scholarly journals Purinergic signaling is enhanced in the absence of UT-A1 and UT-A3

2019 ◽  
Author(s):  
Nathaniel J. Himmel ◽  
Richard T. Rogers ◽  
Sara K. Redd ◽  
Yirong Wang ◽  
Mitsi A. Blount
Keyword(s):  
Purinergic Signaling ◽  
Collecting Duct ◽  
Elevated Serum ◽  
Urinary Concentration ◽  
Mrna Levels ◽  
Urea Transport ◽  
Tissue Samples ◽  
Protein Levels ◽  
Summary Statement ◽  
Renal Tubular

AbstractATP is an important paracrine regulator of renal tubular water and urea transport. The activity of P2Y2, the predominant P2Y receptor of the medullary collecting duct, is mediated by ATP, and modulates urinary concentration. To investigate the role of purinergic signaling in the absence of urea transport in the collecting duct, we housed wild-type (WT) and UT-A1/A3 null (UT-A1/A3 KO) mice in metabolic cages to monitor urine output, and collected tissue samples for analysis. We confirmed that UT-A1/A3 KO mice are polyuric, and concurrently observed lower levels of urinary cAMP as compared to WT, despite elevated serum vasopressin (AVP) levels. Because P2Y2 inhibits AVP-stimulated transport by dampening cAMP synthesis, we suspected that, similar to other models of AVP-resistant polyuria, purinergic signaling is increased in UT-A1/A3 KO mice. In fact, we observed that both urinary ATP and purinergic-mediated prostanoid (PGE2) levels were elevated. Tissue analysis shows that P2Y2 mRNA levels remain unchanged, while P2Y2 protein levels are elevated in KO mice. Collectively, our data suggest that the reduction of medullary osmolality due to the lack of UT-A1 and UT-A3 induces an AVP-resistant polyuria that is possibly exacerbated by, or at least correlated with, enhanced purinergic signaling.Summary statementPhysiological analyses suggest that mice lacking urea transporters have increased renal purinergic signaling, perhaps contributing to previously observed vasopressin-resistant polyurias.

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 Determination of CYP450 Expression Levels in the Human Small Intestine by Mass Spectrometry-Based Targeted Proteomics

2021 ◽  
Vol 22 (23) ◽  
pp. 12791
Author(s):  
Alexia Grangeon ◽  
Valérie Clermont ◽  
Azemi Barama ◽  
Fleur Gaudette ◽  
Jacques Turgeon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Intestine ◽  
Protein Expression ◽  
Mrna Levels ◽  
Targeted Proteomics ◽  
Tissue Samples ◽  
Human Organ ◽  
Expression Levels ◽  
Protein Levels ◽  
Human Small Intestine

The human small intestine can be involved in the first-pass metabolism of drugs. Under this condition, members of the CYP450 superfamily are expected to contribute to drug presystemic biotransformation. The aim of this study was to quantify protein expression levels of 16 major CYP450 isoforms in tissue obtained from nine human organ donors in seven subsections of the small intestine, i.e., duodenum (one section, N = 7 tissue samples), jejunum (three subsections (proximal, mid and distal), N = 9 tissue samples) and ileum (three subsections, (proximal, mid and distal), N = 9 tissue samples), using liquid chromatography tandem mass spectrometry (LC-MS/MS) based targeted proteomics. CYP450 absolute protein expression levels were compared to mRNA levels and enzyme activities by using established probe drugs. Proteins corresponding to seven of sixteen potential CYP450 isoforms were detected and quantified in various sections of the small intestine: CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, CYP3A5 and CYP4F2. Wide inter-subject variability was observed, especially for CYP2D6. CYP2C9 (p = 0.004) and CYP2C19 (p = 0.005) expression levels decreased along the small intestine. From the duodenum to the ileum, CYP2J2 (p = 0.001) increased, and a trend was observed for CYP3A5 (p = 0.13). CYP3A4 expression was higher in the jejunum than in the ileum (p = 0.03), while CYP4F2 expression was lower in the duodenum compared to the jejunum and the ileum (p = 0.005). CYP450 protein levels were better correlated with specific isoform activities than with mRNA levels. This study provides new data on absolute CYP450 quantification in human small intestine that could improve physiologically based pharmacokinetic models. These data could better inform drug absorption profiles while considering the regional expression of CYP450 isoforms.

