Fluconazole inhibits human adrenocortical steroidogenesis in vitro

The antifungal agent ketoconazole is often used to suppress cortisol production in patients with Cushing's syndrome (CS). However, ketoconazole has serious side effects and is hepatotoxic. Here, the in vitro effects of ketoconazole and fluconazole, which might be less toxic, on human adrenocortical steroidogenesis were compared. The effects on steroidogenesis were examined in primary cultures of nine human adrenocortical tissues and two human adrenocortical carcinoma cell lines. Moreover, the effects on mRNA expression levels of steroidogenic enzymes and cell growth were assessed. Ketoconazole significantly inhibited 11-deoxycortisol (H295R cells; maximum inhibition 99%; EC50 0.73 μM) and cortisol production (HAC15 cells; 81%; EC50 0.26 μM and primary cultures (mean EC50 0.75 μM)). In cultures of normal adrenal cells, ketoconazole increased pregnenolone, progesterone, and deoxycorticosterone levels, while concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, DHEA, and androstenedione decreased. Fluconazole also inhibited 11-deoxycortisol production in H295R cells (47%; only at 1 mM) and cortisol production in HAC15 cells (maximum inhibition 55%; EC50 35 μM) and primary cultures (mean EC50 67.7 μM). In the cultures of normal adrenals, fluconazole suppressed corticosterone, 17-hydroxypregnenolone, and androstenedione levels, whereas concentrations of progesterone, deoxycorticosterone, and 11-deoxycortisol increased. Fluconazole (1 mM) slightly increased STAR mRNA expression in both cell lines. Neither compound affected mRNA levels of other steroidogenic enzymes or cell number. In conclusion, by inhibiting 11β-hydroxylase and 17-hydroxylase activity, pharmacological concentrations of fluconazole dose dependently inhibit cortisol production in human adrenocortical cells in vitro. Although fluconazole seems less potent than ketoconazole, it might become an alternative for ketoconazole to control hypercortisolism in CS. Furthermore, patients receiving fluconazole because of mycosis might be at risk for developing adrenocortical insufficiency.

Download Full-text

Age-related changes in human Leydig cell status

Human Reproduction ◽

10.1093/humrep/deaa271 ◽

2020 ◽

Vol 35 (12) ◽

pp. 2663-2676

Author(s):

Valentina Mularoni ◽

Valentina Esposito ◽

Sara Di Persio ◽

Elena Vicini ◽

Gustavo Spadetta ◽

...

Keyword(s):

Mrna Expression ◽

Sertoli Cell ◽

Leydig Cell ◽

Leydig Cells ◽

Cell Number ◽

Organ Donors ◽

Steroidogenic Enzymes ◽

Age Related ◽

Age Range

Abstract STUDY QUESTION What are the consequences of ageing on human Leydig cell number and hormonal function? SUMMARY ANSWER Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. WHAT IS KNOWN ALREADY There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. STUDY DESIGN, SIZE, DURATION This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS Testis biopsies were obtained from 15 heart beating organ donors (age range: 19–85 years) and 24 patients (age range: 19–45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. MAIN RESULTS AND THE ROLE OF CHANCE Increasing age was negatively associated with Leydig cell number (R = −0.49; P < 0.01) and concomitantly with the Sertoli cell population size (R= −0.55; P < 0.001). A positive correlation (R = 0.57; P < 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= −0.52; P < 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men. LIMITATIONS, REASONS FOR CAUTION In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. WIDER IMPLICATIONS OF THE FINDINGS This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Download Full-text

The ABCC6 Transporter Plays a Significant Role in the Efflux of Nilotinib and Dasatinib, and May Contribute to Tyrosine Kinase Inhibitor Resistance

Blood ◽

10.1182/blood.v128.22.4237.4237 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4237-4237

Author(s):

Laura N Eadie ◽

Jarrad M Goyne ◽

Timothy P. Hughes ◽

Deborah L White

Keyword(s):

