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The Role of Mitochondrial Uncoupling in the Development of Diabetic Nephropathy
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Diabetes is closely associated with increased oxidative stress, especially originating from the mitochondria. A mechanism to reduce increased mitochondria superoxide production is to reduce the mitochondria membrane potential by releasing protons across the mitochondria membrane. This phenomenon is referred to as mitochondria uncoupling since oxygen is consumed independently of ATP being produced and can be mediated by Uncoupling Proteins (UCPs). However, increased oxygen consumption is potentially detrimental for the kidney since it can cause tissue hypoxia. Therefore, this thesis aimed to investigate the role of mitochondria uncoupling for development of diabetic nephropathy.

     UCP-2 was demonstrated to be the only isoform expressed in the kidney, and localized to tubular segments performing the majority of tubular electrolyte transport. Streptozotocin-induced diabetes in rats increased UCP-2 protein expression and correlated to increased non-transport dependent oxygen consumption in isolated proximal tubular cells. These effects were prevented by intense insulin treatment to the diabetic animals demonstrating a pivotal role of hyperglycemia. Importantly, elevated UCP-2 protein expression increased mitochondria uncoupling in mitochondria isolated from diabetic kidneys. Mitochondria uncoupling and altered morphology was also evident in kidneys from db/db-mice, a model of type-2 diabetes, together with proteinuria and glomerular hyperfiltration which are both clinical manifestations of diabetic nephropathy. Treatment with the antioxidant coenzyme Q10 prevented mitochondria uncoupling as well as morphological and functional alterations in these kidneys. Acute knockdown of UCP-2 paradoxically increased mitochondria uncoupling in a mechanism involving the adenosine nucleotide transporter. Increased uncoupling via adenosine nucleotide transporter decreased mitochondria membrane potential and kidney oxidative stress but did not affect glomerular filtration rate, renal blood flow, total kidney oxygen consumption or intrarenal tissue oxygen tension.

     The role of increased mitochondria oxygen consumption was investigated by administering the chemical uncoupler dinitrophenol to healthy rats. Importantly, increased mitochondria oxygen consumption resulted in kidney tissue hypoxia, proteinuria and increased staining of the tubular injury marker vimentin, demonstrating a crucial role of increased oxygen consumption per se and the resulting kidney tissue hypoxia for the development of nephropathy.

     Taken together, the data presented in this thesis establishes an important role of mitochondria uncoupling for the development of diabetic nephropathy.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2012. , 74 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 738
Keyword [en]
Kidney, mitochondria, Uncoupling Protein-2, Adenosine Nucleotide Transporter, uncoupling, diabetes, diabetic nephropathy, db/db, dinitrophenol, Coenzyme Q10, oxygen, rats, mice
National Category
Physiology
Research subject
Physiology; Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-167815ISBN: 978-91-554-8266-4 (print)OAI: oai:DiVA.org:uu-167815DiVA: diva2:488170
Public defence
2012-03-16, B42, Biomedical Center, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-02-24 Created: 2012-02-01 Last updated: 2012-03-01Bibliographically approved
List of papers
1. Identification and distribution of uncoupling protein isoforms in the normal and diabetic rat kidney
Open this publication in new window or tab >>Identification and distribution of uncoupling protein isoforms in the normal and diabetic rat kidney
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2009 (English)In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 645, 205-212 p.Article in journal (Refereed) Published
Abstract [en]

Uncoupling protein (UCP)-2 and -3 are ubiquitously expressed throughout the body but there is currently no information regarding the expression and distribution of the different UCP isoforms in the kidney. Due to the known cross-reactivity of the antibodies presently available for detection of UCP-2 and -3 proteins, we measured the mRNA expression of UCP-1, -2 and -3 in the rat kidney in order to detect the kidney-specific UCP isoforms. Thereafter, we determined the intrarenal distribution of the detected UCP isoforms using immunohistochemistry. Thereafter, we compared the protein levels in control and streptozotocin-induced diabetic rats using Western blot. Expressions of the UCP isoforms were also performed in brown adipose tissue and heart as positive controls for UCP-1 and 3, respectively. UCP-2 mRNA was the only isoform detected in the kidney. UCP-2 protein expression in the kidney cortex was localized to proximal tubular cells, but not glomerulus or distal nephron. In the medulla, UCP-2 was localized to cells of the medullary thick ascending loop of Henle, but not to the vasculature or parts of the nephron located in the inner medulla. Western blot showed that diabetic kidneys have about 2.5-fold higher UCP-2 levels compared to controls. In conclusion, UCP-2 is the only isoform detectable in the kidney and UCP-2 protein can be detected in proximal tubular cells and cells of the medullary thick ascending loop of Henle. Furthermore, diabetic rats have increased UCP-2 levels compared to controls, but the mechanisms underlying this increase and its consequences warrants further studies.

