Abstract 134: Activation of the Mineralocorticoid Receptor by O-glycosylation Through Hyperglycemia

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Maniselvan Kuppusamy ◽  
Elise Gomez-Sanchez ◽  
Celso Gomez-Sanchez
Keyword(s):  
High Glucose ◽  
Mineralocorticoid Receptor ◽  
Transcriptional Activity ◽  
Collecting Duct ◽  
Cardiovascular Complications ◽  
Luciferase Reporter ◽  
Cortical Collecting Duct ◽  
Luciferase Reporter Gene ◽  
Reporter Activity ◽  
The Metabolic Syndrome

Introduction: Hypertension and diabetes are independent risk factors for cardiovascular disease, however why they frequently occur together is not clear. Inappropriate mineralocorticoid receptor (MR) activation is associated with diabetes and the metabolic syndrome, as well as hypertension and abnormal cardiovascular remodeling. MR antagonists are effective in reducing hypertension and delaying the onset of renal and cardiovascular complications in diabetes despite circulating aldosterone levels that are within normal limits. Glucose concentrations increase O-glycosylation of many proteins, thus alter their function. Hence, we hypothesized that increased O-GlcNac modification of the MR by high glucose enhances MR activation. Methods: MR transcriptional activity was studied in a mouse cortical collecting duct (M1) cell line stably transfected with a cDNA construct including the MR and one with a hormone-response element driving a Gaussia luciferase reporter gene. The cells were incubated for 48 h with low (5mM) or high (25mM) glucose media with and without Thiamet-G (TMG), an O-GlcNAcase inhibitor to inhibit deglycosylation, and 6-diazo-5-oxonorleucine (DON), a glucosamine-fructose-6-phosphate amidotransferase inhibitor (GFAT) to reduce O-GlcNAc levels. Additionally, MR and GR antagonists were used to identify receptor specificity under low and high glucose conditions. O-GlcNac-modified MR was co-immunoprecipitated with an MR antibody and detected with an O-GlcNAc antibody. Results: 1. Co-immunoprecipitation assays showed that high glucose and TMG increased O-GlcNac-MR by 3-fold. 2. Compared to low glucose, treatment with high glucose and with TMG increased the transcriptional activity of MR by 300%. 3. DON decreased MR-reporter activity by 75%. 4. High glucose alone had no significant basal effect but significantly increased MR activation by aldosterone. 5. MR reporter activity was increased similarly by aldosterone and corticosterone. Conclusion: High glucose increased glycosylation of the MR, augmenting its transcriptional activity. Enhancement of MR activation by hyperglycemia may explain how MRs play a significant role in the cardiorenal pathology in Diabetes.

scholarly journals MicroRNA-194 (miR-194) regulates ROMK channel activity by targeting intersectin 1

2014 ◽  
Vol 306 (1) ◽  
pp. F53-F60 ◽  
Author(s):  
Dao-Hong Lin ◽  
Peng Yue ◽  
Chengbiao Zhang ◽  
Wen-Hui Wang
Keyword(s):  
Collecting Duct ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Seed Sequence ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Luciferase Reporter Gene ◽  
High K ◽  
Low K

The aim of the study is to explore the role of miR-194 in mediating the effect of high-K (HK) intake on ROMK channel. Northern blot analysis showed that miR-194 was expressed in kidney and that HK intake increased while low-K intake decreased the expression of miR-194. Real-time PCR analysis further demonstrated that HK intake increased the miR-194 expression in the cortical collecting duct. HK intake decreased the expression of intersectin 1 (ITSN1) which enhanced With-No-Lysine Kinase (WNK)-induced endocytosis of ROMK. Expression of miR-194 mimic decreased luciferase reporter gene activity in HEK293 T cells transfected with ITSN-1–3′UTR containing the complementary seed sequence for miR-194. In contrast, transfection of miR-194 inhibitor increased the luciferase activity. This effect was absent in the cells transfected with mutated 3′UTR of ITSN1 in which the complimentary seed sequence was deleted. Moreover, the inhibition of miR-194 expression increased the protein level of endogenous ITSN1 in HEK293T cells. Expression of miR-194 mimic also decreased the translation of exogenous ITSN1 in the cells transfected with the ITSN1 containing 3′UTR but not with 3′UTR-free ITSN1. Expression of pre-miR-194 increased K currents and ROMK expression in the plasma membrane in ROMK-transfected cells. Coexpression of ITSN1 reversed the stimulatory effect of miR-194 on ROMK channels. This effect was reversed by coexpression of ITSN1. We conclude that miR-194 regulates ROMK channel activity by modulating ITSN1 expression thereby enhancing ITSN1/WNK-dependent endocytosis. It is possible that miR-194 is involved in mediating the effect of a HK intake on ROMK channel activity.

