Induction of calcyclin after ischemic injury to rat kidney

1997 ◽  
Vol 273 (3) ◽  
pp. F380-F385 ◽  
Author(s):  
A. J. Lewington ◽  
B. J. Padanilam ◽  
M. R. Hammerman
Keyword(s):  
Protein Expression ◽  
Calcium Binding ◽  
Messenger Rna ◽  
Rat Kidney ◽  
Ischemic Injury ◽  
Recovery Process ◽  
Thick Ascending Limb ◽  
Mrna And Protein Expression ◽  
Polymerase Chain

Genes differentially expressed after acute renal ischemic injury were identified using differential display-polymerase chain reaction (DD-PCR). Messenger RNA for calcyclin, a member of the S100 family of calcium-binding proteins, is increased in kidneys by 6 h following ischemic injury to rats compared with sham surgery. The level of calcyclin mRNA is increased 10-fold by 1 day postinjury and declines thereafter. In situ hybridization demonstrates little calcyclin mRNA in kidneys of sham-operated rats. However, calcyclin protein is present in glomeruli and distal tubules (DT). Compared with kidneys from sham-operated controls, both calcyclin mRNA and protein expression are increased at 1-3 days following ischemic injury in the thick ascending limb of Henle, the DT, and in damaged regenerating segments of proximal tubules. By 7 days postischemia there is a reduction in mRNA and protein expression. Calcyclin could play a role in the regulation of renal cell proliferation and regeneration in the recovery process after acute ischemic injury.

High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

1996 ◽  
Vol 54 ◽  
pp. 896-897
Author(s):  
G. W. Hacker ◽  
I. Zehbe ◽  
J. Hainfeld ◽  
A.-H. Graf ◽  
C. Hauser-Kronberger ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
High Performance ◽  
Detection System ◽  
Direct Methods ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

scholarly journals Novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Safia Akhtar ◽  
Silas A. Culver ◽  
Helmy M. Siragy
Keyword(s):  
Protein Expression ◽  
High Fat Diet ◽  
Normal Diet ◽  
Receptor Expression ◽  
Wild Type ◽  
High Fat ◽  
Renal Gluconeogenesis ◽  
Mrna And Protein Expression ◽  
Polymerase Chain ◽  
Renal Expression

AbstractRecent studies suggested that renal gluconeogenesis is substantially stimulated in the kidney in presence of obesity. However, the mechanisms responsible for such stimulation are not well understood. Recently, our laboratory demonstrated that mice fed high fat diet (HFD) exhibited increase in renal Atp6ap2 [also known as (Pro)renin receptor] expression. We hypothesized that HFD upregulates renal gluconeogenesis via Atp6ap2-PGC-1α and AKT pathway. Using real-time polymerase chain reaction, western blot analysis and immunostaining, we evaluated renal expression of the Atp6ap2 and renal gluconeogenic enzymes, PEPCK and G6Pase, in wild type and inducible nephron specific Atp6ap2 knockout mice fed normal diet (ND, 12 kcal% fat) or a high-fat diet (HFD, 45 kcal% fat) for 8 weeks. Compared with ND, HFD mice had significantly higher body weight (23%) (P < 0.05), renal mRNA and protein expression of Atp6ap2 (39 and 35%), PEPCK (44 and 125%) and G6Pase (39 and 44%) respectively. In addition, compared to ND, HFD mice had increased renal protein expression of PGC-1α by 32% (P < 0.05) and downregulated AKT by 33% (P < 0.05) respectively in renal cortex. Atp6ap2-KO abrogated these changes in the mice fed HFD. In conclusion, we identified novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway.

Localization of rat CLC-K2 chloride channel mRNA in the kidney

1999 ◽  
Vol 276 (4) ◽  
pp. F552-F558 ◽  
Author(s):  
Momono Yoshikawa ◽  
Shinichi Uchida ◽  
Atsushi Yamauchi ◽  
Akiko Miyai ◽  
Yujiro Tanaka ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chloride Channel ◽  
Rat Kidney ◽  
Water Channel ◽  
Physiological Role ◽  
Thick Ascending Limb ◽  
Nephron Segments ◽  
Henle's Loop ◽  
Collecting Ducts

To gain insight into the physiological role of a kidney-specific chloride channel, CLC-K2, the exact intrarenal localization was determined by in situ hybridization. In contrast to the inner medullary localization of CLC-K1, the signal of CLC-K2 in our in situ hybridization study was highly evident in the superficial cortex, moderate in the outer medulla, and absent in the inner medulla. To identify the nephron segments where CLC-K2 mRNA was expressed, we performed in situ hybridization of CLC-K2 and immunohistochemistry of marker proteins (Na+/Ca2+exchanger, Na+-Cl−cotransporter, aquaporin-2 water channel, and Tamm-Horsfall glycoprotein) in sequential sections of a rat kidney. Among the tubules of the superficial cortex, CLC-K2 mRNA was highly expressed in the distal convoluted tubules, connecting tubules, and cortical collecting ducts. The expression of CLC-K2 in the outer and inner medullary collecting ducts was almost absent. In contrast, a moderate signal of CLC-K2 mRNA was observed in the medullary thick ascending limb of Henle’s loop, but the signal in the cortical thick ascending limb of Henle’s loop was low. These results clearly demonstrated that CLC-K2 was not colocalized with CLC-K1 and that its localization along the nephron segments was relatively broad compared with that of CLC-K1.

