A mouse line expressing Sall1‐driven inducible Cre recombinase in the kidney mesenchyme

Sall1 is expressed in the metanephric mesenchyme in the developing kidney, and mice deficient in Sall1 show kidney agenesis or dysgenesis. Sall1 is also expressed elsewhere, including in the limb buds, anus, heart, and central nervous system. Dominant‐negative mutations of Sall1 in mice and humans lead to developmental defects in these organs. Here, we generated a mouse line expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the endogenous Sall1 promoter. Upon tamoxifen treatment, these mice showed genomic recombination in the tissues where endogenous Sall1 is expressed. When CreER^T2 mice were crossed with the floxed Sall1 allele, tamoxifen administration during gestation led to a significant decrease in Sall1 expression and small kidneys at birth, suggesting that Sall1 functions were disrupted. Furthermore, Sall1 expression in the kidney was significantly reduced by neonatal tamoxifen treatment. The Sall1CreER^T2 mouse is a valuable tool for in vivo time‐dependent and region‐specific knockout and overexpression studies. genesis 48:207–212, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of apolipoproteinA1 reduction in the polycystic kidney by proteomics analysis of the Mxi1‐deficient mouse

Autosomal dominant polycystic kidney disease is one of the most common human monogenic diseases in which extensive epithelial‐lined cysts develop in kidney and other organs. Affected kidneys are not only characterized by the formation of cysts, but also by changes associated with the extracellular matrix and interstitial inflammation, which can progress to fibrosis and loss of renal function. Mxi1 protein, which is a c‐myc antagonist, may be essential in controlling cellular growth and differentiation. Previously, multiple tubular cysts were observed in kidney of Mxi1‐deficient mice aged 6 months and more. Presently, 2‐DE and MALDI‐TOF MS was employed to identify the differentially expressed proteins in the kidney. Several proteins were identified, among them, apolipoproteinA1 which is a major component of the high‐density lipoprotein complex and has anti‐inflammation effects, was significantly decreased in the Mxi1‐deficient mouse. We confirm the development of inflammation and renal fibrosis and the expression of extracellular matrix molecules including transforming growth factor were also increased in cystic kidney. These results indicate that expression of proteins related with inflammation and renal fibrosis changes by Mxi1 inactivation in polycystic kidney.     

Rapamycin treatment dose‐dependently improves the cystic kidney in a new ADPKD mouse model via the mTORC1 and cell‐cycle‐associated CDK1/cyclin axis

Although translational research into autosomal dominant polycystic kidney disease (ADPKD) and its pathogenesis has made considerable progress, there is presently lack of standardized animal model for preclinical trials. In this study, we developed an orthologous mouse model of human ADPKD by cross‐mating Pkd2 conditional‐knockout mice (Pkd2^f3) to Cre transgenic mice in which Cre is driven by a spectrum of kidney‐related promoters. By systematically characterizing the mouse model, we found that Pkd2^f3/f3 mice with a Cre transgene driven by the mouse villin‐1 promoter (Vil‐Cre;Pkd2^f3/f3) develop overt cysts in the kidney, liver and pancreas and die of end‐stage renal disease (ESRD) at 4–6 months of age. To determine whether these Vil‐Cre;Pkd2^f3/f3 mice were suitable for preclinical trials, we treated the mice with the high‐dose mammalian target of rapamycin (mTOR) inhibitor rapamycin. High‐dose rapamycin significantly increased the lifespan, lowered the cystic index and kidney/body weight ratio and improved renal function in Vil‐Cre;Pkd2^f3/f3 mice in a time‐ and dose‐dependent manner. In addition, we further found that rapamycin arrested aberrant epithelial‐cell proliferation in the ADPKD kidney by down‐regulating the cell‐cycle‐associated cyclin‐dependent kinase 1 (CDK1) and cyclins, namely cyclin A, cyclin B, cyclin D1 and cyclin E, demonstrating a direct link between mTOR signalling changes and the polycystin‐2 dysfunction in cystogenesis. Our newly developed ADPKD model provides a practical platform for translating in vivo preclinical results into ADPKD therapies. The newly defined molecular mechanism by which rapamycin suppresses proliferation via inhibiting abnormally elevated CDK1 and cyclins offers clues to new molecular targets for ADPKD treatment.     

Long interspersed nucleotide element‐1 hypomethylation in folate‐deficient mouse embryonic stem cells

