scholarly journals Structure and regulation of the mDot1 gene, a mouse histone H3 methyltransferase

2004 ◽  
Vol 377 (3) ◽  
pp. 641-651 ◽  
Author(s):  
Wenzheng ZHANG ◽  
Yoshihide HAYASHIZAKI ◽  
Bruce C. KONE
Keyword(s):  
Alternative Splicing ◽  
Fluorescent Protein ◽  
Expressed Sequence Tag ◽  
Collecting Duct ◽  
Signal Transduction Pathway ◽  
Dependent Manner ◽  
Coding Region ◽  
Duct Cells ◽  
Collecting Duct Cells

Recently, a new class of histone methyltransferases that plays an indirect role in chromatin silencing by targeting a conserved lysine residue in the nucleosome core was described, namely the Dot1 (disruptor of telomeric silencing) family [Feng, Wang, Ng, Erdjument-Bromage, Tempst, Struhl and Zhang (2002) Curr. Biol. 12, 1052–1058; van Leeuwen, Gafken and Gottschling (2002) Cell (Cambridge, Mass.) 109, 745–756; Ng, Feng, Wang, Erdjument-Bromage, Tempst, Zhang and Struhl (2002) Genes Dev. 16, 1518–1527]. In the present study, we report the isolation, genomic organization and in vivo expression of a mouse Dot1 homologue (mDot1). Expressed sequence tag analysis identified five mDot1 mRNAs (mDot1a–mDot1e) derived from alternative splicing. mDot1a and mDot1b encode 1540 and 1114 amino acids respectively, whereas mDot1c–mDot1e are incomplete at the 5´-end. mDot1a is closest to its human counterpart (hDot1L), sharing 84% amino acid identity. mDot1b is truncated at its N- and C-termini and contains an internal deletion. The five mDot1 isoforms are encoded by 28 exons on chromosome 10qC1, with exons 24 and 28 further divided into two and four sections respectively. Alternative splicing occurs in exons 3, 4, 12, 24, 27 and 28. Northern-blot analysis with probes corresponding to the methyltransferase domain or the mDot1a-coding region detected 7.6 and 9.5 kb transcripts in multiple tissues, but only the 7.6 kb transcript was evident in mIMCD3-collecting duct cells. Transfection of mDot1a–EGFP constructs (where EGFP stands for enhanced green fluorescent protein) into human embryonic kidney (HEK)-293T or mIMCD3 cells increased the methylation of H3-K79 but not H3-K4, -K9 or -K36. Furthermore, DMSO induced mDot1 gene expression and methylation specifically at H3-K79 in mIMCD3 cells in a time- and dose-dependent manner. Collectively, these results add new members to the Dot1 family and show that mDot1 is involved in a DMSO-mediated signal-transduction pathway in collecting duct cells.

Is the function of the renal papilla coupled exclusively to an anaerobic pattern of metabolism?

1979 ◽  
Vol 236 (5) ◽  
pp. F423-F433 ◽  
Author(s):  
J. J. Cohen
Keyword(s):  
Oxidative Metabolism ◽  
Aerobic Glycolysis ◽  
Collecting Duct ◽  
High Rate ◽  
O2 Uptake ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Mitochondrial Oxidative Metabolism

It is widely accepted that in vivo the function of the papilla of the mammalian kidney is supported primarily by anaerobic metabolism. As a result, the major source of energy for support of function in the papilla is considered to be derived from glycolysis. This orientation originates from two concepts: 1) that in vivo the gaseous environment of the papilla has such a low PO2 that O2 availability limits O2 consumption, and 2) that papillary tissue has a high rate of glycolysis when compared with either cortical tissue or extrarenal tissues. It has also been tacitly assumed that papillary tissue has a "low" O2 uptake. Review of the measurements of PO2 of papillary tissue and of urine PO2 indicates that the PO2 of papillary tissue should not limit its aerobic mitochondrial oxidative metabolism. While the rate of aerobic glycolysis in papillary tissue is high, simultaneously papillary tissue has a rate of O2 uptake similar to that of liver and higher than that of muscle. The major (two-thirds) source of energy for papillary tissue in vitro is from O2 uptake. That papillary tissue is not exclusively dependent on glucose for its energy requirements is indicated by the greater stimulation of papillary tissue QO2 by succinate than by glucose. Thus, papillary tissue has both a high aerobic mitochondrial oxidative metabolism and a high aerobic glycolytic metabolism. It is suggested that the mechanism for the high rate of aerobic glycolysis in the presence of an adequate O2 supply is due to the relatively small mass of mitochondria in papillary tissue in relation to the amount of work done by the tissue. As a result of the limited rate of ATP production by the mitochondrial electron transport chain, the phosphorylation state ([ATP]/[ADP][Pi]) is reduced and the cytoplasmic redox state ([NAD+]/[NADH]) of the papillary collecting duct cells also becomes more reduced; changes in both ratios enhance the rate of glycolysis. This limited metabolic capacity of the collecting duct cells may permit an excess volume of solute and water to be excreted during volume expansion diuresis. The metabolic characteristics of the papilla, when compared to cortex, also provide a basis for the observed differences in substrate selectivity of cortex and medulla with respect to utilization of glucose and lactate. The experimental approaches that may provide information bearing on the suggested mechanisms for regulation of papillary metabolism in relation to tubular work functions are indicated.

