respectively (p = 0.11). The Kt/V of the model was estimated at 0.23. Sinistrin quantity in the ultrafiltrate raised steadily during the dialysis session. After 2 h, the median quantity was 149.2 µg (95% CI 99.7-250.3 µg). CONCLUSIONS: This hemodialysis model is an acceptable compromise between the requirement of hemodynamic tolerance which implies reducing extracorporeal blood volume (using a small dialyzer) and the demonstration that diffusion of molecules through the membrane is achieved.

A recombinant E1-deleted canine adenoviral vector capable of transduction and expression of a transgene in human-derived cells and in vivo.

Human adenovirus (HAV) serotypes 2 and 5 are commonly used as vector backbones for adenovirus-mediated gene transfer. However, HAVs were chosen as a backbone for the vectors for historical reasons and have a number of significant disadvantages when used as a shuttle for gene transfer in humans. As an initial trial to circumvent some of the shortcomings of HAV vectors, we have produced an E1-deleted canine adenovirus type 2 (CAV-2) vector for gene transfer. Initially, we demonstrated that CAV-2 undergoes an abortive viral cycle in a wide range of human-derived cell lines. Second, we assayed human sera containing HAV-5 neutralizing antibodies for their ability to inhibit CAV-2-induced plaques on permissive cells. In the cohort tested, our data demonstrate that the humoral response directed against HAV-5 does not inhibit CAV-2 plaque formation in the majority of cases. Canine cell lines expressing the E1 region of CAV-2 were generated and characterized. A recombinant CAV vector (CAVRSVbetagal) deleted in the E1 region and harboring lacZ was constructed. We show that CAVRSVbetagal is able to transduce and direct expression of the transgene in vitro in a variety of mammalian cells, most notably primary human-derived cells. In addition, gene transfer is demonstrated in vivo using chick embryos.

Developmental and tissue-specific regulation of the murine cardiac actin gene in vivo depends on distinct skeletal and cardiac muscle-specific enhancer elements in addition to the proximal promoter.

Cardiac actin is an early marker of cardiac and skeletal muscle lineages in the mouse. After birth, the gene is down-regulated in skeletal muscle. High-level expression of the murine cardiac actin gene in skeletal myotubes in vitro involves distal (-7.8/-7.0 kb) and proximal (-5.4/-3.5 kb) enhancer sequences as well as the proximal promoter (-0.7/+0.1 kb). Transgenic mice carrying an nlacZ reporter gene under the control of different fragments of the upstream region of the cardiac actin gene were generated. This analysis led to the conclusions that (1) the proximal promoter is a weak but tissue specific element in vivo, (2) consistent high-level expression in skeletal muscle depends on the presence of at least one of the enhancers, (3) expression in adult cardiac muscle requires a cardiac enhancer located in the (-5.4/-0.7 kb) region, and (4) a construct containing these three elements gives a strong specific expression of the transgene in the heart throughout the life of the animal and in embryonic skeletal muscle. All transgenes tested reproduce the down-regulation observed in adult skeletal muscle for the cardiac actin gene. Nonuniform expression of these transgenes in the heart may mark cardiomyocytes derived from different cardiac progenitors.

Myf5 is a novel early axonal marker in the mouse brain and is subjected to post-transcriptional regulation in neurons.

Myf5 is a key basic Helix-Loop-Helix transcription factor capable of converting many non-muscle cells into muscle. Together with MyoD it is essential for initiating the skeletal muscle programme in the embryo. We previously identified unexpected restricted domains of Myf5 transcription in the embryonic mouse brain, first revealed by Myf5-nlacZ(+/)(-) embryos (Tajbakhsh, S. and Buckingham, M. (1995) Development 121, 4077-4083). We have now further characterized these Myf5 expressing neurons. Retrograde labeling with diI, and the use of a transgenic mouse line expressing lacZ under the control of Myf5 regulatory sequences, show that Myf5 transcription provides a novel axonal marker of the medial longitudinal fasciculus (mlf) and the mammillotegmental tract (mtt), the earliest longitudinal tracts to be established in the embryonic mouse brain. Tracts projecting caudally from the developing olfactory system are also labelled. nlacZ and lacZ expression persist in the adult brain, in a few ventral domains such as the mammillary bodies of the hypothalamus and the interpeduncular nucleus, potentially derived from the embryonic structures where the Myf5 gene is transcribed. To investigate the role of Myf5 in the brain, we monitored Myf5 protein accumulation by immunofluorescence and immunoblotting in neurons transcribing the gene. Although Myf5 was detected in muscle myotomal cells, it was absent in neurons. This would account for the lack of myogenic conversion in brain structures and the absence of a neural phenotype in homozygous null mutants. RT-PCR experiments show that the splicing of Myf5 primary transcripts occurs correctly in neurons, suggesting that the lack of Myf5 protein accumulation is due to regulation at the level of mRNA translation or protein stability. In the embryonic neuroepithelium, Myf5 is transcribed in differentiated neurons after the expression of neural basic Helix-Loop-Helix transcription factors. The signalling molecules Wnt1 and Sonic hedgehog, implicated in the activation of Myf5 in myogenic progenitor cells in the somite, are also produced in the viscinity of the Myf5 expression domain in the mesencephalon. We show that cells expressing Wnt1 can activate neuronal Myf5-nlacZ gene expression in dissected head explants isolated from E9.5 embryos. Furthermore, the gene encoding the basic Helix-Loop-Helix transcription factor mSim1 is expressed in adjacent cells in both the somite and the brain, suggesting that signalling molecules necessary for the activation of mSim1 as well as Myf5 are present at these different sites in the embryo. This phenomenon may be widespread and it remains to be seen how many other potentially potent regulatory genes, in addition to Myf5, when activated do not accumulate protein at inappropriate sites in the embryo.

Modular long-range regulation of Myf5 reveals unexpected heterogeneity between skeletal muscles in the mouse embryo.

The myogenic factor Myf5 plays a key role in muscle cell determination, in response to signalling cascades that lead to the specification of muscle progenitor cells. We have adopted a YAC transgenic approach to identify regulatory sequences that direct the complex spatiotemporal expression of this gene during myogenesis in the mouse embryo. Important regulatory regions with distinct properties are distributed over 96 kb upstream of the Myf5 gene. The proximal 23 kb region directs early expression in the branchial arches, epaxial dermomyotome and in a central part of the myotome, the epaxial intercalated domain. Robust expression at most sites in the embryo where skeletal muscle forms depends on an enhancer-like sequence located between -58 and -48 kb from the Myf5 gene. This element is active in the epaxial and hypaxial myotome, in limb muscles, in the hypoglossal chord and also at the sites of Myf5 transcription in prosomeres p1 and p4 of the brain. However later expression of Myf5 depends on a more distal region between -96 and -63 kb, which does not behave as an enhancer. This element is necessary for expression in head muscles but strikingly only plays a role in a subset of trunk muscles, notably the hypaxially derived ventral body muscles and also those of the diaphragm and tongue. Transgene expression in limb muscle masses is not affected by removal of the -96/-63 region. Epaxially derived muscles and some hypaxial muscles, such as the intercostals and those of the limb girdles, are also unaffected. This region therefore reveals unexpected heterogeneity between muscle masses, which may be related to different facets of myogenesis at these sites. Such regulatory heterogeneity may underlie the observed restriction of myopathies to particular muscle subgroups.