scholarly journals Insulin directly stimulates VEGF-A production in the glomerular podocyte

2013 ◽  
Vol 305 (2) ◽  
pp. F182-F188 ◽  
Author(s):  
L. J. Hale ◽  
J. Hurcombe ◽  
A. Lay ◽  
B. Santamaría ◽  
A. M. Valverde ◽  
...  
Keyword(s):  
Mrna Levels ◽  
Wild Type ◽  
Hairpin Rna ◽  
Glomerular Filtration Barrier ◽  
Insulin Resistant ◽  
Filtration Barrier ◽  
Resistance Modulation ◽  
Rna Knockdown

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.

scholarly journals Klotho Deficiency Aggravated Diabetes-induced Podocyte injury

2020 ◽  
Author(s):  
Zhi Chen ◽  
Qing Zhou ◽  
Cong Liu ◽  
Yiping Zeng ◽  
Shaolong Yuan
Keyword(s):  
Dna Damage ◽  
Progressive Disease ◽  
Oxygen Species ◽  
Wild Type ◽  
Glomerular Filtration Barrier ◽  
Podocyte Injury ◽  
Albumin Creatinine Ratio ◽  
Filtration Barrier

Abstract Background: Diabetic nephropathy (DN) is a progressive disease, the main pathogeny of which is podocyte injury inducing glomerular filtration barrier and proteinuria. The occurrence and development of DN could be partly attributed to the reactive oxygen species (ROS) generated by mitochondria. However, researches on how mitochondrial dysfunction (MtD) ultimately causes DNA damage is poor.Methods: We generated streptozotocin (STZ)-induced diabetic mice with wild-type(C57BL/6J) or Klotho deficiency mice (KL+/-) and treated podocytes with high glucose (HG) to investigated the function of Klotho on HG-induced podocyte injury in vivo and in vitro.Results: The absence of Klotho aggravated diabetic phenotypes indicated by podocyte injury accompanied by elevated urea albumin creatinine ratio (UACR), creatinine, urea nitrogen. Then, Klotho deficiency could significantly aggravate DNA damage by increasing 8-OHdG and reducing OGG1. Finally, Klotho deficiency may promote MtD to promote 8-OHdG-induced podocyte injury.Conclusions: Klotho deficiency may promote diabetes-induced podocytic MtD and aggravate 8-OHdG-induced DNA damage by affecting OOG1.

scholarly journals Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
Author(s):  
Gary M. Leong ◽  
Sofia Moverare ◽  
Jesena Brce ◽  
Nathan Doyle ◽  
Klara Sjögren ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Hepatic Expression ◽  
Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals TNF Is Necessary for Castration-Induced Prostate Regression, Whereas TRAIL and FasL Are Dispensable

2011 ◽  
Vol 25 (4) ◽  
pp. 611-620 ◽  
Author(s):  
Jennifer S. Davis ◽  
Kent L. Nastiuk ◽  
John J. Krolewski
Keyword(s):  
Mrna Levels ◽  
Wild Type ◽  
Mouse Prostate ◽  
Epithelial Cell Apoptosis ◽  
Androgen Withdrawal ◽  
Fas Signaling ◽  
Induced Apoptosis ◽  
Physiologic Role

TNF, a proinflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. However, there have been few examples of a physiologic role for TNF-induced apoptosis in vivo. Here, we describe a novel role for TNF in prostate epithelial cell apoptosis after androgen withdrawal. Employing high-resolution serial magnetic resonance imaging to measure mouse prostate volume changes over time, we demonstrate that the extent of castration-induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes but not mice deficient for TNF-related apoptosis-inducing ligand or Fas signaling. Wild-type mice receiving soluble TNF (sTNF) receptor 2 (to bind TNF and block signaling) before castration exhibit an identical reduction of prostate regression. Together, these data indicate that uniquely among known extrinsic death signals, TNF is required for castration-induced prostate regression. Additionally, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate after castration. This is consistent with a paracrine role for TNF in prostate regression. When injected into the peritoneum of Tnf−/− mice at the time of castration, sTNF restores normal levels of prostate regression. However, wild-type mice receiving sTNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient but acts in the context of additional castration-induced signals. These findings support a physiologic role for TNF in prostate regression after androgen withdrawal. Understanding this role may lead to novel therapies for prostate cancer.

scholarly journals “Zebrafishing” for Novel Genes Relevant to the Glomerular Filtration Barrier

