Recovery following relief of unilateral ureteral obstruction in the neonatal rat.
BACKGROUND: Obstructive nephropathy is a primary cause of renal insufficiency in infants and children. This study was designed to distinguish the reversible and irreversible cellular consequences of temporary unilateral ureteral obstruction (UUO) on the developing kidney. METHODS: Rats were subjected to UUO or sham operation in the first 48 hours of life, and the obstruction was removed five days later (or was left in place). Kidneys were removed for study 14 or 28 days later. In additional groups, kidneys were removed at the end of five days of obstruction. Immunoreactive distribution of renin was determined in arterioles, and the distribution of epidermal growth factor, transforming growth factor-beta1, clusterin, vimentin, and alpha-smooth muscle actin was determined in tubules and/or interstitium. The number of glomeruli, glomerular maturation, tubular atrophy, and interstitial collagen deposition was determined by morphometry. Renal cellular proliferation and apoptosis were measured by proliferating cell nuclear antigen and the TdT uridine-nick-end-label technique, respectively. The glomerular filtration rate was measured by inulin clearance. RESULTS: Renal microvascular renin maintained a fetal distribution with persistent UUO; this was partially reversed by the relief of obstruction. Although glomerular maturation was also delayed and glomerular volume was reduced by UUO, the relief of obstruction prevented the reduction in glomerular volume. Although relief of obstruction did not reverse a 40% reduction in the number of nephrons, the glomerular filtration rate of the postobstructed kidney was normal. The relief of obstruction did not improve tubular cell proliferation and only partially reduced apoptosis induced by UUO. This was associated with a persistent reduction in the tubular epidermal growth factor. In addition, the relief of obstruction reduced but did not normalize tubular expression of transforming growth factor-beta1, clusterin, and vimentin, all of which are evidence of persistent tubular injury. The relief of obstruction significantly reduced interstitial fibrosis and expression of alpha-smooth muscle actin by interstitial fibroblasts, but not to normal levels. CONCLUSIONS: The relief of obstruction in the neonatal rat attenuates, but does not reverse, renal vascular, glomerular, tubular, and interstitial injury resulting from five days of UUO. Hyperfiltration by remaining nephrons and residual tubulointerstitial injury in the postobstructed kidney are likely to lead to deterioration of renal function later in life. (+info)
Clusterin has chaperone-like activity similar to that of small heat shock proteins.
Clusterin is a highly conserved protein which is expressed at increased levels by many cell types in response to a broad variety of stress conditions. A genuine physiological function for clusterin has not yet been established. The results presented here demonstrate for the first time that clusterin has chaperone-like activity. At physiological concentrations, clusterin potently protected glutathione S-transferase and catalase from heat-induced precipitation and alpha-lactalbumin and bovine serum albumin from precipitation induced by reduction with dithiothreitol. Enzyme-linked immunosorbent assay data showed that clusterin bound preferentially to heat-stressed glutathione S-transferase and to dithiothreitol-treated bovine serum albumin and alpha-lactalbumin. Size exclusion chromatography and SDS-polyacrylamide gel electrophoresis analyses showed that clusterin formed high molecular weight complexes (HMW) with all four proteins tested. Small heat shock proteins (sHSP) also act in this way to prevent protein precipitation and protect cells from heat and other stresses. The stoichiometric subunit molar ratios of clusterin:stressed protein during formation of HMW complexes (which for the four proteins tested ranged from 1.0:1.3 to 1.0:11) is less than the reported ratios for sHSP-mediated formation of HMW complexes (1.0:1.0 or greater), indicating that clusterin is a very efficient chaperone. Our results suggest that clusterin may play a sHSP-like role in cytoprotection. (+info)
Clusterin gene expression mediates resistance to apoptotic cell death induced by heat shock and oxidative stress.