Abstract 09: Mtor Inhibition Decreases Ligand-induced Mineralocorticoid Receptor Activation

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Yusuf Ali ◽  
Elise P Gomez-sanchez ◽  
Celso E Gomez-sanchez
Keyword(s):  
Reporter Gene ◽  
Transcriptional Activity ◽  
Collecting Duct ◽  
Receptor Activation ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mtor Inhibition ◽  
Positive Effects ◽  
Mtor Phosphorylation

Introduction: ULK1 phosphorylates the MR at S843, decreasing its ligand binding and transcriptional activity. Angiotensin II-induced mTOR phosphorylation of ULK1 inactivates ULK1, preventing its phosphorylation of MR. Aim: Further elucidate the role of mTOR in the regulation of MR transcriptional activity. Methods: M1 mouse cortical collecting duct cells stably transduced with the rat MR cDNA and a MMTV- Gaussia luciferase reporter gene, were incubated with an mTOR activator and several inhibitors, +/- aldosterone or corticosterone. Similar studies were done after lentiviral transduction of CRISPR/gRNA for raptor and rictor genes or mutated MR (mu/S843A) cDNA. Results: mTOR inhibition significantly decreased ligand activation of the MR reporter gene, while the mTOR activator MHY1485 had no effect suggesting that mTOR is tonically active. MR activation induced by aldosterone and corticosterone was also decreased by CRISPR/gRNA gene knockdown of raptor and rictor, the adaptors of mTOR complex 1 and 2, respectively, supporting a role for mTOR. The mTOR inhibitor AZD8055 (AZD) reduced phospho-ULK1 and attenuated ligand-mediated MR transactivation in a dose-dependent manner. The ULK1 inhibitor MRT68921 increased MR transactivation. We speculated that mTOR decreased ULK1 activity by phosphorylating it, thereby preventing ULK1 phosphorylation of the MR at Serine (S843). However, when M1 cells were transduced with an MR cDNA in which S843 was replaced with Alanine that cannot be phosphorylated, ligand-induced activation of the mu/S843A MR was still decreased by AZD, but unchanged by MRT68921. This suggests that mTOR has an additional effect on MR activity unrelated to ULK1 activity. AZD also decreased P70S6K and AKT phosphorylation in these cells. Conclusions: mTOR phosphorylation of ULK1 prevents its phosphorylation of the MR and reduction of MR transcriptional activity. mTOR inhibitors and deletion of raptor and rictor decreased MR transcriptional activity. mTOR has additional positive effects on MR activity possibly related to its phosphorylation of AKT and P70S6K. Inhibition of mTOR action may be a useful target for mitigating excessive MR activation.

scholarly journals Agonistic and antagonistic properties of progesterone metabolites at the human mineralocorticoid receptor

2002 ◽  
pp. 789-799 ◽  
Author(s):  
M Quinkler ◽  
B Meyer ◽  
C Bumke-Vogt ◽  
C Grossmann ◽  
U Gruber ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Mineralocorticoid Receptor ◽  
Antagonistic Activity ◽  
Luciferase Reporter ◽  
Protective Mechanism ◽  
Luciferase Reporter Gene ◽  
Binding Characteristics ◽  
Rabbit Reticulocyte Lysate System ◽  
Progesterone Metabolites ◽  
Agonistic Activity

