Abstract P611: Immortalization of Sheep Proximal Tubule Cells Retain the Ability to Internalize Angiotensinogen that Traffics to the Mitochondria and Nucleus

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Nildris Cruz-Diaz ◽  
Yixin Su ◽  
James C Rose ◽  
Bryan A Wilson ◽  
Mark C Chappell
Keyword(s):  
Cell Line ◽  
Time Course ◽  
Secretory Pathway ◽  
Protein A ◽  
Renin Angiotensin System ◽  
Subcellular Fractionation ◽  
Cell System ◽  
Ang Ii ◽  
Mitochondrial Fractions ◽  
Intracellular Expression

Although there is compelling evidence for an intracellular renin-angiotensin system (RAS) that includes localization of AT1, AT2 and AT7/Mas receptors (R) on the nucleus and mitochondria of various cell types, the mechanism for the intracellular expression of angiotensins remains equivocal as the precursor protein angiotensinogen (Aogen) enters the secretory pathway upon synthesis. Proximal tubules (PTs) of the kidney present a unique cell system since the PTs internalize Aogen and transgenic mice lacking either the PT protein transporter megalin or liver Aogen exhibit reduced renal content of both Aogen and Ang II. We reported that isolated sheep PTs readily internalize Aogen, and subcellular fractionation revealed that Aogen was evident in the nuclear and mitochondrial fractions. The present study sought to establish a permanent cell line derived from the sheep PT to facilitate the characterization of Aogen internalization and processing. Sheep PT cells were isolated by protease digestion and Percoll density gradient separation, maintained in culture to promote epithelial cell growth and immortalized by SV-40 transfection. A clone (SPT-1) was obtained that expressed the SGLT-2 protein, a selective PT marker. SPT-1 cells were incubated with recombinant 125 I-Aogen at 37°C in DMEM/F12 media. A time course [0.5 to 6 hrs] revealed linear uptake of Aogen [r = 0.995] that did not saturate by 6 hrs. Pre-treatment of the SPT-1 cells with renin/ACE/neprilysin/chymase inhibitors [INHIB] or AT1R/AT2R/AT7/MasR antagonists [ANTAG] failed to attenuate Aogen internalization [Control: 209 ± 22; INHIB: 200 ± 21; ANTAG: 217 ± 15 fmol/hr/mg, n=3] while Ang II or Ang-(1-7) [10 μM, each] also did not inhibit, but tended to increase Aogen uptake [238 ± 24 and 244 ± 15 fmol/hr/mg, respectively, n=3]. Subcellular fractionation studies revealed that 12.0 ± 0.2% [n=3] of the total internalized Aogen was localized to the mitochondrial fraction with a higher content in the nucleus following an 18 hr uptake. We conclude that the established SPT-1 cell line which retains the capacity to internalize Aogen and expresses a similar pattern of protein trafficking to isolated PTs, may constitute a relevant model to elucidate the pathway for intracellular expression of angiotensins.

Temperature-sensitive SV40 immortalized rat proximal tubule cell line has functional renin-angiotensin system

1995 ◽  
Vol 268 (3) ◽  
pp. F435-F446 ◽  
Author(s):  
S. S. Tang ◽  
F. Jung ◽  
D. Diamant ◽  
D. Brown ◽  
D. Bachinsky ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Renin Angiotensin System ◽  
Temperature Sensitive ◽  
Proximal Tubule Cell ◽  
Permissive Temperature ◽  
Ang Ii ◽  
Tubule Cell ◽  
Angiotensin System ◽  
Rat Proximal Tubule

Immortalized rat proximal tubule cell (IRPTC) lines should be useful for investigation of proximal tubule (PT) regulation and function but previously have been unavailable. We now report the establishment and characterization of an immortalized transformed, temperature-sensitive IRPTC cell line containing renin-angiotensin system (RAS) components. Primary PT cells prepared from male Wistar rats (4-5 wk old) after collagenase digestion, sieving, and Percoll gradient were cultured on collagen-coated T-75 flasks in Dulbecco's modified Eagle's medium containing 5% fetal calf serum. Subconfluent PT cells were transfected with the temperature-sensitive SV40 mutant viruses (tsA SV40) by direct exposure. After 7-8 wk, several clones were obtained, from which one has been characterized and designated as line 3-2. This cell line appears stable up to 45 passages. Clonal cells transformed with this virus exhibit a transformed phenotype at a permissive temperature of 34 degrees C and grow in multiple layers. When the cells are subsequently placed at a nonpermissive temperature of 41 degrees C, they return to morphology similar to that of untransformed cells of the same lineage. At either 34 degrees C or 41 degrees C, this cell line expresses a variety of PT markers including alkaline phosphatase, cytokeratin, carbonic anhydrase, and glucose transporter isoform 2 (GLUT2), while not expressing factor VIII. Uniquely, these cells also appear to express PT proteins gp330 and CHIP28, markers which are usually lost in cultured cells. Furthermore, the cell line expresses protein and mRNA components of RAS, including angiotensinogen, angiotensin converting enzyme, and renin. The IRPTC cell line expresses few angiotensin II (ANG II) receptors at 34 degrees C, the permissive temperature. However, at the nonpermissive temperature, 41 degrees C, IRPTC expresses ANG II receptor (dissociation constant of 0.7 nM; maximum binding capacity of 265 fmol/mg protein). ANG II (10(-8) M) induced a transient rise in cytoplasmic Ca2+ concentration, which was nearly abolished with losartan but not PD-123319, suggesting this finding is AT1 receptor mediated. This cell line should provide an excellent model of PT and should make it possible to study the cell and molecular biology of the RAS, as well as other regulatory systems of the PT.