scholarly journals Urinary Reabsorption in the Rat Kidney by Anticholinergics

2021 ◽  
Author(s):  
Hideki Oe ◽  
Hatsumi Yoshiki ◽  
Xinmin Zha ◽  
Hisato Kobayashi ◽  
Yoshitaka Aoki ◽  
...  
Keyword(s):  
Absorption Rate ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Bladder Epithelium ◽  
Duct Cells ◽  
Urine Production ◽  
Saline Administration ◽  
Collecting Duct Cells

Abstract Anticholinergics, therapeutic agents for overactive bladder, are clinically suggested to reduce urine output. We investigated whether this effect is due to bladder or kidney urine reabsorption. Various solutions were injected into the bladder of urethane-anesthetized SD rats. The absorption rate for 2 hr was examined following the intravenous administration of the anticholinergics imidafenacin (IM), atropine(AT), and tolterodine(TO). The bilateral ureter was then canulated and saline was administered to obtain a diuretic state. Anticholinergics or 1-deamino-[8-D-arginine]-vasopressin (dDAVP) were intravenously administered. After the IM and dDAVP administrations, the rat kidneys were immunostained with AQP2 antibody, and intracellular cAMP was measured. The absorption rate was ~10% of the saline injected into the bladder and constant even when anticholinergics were administered. The renal urine among peaked 2 hr after the saline administration. Each of the anticholinergics significantly suppressed the urine production in a dose-dependent manner, as did dDAVP. IM and dDAVP increased the intracellular cAMP levels and caused the AQP2 molecule to localize to the collecting duct cells' luminal side. The urinary reabsorption mechanism through the bladder epithelium was not activated by anticholinergic administration. Thus, anticholinergics suppress urine production via an increase in urine reabsorption in the kidneys' collecting duct cells via AQP2.

334 CONSTRUCTION OF A GENETICALLY ENGINEERED PIG EXPRESSING GREEN FLUORESCENT PROTEIN (GFP)-LABELLED PROTEASOMES

2011 ◽  
Vol 23 (1) ◽  
pp. 263 ◽  
Author(s):  
C. W. O'Gorman ◽  
J. Zhao ◽  
M. S. Samuel ◽  
E. M. Walters ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Fluorescent Protein ◽  
Expressed Sequence Tag ◽  
Stop Codon ◽  
Full Length ◽  
Matrix Attachment Region ◽  
Coding Region ◽  
Public Data ◽  
Fetal Fibroblasts

Proteasomes are large protein complexes involved in protein degradation in eukaryotes and undergo dynamic redistribution between cellular compartments. Characterising the cellular localization of proteasomes at various stages of development and in response to stimuli is of interest. We hypothesised that porcine proteasomes could be visualised in vivo via a ubiquitously expressed transgene fusion comprising a proteasomal subunit and green florescent protein (GFP). The full-length sequence for porcine PSMA-1 was first constructed in silico from public data and was used to retrieve a GenBank expressed sequence tag (EST) sequence that appeared to be full length (accession CO946059; kind gift from R. S. Prather). Primers were designed to remove the stop codon and create homology for cloning with InFusion (Clontech, Palo Alto, CA, USA). The amplimer was inserted into pCAG-CreGFP (Addgene plasmid 13776) in place of the Cre coding region. The resulting plasmid (pKW14) was screened via restriction digest and sequenced for confirmation. This plasmid was confirmed functional in porcine fetal fibroblasts. After removal of the plasmid backbones, pKW14, a G418 resistance cassette (NEO), and the chicken egg white matrix attachment region were co-electroporated into male fetal fibroblasts (10 μg of total DNA, 5:2:2 ratio, respectively). Cells were grown in DMEM with 10% fetal bovine serum (FBS) and selection was initiated 36 h after transfection. Following 12 days of selection at 400 mg L–1 G418, colonies were screened by epifluorescence. Positive colonies were harvested and confirmed transgenic for all 3 input DNAs. Positive colonies were randomly pooled as sets of 3 independent integration events. Embryos were reconstructed via SCNT and transferred to 2 recipients. The fusion rates were 70 and 78%, respectively, with transfer numbers of 120 and 125 fused couplets being transferred into synchronized recipients on Day 0 of heat. Both recipients became pregnant and delivered 2 piglets each on Day 114 by Caesarean section. One live piglet was produced from each litter. Of the 2 live-born piglets, 1 survived beyond Day 3 and continues to be healthy. Transgenic status was verified by PCR. Expression was confirmed by epifluorescence of GFP-labelled proteasomes. This founder will be used to establish a model to evaluated cellular localization of proteasomes in vivo and in culture.