Mrna Expression ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

K562 Cells ◽

Mrna Levels ◽

Advisory Committees ◽

Ku812 Cells

Abstract Efflux transporters ABCB1 and ABCG2 interact with tyrosine kinase inhibitors (TKIs) and mediate drug resistance, however, evidence of the interaction of other potentially relevant drug transporters with TKIs is lacking. We investigated the involvement of the closely related transporter ABCC6, in imatinib (IM), nilotinib (NIL) and dasatinib (DAS) transport and also the role of ABCC6 in NIL resistance. The impact of short-term (overnight) exposure to NIL on mRNA expression of ABC transporters in three BCR-ABL1+ cell lines was assessed by Taqman transporter array: K562, K562-Dox and KU812 cells. Several transporters of interest were identified, including ABCC6, based on alterations in mRNA expression. In order to elucidate the importance of ABCC6 in the development of NIL resistance, ABCC6 mRNA levels were determined by RT-PCR in K562 and K562-Dox NIL-resistant lines generated in vitro and compared with ABCC6 mRNA levels in respective parental control cells. ABCC6 protein expression was confirmed by western blot. p-Crkl dependent IC50 experiments in the absence and presence of three ABCC6 inhibitors (indomethacin, INDO; probenecid, PRO; pantoprazole, PP) were performed in patient mononuclear cells (MNCs) and BCR-ABL1+ cell lines to assess the role of ABCC6 in NIL, IM and DAS transport. A marked increase in ABCC6 mRNA expression in response to short-term in vitro NIL exposure occurred: in K562 and KU812 cells ABCC6 mRNA levels increased 9.5- and 9.7-fold in response to overnight NIL exposure respectively. Increased expression of ABCC6 was also observed in cells subjected to long-term NIL exposure during development of NIL resistance in vitro. NIL-resistant K562 cells demonstrated up to 57-fold higher levels of ABCC6 mRNA compared with control cells (p=0.002). Analogous results were observed in NIL-resistant K562-Dox cells (up to 33-fold higher levels of ABCC6 mRNA p=0.002). In order to determine the relevance of ABCC6 in patient cells, p-Crkl dependent IC50 experiments were performed in MNCs from de novo CML patients in the absence and presence of ABCC6 inhibition. Results demonstrated a significant reduction in IC50NIL in the presence of all three ABCC6 inhibitors compared with IC50NIL in the absence of inhibitors. Similar results were observed for IC50DAS but not IC50IM. Experiments in three parental BCR-ABL1+ cell lines confirmed these findings (Table 1). Notably, comparison of IC50 values in the absence of ABCC6 inhibition in KU812 vs. K562 cells revealed that KU812 cells demonstrated increased IC50NIL (307 vs. 257 nM, p=0.0493) and IC50DAS (14 vs 8 nM, p=0.0005). This was unexpected given both cell lines demonstrate negligible expression of ABCB1 (a transporter known to interact with both NIL and DAS). However, assessment of ABCC6 protein levels by western blotting revealed KU812 cells have greater levels of ABCC6 when compared with K562 cells: 53% in KU812 vs. 24% in K562 (ABCC6 normalised to β-actin). A greater %reduction in IC50NIL and IC50DAS in the presence of ABCC6 inhibition was also observed in KU812 cells compared with K562 cells confirming the role of ABCC6 in the transport of NIL and DAS. Combined, these studies highlight the importance of ABCC6 in the export of NIL and DAS from patient MNCs and BCR-ABL1+ cell lines. This is the first report of ABCC6 involvement in TKI transport and results suggest ABCC6 overexpression may also contribute to NIL resistance. The addition of ABCC6 inhibitors to NIL and DAS therapy may enhance the efficacy of these TKIs in the treatment of CML. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group. White:Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

Download Full-text

YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits

International Journal of Molecular Sciences ◽

10.3390/ijms22137226 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7226

Author(s):

Violeta Stojanovska ◽

Aneri Shah ◽

Katja Woidacki ◽

Florence Fischer ◽

Mario Bauer ◽

...

Keyword(s):

Cell Lines ◽

Cancer Progression ◽

Cell Function ◽

Trophoblast Cell ◽

Mrna Levels ◽

Trophoblast Cells ◽

Invasion And Migration ◽

And Migration ◽

Apoptosis And Inflammation

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

Download Full-text

Dengue Virus Induces the Expression and Release of Endocan from Endothelial Cells by an NS1–TLR4-Dependent Mechanism

Microorganisms ◽

10.3390/microorganisms9061305 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1305

Author(s):

Carlos Alonso Domínguez-Alemán ◽

Luis Alberto Sánchez-Vargas ◽

Karina Guadalupe Hernández-Flores ◽

Andrea Isabel Torres-Zugaide ◽

Arturo Reyes-Sandoval ◽

...