scholarly journals The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer.

1985 ◽  
Vol 101 (2) ◽  
pp. 639-645 ◽  
Author(s):  
T L Burgess ◽  
C S Craik ◽  
R B Kelly
Keyword(s):  
Gene Transfer ◽  
Cell Line ◽  
Anterior Pituitary ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Tumor Cell Line ◽  
Subcellular Fractionation ◽  
Regulated Secretory Pathway ◽  
Stimulation Of

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br-cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.

scholarly journals Review: Novel roles of nuclear angiotensin receptors and signaling mechanisms

2012 ◽  
Vol 302 (5) ◽  
pp. R518-R530 ◽  
Author(s):  
TanYa M. Gwathmey ◽  
Ebaa M. Alzayadneh ◽  
Karl D. Pendergrass ◽  
Mark C. Chappell
Keyword(s):  
Cardiovascular Disease ◽  
Renin Angiotensin System ◽  
Angiotensin Receptors ◽  
Major Influence ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
Physiological Regulation ◽  
Intracellular Expression ◽  
And Function

The renin-angiotensin system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT1R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type 1 receptor (AT1R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT2R) and ANG-(1–7) (AT7R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2—the enzyme that converts ANG II to ANG-(1–7)—or the AT7 receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT2 and AT7 stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease.

Ultrastructure of Choriocarcinoma in Vitro

1969 ◽  
Vol 27 ◽  
pp. 362-363
Author(s):  
John C. Garancis ◽  
R. A. Pattillo
Keyword(s):  
Cell Line ◽  
Physiological Function ◽  
Cell System ◽  
Bewo Cell ◽  
Bewo Cells ◽  
Gestational Choriocarcinoma ◽  
Bewo Cell Line

Growth of cell system (BeWo-cell line) derived from human gestational choriocarcinoma has been established and continuously maintained in-vitro. Furthermore, it is evident from the previous studies that this cell line has retained the physiological function of the placental trophoblasts, namely the synthesis of human chorionic gonadotrophil(HCG).The BeWo cells were relatively small and possessed single nuclei, thus indicating that this cell line consists exclusively of cytotrophoblasts. In some instances cells appeared widely separated and their lateral surfaces were provided with numerous microvilli (Fig.1).

ACE2 as therapeutic agent

2020 ◽  
Vol 134 (19) ◽  
pp. 2581-2595
Author(s):  
Qiuhong Li ◽  
Maria B. Grant ◽  
Elaine M. Richards ◽  
Mohan K. Raizada
Keyword(s):  
Therapeutic Agent ◽  
Renin Angiotensin System ◽  
Peptide Hormone ◽  
Experimental Models ◽  
Electrolyte Balance ◽  
Ang Ii ◽  
Angiotensin Converting Enzyme 2 ◽  
Beneficial Effects ◽  
Overwhelming Evidence ◽  
Future Challenges

Abstract The angiotensin-converting enzyme 2 (ACE2) has emerged as a critical regulator of the renin–angiotensin system (RAS), which plays important roles in cardiovascular homeostasis by regulating vascular tone, fluid and electrolyte balance. ACE2 functions as a carboxymonopeptidase hydrolyzing the cleavage of a single C-terminal residue from Angiotensin-II (Ang-II), the key peptide hormone of RAS, to form Angiotensin-(1-7) (Ang-(1-7)), which binds to the G-protein–coupled Mas receptor and activates signaling pathways that counteract the pathways activated by Ang-II. ACE2 is expressed in a variety of tissues and overwhelming evidence substantiates the beneficial effects of enhancing ACE2/Ang-(1-7)/Mas axis under many pathological conditions in these tissues in experimental models. This review will provide a succinct overview on current strategies to enhance ACE2 as therapeutic agent, and discuss limitations and future challenges. ACE2 also has other functions, such as acting as a co-factor for amino acid transport and being exploited by the severe acute respiratory syndrome coronaviruses (SARS-CoVs) as cellular entry receptor, the implications of these functions in development of ACE2-based therapeutics will also be discussed.

