Effects of expression of p53 and Gadd45 on osmotic tolerance of renal inner medullary cells

2006 ◽  
Vol 291 (2) ◽  
pp. F341-F349 ◽  
Author(s):  
Qi Cai ◽  
Natalia I. Dmitrieva ◽  
Joan D. Ferraris ◽  
Luis F. Michea ◽  
Jesus M. Salvador ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Collecting Duct ◽  
Wild Type ◽  
Growth And Survival ◽  
Osmotic Tolerance ◽  
Rate Of Increase ◽  
Gene Knockout Mice ◽  
Collecting Duct Cells ◽  
Urinary Concentrating Ability

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45b−/−, Gadd45g−/−, and Gadd45bg−/− mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. p53−/− Cells do not tolerate as high a concentration of NaCl (or urea) as p53+/+ cells, but urinary concentrating ability of p53−/− mice is normal, as is the histology of inner medullas from p53−/− and Gadd45abg−/− mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ.

scholarly journals The Alopecia Areata Phenotype Is Induced by the Water Avoidance Stress Test In cchcr1-Deficient Mice

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 840
Author(s):  
Qiaofeng Zhao ◽  
Satoshi Koyama ◽  
Nagisa Yoshihara ◽  
Atsushi Takagi ◽  
Etsuko Komiyama ◽  
...  
Keyword(s):  
Alopecia Areata ◽  
Gene Knockout ◽  
Stress Test ◽  
Coiled Coil ◽  
Risk Haplotype ◽  
Gene Expression Microarray ◽  
Wild Type ◽  
Gene Knockout Mice ◽  
Deficient Mice ◽  
Gene Expression Microarray Analysis

We recently discovered a nonsynonymous variant in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene within the alopecia areata (AA) risk haplotype. We also reported that the engineered mice with this risk allele exhibited. To investigate more about the involvement of the CCHCR1 gene in AA pathogenesis, we developed an AA model using C57BL/6N cchcr1 gene knockout mice. In this study, mice (6–8 weeks) were divided into two groups: cchcr1−/− mice and wild-type (WT) littermates. Both groups were subjected to a water avoidance stress (WAS) test. Eight weeks after the WAS test, 25% of cchcr1−/− mice exhibited non-inflammatory foci of alopecia on the dorsal skin. On the other hand, none of wild-type littermates cause hair loss. The foci resembled human AA in terms of gross morphology, trichoscopic findings and histological findings. Additionally, gene expression microarray analysis of cchcr1−/− mice revealed abnormalities of hair related genes compared to the control. Our results strongly suggest that CCHCR1 is associated with AA pathogenesis and that cchcr1−/− mice are a good model for investigating AA.

scholarly journals Potential role of purinergic signaling in urinary concentration in inner medulla: insights from P2Y2 receptor gene knockout mice

2008 ◽  
Vol 295 (6) ◽  
pp. F1715-F1724 ◽  
Author(s):  
Yue Zhang ◽  
Jeff M. Sands ◽  
Donald E. Kohan ◽  
Raoul D. Nelson ◽  
Christopher F. Martin ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Purinergic Signaling ◽  
Collecting Duct ◽  
Receptor Gene ◽  
Urinary Concentration ◽  
P2y2 Receptor ◽  
Concentration Gradients ◽  
Gene Knockout Mice ◽  
Medullary Collecting Duct

