Quantification of TGF-β1 mRNA along rat nephron in obstructive nephropathy

Unilateral ureteral obstruction (UUO) leads to interstitial fibrosis of the obstructed kidney, and transforming growth factor-β1 (TGF-β1) is thought to play an important role in this process. Although increased TGF-β1 mRNA expression in the obstructed kidney has been demonstrated, the source of the increased TGF-β1 remains to be elucidated. To determine the precise localization of TGF-β1 in the obstructed kidney, we examined TGF-β1 mRNA expression using in situ hybridization and competitive RT-PCR in rats with UUO. In situ hybridization demonstrated that TGF-β1 mRNA expression was preferentially increased in tubular epithelial cells and to a lesser degree in infiltrating macrophages in obstructed kidneys. Quantitative analysis using competitive RT-PCR in microdissected nephron segments revealed that levels of TGF-β1 mRNA in obstructed kidneys relative to control kidneys increased significantly in proximal tubules, thick ascending limbs of Henle, and distal convoluted tubules, whereas those in glomeruli and collecting ducts did not change significantly. Of the tubular segments, the proximal tubules appeared to predominantly contribute to increased TGF-β1 mRNA. Our findings suggest that renal tubules, particularly proximal tubules, are the main contributors to increased TGF-β1 mRNA expression in obstructed kidneys and to the subsequent interstitial fibrosis.

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Expression of transforming growth factor alpha during development

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y88-183 ◽

1988 ◽

Vol 66 (8) ◽

pp. 1113-1121 ◽

Cited By ~ 10

Author(s):

V. K. M. Han ◽

A. J. D'Ercole ◽

D. C. Lee

Keyword(s):

In Situ Hybridization ◽

Growth Factor ◽

Mrna Expression ◽

Transforming Growth Factor ◽

Transforming Growth Factor Alpha ◽

Egf Receptors ◽

Hybridization Histochemistry ◽

In Situ Hybridization Histochemistry ◽

Factor Alpha

Transforming growth factors (TGFs) are polypeptides that are produced by transformed and tumour cells, and that can confer phenotypic properties associated with transformation on normal cells in culture. One of these growth-regulating molecules, transforming growth factor alpha (TGF-α), is a 50 amino acid polypeptide that is related to epidermal growth factor (EGF) and binds to the EGF receptor. Previous studies have shown that TGF-α is expressed during rodent embryogenesis between 7 and 14 days gestation. To investigate the cellular sites of TGF-α mRNA expression during development, we have performed Northern analyses and in situ hybridization histochemistry on the conceptus and maternal tissues at various gestational ages. Contrary to previous reports, both Northern analyses and in situ hybridization histochemistry indicate that TGF-α mRNA is predominantly expressed in the maternal decidua and not in the embryo. Decidual expression is induced following implantation, peaks at day 8, and declines through day 15 when the decidua is being resorbed. In situ hybridization revealed that expression of TGF-α mRNA is highest in the region of decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and embryo. In addition, we could not detect TGF-α mRNA expression in other maternal tissues, indicating that the induction of TGF-α transcripts in the decidua is tissue specific, and not a pleiotropic response to changes in hormonal milieu that occur during pregnancy. The developmentally regulated expression of TGF-α mRNA in the decidua, together with the presence of EGF receptors in this tissue, suggests that this peptide may stimulate mitosis and angiogenesis locally by an autocrine mechanism. Because EGF receptors are also present in the embryo and placenta, TGF-α may act on these tissues by a paracrine or endocrine mechanism.