scholarly journals AT1 and AT2 receptors modulate renal tubular cell necroptosis in angiotensin II-infused renal injury mice

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yongjun Zhu ◽  
Hongwang Cui ◽  
Jie Lv ◽  
Haiqin Liang ◽  
Yanping Zheng ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Renal Injury ◽  
Critical Role ◽  
Kidney Injury ◽  
Renin Angiotensin System ◽  
Tubular Cell ◽  
Tubular Damage ◽  
Renal Tubular Cell ◽  
Ang Ii ◽  
Renal Tubular

AbstractAbnormal renin-angiotensin system (RAS) activation plays a critical role in the initiation and progression of chronic kidney disease (CKD) by directly mediating renal tubular cell apoptosis. Our previous study showed that necroptosis may play a more important role than apoptosis in mediating renal tubular cell loss in chronic renal injury rats, but the mechanism involved remains unknown. Here, we investigate whether blocking the angiotensin II type 1 receptor (AT1R) and/or angiotensin II type 2 receptor (AT2R) beneficially alleviates renal tubular cell necroptosis and chronic kidney injury. In an angiotensin II (Ang II)-induced renal injury mouse model, we found that blocking AT1R and AT2R effectively mitigates Ang II-induced increases in necroptotic tubular epithelial cell percentages, necroptosis-related RIP3 and MLKL protein expression, serum creatinine and blood urea nitrogen levels, and tubular damage scores. Furthermore, inhibition of AT1R and AT2R diminishes Ang II-induced necroptosis in HK-2 cells and the AT2 agonist CGP42112A increases the percentage of necroptotic HK-2 cells. In addition, the current study also demonstrates that Losartan and PD123319 effectively mitigated the Ang II-induced increases in Fas and FasL signaling molecule expression. Importantly, disruption of FasL significantly suppressed Ang II-induced increases in necroptotic HK-2 cell percentages, and necroptosis-related proteins. These results suggest that Fas and FasL, as subsequent signaling molecules of AT1R and AT2R, might involve in Ang II-induced necroptosis. Taken together, our results suggest that Ang II-induced necroptosis of renal tubular cell might be involved both AT1R and AT2R and the subsequent expression of Fas, FasL signaling. Thus, AT1R and AT2R might function as critical mediators.

scholarly journals Toxicity Induced by Cytokines, Glucose, and Lipids Increase Apoptosis and Hamper Insulin Secretion in the 1.1E7 Beta Cell-Line

2021 ◽  
Vol 22 (5) ◽  
pp. 2559
Author(s):  
Antonia Diaz-Ganete ◽  
Aranzazu Quiroga-de-Castro ◽  
Rosa M. Mateos ◽  
Francisco Medina ◽  
Carmen Segundo ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
High Glucose ◽  
Secretory Pathway ◽  
Early Stage ◽  
Hybrid Cell ◽  
Basic Research ◽  
Stress Markers ◽  
Beta Cell Line ◽  
Starting Point

Basic research on types 1 and 2 diabetes mellitus require early stage studies using beta cells or cell lines, ideally of human origin and with preserved insulin secretion in response to glucose. The 1.1E7 cells are a hybrid cell line resulting from the electrofusion of dispersed human islets and PANC-1 cells, capable of secreting insulin in response to glucose, but their survival and function under toxic conditions remains untested. This characterization is the purpose of the present study. We treated these cells with a cytokine mix, high glucose, palmitate, and the latter two combined. Under these conditions, we measured cell viability and apoptosis (MTT, Caspase Glo and TUNEL assays, as well as caspase-8 and -9 levels by Western blotting), endoplasmic reticulum stress markers (EIF2AK3, HSPA4, EIF2a, and HSPA5) by real-time PCR, and insulin secretion with a glucose challenge. All of these stimuli (i) induce apoptosis and ER stress markers expression, (ii) reduce mRNA amounts of 2–5 components of genes involved in the insulin secretory pathway, and (iii) abrogate the insulin release capability of 1.1E7 cells in response to glucose. The most pronounced effects were observed with cytokines and with palmitate and high glucose combined. This characterization may well serve as the starting point for those choosing this cell line for future basic research on certain aspects of diabetes.

