Novel molecular mechanisms underlying endothelial and kidney dysfunction in diabetes
Garcia Vaz, Eliana LU
(2020)
In Lund University, Faculty of Medicine Doctoral Dissertation Series
2020:38.
- Abstract
- Diabetes is a chronic disease that is associated with devastating complications. Central to the development of diabetic macro-and microvascular disease is endothelial dysfunction, which appears well before any clinical sign, but importantly, is potentially reversible. We previously demonstrated that hyperglycemia activates the Ca2+/calcineurin transcription factor NFAT in conduit and medium-sized resistance arteries and that in vivo blockade of NFAT signaling abolishes diabetes-driven atherosclerosis. In Paper I we test whether NFAT is involved in the development of endothelial dysfunction in diabetes. NFAT-dependent transcriptional activity was elevated in skin microvessels of diabetic Akita (Ins2+/-) mice when compared to non-diabetic... (More)
- Diabetes is a chronic disease that is associated with devastating complications. Central to the development of diabetic macro-and microvascular disease is endothelial dysfunction, which appears well before any clinical sign, but importantly, is potentially reversible. We previously demonstrated that hyperglycemia activates the Ca2+/calcineurin transcription factor NFAT in conduit and medium-sized resistance arteries and that in vivo blockade of NFAT signaling abolishes diabetes-driven atherosclerosis. In Paper I we test whether NFAT is involved in the development of endothelial dysfunction in diabetes. NFAT-dependent transcriptional activity was elevated in skin microvessels of diabetic Akita (Ins2+/-) mice when compared to non-diabetic littermates. Treatment of diabetic mice with the NFAT blocker A-285222 reduced NFATc3 nuclear accumulation and NFAT-luciferase transcriptional activity in skin
microvessels, resulting in improved microvascular function. A-285222 treatment enhanced dermis eNOS expression and plasma NO
levels of diabetic mice. It also prevented induction of inflammatory cytokines IL-6 and osteopontin, lowered plasma Et-1 and blood
pressure, and improved mice survival without affecting blood glucose. In vivo inhibition of NFAT may represent a novel therapeutic modality to preserve endothelial function in diabetes. Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. In Paper II we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and iethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. We also found that amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs,which determines clathrin-mediated endocytosis. Results provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.
Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). COX-2 is also constitutively expressed, in the absence of overt
nflammation, and its expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. In Paper III we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with NFAT activity and was sensitive to inhibition of calcineurin (CaN)-dependent NFAT activation. However, CaN/NFAT regulation did not contribute to constitutive expression elsewhere or to nflammatory COX-2 induction at any site. Results suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system. One of the major bottlenecks in diabetes research has been the lack of animal models that mimic diabetic complications as it is seen in humans. In Paper IV we characterized the novel BBDR.cg-lepr.cp rat model that we recently developed. BBDR.cg-lepr.cp rats were obese and had larger livers, hearts and kidneys when compared to control littermates. Two subgroups of male BBDR.cg-lepr.cp rats could be distinguished based on their blood glucose, one severely diabetic and one moderately or pre-diabetic. Serum insulin, leptin, triglycerides and cholesterol were increased in all BBDR.cg-lepr.cp rats when compared to controls; with triglycerides and cholesterol being higher in the severely diabetic rats, and leptin and insulin being higher in the pre-diabetic rats. Albumin/creatinine ratio was dramatically increased and histological examination of kidneys displayed loss of glomerular podocin expression, dilated and filled tubuli and interstitial fibrosis, all of which was more pronounced in the severely diabetic rats. BBDR.cg-lepr.cp rats had significantly higher systolic blood pressure, but surprisingly only the moderately diabetic rats had increased aortic mRNA expression of endothelial activation and inflammatory markers. Our findings show that BBDR.cg-lepr.cp rats represent a promising new model that can be used for pathogenic studies as well as for the development and evaluation of new therapies. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/928e9741-5933-458f-a272-56d4c5952373
- author
- Garcia Vaz, Eliana
LU
- supervisor
-
- Maria F Gomez LU
- Leif Groop LU
- opponent
-
- Dr. Porter, Karen E., Leeds Institute of Cardiovascular and Metabolic Medicine, Leeds University
- organization
- publishing date
- 2020
- type
- Thesis
- publication status
- published
- subject
- keywords
- NFAT, diabetes, hyperglycemia, endothelial dysfunction, diabetic kidney disease, COX-2, BBDR.cg- lepr.cp
- in
- Lund University, Faculty of Medicine Doctoral Dissertation Series
- volume
- 2020:38
- pages
- 129 pages
- publisher
- Lund University, Faculty of Medicine
- defense location
- Agardh föreläsningssal, CRC, Jan Waldenströms gata 35, Skånes Universitetssjukhus i Malmö
- defense date
- 2020-04-27 09:00:00
- ISSN
- 1652-8220
- ISBN
- 978-91-7619-899-5
- language
- English
- LU publication?