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Expression of Tandem P Domain K+ Channel, TREK-1, in the Rat Carotid Body

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.5a6755.2005 ◽

2006 ◽

Vol 54 (4) ◽

pp. 467-472 ◽

Cited By ~ 16

Author(s):

Y. Yamamoto ◽

K. Taniguchi

Keyword(s):

In Situ Hybridization ◽

Mrna Expression ◽

Carotid Body ◽

Nerve Fibers ◽

K Channel ◽

Membrane Excitability ◽

Rt Pcr ◽

Glomus Cells ◽

Afferent Nerve Endings

TREK-1 is one of the important potassium channels for regulating membrane excitability. To examine the distribution of TREK-1 in the rat carotid body, we performed RT-PCR for mRNA expression and in situ hybridization and immunohistochemistry for tissue distribution of TREK-1. RT-PCR detected mRNA expression of TREK-1 in the carotid body. Furthermore, in situ hybridization revealed the localization of TREK-1 mRNA in the glomus cells. TREK-1 immunoreactivity was mainly distributed in the glomus cells and nerve fibers in the carotid body. TREK-1 may modulate potassium current of glomus cells and/or afferent nerve endings in the rat carotid body.

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Estrogen Receptor-β mRNA Expression in Rat Ovary: Down-Regulation by Gonadotropins

Molecular Endocrinology ◽

10.1210/mend.11.2.9887 ◽

1997 ◽

Vol 11 (2) ◽

pp. 172-182 ◽

Cited By ~ 2

Author(s):

Michael Byers ◽

George G. J. M. Kuiper ◽

Jan-Åke Gustafsson ◽

Ok-Kyong Park-Sarge

Keyword(s):

Estrogen Receptor ◽

In Situ Hybridization ◽

Mrna Expression ◽

Granulosa Cells ◽

Mrna Levels ◽

Rt Pcr ◽

Adult Rats ◽

Mature Adult ◽

Preovulatory Follicles

Abstract We have examined the expression and regulation of the two estrogen receptor (ERα and ERβ) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ERα and ERβ genes as single (∼6.5-kb) and multiple (ranging from ∼1.0-kb to ∼10.0-kb) transcripts, respectively. ERα mRNA is expressed at a level lower than ERβ mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ERβ mRNA levels were decreased. Examination of ERβ mRNA expression at the cellular level, by in situ hybridization, showed that ERβ mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ERβ mRNA was observed in a subset of corpora lutea, and that the decrease in ERβ mRNA during proestrous evening is attributable, at least in part, to down-regulation of ERβ mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ERα mRNA in the ovary. Although whole ovarian content of ERα mRNA was clearly detected by RT-PCR, no apparent modulation of ERα mRNA levels was observed during the estrous cycle. Examination of ERα mRNA expression at the cellular level, by in situ hybridization, showed that ERα mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ERβ mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ERβ mRNA. In situ hybridization for ERβ mRNA shows that small, growing, and preovulatory follicles express ERβ mRNA in the granulosa cells. The preovulatory follicles contain ERβ mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ERβ mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ERβ mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ERβ mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ERα and ERβ genes, although ERβ is the predominant form of estrogen receptor in the ovary, 2) ERβ mRNA is localized predominantly to the granulosa cells of small, growing, and preovulatory follicles, and 3) the preovulatory LH surge down-regulates ERβ mRNA. These results clearly implicate the physiological importance of ERβ in female reproductive functions.

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Generation of Thyrotropin-Releasing Hormone Receptor 1-Deficient Mice as an Animal Model of Central Hypothyroidism

Molecular Endocrinology ◽

10.1210/me.2004-0017 ◽

2004 ◽

Vol 18 (6) ◽

pp. 1450-1460 ◽

Cited By ~ 49

Author(s):

Roland Rabeler ◽

Jens Mittag ◽

Lars Geffers ◽

Ulrich Rüther ◽

Michael Leitges ◽

...