Osmotic reabsorption of water through aquaporin-2 (AQP2) in the inner medulla is largely dependent on the urea concentration gradients generated by urea transporter (UT) isoforms. Vasopressin (AVP) increases expression of both AQP2 and UT-A isoforms. Activation of the P2Y2 receptor (P2Y2-R) in the medullary collecting duct inhibits AVP-induced water flow. To gain further insights into the overarching effect of purinergic signaling on urinary concentration, we compared the protein abundances of AQP2 and UT-A isoforms between P2Y2-R knockout (KO) and wild-type (WT) mice under basal conditions and following AVP administration. Under basal conditions (a gel diet for 10 days), KO mice concentrated urine to a significantly higher degree, with 1.8-, 1.66-, and 1.29-fold higher protein abundances of AQP2, UT-A1, and UT-A2, respectively, compared with WT, despite comparable circulating AVP levels in both groups. Infusion of 1-desamino-8-d-arginine vasopressin (dDAVP; desmopressin; 1 ng/h sc) for 5 days resulted in 2.14-, 2.6-, and 2.22-fold higher protein abundances of AQP2, AQP3, and UT-A1, respectively, in the inner medullas of KO mice compared with WT mice. In response to acute (45 min) stimulation by AVP (0.2 unit/mouse sc), UT-A1 protein increased by 1.39- and 1.54-fold in WT and KO mice, respectively. These data suggest that genetic deletion of P2Y2-R results in increased abundances of key proteins involved in urinary concentration in the inner medulla, both under basal conditions and following AVP administration. Thus purinergic regulation may play a potential overarching role in balancing the effect of AVP on the urinary concentration mechanism.

scholarly journals Experimental Pseudomonas aeruginosa Keratitis in Interleukin-10 Gene Knockout Mice

2003 ◽  
Vol 71 (3) ◽  
pp. 1328-1336 ◽  
Author(s):  
Nerida Cole ◽  
Mark Krockenberger ◽  
Fiona Stapleton ◽  
Shamila Khan ◽  
Emma Hume ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gene Knockout ◽  
Bacterial Load ◽  
Interleukin 10 ◽  
Wild Type ◽  
Cytokine Levels ◽  
Infiltrating Cells ◽  
Gene Knockout Mice ◽  
Corneal Angiogenesis ◽  
Kinetics Of

ABSTRACT Pseudomonas aeruginosa keratitis is one of the most destructive diseases of the cornea. The host response to this infection is critical to the outcome. The cytokine interleukin-10 (IL-10) is thought to play an important role in modulating excessive inflammation and antimicrobial defenses. We have found that in IL-10−/− mice there is a significant decrease in bacterial load in corneas at 7 days postchallenge with P. aeruginosa. This decrease was accompanied by a reduction in neutrophil numbers in the cornea and changes in cytokine levels compared to those of wild-type mice. A characteristic increase in neovascularization in the cornea was found in the IL-10−/− mice. This increased angiogenesis correlated with an increased expression of KC, whereas the kinetics of macrophage inflammatory peptide 2 expression correlated with neutrophil numbers. This finding suggests that KC may play a role in corneal angiogenesis. The source of IL-10 in mouse corneas was identified as a subpopulation of infiltrating cells and keratocytes. This study demonstrates that IL-10 plays an important role in regulating the balance of inflammatory mediators during P. aeruginosa infection of the cornea.

scholarly journals Critical Role of Cytotoxic T Lymphocytes in Immune Clearance of Rickettsial Infection

2001 ◽  
Vol 69 (3) ◽  
pp. 1841-1846 ◽  
Author(s):  
David H. Walker ◽  
Juan P. Olano ◽  
Hui-Min Feng
Keyword(s):  
T Lymphocytes ◽  
Knockout Mice ◽  
Gene Knockout ◽  
Wild Type ◽  
Spleen Cells ◽  
Rickettsial Infection ◽  
Cd8 T Lymphocytes ◽  
Gene Knockout Mice ◽  
Ifn Γ ◽  
Ctl Activity