Reduced urinary corin levels in patients with chronic kidney disease

2013 ◽  
Vol 124 (12) ◽  
pp. 709-717 ◽  
Author(s):  
Chaodong Fang ◽  
Lei Shen ◽  
Liang Dong ◽  
Meng Liu ◽  
Sensen Shi ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Human Urine ◽  
Animal Studies ◽  
Kidney Tissue ◽  
Renal Tubules ◽  
Tissue Samples ◽  
Protein Levels ◽  
Normal Individuals ◽  
Renal Tubular

Corin is a cardiac protease that regulates BP (blood pressure) by activating natriuretic peptides. Recent animal studies identified corin expression in the kidney where it may regulate renal function. In the present study, we tested the hypothesis that corin may be present in human urine and that urinary corin levels may be altered in patients with kidney disease. We obtained urine and kidney tissue samples from normal individuals and CKD (chronic kidney disease) patients. Using ELISA, we detected corin protein in human urine. In normal individuals, urinary corin levels did not correlate with that of plasma, indicating that urinary corin is probably of kidney origin. Compared with normal controls, CKD patients had markedly reduced urinary corin levels and this reduction correlated with disease severity. By immunostaining, human corin protein was identified on the epithelial cell surface in renal tubules. The renal corin mRNA and protein levels were significantly lower in CKD patients than non-CKD controls. The results indicate that renal tubular corin may be shed into urine and that urinary and renal corin levels were reduced in CKD patients. These data suggest that reduced corin levels in the kidney may reflect the underlying pathology in CKD.

Downregulation of SGK1 by nucleotides in renal tubular epithelial cells

2007 ◽  
Vol 293 (5) ◽  
pp. F1751-F1757 ◽  
Author(s):  
Liuzhe Li ◽  
Charles S. Wingo ◽  
Shen-Ling Xia
Keyword(s):  
Epithelial Cells ◽  
Purinergic Receptors ◽  
Kinase Activity ◽  
Purinergic Signaling ◽  
Collecting Duct ◽  
Collecting Duct Cells ◽  
Renal Collecting Duct ◽  
Renal Tubular ◽  
First Time ◽  
Dependent Pathway

This study determined whether nucleotides that bind to purinergic receptors (P2R) regulate the expression or function of serum- and glucocorticoid-inducible kinase-1 (SGK1) in mouse renal inner medullar collecting duct cells (mIMCD-3). The SGK1 protein was detected by Western blotting. A significant reduction of cytosolic SGK1 expression was observed in the cells pretreated with P2R agonist adenosine 5′- O-(3-thiotriphosphate) (ATPγS), and the reduction could be reversed by P2R antagonists. This reduction was also observed in cells that were pretreated with agonists for P2R subtypes. Using ELISA, we observed a reduced SGK1 kinase activity in ATPγS-pretreated cells. This effect was reversed by P2R antagonists. Furthermore, an increase of SGK1 kinase activity in aldosterone-pretreated cells was suppressed by ATPγS. These studies demonstrate for the first time that SGK1 can be downregulated by nucleotides in renal collecting duct epithelial cells, likely via the activation of P2R, and suggest that activation of renal purinergic signaling regulates a SGK1-dependent pathway that is known to modulate ion transport in the renal collecting duct.

Development of urinary concentrating capacity: role of aquaporin-2

1996 ◽  
Vol 271 (2) ◽  
pp. F461-F468 ◽  
Author(s):  
M. Yasui ◽  
D. Marples ◽  
R. Belusa ◽  
A. C. Eklof ◽  
G. Celsi ◽  
...  
Keyword(s):  
Single Injection ◽  
Collecting Duct ◽  
Water Channel ◽  
Urine Osmolality ◽  
Postnatal Life ◽  
Mrna Levels ◽  
Adult Rats ◽  
Protein Levels ◽  
Aquaporin 2 ◽  
Low Expression

The capacity to concentrate urine develops progressively during postnatal life in most mammalian species. Here we have examined whether low expression of the arginine vasopressin (AVP)-activated water channel aquaporin-2 (AQP-2) may be a limiting factor for the concentrating capacity in the infant rats. Urine osmolality in response to 24-h dehydration increased significantly from 10 to 40 days of age. The most rapid increase occurred during the weaning period, i.e., days 15-20. A similar developmental pattern was observed for AQP-2 mRNA levels in the renal medulla. AQP-2 protein levels also increased markedly from day 10 to 40. Immunohistochemistry revealed that AQP-2 was exclusively located in collecting duct principal cells both in infant and adult rats but that the signal was much weaker in infants. To further examine the relationship between urinary concentrating capacity and AQP-2 expression, we treated rats with a single injection of betamethasone, which is known to accelerate maturation in several organs. Twenty-four hours after treatment, there was an increase in urine osmolality, renal medullary AQP-2 mRNA, and AQP-2 protein levels in infant but not in adult rats. A single injection of a specific V2 agonist caused within 6 h significant increase of AQP-2 mRNA in both infant and adult. The expression of the mRNA of three other transporters involved in the concentrating process, medullary Na(+)-K(+)-ATPase alpha-subunit, Na-K-2Cl cotransporter, and epithelial chloride channel also increased during the weaning period and were upregulated by glucocorticoids. We conclude that there is a well-synchronized development of the many of the components that determine the concentrating capacity and that the low expression of AQP-2 is one of the limiting factors for low concentrating capacity in infants.