scholarly journals Urinary reabsorption in the rat kidney by anticholinergics

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hideki Oe ◽  
Hatsumi Yoshiki ◽  
Xinmin Zha ◽  
Hisato Kobayashi ◽  
Yoshitaka Aoki ◽  
...  
Keyword(s):  
Absorption Rate ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Bladder Epithelium ◽  
Duct Cells ◽  
Urine Production ◽  
Saline Administration ◽  
Collecting Duct Cells

AbstractAnticholinergics, therapeutic agents for overactive bladder, are clinically suggested to reduce urine output. We investigated whether this effect is due to bladder or kidney urine reabsorption. Various solutions were injected into the bladder of urethane-anesthetized SD rats. The absorption rate for 2 h was examined following the intravenous administration of the anticholinergics imidafenacin (IM), atropine (AT), and tolterodine (TO). The bilateral ureter was then canulated and saline was administered to obtain a diuretic state. Anticholinergics or 1-deamino-[8-D-arginine]-vasopressin (dDAVP) were intravenously administered. After the IM and dDAVP administrations, the rat kidneys were immunostained with AQP2 antibody, and intracellular cAMP was measured. The absorption rate was ~ 10% of the saline injected into the bladder and constant even when anticholinergics were administered. The renal urine among peaked 2 h after the saline administration. Each of the anticholinergics significantly suppressed the urine production in a dose-dependent manner, as did dDAVP. IM and dDAVP increased the intracellular cAMP levels and caused the AQP2 molecule to localize to the collecting duct cells' luminal side. The urinary reabsorption mechanism through the bladder epithelium was not activated by anticholinergic administration. Thus, anticholinergics suppress urine production via an increase in urine reabsorption in the kidneys' collecting duct cells via AQP2.

scholarly journals Loss of primary cilia increases polycystin-2 and TRPV4 and the appearance of a nonselective cation channel in the mouse cortical collecting duct

2019 ◽  
Vol 317 (3) ◽  
pp. F632-F637 ◽  
Author(s):  
Takamitsu Saigusa ◽  
Qiang Yue ◽  
Marlene A. Bunni ◽  
P. Darwin Bell ◽  
Douglas C. Eaton
Keyword(s):  
Collecting Duct ◽  
Cation Channel ◽  
Conditional Knockout ◽  
Nonselective Cation Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Duct Cells ◽  
Collecting Ducts ◽  
Collecting Duct Cells

Flow-related bending of cilia results in Ca2+ influx through a polycystin-1 (Pkd1) and polycystin-2 (Pkd2) complex, both of which are members of the transient receptor potential (TRP) family (TRPP1 and TRPP2, respectively). Deletion of this complex as well as cilia result in polycystic kidney disease. The Ca2+ influx pathway has been previously characterized in immortalized collecting duct cells without cilia and found to be a 23-pS channel that was a multimere of TRPP2 and TRPV4. The purpose of the present study was to determine if this TRPP2 and TRPV4 multimere exists in vivo. Apical channel activity was measured using the patch-clamp technique from isolated split-open cortical collecting ducts from adult conditional knockout mice with ( Ift88flox/flox) or without ( Ift88−/−) cilia. Single tubules were isolated for measurements of mRNA for Pkd1, Pkd2, Trpv4, and epithelial Na+ channel subunits. The predominant channel activity from Ift88flox/flox mice was from epithelial Na+ channel [5-pS Na+-selective channels with long mean open times (475.7 ± 83.26 ms) and open probability > 0.2]. With the loss of cilia, the predominant conductance was a 23-pS nonselective cation channel (reversal potential near 0) with a short mean open time (72 ± 17 ms), open probability < 0.08, and a characteristic flickery opening. Loss of cilia increased mRNA levels for Pkd2 and Trpv4 from single isolated cortical collecting ducts. In conclusion, 23-pS channels exist in vivo, and activity of this channel is elevated with loss of cilia, consistent with previous finding of an elevated-unregulated Ca2+-permeable pathway at the apical membrane of collecting duct cells that lack cilia.