Folate is thought to contribute to health and development by methylation regulation. Long interspersed nucleotide element‐1 (LINE‐1), which is regulated by methylation modification, plays an important role in sculpting the structure and function of genomes. Some studies have shown that folate concentration is related to LINE‐1 methylation. However, the direct association between LINE‐1 methylation and folate deficiency remains unclear. To explore whether folate deficiency directly induced LINE‐1 hypomethylation and to analyze the relationship between folate concentration and the LINE‐1 methylation level, mouse ESCs were treated with various concentrations of folate which was measured by chemiluminescent immunoassay, and the homocysteine content was detected by ELISA. LINE‐1 methylation was examined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry at various time points. Concurrently, cell proliferation and differentiation were observed. The result showed that the intracellular folate decreases under folate‐deficient condition, conversely, homocysteine content increased gradually and there was a negatively correlated between them. Folate insufficiency induced LINE‐1 hypomethylation at the lowest levels in folate‐free group and moderate in folate‐deficient group, compared with that in the folate‐normal group at day 18. Moreover, LINE‐1 methylation level was positively correlated with folate content, and negatively correlated with homocysteine content. At corresponding time points, proliferation and differentiation of mouse ESCs showed no alteration in all groups. Our data indicated that folate deficiency affected the homeostasis of folate‐mediated one‐carbon metabolism, leading to reduced LINE‐1 methylation in mouse ESCs. This study provides preliminary evidence of folate deficiency affecting early embryonic development. J. Cell. Biochem. 114: 1549–1558, 2013. © 2013 Wiley Periodicals, Inc.     

SIP1 expression patterns in brain investigated by generating a SIP1‐EGFP reporter knock‐in mouse

A loss of function of SIP1 (Smad interacting protein 1) in the mouse as well as in human of Mowat–Wilson syndrome results in severe and multiple defects in neural tissue development, especially in the brain. However, no detailed expression analysis of SIP1 during brain development has been previously reported. In this study, we describe the generation of an EGFP knock‐in reporter mouse for the Sip1 locus and our subsequent analysis of SIP1‐EGFP fusion protein expression during brain development. SIP1‐EGFP expression was observed in the pyramidal neurons of the hippocampus, the dentate gyrus, and the postmitotic neurons in the cerebral cortex. In layer 5 of the cerebral cortex, SIP1‐EGFP expression was complementary to the Ctip2‐expressing neurons, most of which are thought to be the cortico‐spinal neurons. This suggested that SIP1‐EGFP expressing cells might have the specific trajectory targets other than the spinal region. We further observed SIP1‐EGFP expression in oligodendrocytes of the corpus callosum and fimbria, Bergmann glial cells of the cerebellum, the olfactory bulb, and in the serotonergic and dopaminergic neurons of the raphe nuclei in the brainstem. These findings may help to clarify the unknown roles of SIP1 in these cells and the pathoetiology of Mowat‐Wilson syndrome. genesis 52:56–67, 2014. © 2013 Wiley Periodicals, Inc.     

Metabolomics of the interaction between PPAR‐α and age in the PPAR‐α‐null mouse

Regulation between the fed and fasted states in mammals is partially controlled by peroxisome proliferator‐activated receptor‐α (PPAR‐α). Expression of the receptor is high in the liver, heart and skeletal muscle, but decreases with age. A combined ¹H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography‐mass spectrometry metabolomic approach has been used to examine metabolism in the liver, heart, skeletal muscle and adipose tissue in PPAR‐α‐null mice and wild‐type controls during ageing between 3 and 13 months. For the PPAR‐α‐null mouse, multivariate statistics highlighted hepatic steatosis, reductions in the concentrations of glucose and glycogen in both the liver and muscle tissue, and profound changes in lipid metabolism in each tissue, reflecting known expression targets of the PPAR‐α receptor. Hepatic glycogen and glucose also decreased with age for both genotypes. These findings indicate the development of age‐related hepatic steatosis in the PPAR‐α‐null mouse, with the normal metabolic changes associated with ageing exacerbating changes associated with genotype. Furthermore, the combined metabolomic and multivariate statistics approach provides a robust method for examining the interaction between age and genotype.     

Generation of an Fsp1 (fibroblast‐specific protein 1)‐Flpo transgenic mouse strain

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.     

Role of endothelial‐to‐mesenchymal transition induced by TGF‐β1 in transplant kidney interstitial fibrosis

Chronic allograft dysfunction (CAD) induced by kidney interstitial fibrosis is the main cause of allograft failure in kidney transplantation. Endothelial‐to‐mesenchymal transition (EndMT) may play an important role in kidney fibrosis. We, therefore, undertook this study to characterize the functions and potential mechanism of EndMT in transplant kidney interstitial fibrosis. Proteins and mRNAs associated with EndMT were examined in human umbilical vein endothelial cells (HUVECs) treated with transforming growth factor‐beta1 (TGF‐β1) at different doses or at different intervals with western blotting, qRT‐PCR and ELISA assays. Cell motility and migration were evaluated with motility and migration assays. The mechanism of EndMT induced by TGF‐β1 was determined by western blotting analysis of factors involved in various canonical and non‐canonical pathways. In addition, human kidney tissues from control and CAD group were also examined for these proteins by HE, Masson's trichrome, immunohistochemical, indirect immunofluorescence double staining and western blotting assays. TGF‐β1 significantly promoted the development of EndMT in a time‐dependent and dose‐dependent manner and promoted the motility and migration ability of HUVECs. The TGF‐β/Smad and Akt/mTOR/p70S6K signalling pathways were found to be associated with the pathogenesis of EndMT induced by TGF‐β1, which was also proven in vivo by the analysis of specimens from the control and CAD groups. EndMT may promote transplant kidney interstitial fibrosis by targetting the TGF‐β/Smad and Akt/mTOR/p70S6K signalling pathways, and hence, result in the development of CAD in kidney transplant recipients.     

