Phosphorylation of PrxII promotes JNK-dependent apoptosis in adult cloned pig kidney

Organ transplantation is the most effective medical therapy for end-stage renal disease patients; however, there is a critical shortage of human donor organs. Therefore, xenotransplantation using genetically modified cloned porcine kidney is considered as a viable solution, but its fundamental therapeutic mechanism and difference from non-cloned porcine or human kidney for its clinical application is not well known. Here, we performed proteomic analysis to investigate the differentially expressed molecules in kidney tissue obtained from cloned porcine by SCNT, when compared with normal porcine kidney in same age as a control. A total of 80 protein spots were differentially expressed between cloned porcine kidney and control kidney, including apoptotic proteins, structural and anti-oxidant related proteins. Furthermore, very interestingly, the differential expression pattern of PrxII in the cloned porcine kidney was distinguishable from that in the control kidney in terms of the pI and molecular weight. Along with this, apoptotic marker proteins were up-regulated in the cloned porcine kidney. We suggested that these alterations were induced by post-translational modification such as phosphorylation in PrxII and could be mediated by JNK. With this result, we also observed that the down-regulation of JNK activity was caused by blockage of phosphorylation in PrxII T89A region. Taken together, cloned porcine kidney is more susceptible in JNK-induced apoptosis caused by PrxII phosphorylation, in oxidative stress condition. These results will be helpful in the application of cloned porcine xeno-transplants for treating end-stage renal disease patients in a clinical setting.     

Determinants of RNA Binding and Translational Repression by the Bicaudal-C Regulatory Protein

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.     

LGR4/GPR48 Inactivation Leads to Aniridia-Genitourinary Anomalies-Mental Retardation Syndrome Defects

AGR syndrome (the clinical triad of aniridia, genitourinary anomalies, and mental retardation, a subgroup of WAGR syndrome for Wilm''s tumor, aniridia, genitourinary anomalies, and mental retardation) is a rare syndrome caused by a contiguous gene deletion in the 11p13–14 region. However, the mechanisms of WAGR syndrome pathogenesis are elusive. In this study we provide evidence that LGR4 (also named GPR48), the only G-protein-coupled receptor gene in the human chromosome 11p12–11p14.4 fragment, is the key gene responsible for the diseases of AGR syndrome. Deletion of Lgr4 in mouse led to aniridia, polycystic kidney disease, genitourinary anomalies, and mental retardation, similar to the pathological defects of AGR syndrome. Furthermore, Lgr4 inactivation significantly increased cell apoptosis and decreased the expression of multiple important genes involved in the development of WAGR syndrome related organs. Specifically, deletion of Lgr4 down-regulated the expression of histone demethylases Jmjd2a and Fbxl10 through cAMP-CREB signaling pathways both in mouse embryonic fibroblast cells and in urinary and reproductive system mouse tissues. Our data suggest that Lgr4, which regulates eye, kidney, testis, ovary, and uterine organ development as well as mental development through genetic and epigenetic surveillance, is a novel candidate gene for the pathogenesis of AGR syndrome.     

Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation

Immunohistochemical analysis was used to define the precise cell-specific localization of Glucose-6-phosphatase (Glc6Pase) and cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) in the digestive system (liver, small intestine and pancreas) and the kidney. Co-expression of Glc6Pase and PEPCK-C was shown to take place in hepatocytes, in proximal tubules of the cortex kidney and at the top of the villi of the small intestine suggesting that these tissues are all able to perform complete gluconeogenesis. On the other hand, intrahepatic bile ducts, collecting tubes of the nephron and the urinary epithelium in the calices of the kidney, as well as the crypts of the small intestine, express Glc6Pase without significant levels of PEPCK-C. In such cases, the function of Glc6Pase could be related to the transepithelial transport of glucose characteristic of these tissues, rather than to the neoformation of glucose. Lastly, PEPCK-C expression in the absence of Glc6Pase was noted in both the exocrine pancreas and the endocrine islets of Langerhans. Possible roles of PEPCK-C in exocrine pancreas might be the provision of gluconeogenic intermediates for further conversion into glucose in the liver, whereas PEPCK-C would be instrumental in pyruvate cycling, which has been suggested to play a regulatory role in insulin secretion by the β-cells of the islets. An erratum to this article can be found at     

从"补脾强肾"小儿推拿手法论治小儿脑瘫

目的:从中医的角度,探究"补脾强肾"小儿推拿手法论治小儿脑瘫的作用机制。方法:采用回顾性的分析,收集中西医治疗小儿脑瘫的相关文章,分析小儿脑瘫的原因、治疗方法及作用机制。结果:在中医中脑瘫病位在脑、脾、肾,脑瘫患儿普遍存在脾肾亏虚,表现为肌肉瘦削,筋骨痿软,故属于"五迟"、"五软"范畴。这与现代医学所观察到的脑瘫患儿肢体运动障碍和营养吸收障碍相符。因此该病除了应用现代康复技术手段外,还应从补益脾肾的角度开展相应的中医治疗。临床中不能接受内服药物治疗的患儿,可以采用小儿推拿治疗,治疗效果显著。结论:在脑瘫康复训练中配合补脾强肾小儿推拿手法,同补先后天之本,可提高营养生长水平,缩短康复疗程,提升康复疗效,最终达到全面促进脑瘫患儿康复的目的。     

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.     

Caffeic acid phenethyl ester changes the indices of oxidative stress in serum of rats with renal ischaemia–reperfusion injury

Oxygen‐derived free radicals have been implicated in the pathogenesis of renal injury after ischaemia–reperfusion. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, exhibits antioxidant properties. To investigate whether treatment with either CAPE or alpha‐tocopherol modifies the levels of the endogenous indices of oxidant stress, we examined their effects on an in vivo model of renal ischaemia–reperfusion injury in rats. CAPE at 10 μmol kg^?1 or alpha‐tocopherol at 10 mg kg^?1 was administered intraperitoneally before reperfusion. Acute administration of both CAPE and alpha‐tocopherol altered the indices of oxidative stress differently in renal ischaemia–reperfusion injury. Copyright © 2001 John Wiley & Sons, Ltd.     

Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.     

Proteomic identification of tyrosine nitration targets in kidney of spontaneously hypertensive rats

Nitrosative and oxidative stress are implicated in the development of hypertension. Events in the renal medulla may play a key role in the development and progression of hypertension. This may arise through disruption of nitric oxide signalling in the medulla and be accompanied by enhanced nitrosative and oxidative stress as indicated by the presence of proteins containing 3-nitrotyrosine. Here we demonstrate enhanced protein nitration in the medulla of spontaneously hypertensive rats. We have identified several nitrated proteins with both varied subcellular location and functional roles. These proteins are involved in nitric oxide signalling, antioxidant defense and energy metabolism. Moreover, increased nitration was observed in conjunction with enhanced oxidative damage as evidenced by the presence of protein carbonyl oxidative stress biomarkers. Our results suggest that kidney medulla is subject to enhanced nitrosative and oxidative stress, and that resulting protein modifications may contribute to the progression of hypertension.