(Pro)renin receptor is required for prorenin-dependent and -independent regulation of vacuolar H+-ATPase activity in MDCK.C11 collecting duct cells

2013 ◽  
Vol 305 (3) ◽  
pp. F417-F425 ◽  
Author(s):  
Xifeng Lu ◽  
Ingrid M. Garrelds ◽  
Carsten A. Wagner ◽  
A. H. Jan Danser ◽  
Marcel E. Meima
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Dependent Manner ◽  
Atpase Inhibitor ◽  
Duct Cells ◽  
Prorenin Receptor ◽  
Receptor Blockers ◽  
Collecting Duct Cells ◽  
Interfering Rna

Prorenin binding to the prorenin receptor [(P)RR] results in nonproteolytic activation of prorenin but also directly (i.e., independent of angiotensin generation) activates signal transduction cascades that can lead to the upregulation of profibrotic factors. The (P)RR is an accessory protein of vacuolar-type H+-ATPase (V-ATPase) and is required for V-ATPase integrity. In addition, in collecting duct cells, prorenin-induced activation of Erk depends on V-ATPase activity. However, whether prorenin binding to the (P)RR directly regulates V-ATPase activity is as yet unknown. Here, we studied the effect of prorenin on plasma membrane V-ATPase activity in Madin-Darby canine kidney clone 11 (MDCK.C11) cells, which resemble intercalated cells of the collecting duct. Prorenin increased V-ATPase activity at low nanomolar concentrations, and the V-ATPase inhibitor bafilomycin A1, but not the angiotensin II type 1 and 2 receptor blockers irbesartan and PD-123319, prevented this. Increased, but not basal, V-ATPase activity was abolished by small interfering RNA depletion of the (P)RR. Unexpectedly, the putative peptidic (P)RR blocker handle region peptide also increasedV-ATPase activity in a (P)RR-dependent manner. Finally, [Arg8]-vasopressin-stimulated V-ATPase activity and cAMP production were also abolished by (P)RR depletion. Our results show that in MDCK.C11 cells, the (P)RR is required for prorenin-dependent and -independent regulation of V-ATPase activity.

Stanniocalcin-1 secretion and receptor regulation in kidney cells

2008 ◽  
Vol 294 (4) ◽  
pp. F788-F794 ◽  
Author(s):  
Olga Sazonova ◽  
Kathi A. James ◽  
Christopher R. McCudden ◽  
Daniel Segal ◽  
Asghar Talebian ◽  
...  
Keyword(s):  
Cell Lines ◽  
Collecting Duct ◽  
Signal Transduction Pathway ◽  
Distal Tubule ◽  
Receptor Regulation ◽  
Thick Ascending Limb ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Pkc Activation

Kidney collecting duct principal cells are the main source of stanniocalcin-1 (STC-1) production and secretion. From there, the hormone targets thick ascending limb and distal convoluted tubule cells, as well as collecting duct cells. More specifically, STC-1 targets their mitochondria to exert putative antiapoptotic effects. Two distal tubule cell lines serve as models of STC-1 production and/or mechanism of action. Madin-Darby canine kidney-1 (MDCK-1) cells mimic collecting duct cells in their synthesis of STC-1 ligand and receptor, whereas inner medullary collecting duct-3 (IMCD-3) cells respond to additions of STC-1 by increasing their respiration rate. In the present study, MDCK cell STC-1 secretion was examined under normal and hypertonic conditions, vectorally, and in response to hormones and signal transduction pathway activators/inhibitors. STC-1 receptor regulation was monitored in both cell lines in response to changing ligand concentration. The results showed that NaCl-induced hypertonicity had concentration-dependent stimulatory effects on STC-1 secretion, as did the PKC activator TPA. Calcium and ionomycin were inhibitory, whereas calcium receptor agonists had no effect. Angiotensin II, aldosterone, atrial natriuretic factor, antidiuretic hormone, and forskolin also had no effects. Moreover, STC-1 secretion exhibited no vectoral preference. STC-1 receptors were insensitive to homologous downregulation in both cell lines. In contrast, they were upregulated when STC-1 secretion was inhibited by calcium. The findings suggest that hypertonicity-induced STC-1 secretion is regulated through PKC activation and that high intracellular calcium levels are a potent inhibitor of release. More intriguingly, the results suggest that the receptor may not accompany STC-1 in its passage to the mitochondria.

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