Identification and localization of extracellular Ca2+-sensing receptor in rat intestine

1998 ◽  
Vol 274 (1) ◽  
pp. G122-G130 ◽  
Author(s):  
Naibedya Chattopadhyay ◽  
Ivan Cheng ◽  
Kimberly Rogers ◽  
Daniela Riccardi ◽  
Amy Hall ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Large Intestine ◽  
Rat Kidney ◽  
Cell Types ◽  
Northern Analysis ◽  
Reaction Products ◽  
Polymerase Chain ◽  
Small Intestinal ◽  
Small And Large Intestine

The extracellular calcium ([Formula: see text])-sensing receptor (CaR) plays vital roles in [Formula: see text] homeostasis, but no data are available on its expression in small and large intestine. Polymerase chain reaction products amplified from reverse-transcribed duodenal RNA using CaR-specific primers showed >99% homology with the rat kidney CaR. Northern analysis with a CaR-specific cRNA probe demonstrated 4.1- and 7.5-kb transcripts in all intestinal segments. Immunohistochemistry with CaR-specific antisera showed clear basal staining of epithelial cells of small intestinal villi and crypts and modest apical staining of the former, whereas there was both basal and apical staining of colonic crypt epithelial cells. In situ hybridization and immunohistochemistry also demonstrated CaR expression in Auerbach’s myenteric plexus of small and large intestines and in the submucosa in the region of Meissner’s plexus. Our results reveal CaR expression in several cell types of small and large intestine, in which it may modulate absorptive and/or secretomotor functions.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

5-HT2B receptors are expressed on astrocytes from brain and in culture and are a chronic target for all five conventional ‘serotonin-specific reuptake inhibitors’

2010 ◽  
Vol 6 (2) ◽  
pp. 113-125 ◽  
Author(s):  
Shiquen Zhang ◽  
Baoman Li ◽  
Ditte Lovatt ◽  
Junnan Xu ◽  
Dan Song ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Sorting ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Calcium Dependent ◽  
Mrna And Protein Expression ◽  
Polymerase Chain ◽  
Well Differentiated

In well-differentiated primary cultures of mouse astrocytes, which express no serotonin transporter (SERT), the ‘serotonin-specific reuptake inhibitor’ (SSRI) fluoxetine leads acutely to 5-HT2B receptor-mediated, transactivation-dependent phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) with an EC50 of ~5 μM, and chronically to ERK1/2 phosphorylation-dependent upregulation of mRNA and protein expression of calcium-dependent phospholipase A2 (cPLA2) with ten-fold higher affinity. This affinity is high enough that fluoxetine given therapeutically may activate astrocytic 5-HT2B receptors (Li et al., 2008, 2009). We now confirm the expression of 5-HT2B receptors in astrocytes freshly dissociated from mouse brain and isolated by fluorescence-activated cell sorting (FACS) and investigate in cultured cells if the effects of fluoxetine are shared by all five conventional SSRIs with sufficiently high affinity to be relevant for mechanism(s) of action of SSRIs. Phosphorylated and total ERK1/2 and mRNA and protein expression of cPLA2a were determined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Paroxetine, which differs widely from fluoxetine in affinity for SERT and for another 5-HT2 receptor, the 5-HT2C receptor, acted acutely and chronically like fluoxetine. One micromolar of paroxetine, fluvoxamine or sertraline increased cPLA2a expression during chronic treatment; citalopram had a similar effect at 0.1–0.5 μM; these are therapeutically relevant concentrations.

A comparison of PGC-1α mRNA and protein expression in response to 1-week endurance training on alternate days or 4 consecutive days

2015 ◽  
Vol 40 (11) ◽  
pp. 1210-1213 ◽  
Author(s):  
Li-Ping Huang ◽  
Min Yao ◽  
Ya-Li Wang ◽  
Allan Davie ◽  
Shi Zhou
Keyword(s):  
Protein Expression ◽  
Endurance Training ◽  
Messenger Rna ◽  
Gastrocnemius Muscle ◽  
Molecular Mechanisms ◽  
Treadmill Exercise ◽  
Cumulative Effect ◽  
Mrna And Protein Expression ◽  
Mechanisms Of Adaptation ◽  
Training Protocols

To understand the molecular mechanisms of adaptation to different training protocols, this study compared the effects of 4 sessions of 90 min treadmill exercise on alternate days or consecutive days in 1 week on messenger RNA (mRNA) and protein expression of proliferator-activated receptor-γ coactivator 1-α in rat gastrocnemius muscle. The mRNA significantly increased by 25.8-fold after alternate-day and 10.1-fold after consecutive-day training, while the protein showed no significant cumulative effect, 1.5–1.7-fold above baseline, in the 2 protocols.