OBJECTIVE: Progesterone binds to the human mineralocorticoid receptor (hMR) with nearly the same affinity as do aldosterone and cortisol, but confers only low agonistic activity. It is still unclear how aldosterone can act as a mineralocorticoid in situations with high progesterone concentrations, e.g. pregnancy. One mechanism could be conversion of progesterone to inactive compounds in hMR target tissues. DESIGN: We analyzed the agonist and antagonist activities of 16 progesterone metabolites by their binding characteristics for hMR as well as functional studies assessing transactivation. METHODS: We studied binding affinity using hMR expressed in a T7-coupled rabbit reticulocyte lysate system. We used co-transfection of an hMR expression vector together with a luciferase reporter gene in CV-1 cells to investigate agonistic and antagonistic properties. RESULTS: Progesterone and 11beta-OH-progesterone (11beta-OH-P) showed a slightly higher binding affinity than cortisol, deoxycorticosterone and aldosterone. 20alpha-dihydro(DH)-P, 5alpha-DH-P and 17alpha-OH-P had a 3- to 10-fold lower binding potency. All other progesterone metabolites showed a weak affinity for hMR. 20alpha-DH-P exhibited the strongest agonistic potency among the metabolites tested, reaching 11.5% of aldosterone transactivation. The agonistic activity of 11beta-OH-P, 11alpha-OH-P and 17alpha-OH-P was 9, 5.1 and 4.1% respectively. At a concentration of 100 nmol/l, progesterone, 17alpha-OH-P and 20alpha-DH-P inhibit nearly 75, 40 and 35% of the transactivation by aldosterone respectively. All other progesterone metabolites tested demonstrate weaker affinity, and agonistic and antagonistic potency. CONCLUSIONS: The binding affinity for hMR and the agonistic and antagonistic activity diminish with increasing reduction of the progesterone molecule at C20, C17 and at ring A. We assume that progesterone metabolism to these compounds is a possible protective mechanism for hMR. 17alpha-OH-P is a strong hMR antagonist and could exacerbate mineralocorticoid deficiency in patients with congenital adrenal hyperplasia.

Critical role of the mineralocorticoid receptor in aldosterone-dependent and aldosterone-independent regulation of ENaC in the distal nephron

2021 ◽  
Author(s):  
Viatcheslav Nesterov ◽  
Marko Bertog ◽  
Jérémie Canonica ◽  
Edith Hummler ◽  
Richard Coleman ◽  
...  
Keyword(s):  
Mineralocorticoid Receptor ◽  
Collecting Duct ◽  
Critical Role ◽  
Sodium Absorption ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Patch Clamp Technique ◽  
Transition Zones ◽  
Rate Limiting Step

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the collecting duct. Previously, we demonstrated that ENaC activity in the DCT2/CNT transition zone is constitutively high and independent of aldosterone, in contrast to its aldosterone dependence in the late CNT and initial cortical collecting duct (CNT/CCD). The mineralocorticoid receptor (MR) is expressed in the entire ASDN. Its activation by glucocorticoids is prevented through 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) abundantly expressed in the late but probably not the early part of ASDN. We hypothesized that ENaC function in the early part of the ASDN is aldosterone-independent but may depend on MR activated by glucocorticoids due to low 11β-HSD2 abundance. To test this hypothesis, we used doxycycline-inducible nephron-specific MR-deficient mice (MR KO). Whole-cell ENaC currents were investigated in isolated nephron fragments from DCT2/CNT or CNT/CCD transition zones using the patch-clamp technique. ENaC activity was detectable in CNT/CCD of control mice but absent or barely detectable in the majority of CNT/CCD preparations from MR KO mice. Importantly, ENaC currents in DCT2/CNT were greatly reduced in MR KO mice compared to ENaC currents in DCT2/CNT of control mice. Immunofluorescence for 11β-HSD2 was abundant in CCD, less prominent in CNT and very low in DCT2. We conclude that MR is critically important for maintaining aldosterone-independent ENaC activity in DCT2/CNT. Aldosterone-independent MR activation is probably mediated by glucocorticoids due to low expression of 11β-HSD2.