ABSTRACT Cytotoxic T-lymphocyte (CTL) activity developed against the major infected target cells of rickettsial infections, endothelial cells and macrophages. Spleen cells from mice immune to Rickettsia conorii exerted specific major histocompatibility complex (MHC) class I-matched CTL activity against R. conorii-infected SVEC-10 endothelial cells, with peak activity on day 10. Similarly, spleen cells from Rickettsia australis-immune mice exerted specific CTL activity against an R. australis-infected macrophage-like cell line. Gamma interferon (IFN-γ) gene knockout mice were more than 100-fold more susceptible to R. australis infection than wild-type C57BL/6 mice. MHC class I gene knockout mice were the most susceptible, more than 50,000-fold more susceptible to a lethal outcome of R. australis infection than wild-type C57BL/6 mice. These results indicate that CTL activity was more critical to recovery from rickettsial infection than were the effects of IFN-γ. The observation that perforin gene knockout mice were more than 100-fold more susceptible than wild-type C57BL/6 mice indicates that perforin-mediated activity accounts for a large component, but not all, of the CTL-mediated antirickettsial effect. CTL activity was expressed by immune CD8 T lymphocytes. Adoptive transfer of immune CD8 T lymphocytes from IFN-γ gene knockout mice intoR. australis-infected IFN-γ gene knockout mice dramatically reduced the infectious rickettsial content in the organs, confirming that CD8 T lymphocytes provide immunity against rickettsiae besides that provided by the secretion of IFN-γ. CTLs appear to be crucial to recovery from rickettsial infection.

scholarly journals Phagocyte-Derived Reactive Oxygen Species Do Not Influence the Progression of Murine Blood-Stage Malaria Infections

2005 ◽  
Vol 73 (8) ◽  
pp. 4941-4947 ◽  
Author(s):  
S. M. Potter ◽  
A. J. Mitchell ◽  
W. B. Cowden ◽  
L. A. Sanni ◽  
M. Dinauer ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Knockout Mice ◽  
Plasmodium Berghei ◽  
Gene Knockout ◽  
Reactive Oxygen ◽  
Plasmodium Yoelii ◽  
Oxygen Species ◽  
Wild Type ◽  
Gene Knockout Mice

ABSTRACT Phagocyte-derived reactive oxygen species have been implicated in the clearance of malaria infections. We investigated the progression of five different strains of murine malaria in gp91phox−/− mice, which lack a functional NADPH oxidase and thus the ability to produce phagocyte-derived reactive oxygen species. We found that the absence of functional NADPH oxidase in the gene knockout mice had no effect on the parasitemia or total parasite burden in mice infected with either resolving (Plasmodium yoelii and Plasmodium chabaudi K562) or fatal (Plasmodium berghei ANKA, Plasmodium berghei K173 and Plasmodium vinckei vinckei) strains of malaria. This lack of effect was apparent in both primary and secondary infections with P. yoelii and P. chabaudi. There was also no difference in the presentation of clinical or pathological signs between the gp91phox−/− or wild-type strains of mice infected with malaria. Progression of P. berghei ANKA and P. berghei K173 infections was unchanged in glutathione peroxidase-1 gene knockout mice compared to their wild-type counterparts. The rates of parasitemia progression in gp91phox−/− mice and wild-type mice were not significantly different when they were treated with l-N G -methylarginine, an inhibitor of nitric oxide synthase. These results suggest that phagocyte-derived reactive oxygen species are not crucial for the clearance of malaria parasites, at least in murine models.

Defining an inhibitory domain in the α-subunit of the epithelial sodium channel

2008 ◽  
Vol 294 (1) ◽  
pp. F47-F52 ◽  
Author(s):  
Marcelo D. Carattino ◽  
Christopher J. Passero ◽  
Carlos A. Steren ◽  
Ahmad B. Maarouf ◽  
Joseph M. Pilewski ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Collecting Duct ◽  
Airway Epithelial Cells ◽  
Xenopus Laevis Oocytes ◽  
Airway Epithelial ◽  
Wild Type ◽  
Epithelial Sodium Channels ◽  
Α Subunit ◽  
Collecting Duct Cells ◽  
Inhibitory Domain