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Nils Hanke ◽  
Lynne Staggs ◽  
Patricia Schroder ◽  
Jennifer Litteral ◽  
Susanne Fleig ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Barrier Function ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Glomerular Filtration Barrier ◽  
Novel Genes ◽  
Zebrafish Model ◽  
Filtration Barrier

Data for genes relevant to glomerular filtration barrier function or proteinuria is continually increasing in an era of microarrays, genome-wide association studies, and quantitative trait locus analysis. Researchers are limited by published literature searches to select the most relevant genes to investigate. High-throughput cell cultures and otherin vitrosystems ultimately need to demonstrate proof in anin vivomodel. Generating mammalian models for the genes of interest is costly and time intensive, and yields only a small number of test subjects. These models also have many pitfalls such as possible embryonic mortality and failure to generate phenotypes or generate nonkidney specific phenotypes. Here we describe anin vivozebrafish model as a simple vertebrate screening system to identify genes relevant to glomerular filtration barrier function. Using our technology, we are able to screen entirely novel genes in 4–6 weeks in hundreds of live test subjects at a fraction of the cost of a mammalian model. Our system produces consistent and reliable evidence for gene relevance in glomerular kidney disease; the results then provide merit for further analysis in mammalian models.

scholarly journals Adrenocortical function and regulation of the steroid 21-hydroxylase gene in NGFI-B-deficient mice.

1995 ◽  
Vol 15 (8) ◽  
pp. 4331-4316 ◽  
Author(s):  
P A Crawford ◽  
Y Sadovsky ◽  
K Woodson ◽  
S L Lee ◽  
J Milbrandt
Keyword(s):  
Early Gene ◽  
Adrenocortical Function ◽  
Mrna Levels ◽  
Orphan Nuclear Receptor ◽  
Wild Type ◽  
Gene Encoding ◽  
Serum Corticosterone ◽  
B Mice

The immediate-early gene NGFI-B encodes an orphan nuclear receptor that binds DNA as a monomer and activates transcription through a canonical response element (NBRE). NGFI-B is expressed under basal conditions and in response to external stimuli in many mammalian tissues. In particular, NGFI-B expression is dramatically elevated in the adrenal cortex in response to stress and in Y1 adrenocortical cells in response to adrenocorticotropin. NGFI-B activates transcription through an NBRE of the gene encoding 21-hydroxylase (P450c21) in Y1 cells. Steroidogenic factor 1 (SF-1), a homolog of NGFI-B, also activates the P450c21 promoter. To examine the influence of these factors on P450c21 expression in vivo and the function of the hypothalamic-pituitary-adrenocortical axis as a whole, we generated NGFI-B (-/-) mice. These mice thrive and reproduce normally and maintain normal basal adrenocorticotropin, corticosterone, and P450c21 mRNA levels. In response to increases in adrenocorticotropin, NGFI-B (-/-) and wild-type mice demonstrated equivalent increases in serum corticosterone levels. Furthermore, and in contrast to in vitro results, no increases in P450c21 mRNA levels were observed in response to increases in adrenocorticotropin in NGFI-B (-/-) or wild-type mice. While SF-1 mRNA levels were not increased with increased steroidogenic demand, adrenal expression of Nurr1, a close homolog of NGFI-B, was induced to a greater extent by lipopolysaccharide in NGFI-B (-/-) mice than in wild-type mice. Finally, when the administration of dexamethasone for suppression was stopped, P450c21 mRNA and serum corticosterone levels recovered at the same rate in wild-type and NGFI-B (-/-) mice. Thus, while NGFI-B appears poised to affect the structure and function of the adrenal gland, the gland functions normally in its absence, suggesting that other factors, including Nurr1 and SF-1, are sufficient to drive P450c21 expression in mice and maintain normal steroidogenesis.

scholarly journals β-Defensins Coordinate In Vivo to Inhibit Bacterial Infections of the Trachea

Vaccines ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 57 ◽  
Author(s):  
Lisa Ryan ◽  
Jichuan Wu ◽  
Kyell Schwartz ◽  
Sunghan Yim ◽  
Gill Diamond
Keyword(s):  
Epithelial Cells ◽  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Airway Epithelial Cells ◽  
Mrna Levels ◽  
Wild Type ◽  
Type Iii ◽  
Primary Mouse