In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

1999 ◽  
Vol 276 (4) ◽  
pp. C883-C891 ◽  
Author(s):  
Carola E. Wright ◽  
F. Haddad ◽  
A. X. Qin ◽  
P. W. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Reporter Gene ◽  
Normal Control ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Construct ◽  
Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

scholarly journals Xiao Yao San against Corticosterone-Induced Stress Injury via Upregulating Glucocorticoid Receptor Reaction Element Transcriptional Activity

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Guoping Cao ◽  
Shenglan Gong ◽  
Fengxue Zhang ◽  
Wenjun Fu
Keyword(s):  
Hippocampal Neurons ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Stress Injury ◽  
Induced Stress

Previous studies have revealed that uncontrollable stress can impair the synaptic plasticity and firing property of hippocampal neurons, which influenced various hippocampal-dependent tasks including memory, cognition, behavior, and mood. In this work, we had investigated the effects and mechanisms of the Chinese herbal medicine Xiao Yao San (XYS) against corticosterone-induced stress injury in primary hippocampal neurons (PHN) cells. We found that XYS and RU38486 could increase cell viabilities and decrease cell apoptosis by MTT, immunofluorescence, and flow cytometry assays. In addition, we observed that XYS notably inhibited the nuclear translocation of GR and upregulated the mRNA and protein expressions levels of Caveolin-1, GR, BDNF, TrkB, and FKBP4. However, XYS downregulated the FKBP51 expressions. Furthermore, the results of the electrophoretic mobility shift assay (EMSA) and double luciferase reporter gene detection indicated that FKBP4 promotes the transcriptional activity of GR reaction element (GRE) by binding with GR, and FKBP51 processed the opposite action. Thein vivoexperiment also proved the functions of XYS. These results suggested that XYS showed an efficient neuroprotection against corticosterone-induced stress injury in PHN cells by upregulating GRE transcriptional activity, which should be developed as a potential candidate for treating stress injury in the future.

scholarly journals Albuminuria induces a proinflammatory and profibrotic response in cortical collecting ducts via the 24p3 receptor

2013 ◽  
Vol 305 (7) ◽  
pp. F1053-F1063 ◽  
Author(s):  
Eva Dizin ◽  
Udo Hasler ◽  
Stellor Nlandu-Khodo ◽  
Marc Fila ◽  
Isabelle Roth ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Collecting Duct ◽  
Transforming Growth Factor Β ◽  
Luciferase Reporter ◽  
Lipocalin 2 ◽  
Luciferase Reporter Gene ◽  
Puromycin Aminonucleoside ◽  
Gene Assay ◽  
Albumin Endocytosis

Albuminuria is strongly associated with progressive kidney tubulo-interstitial damage and chronic kidney disease (CKD) progression. In proteinuric nephropathies, albumin reabsorption by the proximal tubule is saturated and the distal nephron is exposed to high concentrations of luminal albumin that may produce adverse effects. Since proximal tubular cells exposed to albuminuria exhibit a proinflammatory and profibrotic response, we assessed the effect of albuminuria in the collecting duct (CD). With the use of kidney sections and isolated cortical CDs (CCDs) from puromycin-aminonucleoside-induced nephrotic rats (PAN rats) exhibiting proteinuria, immunofluorescence microscopy revealed internalized albumin in CD cells. In these proteinuric rats, increased expression levels of cytokines and profibrotic signaling markers were detected in isolated CCDs and bands of inflammatory fibrosis could be observed around CDs. Albumin endocytosis was confirmed by FITC-albumin uptake in cultured murine CCD (mCCDcl1) cells. Exposure of mCCDcl1 cells to albumin induced NF-κB activation as assessed by luciferase reporter gene assay, nuclear translocation of NF-κB p65 subunit, and increased NF-κB target gene expression. Moreover, albuminuria-like condition results in transforming growth factor-β1 (TGF-β1) overexpression and the upregulation of profibrotic signaling markers such as Snail or vimentin via an autocrine mechanism. In mCCDcl1 cells, neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2/24p3 receptor (24p3R) mediates albumin endocytosis as well as activation of NF-κB and TGF-β1 signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.