Keyword(s):

Animal Model ◽

In Situ Hybridization ◽

Mrna Expression ◽

Pcr Analysis ◽

Target Cells ◽

Mrna Levels ◽

Rt Pcr ◽

Central Hypothyroidism ◽

R1 Gene

Abstract To provide an animal model of central hypothyroidism, mice deficient in the TRH-receptor 1 (TRH-R1) gene were generated by homologous recombination. The pituitaries of TRH-R1−/− mice are devoid of any TRH-binding capacity, demonstrating that TRH-R1 is the only receptor localized on TRH target cells of the pituitary. With the exception of some retardation in growth rate, TRH-R1−/− mice appear normal, but compared with control animals they exhibit a considerable decrease in serum T3, T4, and prolactin (PRL) levels but not in serum TSH levels. In situ hybridization histochemistry and real-time RT-PCR analysis revealed that in adult TRH-R1−/− animals TSHβ-mRNA expression is not impaired whereas PRL mRNA and GH mRNA levels are considerably reduced compared with control mice. The numbers of thyrotropes, somatotropes, and lactotropes, however, are not affected by the deletion of the TRH-R1 gene. The mutant mice are fertile, and the dams nourish their pups well, indicating that TRH is not a decisive factor for suckling-induced PRL release. In situ hybridization and quantitative RT-PCR analysis, furthermore, revealed that, as in control animals, pituitary PRL-mRNA expression in TRH-R1−/− is considerably increased during lactation, albeit strongly reduced as compared with lactating control animals.

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Dendrobium Mixture Improved Diabetic Nephropathy in db/db Mice by Regulating TGF-β1/Smads Signal Transduction

10.21203/rs.3.rs-177708/v1 ◽

2021 ◽

Author(s):

Yong Chen ◽

Xiaohui Lin ◽

Yanfang Zheng ◽

Wenzhen Yu ◽

Fan Lin ◽

...

Keyword(s):

Signal Transduction ◽

Diabetic Nephropathy ◽

Mrna Expression ◽

Signaling Pathway ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Fasting Blood Glucose ◽

Blood Lipid ◽

Control Group ◽

Tgf Β1

Abstract BackgroundDendrobium mixture (DMix) is an effective treatment for diabetic nephropathy (DN), but the underlying molecular mechanism remains unclear. In this study, we investigated whether DMix regulates the transforming growth factor-β1 (TGF-β1)/Smads signal transduction pathway. MethodsTwenty-four db/db mice were randomly divided into three groups: the model, DMix, and gliquidone groups, while eight db/m mice were selected as the normal control group. The drug was administered by continuous gavage for 8 weeks. Body weight (BW), kidney weight (KW), kidney index, fasting blood glucose (FBG), blood lipid, 24-hour urinary albumin excretion rate, blood urea nitrogen, and serum creatinine levels were measured. Pathological changes in the renal tissue were observed using a light microscope. Real-time quantitative PCR and immunohistochemical staining were used to detect mRNA expression of TGF-β1 and alpha-smooth muscle actin (α-SMA) genes and proteins, respectively, in renal tissues. TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3, and α-SMA expression levels were measured using western blotting. ResultsDMix significantly reduced FBG level, BW, KW, and blood lipid level, and improved renal function in db/db mice. Histopathology showed that DMix alleviated glomerular mesangial cell proliferation and renal interstitial fibrosis in db/db mice. Additionally, DMix reduced protein and mRNA expression of TGF-β1 and α-SMA, and inhibited Smad2 and Smad3 phosphorylation. ConclusionsThe findings suggest that DMix may inhibit renal fibrosis and delay the progression of DN by regulating the TGF-β1/Smads signaling pathway. Key words: Diabetic nephropathy, Dendrobium mixture, TGF-β1/Smads signaling pathway

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CD44v3-v10 reduces the profibrotic effects of TGF-β1 and attenuates tubular injury in the early stage of chronic obstructive nephropathy

AJP Renal Physiology ◽

10.1152/ajprenal.00340.2013 ◽

2013 ◽

Vol 305 (10) ◽

pp. F1445-F1454 ◽

Cited By ~ 5

Author(s):

Elena Rampanelli ◽

Kasper Rouschop ◽

Gwendoline J. D. Teske ◽

Nike Claessen ◽

Jaklien C. Leemans ◽

...