Sirt1–hypoxia‐inducible factor‐1α interaction is a key mediator of tubulointerstitial damage in the aged kidney

Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia‐inducible factor (HIF)‐1α is largely unknown. In this study, we investigated whether HIF‐1α could be a deacetylation target of Sirt1 and the effect of their interaction on age‐associated renal injury. Five‐week‐old (young) and 24‐month‐old (old) C57Bl/6J mice were assessed for their age‐associated changes. Kidneys from aged mice showed increased infiltration of CD68‐positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF‐1α. The level of Bcl‐2/adenovirus E1B‐interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor‐β1, which are regulated by HIF‐1α, was significantly higher in aged mice suggesting that HIF‐1α activity was increased. In HK‐2 cells, Sirt1 inhibitor sirtinol and siRNA‐mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down‐regulated, which allowed the acetylation and activation of HIF‐1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia‐induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF‐1α activity by Sirt1‐induced deacetylation of HIF‐1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF‐1α‐transfected HK‐2 cells. Finally, we confirmed that chronic activation of HIF‐1α promoted apoptosis and fibrosis, using tubular cell‐specific HIF‐1α transgenic mice. Taken together, our data suggest that Sirt1‐induced deacetylation of HIF‐1α may have protective effects against tubulointerstitial damage in aged kidney.     

Spontaneous development of Alzheimer's disease‐associated brain pathology in a Shugoshin‐1 mouse cohesinopathy model

Spontaneous late‐onset Alzheimer's disease (LOAD) accounts for more than 95% of all human AD. As mice do not normally develop AD and as understanding on molecular processes leading to spontaneous LOAD has been insufficient to successfully model LOAD in mouse, no mouse model for LOAD has been available. Existing mouse AD models are all early‐onset AD (EOAD) models that rely on forcible expression of AD‐associated protein(s), which may not recapitulate prerequisites for spontaneous LOAD. This limitation in AD modeling may contribute to the high failure rate of AD drugs in clinical trials. In this study, we hypothesized that genomic instability facilitates development of LOAD and tested two genomic instability mice models in the brain pathology at the old age. Shugoshin‐1 (Sgo1) haploinsufficient (?) mice, a model of chromosome instability (CIN) with chromosomal and centrosomal cohesinopathy, spontaneously exhibited a major feature of AD pathology; amyloid beta accumulation that colocalized with phosphorylated Tau, beta‐secretase 1 (BACE), and mitotic marker phospho‐Histone H3 (p‐H3) in the brain. Another CIN model, spindle checkpoint‐defective BubR1^?/+ haploinsufficient mice, did not exhibit the pathology at the same age, suggesting the prolonged mitosis‐origin of the AD pathology. RNA‐seq identified ten differentially expressed genes, among which seven genes have indicated association with AD pathology or neuronal functions (e.g., ARC, EBF3). Thus, the model represents a novel model that recapitulates spontaneous LOAD pathology in mouse. The Sgo1^?/+ mouse may serve as a novel tool for investigating mechanisms of spontaneous progression of LOAD pathology, for early diagnosis markers, and for drug development.     

Recombinant PBD‐1 (porcine beta‐defensin 1) expressed in the milk by transplanting transgenic mES‐like‐derived cells into mouse mammary gland

ES (embryonic stem)‐derived cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES‐derived cells to improve functional outcome following partially damaged breast and also the modification of mammary tissue to produce costly proteins. This study investigates the feasibility of implanting mES‐dK (mouse ES‐derived keratinocytes‐like) cells stably transfected with a mammary gland special expression vector for the PBD‐1 (porcine beta‐defensin 1) in developing mammary glands. Our aim was to assess the ability of cell grafting to improve functional outcome following partial damage of the breast, also on the breast modification mammary tissue in mice for the production of PBD‐1 protein secreted in the milk. Our results showed that the ratios of the surviving cells labelled with the myoepithelial or luminal cell markers, EMA (epithelial membrane antigen) and CALLA, were 41.7±15.2% and 28.4±9.6%, respectively, which revealed that transplanted mES‐dK cells survived, integrated in vivo and differentiated into myoepithelial or luminal cells. In addition, Western blot analysis showed that 37.5% (3 out of 8) female transplanted mice had PBD‐1 expression in their milk and reached 0.4998, 0.5229 and 0.5195 μg/ml, respectively.     

Defective ATM‐Kap‐1‐mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model

ATM‐mediated phosphorylation of KAP‐1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24^?/? mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM‐Kap‐1 signaling is compromised in Zmpste24^?/? MEFs, leading to defective DNA damage‐induced chromatin remodeling. Knocking down Kap‐1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24^?/? MEFs. Thus, ATM‐Kap‐1‐mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.     

Roles of mouse sperm‐associated alpha‐L‐fucosidases in fertilization

Sperm‐associated α‐L ‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L ‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L ‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L ‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L ‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L ‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L ‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L ‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L ‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L ‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L ‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L ‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013. © 2013 Wiley Periodicals, Inc.