Up-Regulation of Protease-Activated Receptor 2 in Allergic Rhinitis

2007 ◽  
Vol 116 (7) ◽  
pp. 554-558 ◽  
Author(s):  
Heung-Man Lee ◽  
Hyo Yeol Kim ◽  
Hee Joon Kang ◽  
Jeong Soo Woo ◽  
Sung Won Chae ◽  
...  
Keyword(s):  
Allergic Rhinitis ◽  
Nasal Mucosa ◽  
Messenger Rna ◽  
Tissue Sections ◽  
Submucosal Glands ◽  
Mrna And Protein Expression ◽  
Polymerase Chain ◽  
Nasal Inflammation ◽  
Protease Activated Receptor 2 ◽  
Normal Controls

Objectives: We compared the patterns of PAR-2 messenger RNA (mRNA) and protein expression in the nasal mucosa of subjects with and without allergic rhinitis. Methods: Biopsy specimens were obtained from 10 patients with allergic rhinitis and 10 normal controls. RNA was extracted from the nasal mucosa, and semiquantitative reverse transcription-polymerase chain reaction was performed for PAR-2. Tissue sections were immunostained for PAR-2 by use of specific antibody. Results: The expression levels of PAR-2 mRNA in allergic rhinitis nasal mucosa were significantly up-regulated as compared with those in normal nasal mucosa. PAR-2 immunoreactivity was observed in the epithelium and submucosal glands in both normal controls and subjects with allergic rhinitis. Stronger immunoreactivity for PAR-2 was observed in allergic rhinitis nasal mucosa as compared with normal nasal mucosa. Conclusions: These results suggest that PAR-2 may be involved in allergic nasal inflammation.

Wnt/β-Catenin and Hippo Pathway Deregulation in Mammary Tumors of Humans, Dogs, and Cats

2020 ◽  
Vol 57 (6) ◽  
pp. 774-790
Author(s):  
Alessandro Sammarco ◽  
Chiara Gomiero ◽  
Roberta Sacchetto ◽  
Giorgia Beffagna ◽  
Silvia Michieletto ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Level ◽  
Hippo Pathway ◽  
Mammary Tumors ◽  
Breast Cancers ◽  
Tumor Tissues ◽  
Mrna And Protein Expression ◽  
Polymerase Chain

Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/β-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for β-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and β-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, β-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active β-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of β-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of β-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/β-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/β-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.

146 DIFFERENTIAL EXPRESSION OF TRANSCELLULAR CALCIUM TRANSPORT AND PARACELLULAR TIGHT JUNCTION GENES IN THE PLACENTAE OF CALBINDIN-D9k, -D28k AND -D9k/D28k KNOCKOUT MICE

2012 ◽  
Vol 24 (1) ◽  
pp. 185
Author(s):  
I. H. Hwang ◽  
E. B. Jeung
Keyword(s):  
Protein Expression ◽  
Tight Junction ◽  
Calcium Binding ◽  
Calcium Transport ◽  
Binding Proteins ◽  
Calcium Binding Proteins ◽  
Paracellular Pathway ◽  
Calbindin D9k ◽  
Transcellular Pathway ◽  
Mrna And Protein Expression

The placenta has many essential roles in the maintenance of pregnancy and homeostasis. Calcium transport and regulation are also controlled by the placenta. In general, calcium transport is divided into an active transcellular pathway and a passive paracellular pathway. Transient receptor potential cation channel subfamily V member 5/6 (TRPV5/6), calbindin-D9k/-28k (CaBP-9k/-28k) and Na+/Ca2+ exchanger (NCX1) are involved in the transcellular pathway. The paracellular pathway is determined by the expression of tight junction genes, such as occludin, claudins and ZO-1. In this study, we analysed the difference in calcium transport in the placentae of CaBP-9k and CaBP-28k knockout (KO) mice compared with that of wild-type (WT) mice. Placentae were collected and used for mRNA and protein evaluation from 9 mice of each type (a total of 36 mice). All mice were killed on gestational Day 19. We confirmed mRNA expression by RT-qPCR and protein expression by Western blot analysis. The data were statistically analysed by one-way ANOVA using Tukey's test. In the transcellular pathway, the expression levels of NCX1 and TPRV6 were shown to be significantly increased in KO mice compared with WT mice. In the paracellular pathway, occluding, which is directly related to permeability, mRNA and protein expression was significantly increased in single KO mice compared with WT, but not in double KO mice. Claudin-4, which is a cation barrier, mRNA and protein expression patterns were significantly decreased in single KO mice compared with WT, but not in double KO mice. These results imply that the disability of calcium buffering due to CaBP-9k or CaBP-28k KO may lead to an accelerated transcellular pathway. In addition, a single KO of calcium-binding proteins may lead to decreased paracellular permeability to weaken the leakage of calcium through the tight junction and may also lead to increased cation selectivity of tight junctions. Taken together, these results might indicate that KO of calcium-binding proteins may induce activation of a compensating transcellular pathway in the placenta of mice and a paracellular pathway may support the maintenance of calcium homeostasis.

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