- yes
- id
- 928e9741-5933-458f-a272-56d4c5952373
- date added to LUP
- 2020-02-28 10:50:32
- date last changed
- 2024-09-09 07:44:30
@phdthesis{928e9741-5933-458f-a272-56d4c5952373,
abstract = {{Diabetes is a chronic disease that is associated with devastating complications. Central to the development of diabetic macro-and microvascular disease is endothelial dysfunction, which appears well before any clinical sign, but importantly, is potentially reversible. We previously demonstrated that hyperglycemia activates the Ca2+/calcineurin transcription factor NFAT in conduit and medium-sized resistance arteries and that in vivo blockade of NFAT signaling abolishes diabetes-driven atherosclerosis. In Paper I we test whether NFAT is involved in the development of endothelial dysfunction in diabetes. NFAT-dependent transcriptional activity was elevated in skin microvessels of diabetic Akita (Ins2+/-) mice when compared to non-diabetic littermates. Treatment of diabetic mice with the NFAT blocker A-285222 reduced NFATc3 nuclear accumulation and NFAT-luciferase transcriptional activity in skin <br/>microvessels, resulting in improved microvascular function. A-285222 treatment enhanced dermis eNOS expression and plasma NO <br/>levels of diabetic mice. It also prevented induction of inflammatory cytokines IL-6 and osteopontin, lowered plasma Et-1 and blood <br/>pressure, and improved mice survival without affecting blood glucose. In vivo inhibition of NFAT may represent a novel therapeutic modality to preserve endothelial function in diabetes. Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. In Paper II we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and iethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. We also found that amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs,which determines clathrin-mediated endocytosis. Results provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria. <br/>Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). COX-2 is also constitutively expressed, in the absence of overt <br/> nflammation, and its expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. In Paper III we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with NFAT activity and was sensitive to inhibition of calcineurin (CaN)-dependent NFAT activation. However, CaN/NFAT regulation did not contribute to constitutive expression elsewhere or to nflammatory COX-2 induction at any site. Results suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system. One of the major bottlenecks in diabetes research has been the lack of animal models that mimic diabetic complications as it is seen in humans. In Paper IV we characterized the novel BBDR.cg-lepr.cp rat model that we recently developed. BBDR.cg-lepr.cp rats were obese and had larger livers, hearts and kidneys when compared to control littermates. Two subgroups of male BBDR.cg-lepr.cp rats could be distinguished based on their blood glucose, one severely diabetic and one moderately or pre-diabetic. Serum insulin, leptin, triglycerides and cholesterol were increased in all BBDR.cg-lepr.cp rats when compared to controls; with triglycerides and cholesterol being higher in the severely diabetic rats, and leptin and insulin being higher in the pre-diabetic rats. Albumin/creatinine ratio was dramatically increased and histological examination of kidneys displayed loss of glomerular podocin expression, dilated and filled tubuli and interstitial fibrosis, all of which was more pronounced in the severely diabetic rats. BBDR.cg-lepr.cp rats had significantly higher systolic blood pressure, but surprisingly only the moderately diabetic rats had increased aortic mRNA expression of endothelial activation and inflammatory markers. Our findings show that BBDR.cg-lepr.cp rats represent a promising new model that can be used for pathogenic studies as well as for the development and evaluation of new therapies.}},
author = {{Garcia Vaz, Eliana}},
isbn = {{978-91-7619-899-5}},
issn = {{1652-8220}},
keywords = {{NFAT, diabetes, hyperglycemia, endothelial dysfunction, diabetic kidney disease, COX-2, BBDR.cg- lepr.cp}},
language = {{eng}},
publisher = {{Lund University, Faculty of Medicine}},
school = {{Lund University}},
series = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
title = {{Novel molecular mechanisms underlying endothelial and kidney dysfunction in diabetes}},
volume = {{2020:38}},
year = {{2020}},
}