Clusterin is a widely expressed, well conserved, secreted glycoprotein, which is highly induced in tissues regressing as a consequence of apoptotic cell death in vivo. It has recently been shown that clusterin expression is only confined to surviving cells following the induction of apoptosis in vitro, suggesting that it is involved in cell survival rather than death. In the hypothesis that clusterin may be implicated in cellular responses to stress, clusterin gene expression was analyzed in the A431 human epidermoid cancer cell line following heat shock and oxidative stress. Our results show that both a transient heat shock (20 min at 42 degrees C) and various oxidative stresses, including hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure, induce a strong increase in clusterin mRNA levels as assessed by northern blot. Nuclear run-on analysis suggests that transcriptional activation is involved in inducing clusterin mRNA in response to heat shock. Using pulse-chase analysis of control and heat shocked cells, it is shown that clusterin mRNA is translated and secreted, thus resulting in increased extracellular levels of the protein following heat shock. To investigate the function of clusterin in response to these stresses, clusterin anti-sense transfectants that stably express virtually no clusterin at the mRNA and protein level were generated in A431 cells. These anti-sense transfectants are shown to be highly sensitive to apoptotic cell death induced by heat shock or oxidative stress compared with wild-type A431 cells or control transfectants. Taken together, our results show that clusterin gene expression is induced in response to heat shock and oxidative stress in human A431 cells, and confers cellular protection against heat shock and oxidative stress. (+info)
Effect of targeted expression of clusterin in photoreceptor cells on retinal development and differentiation.
Clusterin expression is increased in tissues undergoing apoptosis, including neurodegenerative retina, but the causal relationships remain to be clarified. To test the hypothesis that overexpression of clusterin could induce apoptosis in neurons, transgenic mice were generated in which rat clusterin transgene was expressed in photoreceptor cells under the transcriptional control of the human interphotoreceptor retinoid-binding protein (IRBP) promoter. Photoreceptor cell death in the resulting transgenic mice was examined by histology and TUNEL techniques. The expression of the clusterin transgene was confirmed by in situ hybridization in the photoreceptor cells, and results in a complex pattern of clusterin protein distribution in the retina. A reduction in apoptotic staining in the transgenic retinas was observed from birth to postnatal day 15. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor cell death, but may relate to cytoprotective functions. (+info)
Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells.
Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression. (+info)
Clusterin (SGP-2) gene expression is cell cycle dependent in normal human dermal fibroblasts.
In confluent human dermal fibroblasts brought to quiescence (G0) by serum starvation, the S phase peaked at 24 h after serum re-addiction and G2/M phase peaked at 36 h. This was confirmed by titration of h-gas1 mRNA (a marker of G0 phase) and histone H3 (a marker of S phase). Clusterin mRNA accumulation progressively increased in cells proceeding to confluence after seeding and to quiescence upon serum starvation, and peaked at around G0, in parallel with h-gas1 mRNA. At 6 h (roughly G1 phase) clusterin transcript formed a second peak, followed by a gradual decrease until 36 h. Correspondence of clusterin protein accumulation to its mRNA occurred solely with regard to the G0 peak but not to the second one. The possible meaning of the cell cycle related clusterin gene expression is discussed. (+info)
Isolation of Ku70-binding proteins (KUBs).
DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, >10 kb trans-cripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation. (+info)
Targeted disruption of the murine lecithin:cholesterol acyltransferase gene is associated with reductions in plasma paraoxonase and platelet-activating factor acetylhydrolase activities but not in apolipoprotein J concentration.
Lecithin:cholesteryl acyltransferase (LCAT) deficiency resulting from targeted disruption of the Lcat gene in the mouse is associated with dramatic decreases in HDL concentration and the accumulation of nascent HDL in the plasma. We examined whether LCAT deficiency in mice is associated with a concomitant decrease in two antioxidative enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH). In control Lcat (+/+) mice both these enzymes are transported on HDL. Compared to Lcat (+/+) mice, HDL-cholesterol is reduced 94% and apoA-I, 90%, in Lcat (-/-) mice; this reduction in HDL is paralleled by a 71% decrease in PAF-AH activity and in a 58% decrease in PON activity. Apolipoprotein J (apoJ) levels, rather than being decreased, were significantly (P = 0.01) higher (36%) in Lcat (-/-) than in Lcat (+/+) mice, and the apo J/PON ratio was 3-fold greater in Lcat (-/-) than in Lcat (+/+) animals. Even though apolipoprotein A-I (apoA-I) concentration and PON activity were drastically reduced, there was no reduction in apoA-I and PON liver mRNA levels suggesting that post-transcriptional events are responsible for the reduction of plasma PON and apoA-I levels. Fast protein liquid chromatography (FPLC) revealed that in Lcat (+/+) mice both PON and PAF-AH activity is associated with large, apoA-I-containing HDL particles (9.7 nm by non-denaturing gradient gel electrophoresis) while in Lcat (-/-) mice both enzymes are associated with small 8.2 nm particles. We conclude that the concomitant reduction in HDL and apoA-I concentrations and PON and PAF-AH activities is best explained by rapid clearance of the small HDL particles found in LCAT deficiency. (+info)