Repression of hypoxia-inducible factor-1 contributes to increased mitochondrial reactive oxygen species production in diabetes
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Evaluation Summary:
The authors report that hyperglicemia suppresses HIF1a activity in models of diabetic nephropathy, which in turn leads to cell death by increasing mitochondrial ROS. The notion that HIF1a regulates mitochondrial respiration and thus mitochondrial ROS production is well documented in numerous systems. However, this model has not been tested in the context of diabetic nephropathy so far. The study is thus novel, informative and with interesting translational implications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Excessive production of mitochondrial reactive oxygen species (ROS) is a central mechanism for the development of diabetes complications. Recently, hypoxia has been identified to play an additional pathogenic role in diabetes. In this study, we hypothesized that ROS overproduction was secondary to the impaired responses to hypoxia due to the inhibition of hypoxia-inducible factor-1 (HIF-1) by hyperglycemia.
Methods:
The ROS levels were analyzed in the blood of healthy subjects and individuals with type 1 diabetes after exposure to hypoxia. The relation between HIF-1, glucose levels, ROS production and its functional consequences were analyzed in renal mIMCD-3 cells and in kidneys of mouse models of diabetes.
Results:
Exposure to hypoxia increased circulating ROS in subjects with diabetes, but not in subjects without diabetes. High glucose concentrations repressed HIF-1 both in hypoxic cells and in kidneys of animals with diabetes, through a HIF prolyl-hydroxylase (PHD)-dependent mechanism. The impaired HIF-1 signaling contributed to excess production of mitochondrial ROS through increased mitochondrial respiration that was mediated by Pyruvate dehydrogenase kinase 1 (PDK1). The restoration of HIF-1 function attenuated ROS overproduction despite persistent hyperglycemia, and conferred protection against apoptosis and renal injury in diabetes.
Conclusions:
We conclude that the repression of HIF-1 plays a central role in mitochondrial ROS overproduction in diabetes and is a potential therapeutic target for diabetic complications. These findings are timely since the first PHD inhibitor that can activate HIF-1 has been newly approved for clinical use.
Funding:
This work was supported by grants from the Swedish Research Council, Stockholm County Research Council, Stockholm Regional Research Foundation, Bert von Kantzows Foundation, Swedish Society of Medicine, Kung Gustaf V:s och Drottning Victorias Frimurarestifelse, Karolinska Institute’s Research Foundations, Strategic Research Programme in Diabetes, and Erling-Persson Family Foundation for S-B.C.; grants from the Swedish Research Council and Swedish Heart and Lung Foundation for T.A.S.; and ERC consolidator grant for M.M.
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Evaluation Summary:
The authors report that hyperglicemia suppresses HIF1a activity in models of diabetic nephropathy, which in turn leads to cell death by increasing mitochondrial ROS. The notion that HIF1a regulates mitochondrial respiration and thus mitochondrial ROS production is well documented in numerous systems. However, this model has not been tested in the context of diabetic nephropathy so far. The study is thus novel, informative and with interesting translational implications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this study, Zheng and colleagues report the novel findings that in diabetic models hyperglycemia suppress HIF1a in a PHD-dependent manner, and this in turn leads to increased mitochondrial ROS and cell death. Both the increased ROS and the cell death are prevented by increasing HIF1a activity either pharmacologically or genetically.
The paper is novel, informative, and with interesting translational implications. The authors used a variety of in vitro and in vivo models for the testing of their hypothesis, with special emphasis on a model of diabetic nephropathy.
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Reviewer #2 (Public Review):
The manuscript by Zheng et al addresses the question of whether abnormal HIF stabilisation in response to hypoxia could be responsible for the increased ROS generation in diabetes, and whether hyperglycaemia could be the driver for the ROS damage and cell death in hypoxia. Diabetes complications across multiple organs are associated with oxidative damage and are accompanied by restricted oxygen delivery due to vascular dysfunction. Thus, understanding how these two phenotypes are linked by diabetes is an important question for advancing our understanding of diabetes complications and to identify novel therapeutic targets.