Effects of dietary salt changes on renal renin-angiotensin system in rats

2002 ◽  
Vol 283 (5) ◽  
pp. F995-F1002 ◽  
Author(s):  
Catherine Ingert ◽  
Michèle Grima ◽  
Catherine Coquard ◽  
Mariette Barthelmebs ◽  
Jean-Louis Imbs
Keyword(s):  
Renal Cortex ◽  
Salt Content ◽  
Renin Angiotensin System ◽  
Fold Increase ◽  
Kidney Cortex ◽  
Ang Ii ◽  
Dietary Salt ◽  
High Sodium ◽  
Ace Activity ◽  
Wistar Kyoto

Renin (RA) and angiotensin-converting enzyme (ACE) activities and angiotensinogen, ANG I, and ANG II levels were measured in the kidney (cortex and medulla) and plasma of Wistar-Kyoto rats on a low-sodium (LS; 0.025% NaCl; n= 8), normal-sodium (NS; 1% NaCl; n = 7), or high-sodium (HS; 8% NaCl; n = 7) diet for 21 days. RA, ANG I, and ANG II levels increased in a manner inversely related to sodium content of the diet in both plasma and renal tissues. The LS diet resulted in a 16-, 2.8-, and 1.8-fold increase in plasma RA, ANG I, and ANG II levels, respectively, compared with those in HS rats. In the renal cortex and medulla, RA, ANG I, and ANG II levels were also increased by diminution of dietary salt content but, in contrast to plasma, ANG II levels increased much more than RA or ANG I levels [5.4 (cortex)- and 4.7 (medulla)-fold compared with HS rats]. In summary, we demonstrated variations of ANG II levels in the kidney during dietary salt modifications. Our results confirm that RA and ACE activity are not the steps limiting intrarenal ANG II levels. Nevertheless, despite RA and ACE activity differences between renal cortex and medulla, ANG I and ANG II levels are equivalent in these two tissues; these results argue against a compartmentalization of RAS in these two intrarenal areas.

scholarly journals A novel role for miR-133a in centrally mediated activation of the renin-angiotensin system in congestive heart failure

2017 ◽  
Vol 312 (5) ◽  
pp. H968-H979 ◽  
Author(s):  
Neeru M. Sharma ◽  
Shyam S. Nandi ◽  
Hong Zheng ◽  
Paras K. Mishra ◽  
Kaushik P. Patel
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Paraventricular Nucleus ◽  
Renin Angiotensin System ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Ang Ii ◽  
Angiotensin System ◽  
Renin Angiotensin

An activated renin-angiotensin system (RAS) within the central nervous system has been implicated in sympathoexcitation during various disease conditions including congestive heart failure (CHF). In particular, activation of the RAS in the paraventricular nucleus (PVN) of the hypothalamus has been recognized to augment sympathoexcitation in CHF. We observed a 2.6-fold increase in angiotensinogen (AGT) in the PVN of CHF. To elucidate the molecular mechanism for increased expression of AGT, we performed in silico analysis of the 3′-untranslated region (3′-UTR) of AGT and found a potential binding site for microRNA (miR)-133a. We hypothesized that decreased miR-133a might contribute to increased AGT in the PVN of CHF rats. Overexpression of miR-133a in NG108 cells resulted in 1.4- and 1.5-fold decreases in AGT and angiotensin type II (ANG II) type 1 receptor (AT1R) mRNA levels, respectively. A luciferase reporter assay performed on NG108 cells confirmed miR-133a binding to the 3′-UTR of AGT. Consistent with these in vitro data, we observed a 1.9-fold decrease in miR-133a expression with a concomitant increase in AGT and AT1R expression within the PVN of CHF rats. Furthermore, restoring the levels of miR-133a within the PVN of CHF rats with viral transduction resulted in a significant reduction of AGT (1.4-fold) and AT1R (1.5-fold) levels with a concomitant decrease in basal renal sympathetic nerve activity (RSNA). Restoration of miR-133a also abrogated the enhanced RSNA responses to microinjected ANG II within the PVN of CHF rats. These results reveal a novel and potentially unique role for miR-133a in the regulation of ANG II within the PVN of CHF rats, which may potentially contribute to the commonly observed sympathoexcitation in CHF. NEW & NOTEWORTHY Angiotensinogen (AGT) expression is upregulated in the paraventricular nucleus of the hypothalamus through posttranscriptional mechanism interceded by microRNA-133a in heart failure. Understanding the mechanism of increased expression of AGT in pathological conditions leading to increased sympathoexcitation may provide the basis for the possible development of new therapeutic agents with enhanced specificity.

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