scholarly journals Lithium: a versatile tool for understanding renal physiology

2013 ◽  
Vol 304 (9) ◽  
pp. F1139-F1149 ◽  
Author(s):  
Bellamkonda K. Kishore ◽  
Carolyn M. Ecelbarger
Keyword(s):  
Diabetes Insipidus ◽  
Glycogen Synthase ◽  
Purinergic Signaling ◽  
Collecting Duct ◽  
Renal Physiology ◽  
Urinary Concentration ◽  
Paradigm Shifts ◽  
Molecular Physiology ◽  
Versatile Tool

By virtue of its unique interactions with kidney cells, lithium became an important research tool in renal physiology and pathophysiology. Investigators have uncovered the intricate relationships of lithium with the vasopressin and aldosterone systems, and the membrane channels or transporters regulated by them. While doing so, their work has also led to 1) questioning the role of adenylyl cyclase activity and prostaglandins in lithium-induced suppression of aquaporin-2 gene transcription; 2) unraveling the role of purinergic signaling in lithium-induced polyuria; and 3) highlighting the importance of the epithelial sodium channel (ENaC) in lithium-induced nephrogenic diabetes insipidus (NDI). Lithium-induced remodeling of the collecting duct has the potential to shed new light on collecting duct remodeling in disease conditions, such as diabetes insipidus. The finding that lithium inhibits glycogen synthase kinase-3β (GSK3β) has opened an avenue for studies on the role of GSK3β in urinary concentration, and GSK isoforms in renal development. Finally, proteomic and metabolomic profiling of the kidney and urine in rats treated with lithium is providing insights into how the kidney adapts its metabolism in conditions such as acquired NDI and the multifactorial nature of lithium-induced NDI. This review provides state-of-the-art knowledge of lithium as a versatile tool for understanding the molecular physiology of the kidney, and a comprehensive view of how this tool is challenging some of our long-standing concepts in renal physiology, often with paradigm shifts, and presenting paradoxical situations in renal pathophysiology. In addition, this review points to future directions in research where lithium can lead the renal community.

scholarly journals Oxygen regulation of the epithelial Na channel in the collecting duct

2011 ◽  
Vol 300 (2) ◽  
pp. F412-F424 ◽  
Author(s):  
Russell F. Husted ◽  
Hongyan Lu ◽  
Rita D. Sigmund ◽  
John B. Stokes
Keyword(s):  
Kinase Activity ◽  
Collecting Duct ◽  
Hypoxia Inducible Factor ◽  
Na Channel ◽  
Mrna Levels ◽  
Apical Surface ◽  
Amp Kinase ◽  
Na Transport ◽  
Protein Levels ◽  
Epithelial Na Channel

The Po2 within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing Po2 on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O2 concentration from 20 to 8% decreased ENaC activity by 40%; increasing O2 content to 40% increased ENaC activity by 50%. The O2 effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O2 medium. Corticosteroids increased ENaC activity at each O2 concentration; there was no interaction. The pathways for O2 and steroid regulation of ENaC are different since O2 did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2–45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O2 appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O2 concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O2 induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O2; both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.

scholarly journals Elucidating the role of pigment epithelium-derived factor (PEDF) in metabolic PCOS models

2020 ◽  
Vol 244 (2) ◽  
pp. 297-308 ◽  
Author(s):  
Michal Silber ◽  
Irit Miller ◽  
Hadas Bar-Joseph ◽  
Ido Ben-Ami ◽  
Ruth Shalgi
Keyword(s):  
Pigment Epithelium ◽  
Elevated Serum ◽  
Female Offspring ◽  
Mrna Levels ◽  
Prolonged Incubation ◽  
Protein Levels ◽  
Potential Therapeutic Role ◽  
Endothelial Growth Factor

