siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury.

Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t(1/2) for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.

Studies on the cholesterol-free mouse: strong activation of LXR-regulated hepatic genes when replacing cholesterol with desmosterol.

OBJECTIVE: Characterization of cholesterol homeostasis in male mice with a genetic inactivation of 3beta-hydroxysteroid-delta24-reductase, causing replacement of almost all cholesterol with desmosterol. METHODS AND RESULTS: There was an increase in hepatic sterol synthesis and markedly increased fecal loss of neutral sterols. Fecal excretion of bile acids was similar in knockout mice and in controls. The composition of bile acids was changed, with reduced formation of cholic acid. It was shown that both Cyp7a1 and Cyp27a1 are active toward desmosterol, consistent with the formation of normal bile acids from this steroid. The levels of plant sterols were markedly reduced. Hepatic mRNA levels of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase, Srebp-1c, Srebp-2, Cyp7a1, Abcg5, Abcg8, and Fas were all significantly increased. CONCLUSIONS: The changes in hepatic mRNA levels in combination with increased biliary and fecal excretion of neutral steroids, reduced tissue levels of plant sterols, increased plasma levels of triglyceride-rich VLDL, are consistent with a strong activation of LXR-targeted genes. The markedly increased fecal loss of neutral sterols may explain the fact that the Dhcr24-/- mice do not accumulate dietary cholesterol. The study illustrates the importance of the integrity of the cholesterol structure--presence of a double bond in the steroid side-chain is compatible with life but is associated with serious disturbances in sterol homeostasis.

Inhibition of oxygen-induced retinopathy in RTP801-deficient mice.

PURPOSE: Ischemic proliferative retinopathy, which occurs as a complication of diabetes mellitus, prematurity, or retinal vein occlusion, is a major cause of blindness worldwide. In addition to retinal neovascularization, it involves retinal degeneration, of which apoptosis is the main cause. A prior report has described the cloning of a novel HIF-1-responsive gene, RTP801, which displays strong hypoxia-dependent upregulation in ischemic cells of neuronal origin, both in vitro and in vivo. Moreover, inducible overexpression of RTP801 promotes the apoptotic death of differentiated neuron-like PC12 cells and increases their sensitivity to ischemic injury and oxidative stress. The purpose of the study was to examine the potential role of RTP801 in the pathogenesis of retinopathy, using RTP801-deficient mice. METHODS: Wild-type and RTP801-knockout mice were used in a model of retinopathy of prematurity (ROP). Their retinas were collected at postnatal day (P)14 and P17. They were examined by fluorescein angiography and by analysis of VEGF expression, neovascularization, and apoptosis. RESULTS: The expression of RTP801 was induced in the wild-type retina after hypoxia treatment. The retinal expression of VEGF after transfer to normoxic conditions was similarly upregulated in both wild-type and knockout mice. Nevertheless, the retinas of the RTP801-knockout mice in an ROP model showed a significant reduction in retinal neovascularization (P < 0.0001) and in the number of apoptotic cells in the inner nuclear layer (P < 0.0001). CONCLUSIONS: In the absence of RTP801 expression, development of retinopathy in the mouse model of ROP was significantly attenuated, thus implying an important role of RTP801 in the pathogenesis of ROP.

Studies on the transcriptional regulation of cholesterol 24-hydroxylase (CYP46A1): marked insensitivity toward different regulatory axes.

Mammalian CNS contains a disproportionally large and remarkably stable pool of cholesterol. Despite an efficient recycling there is some requirement for elimination of brain cholesterol. Conversion of cholesterol into 24S-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1) is the quantitatively most important mechanism. Based on the protein expression and plasma levels of 24S-hydroxycholesterol, CYP46A1 activity appears to be highly stable in adults. Here we have made a structural and functional characterization of the promoter of the human CYP46A1 gene. No canonical TATA or CAAT boxes were found in the promoter region. Moreover this region had a high GC content, a feature often found in genes considered to have a largely housekeeping function. A broad spectrum of regulatory axes using a variety of promoter constructs did not result in a significant transcriptional regulation. Oxidative stress caused a significant increase in transcriptional activity. The possibility of a substrate-dependent transcriptional regulation was explored in vivo in a sterol-deficient mouse model (Dhcr24 null) in which almost all cholesterol had been replaced with desmosterol, which is not a substrate for CYP46A1. Compared with heterozygous littermates there was no statistically significant difference in the mRNA levels of Cyp46a1. During the first 2 weeks of life in the wild-type mouse, however, a significant increase of Cyp46a1 mRNA levels was found, in parallel with an increase in 24S-hydroxycholesterol level and a reduction of cholesterol synthesis. The failure to demonstrate a significant transcriptional regulation under most conditions is discussed in relation to the turnover of brain and neuronal cholesterol.