Keyword(s):

Endothelial Cell ◽

Mrna Expression ◽

Cell Lines ◽

Cell Activation ◽

Dengue Infection ◽

Elevated Serum ◽

Fold Increase ◽

Endothelial Cell Activation ◽

Stimulation Of

A common hallmark of dengue infections is the dysfunction of the vascular endothelium induced by different biological mechanisms. In this paper, we studied the role of recombinant NS1 proteins representing the four dengue serotypes, and their role in promoting the expression and release of endocan, which is a highly specific biomarker of endothelial cell activation. We evaluated mRNA expression and the levels of endocan protein in vitro following the stimulation of HUVEC and HMEC-1 cell lines with recombinant NS1 proteins. NS1 proteins increase endocan mRNA expression 48 h post-activation in both endothelial cell lines. Endocan mRNA expression levels were higher in HUVEC and HMEC-1 cells stimulated with NS1 proteins than in non-stimulated cells (p < 0.05). A two-fold to three-fold increase in endocan protein release was observed after the stimulation of HUVECs or HMEC-1 cells with NS1 proteins compared with that in non-stimulated cells (p < 0.05). The blockade of Toll-like receptor 4 (TLR-4) signaling on HMEC-1 cells with an antagonistic antibody prevented NS1-dependent endocan production. Dengue-infected patients showed elevated serum endocan levels (≥30 ng/mL) during early dengue infection. High endocan serum levels were associated with laboratory abnormalities, such as lymphopenia and thrombocytopenia, and are associated with the presence of NS1 in the serum.

Download Full-text

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

Parasites & Vectors ◽

10.1186/s13071-021-04618-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Hai-Jun Gao ◽

Xu-Dong Sun ◽

Yan-Ping Luo ◽

Hua-Sheng Pang ◽

Xing-Ming Ma ◽

...

Keyword(s):

Protein Kinase ◽

Mrna Expression ◽

Echinococcus Granulosus ◽

Calcium Content ◽

Mrna Levels ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Download Full-text

Steroidogenic enzymes mRNA expression profile and steroids production in bovine theca cells cultured in vitro and stimulated with angiotensin II

Ciência Rural ◽

10.1590/0103-8478cr20240254 ◽

2015 ◽

Vol 45 (4) ◽

pp. 704-710 ◽

Cited By ~ 2

Author(s):

Melânia Lazzari Rigo ◽

Andressa Minussi Pereira Dau ◽

Werner Giehl Glanzner ◽

Manoel Martins ◽

Renato Zanella ◽

...

Keyword(s):

High Performance ◽

Culture Media ◽

Mrna Levels ◽

Ang Ii ◽

Rt Pcr ◽

Steroidogenic Enzymes ◽

Theca Cells ◽

Mrna Profile ◽

Dose Response Effect

The main objective of this study was to detect the steroidogenic effects of Ang II in bovine theca cells in vitro. Bovine theca cells were obtained from follicles (larger than 10mm of diameter) collected from a local abattoir and submitted to different treatments in a sequence of experiments. In experiment 1, CYP17A1 mRNA profile was evaluated in LH- (10ng ml-1) and Ang II-treated (0.1µM) theca cells. In experiment 2, a dose-response effect of Ang II (0.001; 0.1 e 10µM) plus insulin (100ng ml-1) and LH (100ng ml-1) was evaluated on steroidogenesis of bovine theca cells. Experiment 3 explored the effects of saralasin (an antagonist of Ang II receptors) on steroid production and steroidogenic enzymes regulation in theca cells. After 24 hours, culture media from experiments 2 and 3 was collected to evaluate testosterone and androstenedione levels by High-Performance Liquid Chromatography. In parallel, mRNA levels of key steroidogenic enzymes (HSD3B2, CYP11A1, CYP17A1) and STAR were assessed by RT-PCR. There was no difference in testosterone and androstenedione production between treated and controls groups, as well as in mRNA levels of the evaluated genes. In conclusion, the results suggest that Ang II does not regulate steroidogenesis in bovine theca cells

Download Full-text

Annexin V decreases PS-mediated macrophage efferocytosis and deteriorates elastase-induced pulmonary emphysema in mice

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00066.2012 ◽

2012 ◽

Vol 303 (10) ◽

pp. L852-L860 ◽

Cited By ~ 13

Author(s):

S. Yoshida ◽

N. Minematsu ◽

S. Chubachi ◽

H. Nakamura ◽

M. Miyazaki ◽

...

Keyword(s):

Mrna Expression ◽

Cell Apoptosis ◽

Pulmonary Emphysema ◽

Keratinocyte Growth Factor ◽

Annexin V ◽

Phenotypic Change ◽

Chronic Obstructive ◽

Mrna Levels

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

Download Full-text

Activation of nuclear factor of activated T cells 5 in the peritoneal membrane of uremic patients

AJP Renal Physiology ◽

10.1152/ajprenal.00617.2014 ◽

2015 ◽

Vol 308 (11) ◽

pp. F1247-F1258 ◽

Cited By ~ 7

Author(s):

Daniel Kitterer ◽

Joerg Latus ◽

Christoph Ulmer ◽

Peter Fritz ◽

Dagmar Biegger ◽

...