Epithelial sodium channels (ENaC) are processed by proteases as they transit the biosynthetic pathway. We recently observed that furin-dependent processing of the α-subunit of ENaC at two sites within its extracellular domain is required for channel activation due to release of a 26-residue inhibitory domain. While channels with α-subunits lacking the furin sites are not cleaved and have very low activity, channels lacking the furin consensus sites as well as the tract between these sites (αD206–R231) are active. We analyzed channels with a series of deletions in the tract αD206–R231 and lacking the α-subunit furin consensus sites in Xenopus laevis oocytes. We found an eight-residue tract that, when deleted, restored channel activity to the level found in oocytes expressing wild-type ENaC. A synthetic peptide, LPHPLQRL, representing the tract αL211–L218, inhibited wild-type ENaC expressed in oocytes with an IC50 of 0.9 μM, and inhibited channels expressed in collecting duct cells and human primary airway epithelial cells with an IC50s of between ∼50 and 100 μM. Analyses of peptides with deletions within this inhibitory tract indicate that eight residues is the minimal backbone length that is required for ENaC inhibition. Analyses of 8-mer peptides with conserved and nonconserved substitutions suggest that L1, P2, H3, P4, and L8 are required for inhibitory activity. Our findings suggest that this eight-residue tract is a key conserved inhibitory domain that provides epithelial cells with a reserve of inactive channels that can be activated as required by proteases.

scholarly journals Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption

2018 ◽  
Vol 50 (4) ◽  
pp. 1361-1375 ◽  
Author(s):  
Jie Xu ◽  
Sharon Barone ◽  
Kamyar Zahedi ◽  
Marybeth Brooks ◽  
Manoocher Soleimani
Keyword(s):  
In Situ Hybridization ◽  
Subcellular Localization ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Acid Base ◽  
Duct Cells ◽  
Collecting Ducts ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

Background/Aims: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney. Methods: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice. Results: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis. Conclusion: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.

scholarly journals Impact of High Salt Diet on Cerebral Vascular Function and Stroke in Tff3−/−/C57BL/6N Knockout and WT (C57BL/6N) Control Mice

2019 ◽  
Vol 20 (20) ◽  
pp. 5188 ◽  
Author(s):  
Nataša Kozina ◽  
Zrinka Mihaljević ◽  
Mirela Baus Lončar ◽  
Martina Mihalj ◽  
Mihael Mišir ◽  
...  
Keyword(s):  
Vascular Function ◽  
Gene Knockout ◽  
Innate Immune ◽  
High Salt ◽  
No Production ◽  
Wild Type ◽  
High Salt Diet ◽  
Salt Diet ◽  
Gene Knockout Mice ◽  
Induced Changes

High salt (HS) dietary intake leads to impaired vascular endothelium-dependent responses to various physiological stimuli, some of which are mediated by arachidonic acid (AA) metabolites. Transgenic Tff3−/− gene knockout mice (Tff3−/−/C57BL/6N) have changes in lipid metabolism which may affect vascular function and outcomes of stroke. We aimed to study the effects of one week of HS diet (4% NaCl) on vascular function and stroke induced by transient occlusion of middle cerebral artery in Tff3−/− and wild type (WT/C57BL/6N) mice. Flow-induced dilation (FID) of carotid artery was reduced in WT-HS mice, but not affected in Tff3−/−-HS mice. Nitric oxide (NO) mediated FID. NO production was decreased with HS diet. On the contrary, acetylcholine-induced dilation was significantly decreased in Tff3−/− mice on both diets and WT-HS mice. HS intake and Tff3 gene depletion affected the structural components of the vessels. Proteomic analysis revealed a significant effect of Tff3 gene deficiency on HS diet-induced changes in neuronal structural proteins and acute innate immune response proteins’ expression and Tff3 depletion, but HS diet did not increase the stroke volume, which is related to proteome modification and upregulation of genes involved mainly in cellular antioxidative defense. In conclusion, Tff3 depletion seems to partially impair vascular function and worsen the outcomes of stroke, which is moderately affected by HS diet.

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