scholarly journals Cardiac Hypertrophy and Fibrosis in the Metabolic Syndrome: A Role for Aldosterone and the Mineralocorticoid Receptor

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Eric E. Essick ◽  
Flora Sam
Keyword(s):  
Metabolic Syndrome ◽  
Cardiac Hypertrophy ◽  
Cross Talk ◽  
Mineralocorticoid Receptor ◽  
Ventricular Hypertrophy ◽  
Cardiac Fibrosis ◽  
Cardiovascular Complications ◽  
Left Ventricular ◽  
The Metabolic Syndrome ◽  
Increased Risk

Obesity and hypertension, major risk factors for the metabolic syndrome, render individuals susceptible to an increased risk of cardiovascular complications, such as adverse cardiac remodeling and heart failure. There has been much investigation into the role that an increase in the renin-angiotensin-aldosterone system (RAAS) plays in the pathogenesis of metabolic syndrome and in particular, how aldosterone mediates left ventricular hypertrophy and increased cardiac fibrosis via its interaction with the mineralocorticoid receptor (MR). Here, we review the pertinent findings that link obesity with elevated aldosterone and the development of cardiac hypertrophy and fibrosis associated with the metabolic syndrome. These studies illustrate a complex cross-talk between adipose tissue, the heart, and the adrenal cortex. Furthermore, we discuss findings from our laboratory that suggest that cardiac hypertrophy and fibrosis in the metabolic syndrome may involve cross-talk between aldosterone and adipokines (such as adiponectin).

scholarly journals miR-30e targets GLIPR-2 to modulate diabetic nephropathy: in vitro and in vivo experiments

2017 ◽  
Vol 59 (2) ◽  
pp. 181-190 ◽  
Author(s):  
Dong Zhao ◽  
Jinhua Jia ◽  
Hong Shao
Keyword(s):  
High Glucose ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Related Factors ◽  
Qrt Pcr ◽  
In Vivo Experiments ◽  
Renal Tubular

The objectives of this study are to investigate the effect of miR-30e targeting GLIPR-2 on the pathological mechanism of DN. The renal tissues of db/db and db/m mice at different age of weeks were stained with PAS. qRT-PCR was applied to detect the expression of miR-30e and GLIPR-2, not only in the renal tissues of mice but also in the renal tubular epithelial cells (RTECs). By luciferase reporter gene assays, we found the 3′-UTR of the GLIPR-2 mRNA as a direct target of miR-30e. The RTECs cultured in high glucose were divided into blank control, NC, miR-30e mimics, miR-30e inhibitors, miR-30e inhibitor + si-GLIPR-2 and si-GLIPR-2 groups. MTT and flow cytometry were utilized to measure the proliferation and apoptosis of RTECs, while qRT-PCR and Western blot to detect the expression of GLIPR-2- and EMT-related factors. The following results were obtained: In the renal tissues of over 8-week-old db/db mice and the RTECs cultured for 6 h in high glucose, miR-30e was downexpressed while GLIPR-2 was upregulated in a time-dependent manner. Besides, overexpression of miR-30e and si-GLIPR-2 can not only greatly improve the proliferation of RTECs cultured in high glucose, but also downregulate the apoptosis rate of RTECs and the expressions of GLIPR-2, vimentin, α-SMA, Col-I and FN and upregulate E-cadherin. Moreover, si-GLIPR-2 can reverse the proliferation reduction, GLIPR-2 and EMT occurrence caused by the downexpression of miR-30e in RTECs. In conclusion, miR-30e is downregulated in DN, and the overexpression of miR-30e can inhibit GLIPR-2, promote the proliferation of RTECs and inhibit EMT, ultimately avoid leading to renal fibrosis in DN.

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