Keyword(s):

T Cells ◽

Mrna Expression ◽

Mrna Levels ◽

Nuclear Factor ◽

Activated T Cells ◽

Significant Difference ◽

Ccl2 Mrna ◽

Uremic Patients ◽

High Concentrations

Peritoneal inflammation and fibrosis are responses to the uremic milieu and exposure to hyperosmolar dialysis fluids in patients on peritoneal dialysis. Cells respond to high osmolarity via the transcription factor nuclear factor of activated T cells (NFAT5). In the present study, the response of human peritoneal fibroblasts to glucose was analyzed in vitro. Expression levels of NFAT5 and chemokine (C-C motif) ligand (CCL2) mRNA were quantified in peritoneal biopsies of five nonuremic control patients, five uremic patients before PD (pPD), and eight patients on PD (oPD) using real-time PCR. Biopsies from 5 control patients, 25 pPD patients, and 25 oPD patients were investigated using immunohistochemistry to detect the expression of NFAT5, CCL2, NF-κB p50, NF-κB p65, and CD68. High glucose concentrations led to an early, dose-dependent induction of NFAT5 mRNA in human peritoneal fibroblasts. CCL2 mRNA expression was upregulated by high concentrations of glucose after 6 h, but, most notably, a concentration-dependent induction of CCL2 was present after 96 h. In human peritoneal biopsies, NFAT5 mRNA levels were increased in uremic patients compared with nonuremic control patients. No significant difference was found between the pPD group and oPD group. CCL2 mRNA expression was higher in the oPD group. Immunohistochemistry analysis was consistent with the results of mRNA analysis. CD68-positive cells were significantly increased in the oPD group. In conclusion, uremia results in NFAT5 induction, which might promote early changes of the peritoneum. Upregulation of NFAT5 in PD patients is associated with NFκB induction, potentially resulting in the recruitment of macrophages.

Download Full-text

Abstract 261: Endothelial Cells Contribute to RAS Activation in Microvascular Smooth Muscle Cells

Circulation Research ◽

10.1161/res.111.suppl_1.a261 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Kayoko Miyata ◽

Ryousuke Satou ◽

L Gabriel Navar

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Mrna Expression ◽

Culture Medium ◽

Primary Cultures ◽

Mrna Levels ◽

Ang Ii ◽

Relative Ratio ◽

Renin Mrna ◽

Afferent Arterioles

Introduction: We have demonstrated that Ang II augments angiotensinogen (AGT) expression in rat preglomerular vascular smooth muscle cells (VSMCs). However, it is unclear if endothelial cells (ECs) are involved in augmentation of AGT in renal afferent arterioles. Hypothesis: We assessed the hypothesis that the ECs respond to paracrine signals that Ang II contribute to AGT augmentation in VSMCs. Objective: We established primary cultures of preglomerular ECs and examined the effects of Ang II and/or culture medium from ECs on AGT expression in preglomerular VSMCs. Methods and Results: We established primary cultures of preglomerular ECs, isolated from afferent arterioles of Sprague-Dawley rats. The cells were identified as ECs by being positive for a marker, CD34 and endothelial NOS and negative for alpha-SMA (a marker for VSMCs) and P4H-b (a marker for Fibroblasts) by immnostaining. The expression levels of AGT mRNA and renin mRNA in preglomerular ECs were examined by real-time RT-PCR. Ang II (100 pmol/L) increased AGT mRNA levels (1.34 +/- 0.16, by 100 pmol/L, N=4) and Renin mRNA levels (6.16 +/- 0.96, by 100 nmol/L, N=4) in ECs. On the other hand, the same dose of Ang II suppressed Renin mRNA expression in isolated Juxtaglomerular cells (JGs). These results indicate that preglomerular ECs are respond to Ang II and exclude the possible contamination of JGs into ECs. 100 pmol/L of Ang II increased AGT mRNA expression levels (1.37 +/- 0.03, relative ratio, N=4) in preglomerular VSMCs and the culture medium of ECs without Ang II treatment also more increased AGT mRNA expression (1.62 +/- 0.13, relative ratio, N=4) in preglomerular VSMCs. The AGT mRNA expression augmentation was enhanced when preglomerular VSMCs were treated with culture medium of Ang II-treated preglomerular ECs (2.39 +/- 0.41, relative ratio, N=4). The synergistic effects of Ang II and preglomerular ECs were also observed in PAI-1 expression in preglomerular VSMCs. Conclusion: These data demonstrate that preglomerular ECs contribute to Ang II-upregulation of AGT in renal afferent arterioles leading to further Ang II augmentation, which leads to increases in inflammatory and sclerotic factors in preglomerular VSMCs.

Download Full-text