Strengths
This study uses multiple different techniques to address this question, using cell culture, animal models as well as human samples.
Mechanistic conclusions are based on both …
Reviewer #2 (Public Review):
The manuscript by Zheng et al addresses the question of whether abnormal HIF stabilisation in response to hypoxia could be responsible for the increased ROS generation in diabetes, and whether hyperglycaemia could be the driver for the ROS damage and cell death in hypoxia. Diabetes complications across multiple organs are associated with oxidative damage and are accompanied by restricted oxygen delivery due to vascular dysfunction. Thus, understanding how these two phenotypes are linked by diabetes is an important question for advancing our understanding of diabetes complications and to identify novel therapeutic targets.
Strengths
This study uses multiple different techniques to address this question, using cell culture, animal models as well as human samples.
Mechanistic conclusions are based on both genetic and pharmacological approaches, adding strength to the key findings.
This is an excellent continuation of high-quality work conducted by this group in this field. The conclusions have far reaching consequences within the field.
Weaknesses
Although the authors establish a translational pipeline for their findings, from cells to animals to humans, the findings from the human studies could be interpreted in alternative ways to that presented. Cells cultured for 24hrs in hypoxia have sufficient time to activate the HIF transcription factor, its downstream targets and result in a functional outcome for the cell. Humans exposed to hypoxia for 1 hr will not have the same time to induce the HIF-dependent effects, and other mechanisms will be at play. To some extent this is due to the difficulties of performing mechanistic studies in humans, however, care should be taken in not oversimplifying findings and instead highlighting alternative conclusions.
Reactive oxygen species are a group of molecules comprising different species with different reactivities. It is important to be clear which aspects of ROS and their downstream damage are being measured with these methodologies
Care must be taken to ensure the correct statistical analysis is performed based on the groups assessed, the study design and the scientific question being asked.
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Reviewer #3 (Public Review):
Strengths:
1. Oxidative stress and damage is central to pathology of diabetic complications. As mitochondria are a key generator of ROS-mediated damage, the authors nicely connect glucose-dependent HIF1a deficiency with the development of mitochondrial ROS-mediated damage.
2. Approaches to understand and target oxidant stress in diabetic complications are elusive and the authors nicely delineate a PHD2-dependent mechanism by which HIF1a levels decline, subsequently giving rise to renal dysfunction.
3. Notably, improvement of HIF1a levels by PHD2 pharmacologic or genetic inhibition appears to ameliorate renal injury independent of glycemic control. This could be of high translational value as therapies to ameliorate diabetic nephropathy do not exist currently in the clinic.Weaknesses:
1. The mechanism by …Reviewer #3 (Public Review):
Strengths:
1. Oxidative stress and damage is central to pathology of diabetic complications. As mitochondria are a key generator of ROS-mediated damage, the authors nicely connect glucose-dependent HIF1a deficiency with the development of mitochondrial ROS-mediated damage.
2. Approaches to understand and target oxidant stress in diabetic complications are elusive and the authors nicely delineate a PHD2-dependent mechanism by which HIF1a levels decline, subsequently giving rise to renal dysfunction.
3. Notably, improvement of HIF1a levels by PHD2 pharmacologic or genetic inhibition appears to ameliorate renal injury independent of glycemic control. This could be of high translational value as therapies to ameliorate diabetic nephropathy do not exist currently in the clinic.Weaknesses:
1. The mechanism by which hyperglycemia precipitates PHD2-dependent HIF1a degradation and activation of renal injury is not clear.
2. The induction of mitochondrial damage to elicit mitochondrial ROS and subsequent renal compromise downstream of HIF1a deficiency is proposed to be via PDK1. It would strengthen the paper if the PDK1-dependent mechanism was further solidified.
3. Clinical data on patients with uncontrolled T1D and impaired circulating hypoxia responses are very interesting, but it is unclear how they directly relate to the renal specific findings presented in the remainder of the manuscript. -
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