Place, publisher, year, edition, pages
New York: Springer, 2009
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-102380 (URN)10.1007/978-0-387-85998-9_32 (DOI)000262627100032 ()19227473 (PubMedID)978-0-387-85997-2 (ISBN)
Conference
Annual Conference of the International-Society-on-Oxygen-Transport-to-Tissue
Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2017-12-13
2. Diabetes-induced up-regulation of uncoupling protein-2 results in increased mitochondrial uncoupling in kidney proximal tubular cells
Open this publication in new window or tab >>Diabetes-induced up-regulation of uncoupling protein-2 results in increased mitochondrial uncoupling in kidney proximal tubular cells
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2008 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1777, no 7-8, 935-940 p.Article in journal (Refereed) Published
Abstract [en]

We have previously reported increased O(2) consumption unrelated to active transport by tubular cells and up-regulated mitochondrial uncoupling protein (UCP)-2 expressions in diabetic kidneys. It is presently unknown if the increased UCP-2 levels in the diabetic kidney results in mitochondrial uncoupling and increased O(2) consumption, which we therefore investigated in this study. The presence of UCP-2 in proximal tubular cells was confirmed by immunohistochemistry and found to be increased (western blot) in homogenized tissue and isolated mitochondria from kidney cortex of diabetic rats. Isolated proximal tubular cells had increased total and ouabain-insensitive O(2) consumption compared to controls. Isolated mitochondria from diabetic animals displayed increased glutamate-stimulated O(2) consumption (in the absence of ADP and during inhibition of the ATP-synthase by oligomycin) compared to controls. Guanosine diphosphate, an UCP inhibitor, and bovine serum albumin which removes fatty acids that are essential for UCP-2 uncoupling activity, independently prevented the increased glutamate-stimulated O(2) consumption in mitochondria from diabetic animals. In conclusion, diabetic rats have increased mitochondrial UCP-2 expression in renal proximal tubular cells, which results in mitochondrial uncoupling and increased O(2) consumption. This mechanism may be protective against diabetes-induced oxidative stress, but will increase O(2) usage. The subsequently reduced O(2) availability may contribute to diabetes-induced progressive kidney damage.

Keyword
Diabetes mellitus, kidney, mitochondria, uncoupling protein-2, oxygen consumption
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-110142 (URN)10.1016/j.bbabio.2008.03.030 (DOI)000257696400054 ()18439413 (PubMedID)
Note
Conference Information: 15th European Bioenergetic Conference Trinity Coll, Dublin, IRELAND, JUL 19-24, 2008 Available from: 2009-11-04 Created: 2009-11-04 Last updated: 2017-12-12Bibliographically approved
3. Coenzyme Q10 prevents GDP-sensitive mitochondrial uncoupling, glomerular hyperfiltration and proteinuria in kidneys from db/db-mice as a model of type 2 diabetes
Open this publication in new window or tab >>Coenzyme Q10 prevents GDP-sensitive mitochondrial uncoupling, glomerular hyperfiltration and proteinuria in kidneys from db/db-mice as a model of type 2 diabetes
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2012 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 55, no 5, 1535-1543 p.Article in journal (Refereed) Published
Abstract [en]

Aims/hypothesis: Increased oxygen consumption results in kidney tissue hypoxia, which is proposed to contribute to the development of diabetic nephropathy. Oxidative stress causes increased oxygen consumption in type 1 diabetic kidneys, partly mediated by uncoupling protein-2 (UCP-2)-induced mitochondrial uncoupling. The present study investigates the role of UCP-2 and oxidative stress in mitochondrial oxygen consumption and kidney function in db/db mice as a model of type 2 diabetes.

Methods: Mitochondrial oxygen consumption, glomerular filtration rate and proteinuria were investigated in db/db mice and corresponding controls with and without coenzyme Q10 (CoQ10) treatment.

Results: Untreated db/db mice displayed mitochondrial uncoupling, manifested as glutamate-stimulated oxygen consumption (2.7 +/- 0.1 vs 0.2 +/- 0.1 pmol O-2 s(-1) [mg protein](-1)), glomerular hyperfiltration (502 +/- 26 vs 385 +/- 3 mu l/min), increased proteinuria (21 +/- 2 vs 14 +/- 1, mu g/24 h), mitochondrial fragmentation (fragmentation score 2.4 +/- 0.3 vs 0.7 +/- 0.1) and size (1.6 +/- 0.1 vs 1 +/- 0.0 mu m) compared with untreated controls. All alterations were prevented or reduced by CoQ10 treatment. Mitochondrial uncoupling was partly inhibited by the UCP inhibitor GDP (-1.1 +/- 0.1 pmol O-2 s(-1) [mg protein](-1)). UCP-2 protein levels were similar in untreated control and db/db mice (67 +/- 9 vs 67 +/- 4 optical density; OD) but were reduced in CoQ10 treated groups (43 +/- 2 and 38 +/- 7 OD).