PCOS is the most common endocrinopathy in women; associated with obesity and insulin resistance (IR). IR leads to accumulation of advanced-glycation-end-products (AGEs) and their receptor, RAGE. PCOS patients have increased levels of vascular endothelial growth factor (VEGF), interleukin 6/8 (IL-6/8) and anti-Mϋllerian-hormone (AMH). PEDF is a secreted-glycoprotein known for its anti-angiogenic and anti-inflammatory properties. We aimed to elucidate the role of PEDF in the pathogenesis and treatment of PCOS. We used a prenatal PCOS mouse model and fed the female offspring a high-fat diet, inducing metabolic PCOS (met.PCOS) characteristics. Female offspring were divided into three groups: control; met.PCOS; met.PCOS + recombinant PEDF (rPEDF). Met.PCOS mice gained more weight, had elevated serum IL-6 and higher mRNA levels of AMH, PEDF and RAGE in their granulosa cells (GCs) than met.PCOS + rPEDF mice. An in vitro Met.PCOS model in human GCs (KGN) line was induced by prolonged incubation with insulin/AGEs, causing development of IR. Under the same conditions, we observed an elevation of VEGF, IL-6/8 mRNAs, concomitantly with an increase in PEDF mRNA, intracellular protein levels, and an elevation of PEDF receptors (PEDF-Rs) mRNA and protein. Simultaneously, a reduction in the secretion of PEDF from GCs, was measured in the medium. The addition of rPEDF (5 nM) activated P38 signaling, implying that PEDF-Rs maintained functionality, and negated AGE-induced elevation of IL-6/8 and VEGF mRNAs. Decreased PEDF secretion may be a major contributor to hyperangiogenesis and chronic inflammation, which lie at the core of PCOS pathogenesis. rPEDF treatment may restore physiological angiogenesis inflammatory balance, thus suggesting a potential therapeutic role in PCOS.

scholarly journals Suppression of sphingosine 1-phosphate lyase retards the liver regeneration in mice after partial hepatectomy

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Yuko Kageyama ◽  
Baasanjav Uranbileg ◽  
Yoshika Kusumoto ◽  
Eri Sakai ◽  
Hitoshi Ikeda ◽  
...  
Keyword(s):  
Liver Regeneration ◽  
Partial Hepatectomy ◽  
Nuclear Antigen ◽  
Lipid Mediator ◽  
Mrna Levels ◽  
Tissue Samples ◽  
Protein Levels ◽  
Sphingosine 1 Phosphate ◽  
S1p Lyase

Abstract Background: Liver regeneration is an extremely complicated process that is regulated by a number of signaling pathways. Sphingosine 1-phosphate (S1P), a potent bioactive lipid mediator playing crucial roles in various cellular responses through its receptors, has been attracting attention in the fields of hepatology, where S1P lyase (SPL), an irreversibly degrading enzyme of S1P, reportedly has a stimulatory role in growth of hepatocellular carcinoma (HCC). Aim of the study: To examine whether SPL might play a stimulatory role in liver regeneration. Method: Using in-vivo siRNA technology, we inhibited SPL expression. Seventy percent of the liver was resected in mice as partial hepatectomy (PH). Liver tissue samples were collected and mRNA expression level of the SPL, IHC of the proliferating cell nuclear antigen (PCNA), protein levels of various proliferation factors and lipid measurements were performed in different groups. Results: The mRNA levels of SPL increased in PH mice on the third day after PH surgery. When we suppressed the expression of SPL by in-vivo siRNA, we observed a significant decline of the PCNA positive cell numbers. Furthermore, the Cyclin D1 expressions and phosphorylation of ERK also were decreased in the siSPL injected PH group. Conclusion: We verified the importance of the SPL in liver regeneration, using the mice PH model. SPL might be a potential target to facilitate liver regeneration.

Evaluation of MMP-12 expression in chronic rhinosinusitis with nasal polyposis

2021 ◽  
Vol 0 (0) ◽  
pp. 0-0
Author(s):  
S. Lygeros ◽  
G. Danielides ◽  
G.C. Kyriakopoulos ◽  
K. Grafanaki ◽  
F. Tsapardoni ◽  
...  
Keyword(s):  
Chronic Rhinosinusitis ◽  
Gene Expression Regulation ◽  
Statistical Significance ◽  
Sinus Surgery ◽  
Mrna Levels ◽  
Tissue Samples ◽  
Potential Implication ◽  
Protein Levels ◽  
Healthy Mucosa ◽  
The Difference

Background: The purpose of this study was to evaluate the expression of MMP-12 in patients with chronic rhinosinusitis with polyps (CRSwNP). Methodology: Tissue samples from 37 patients with CRSwNP undergoing functional endoscopic sinus surgery and healthy mucosa specimens from 12 healthy controls were obtained intraoperatively. The mRNA and protein expression levels of MMP-12 were quantified by real-time polymerase chain reaction and Western blotting, respectively. Results: mRNA levels of MMP-12 were significantly elevated in the CRSwNP tissue samples compared to those in control ones. The protein levels of MMP-12 showed a trend of increasing but with no statistical significance. Conclusions: Elevation of MMP-12 in patients with CRSwNP suggests its potential implication in the pathogenesis of the disease. The difference in the expression profile observed between mRNA and protein levels could be due to post-translational gene expression regulation. Our findings provide evidence that MMP-12 along with other MMPs may serve as a biomarker and therapeutic target in the management of the disease.

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