Keyword(s):

Growth Factor ◽

Transgenic Mice ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Kidney Diseases ◽

Early Stage ◽

Obstructive Nephropathy ◽

Chronic Obstructive ◽

Tubular Damage ◽

Tgf Β1

CD44 family members are cell surface glycoproteins, which are expressed on tubular epithelial cells (TEC) solely upon kidney injury and are involved in renal fibrosis development. Renal interstitial fibrosis is the final manifestation of chronic kidney diseases and is regulated by a complex network of cytokines, including the profibrotic factor transforming growth factor-β1 (TGF-β1) and the two antifibrotic cytokines bone morphogenic protein-7 (BMP-7) and hepatocyte growth factor (HGF). The present study investigates the potential role of CD44 standard (CD44s) and CD44v3-v10 (CD44v3) isoforms as modulators of the balance between TGF-β1 and HGF/BMP-7. CD44s is the shortest and most common isoform. CD44v3-v10 (CD44v3) has heparan sulfate moieties, which enable the binding to HGF/BMP-7, and hence, might exert renoprotective effects. Using transgenic mice overexpressing either CD44s or CD44v3 specifically on proximal TEC, we found that in vitro the overexpression of CD44v3 on primary TEC renders cells less susceptible to TGF-β1 profibrotic actions and more sensitive to BMP-7 and HGF compared with TEC overexpressing CD44s. One day after unilateral ureteric obstruction, obstructed kidneys from CD44v3 transgenic mice showed less tubular damage and myofibroblasts accumulation, which was associated with decreased TGF-β1 signaling and increased BMP-7 synthesis and signaling compared with kidneys from wild-type and CD44s transgenic mice. These data suggest that CD44v3 plays a renoprotective role in early stage of chronic obstructive nephropathy.

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Role of Transforming Growth Factor-β1 in Invasion and Metastasis in Gastric Carcinoma

Journal of Clinical Oncology ◽

10.1200/jco.1999.17.2.607 ◽

1999 ◽

Vol 17 (2) ◽

pp. 607-607 ◽

Cited By ~ 106

Author(s):

Yoshihiko Maehara ◽

Yoshihiro Kakeji ◽

Akira Kabashima ◽

Yasunori Emi ◽

Akihiro Watanabe ◽

...

Keyword(s):

Gastric Cancer ◽

In Situ Hybridization ◽

Growth Factor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

Invasion And Metastasis ◽

Serosal Invasion ◽

Tgf Β1

PURPOSE: Transforming growth factor-beta1 (TGF-β1) is a major modulator of cellular proliferation and extracellular matrix formation. We determined the role of TGF-β1 in invasion and metastasis in gastric cancer. MATERIALS AND METHODS: We detected TGF-β1 expression in primary and lymph node metastatic lesions of gastric cancer, using an antibody and in situ hybridization. The plasma TGF-β1 levels in the peripheral vein and in the tumor drainage vein were assayed. RESULTS: In the cytoplasm of cancer cells, TGF-β1 was immunostained in 35.9% (78 of 217) of primary gastric carcinomas, and this expression was confirmed by in situ hybridization. Of 59 gastric carcinomas with a TGF-β1–negative primary tumor, metastatic lymph nodes were positive for TGF-β1 staining in 32 cases (54.2%). Positive staining of TGF-β1 in gastric cancer tissues was closely related to serosal invasion, infiltrative growth, and lymph node metastasis. Multivariate analysis showed that the expression of TGF-β1 was an independent risk factor for serosal invasion and infiltrative growth of the tumor. The plasma level of TGF-β1 did not differ between TGF-β1–negative and –positive groups. There were also no differences in plasma TGF-β1 levels among each tumor stage, between the peripheral and the tumor drainage veins, and between preoperative and postoperative testings. CONCLUSION: Transforming growth factor-β1 is closely related to the invasion and metastasis of gastric cancer, and production of TGF-β1 in the tumor does not contribute to the total amount of TGF-β1 in the blood circulation. We interpret our observations to mean that in a tumor microenvironment, TGF-β1 alters the biologic behavior of the tumor.

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