Altered Endothelial Nitric Oxide Synthase Targeting and Conformation and Caveolin-1 Expression in the Diabetic Kidney

Komers, Radko; Schutzer, William E.; Reed, John; Lindsley, Jessie N.; Oyama, T; Buck, David C.; Mader, Scott L.; Anderson, Sharon

doi:10.2337/db05-1595

Cited by 73 publications

(71 citation statements)

References 50 publications

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“…Tissue preparation and western blot analysis were performed as previously described 17,29 using the primary antibody raised against KDM6A (Abcam) or Ezh2 (Upstate, Billerica, MA, USA).…”

Section: Immunoblottingmentioning

confidence: 99%

Epigenetic changes in renal genes dysregulated in mouse and rat models of type 1 diabetes

Komers

Mar

Denisenko

et al. 2013

Laboratory Investigation

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Epigenetic processes are increasingly being recognized as factors in the pathophysiology of diabetes complications, but few chromatin studies have been done in diabetic nephropathy (DN). We hypothesized that changes in mRNA expression of DN-related genes are associated with epigenetic alterations and aberrant expression of histone-modifying enzymes. RT-PCR and a matrix-chromatin immunoprecipitation platform were used to examine renal mRNA expression, RNA polymerase II (Pol II) recruitment, and epigenetic marks at DN-related genes in the mouse (OVE26) and streptozotocininduced rat models of type 1 diabetes. Diabetes induced renal expression of Cox2, S100A4/FSP-1, and vimentin genes in both the mouse and the rat models of DN. Mcp-1 and laminin g1 (Lamc1) expression were increased in diabetic mice but not in rats. Comparison of mRNA and Pol II levels suggested that the diabetes-induced expression of these transcripts is mediated by transcriptional and posttranscriptional processes. Decreases in histone H3 lysine 27 tri-methylation (H3K27m3, silencing mark) and increases in H3 lysine 4 di-methylation (H3K4m2, activating mark) levels were the most consistent epigenetic alterations in the tested genes. In agreement with these results, immunoblot analysis showed increased protein abundance of renal H3K27m2/3 demethylase KDM6A, but no changes in cognate methyltransferase Ezh2 in kidneys of the OVE26 mice compared with controls. In diabetic rats, Ezh2 expression was higher without changes in KDM6A, demonstrating that mechanisms of DN-induced H3K27m3 loss could be species specific. In summary, we show that altered mRNA expression of some DN-related genes is associated with changes in Pol II recruitment and a corresponding decrease in repressive H3K27m3 at the selected loci, and at least in mice with equivalent changes in renal expression of cognate histone-modifying enzymes. This pattern could contribute to diabetes-mediated transitions in chromatin that facilitate transcriptional changes in the diabetic kidney.

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“…Tissue preparation and western blot analysis were performed as previously described 17,29 using the primary antibody raised against KDM6A (Abcam) or Ezh2 (Upstate, Billerica, MA, USA).…”

Section: Immunoblottingmentioning

confidence: 99%

Epigenetic changes in renal genes dysregulated in mouse and rat models of type 1 diabetes

Komers

Mar

Denisenko

et al. 2013

Laboratory Investigation

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“…The kidneys were homogenized in lysis buffer containing protease and phosphatase inhibitors and analysed by immunoblotting as previously described (Komers et al, 2006), using primary antibodies raised against phospho-serine 19-MLC (P-MLC, 1:800, Cell Signaling, Beverly, MA, USA cat. #3671); and ROCK1 and ROCK2 (1:500, BD Transduction Laboratories™, 2350 Qume Drive, San Jose, CA, USA, cat.…”

Section: Immunoblottingmentioning

confidence: 99%

Effects of systemic inhibition of Rho kinase on blood pressure and renal haemodynamics in diabetic rats

Komers

Oyama

Beard

et al. 2010

British J Pharmacology

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BACKGROUND AND PURPOSE The RhoA/Rho associated kinases (ROCK) pathway has been implicated in the pathophysiology of diabetic nephropathy (DN). Early stages of diabetes are associated with renal haemodynamic changes, contributing to later development of DN. However, the role of RhoA/ROCK, known regulators of vascular tone, in this process has not been studied.EXPERIMENTAL APPROACH Blood pressure (BP), glomerular filtration (GFR), effective renal plasma flow and filtration fraction (FF) in response to the ROCK inhibitors Y27632 (0.1 and 0.5 mg·kg−1) and fasudil (0.3 and 1.5 mg·kg−1) were examined in streptozotocin‐diabetic rats and non‐diabetic controls.KEY RESULTS Diabetic rats demonstrated baseline increases in GFR and FF. In contrast to similar decreases in BP in diabetic and control rats, renal vasodilator effects and a decrease in FF, following ROCK inhibition were observed only in diabetic rats. The vasodilator effects of Y27632 and a further decrease in FF, were also detected in diabetic rats pretreated with the angiotensin antagonist losartan. The effects of ROCK inhibitors in diabetic rats were modulated by prior protein kinase C (PKC)β inhibition with ruboxistaurin, which abolished their effects on FF. Consistent with the renal vasodilator effects, the ROCK inhibitors reduced phosphorylation of myosin light chain in diabetic kidneys.CONCLUSIONS AND IMPLICATIONS The results indicate greater dependence of renal haemodynamics on RhoA/ROCK and beneficial haemodynamic effects of ROCK inhibitors in diabetes, which were additive to the effects of losartan. In this process, the RhoA/ROCK pathway operated downstream of or interacted with, PKCβ in some segments of the renal vascular tree.

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“…For instance, diabetic persons often have poor peripheral circulation, which leads to ischemic injury, impaired wound healing, and impaired vision, all of which involve altered endothelial cell function that relates to endothelial nitric oxide (NO) synthase (eNOS) and the generation of NO (2,21). Several studies have shown that HDL acting through its receptor, SR-BI, can stimulate eNOS to produce NO (4,16,24,36).…”

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Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin

White

Guerin

Swanson

et al. 2008

American Journal of Physiology-Cell Physiology

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Smart EJ. Diabetic HDL-associated myristic acid inhibits acetylcholineinduced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin. Am J Physiol Cell Physiol 294: C295-C305, 2008. First published October 31, 2007 doi:10.1152/ajpcell.00042.2007.-In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients. fatty acid; lipoprotein; diabetes; signaling CARDIOVASCULAR DISEASES are influenced by both genetic and environmental factors that can negatively or positively influence the development and progression of cardiovascular diseases, such as plasma levels of LDL and HDL (25,26). High plasma levels of LDL promote the accumulation of cholesterol in vessel wall macrophages and the formation of atherosclerotic plaque (3,

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Altered Endothelial Nitric Oxide Synthase Targeting and Conformation and Caveolin-1 Expression in the Diabetic Kidney

Cited by 73 publications

References 50 publications

Epigenetic changes in renal genes dysregulated in mouse and rat models of type 1 diabetes

Epigenetic changes in renal genes dysregulated in mouse and rat models of type 1 diabetes

Effects of systemic inhibition of Rho kinase on blood pressure and renal haemodynamics in diabetic rats

Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin

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