β-defensins are predicted to play an important role in innate immunity against bacterial infections in the airway. We previously observed that a type III-secretion product of Bordetella bronchiseptica inhibits the NF-κB-mediated induction of a β-defensin in airway epithelial cells in vitro. To confirm this in vivo and to examine the relative roles of other β-defensins in the airway, we infected wild-type C57BL/6 mice and mice with a deletion of the mBD-1 gene with B. bronchiseptica wild-type strain, RB50 and its mutant strain lacking the type III-secretion system, WD3. The bacteria were quantified in the trachea and the nasal tissue and mRNA levels of mouse β-defensin-3 (mBD-3) were assessed after 24 h. Infection with the wild-type bacterial strain resulted in lower mBD-3 mRNA levels in the trachea than in mice infected with the type III-deficient strain. Furthermore, we observed an increase in bacterial numbers of RB50 only in the tracheas of mBD-1-deficient mice. Neutrophils were also more abundant on the trachea in RB50 infected WT mice but not in the bronchiolar lavage fluid (BAL), compared with WD3 infected WT and mBD-1−/− mice, indicating that the coordination of β-defensin chemotactic effects may be confined to tracheal epithelial cells (TEC). RB50 decreased the ability of mice to mount an early specific antibody response, seven days after infection in both WT and mBD-1−/− mice but there were no differences in titers between RB50-infected WT and mBD-1−/− mice or between WD3-infected WT and mBD-1−/− mice, indicating mBD-1 was not involved in induction of the humoral immune response to the B. bronchiseptica. Challenge of primary mouse TEC in vitro with RB50 and WD3, along with IL-1β, further corroborated the in vivo studies. The results demonstrate that at least two β-defensins can coordinate early in an infection to limit the growth of bacteria in the trachea.

Altered triglyceride-rich lipoprotein production in Zucker diabetic fatty rats

2004 ◽  
Vol 287 (1) ◽  
pp. E42-E49 ◽  
Author(s):  
Doru V. Chirieac ◽  
Heidi L. Collins ◽  
Joanne Cianci ◽  
Janet D. Sparks ◽  
Charles E. Sparks
Keyword(s):  
Insulin Dose ◽  
Mrna Levels ◽  
Insulin Resistant ◽  
Apob Mrna ◽  
Zucker Diabetic Fatty Rats ◽  
Triglyceride Rich Lipoprotein ◽  
Zdf Rats ◽  
And Control

Triglyceride-rich lipoprotein (TRL) production was studied in Zucker diabetic fatty (ZDF) rats, a model of insulin-resistant type 2 diabetes progression. TRL production was measured in vivo by blocking catabolism with Triton WR-1339. Ten-week ZDF rats are hyperinsulinemic with increased TRL production [both triglyceride and apolipoprotein B (apoB)]. Twenty-week ZDF rats are insulinopenic, and TRL production is similar to lean controls. Insulin infusion suppresses glucose and free fatty acids in 10- and 20-wk ZDF rats. Increased TRL production is not reduced by insulin in 10-wk rats; however, at 20 wk, TRL production is suppressed by insulin. In vitro studies with hepatocytes derived from 10-wk ZDF rats showed minimal insulin dose effects on apoB secretion compared with the response and sensitivity of hepatocytes derived from 20-wk ZDF and control lean rats. Hepatic sterol regulatory-binding protein (SREBP)-1c mRNA levels are increased at 10 wk but return to control levels at 20 wk. ApoB mRNA levels are similar to lean controls at 10 and 20 wk. The following two mechanisms for hypertriglyceridemia associated with hyperinsulinemia are suggested: increased TRL synthesis and loss of TRL suppression. Increased triglyceride production in hyperinsulinemic rats likely relates to increased expression of SREBP-1c, whereas increased apoB production involves posttranscriptional processes.

Shock-induced stress induces loss of microvascular endothelial Tie2 in the kidney which is not associated with reduced glomerular barrier function

2009 ◽  
Vol 297 (2) ◽  
pp. F272-F281 ◽  
Author(s):  
Matijs van Meurs ◽  
Neng F. Kurniati ◽  
Francis M. Wulfert ◽  
Sigridur A. Asgeirsdottir ◽  
Inge A. de Graaf ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Barrier Function ◽  
Human Kidney ◽  
Mrna Levels ◽  
Barrier Dysfunction ◽  
Molecular Control ◽  
Filtration Barrier ◽  
Glomerular Barrier