Conclusions/interpretation: db/db mice displayed oxidative stress-mediated activation of UCP-2, which resulted in mitochondrial uncoupling and increased oxygen consumption. CoQ10 prevented altered mitochondrial function and morphology, glomerular hyperfiltration and proteinuria in db/db mice, highlighting the role of mitochondria in the pathogenesis of diabetic nephropathy and the benefits of preventing increased oxidative stress.

Keyword
db/db-mice, Kidney, Mitochondria, Type 2 diabetes, Uncoupling protein-2
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-167777 (URN)10.1007/s00125-012-2469-5 (DOI)000302994600036 ()
Available from: 2012-02-01 Created: 2012-02-01 Last updated: 2017-12-08Bibliographically approved
4. Acute knockdown of uncoupling protein-2 increases mitochondria uncoupling via the adenine nucleotide transporter and decreases oxidative stress in diabetic kidneys
Open this publication in new window or tab >>Acute knockdown of uncoupling protein-2 increases mitochondria uncoupling via the adenine nucleotide transporter and decreases oxidative stress in diabetic kidneys
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, e39635- p.Article in journal (Refereed) Published
Abstract [en]

Increased O2 metabolism resulting in chronic hypoxia is common in models of endstage renal disease. Mitochondrial uncoupling increases O2 consumption but the ensuing reduction in mitochondrial membrane potential may limit excessive oxidative stress. The present study addressed the hypothesis that mitochondrial uncoupling regulates mitochondria function and oxidative stress in the diabetic kidney. Isolated mitochondria from kidney cortex of control and streptozotocin-induced diabetic rats were studied before and after siRNA knockdown of uncoupling protein-2 (UCP-2). Diabetes resulted in increased UCP-2 protein expression and UCP-2-mediated uncoupling, but normal mitochondria membrane potential. This uncoupling was inhibited by GDP, which also increased the membrane potential. siRNA reduced UCP-2 protein expression in controls and diabetics (−30–50%), but paradoxically further increased uncoupling and markedly reduced the membrane potential. This siRNA mediated uncoupling was unaffected by GDP but was blocked by ADP and carboxyatractylate (CAT). Mitochondria membrane potential after UCP-2 siRNA was unaffected by GDP but increased by CAT. This demonstrated that further increased mitochondria uncoupling after siRNA towards UCP-2 is mediated through the adenine nucleotide transporter (ANT). The increased oxidative stress in the diabetic kidney, manifested as increased thiobarbituric acids, was reduced by knocking down UCP-2 whereas whole-body oxidative stress, manifested as increased circulating malondialdehyde, remained unaffected. All parameters investigated were unaffected by scrambled siRNA. In conclusion, mitochondrial uncoupling via UCP-2 regulates mitochondria membrane potential in diabetes. However, blockade of the diabetes-induced upregulation of UCP- 2 results in excessive uncoupling and reduced oxidative stress in the kidney via activation of ANT.

National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-167782 (URN)10.1371/journal.pone.0039635 (DOI)000305966500017 ()
Available from: 2012-02-01 Created: 2012-02-01 Last updated: 2017-12-08Bibliographically approved
5. Kidney function after in vivo gene silencing of Uncoupling Protein-2 in streptozotocin-induced diabetic rats
Open this publication in new window or tab >>Kidney function after in vivo gene silencing of Uncoupling Protein-2 in streptozotocin-induced diabetic rats
2013 (English)In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 765, 217-223 p.Article in journal (Refereed) Published
Abstract [en]

Kidney uncoupling protein 2 (UCP-2) increases in streptozotocin-induced diabetes, resulting in mitochondria uncoupling, i.e., increased oxygen consumption unrelated to active transport. The present study aimed to investigate the role of UCP-2 for normal and diabetic kidney function utilizing small interference RNA (siRNA) to reduce protein expression. Diabetic animals had increased glomerular filtration rate and kidney oxygen consumption, resulting in decreased oxygen tension and transported sodium per consumed oxygen. UCP-2 protein levels decreased 2 and 50% after UCP-2 siRNA administration in control and diabetic animals respectively. Kidney function was unaffected by in vivo siRNA-mediated gene silencing of UCP-2. The reason for the lack of effect of reducing UCP-2 is presently unknown but may involve compensatory mitochondrial uncoupling by the adenosine nucleotide transporter.

Place, publisher, year, edition, pages
Springer-Verlag New York, 2013
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-167783 (URN)10.1007/978-1-4614-4989-8_30 (DOI)000339280100031 ()978-1-4614-4771-9 (ISBN)978-1-4614-4989-8 (ISBN)
Available from: 2012-02-01 Created: 2012-02-01 Last updated: 2017-12-08
6. Increased mitochondria uncoupling results in kidney tissue hypoxia and proteinuria.
Open this publication in new window or tab >>Increased mitochondria uncoupling results in kidney tissue hypoxia and proteinuria.
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(English)Manuscript (preprint) (Other academic)
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-167786 (URN)
Available from: 2012-02-01 Created: 2012-02-01 Last updated: 2012-03-01

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