Both hemorrhagic shock and endotoxemia induce a pronounced vascular activation in the kidney which coincides with albuminuria and glomerular barrier dysfunction. We hypothesized that changes in Tie2, a vascular restricted receptor tyrosine kinase shown to control microvascular integrity and endothelial inflammation, underlie this loss of glomerular barrier function. In healthy murine and human kidney, Tie2 is heterogeneously expressed in all microvascular beds, although to different extents. In mice subjected to hemorrhagic and septic shock, Tie2 mRNA and protein were rapidly, and temporarily, lost from the renal microvasculature, and normalized within 24 h after initiation of the shock insult. The loss of Tie2 protein could not be attributed to shedding as both in mice and healthy volunteers subjected to endotoxemia, sTie2 levels in the systemic circulation did not change. In an attempt to identify the molecular control of Tie2, we activated glomerular endothelial cell cultures and human kidney slices in vitro with LPS or TNF-α, but did not observe a change in Tie2 mRNA levels. In parallel to the loss of Tie2 in vivo, an overt influx of neutrophils in the glomerular compartment, which coincided with proteinuria, was seen. As neutrophil-endothelial cell interactions may play a role in endothelial adaptation to shock, and these effects cannot be mimicked in vitro, we depleted neutrophils before shock induction. While this neutrophil depletion abolished proteinuria, Tie2 was not rescued, implying that Tie2 may not be a major factor controlling maintenance of the glomerular filtration barrier in this model.

C-Cbl Regulates c-Mpl Receptor Trafficking and Its Internalization

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2388-2388
Author(s):  
Sebastian Jonas Saur ◽  
Melanie Märklin ◽  
Manuela Ganser ◽  
Kyle Hoehn ◽  
James E David ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Plasma Levels ◽  
Thrombus Formation ◽  
Surface Expression ◽  
Mrna Levels ◽  
Wild Type ◽  
Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors and cytokines in order to maintain physiological levels of circulating platelets. Thrombopoietin (TPO) signalling via its receptor c-Mpl is a key regulator of megakaryopoiesis driving megakaryocyte differentiation, promoting endomitosis and proplatelet formation. Therefore TPO/c-Mpl signalling needs to be tightly regulated to maintain physiological megakaryopoiesis. One of the most effective mechanisms to permanently disable activated signalling proteins is by targeted degradation via lysosomes or proteasomes. Previous studies have identified c-Cbl as an E3 ligase responsible for the ubiquitination of c-Mpl in cell lines. In this study, we investigated the mechanisms of TPO-mediated c-Mpl degradation in primary mouse cells. In order to determine the potential role of c-Cbl in murine megakaryopoiesis we used a conditional PF4-Cre c-Cbl knockout (ko) mouse model to specifically delete c-Cbl in the megakaryocytic lineage. Megakaryocytes were generated in vitro by culturing bone marrow from WT and PF4-Cre/c-Cbl-floxed (c-Cbl ko) lines for 72 hrs in the presence of rmTPO. C-Cbl ko mice showed significant bone marrow megakaryocyte hyperplasia, however megakaryocyte numbers in the spleen remained unchanged. Platelets counts were significantly elevated as compared to control mice (1.2 x106 vs. 1.7x106 p=0.0001) and in addition, the platelets from the c-Cbl ko mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). Using a method of in vivo double labelling of platelets, we were able to simultaneously follow the survival of both the entire population of platelets and new platelets which were generated during the last 24 hours. There were more new platelets produced within a 24 h period in the c-Cbl ko mice although the half-life of platelets was similar in the both cohorts. Although c-Cbl ko mice exhibited thrombocytosis, they showed a severe defect in thrombus formation using an in vivo thrombus formation model with Fe3Cl. TPO plasma levels, known to be inversely regulated by circulating platelet numbers, were surprisingly increased (250 vs. 420 pg/ml, p=0.005) in the c-Cbl ko mice. There was no difference in liver mRNA levels in the two cohorts. We therefore looked at c-Mpl protein and mRNA expression in megakaryocytes and found c-Cbl ko mice to express more c-Mpl compared with wild type controls. Surprisingly, we found c-Mpl surface expression to be reduced and internalization of the receptor significantly impaired following TPO stimulation in c-Cbl ko mice. Incubating platelets in vitro with TPO for 2 hours to evaluate the TPO uptake capacity of platelets, we found c-Cbl ko platelets to show a severe uptake defect compared with wild type control platelets. Taken together, we have successfully ablated c-Cbl specifically from the megakaryocyte lineage and demonstrated that this has profound effects on platelet counts and size. In addition, we showed that c-Cbl ablation leads to reduced c-Mpl surface expression and impaired internalization, which culminates in increased TPO plasma levels causing increased megakaryopoiesis in the c-Cbl ko mice. In summary, our data enhance our understanding of the regulation of TPO signalling and the physiological role of c-Cbl in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

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