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The role of hypoxia for the development of diabetic nephropathy: Temporal relationship and involvement of endothelin receptor signaling
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Linköping University, Center for Medical Image Science and Visualization (CMIV). (Experimentell Njurmedicin)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Diabetic nephropathy is one of the most common causes of end stage renal disease and develops in approximately one third of all diabetes patients. Disease progression is characterized by deteriorating glomerular filtration rate and escalating urinary albumin/protein excretion; both are used as clinical markers for disease progression. Recently, it has been proposed that intrarenal hypoxia is a unifying mechanism for chronic kidney disease, including diabetic nephropathy. Several mechanistic pathways have been linked to the development of intrarenal hypoxia and diabetic nephropathy including increased angiotensin II signaling, oxidative stress and hyperglycemia per se. Furthermore, pathological endothelin signaling has recently immerged as a possible contributing factor for chronic kidney disease and diabetic nephropathy. The overall aims of this thesis were therefore to determine the temporal relationship between development of intrarenal hypoxia and kidney disease as well as elucidate the potential link between endothelin signaling, intrarenal hypoxia and kidney disease in experimental insulinopenic diabetes.

It is well established that different mouse strains have different susceptibility for kidney and cardiovascular disease. The first step was therefore to compare four commonly used mouse strains with regards to development of kidney disease after onset of insulinopenic diabetes. From the results of this study, we concluded that the NMRI mouse strain has a disease progression closest to the human disease and this strain was chosen in the subsequent studies in mice.

The next step was to adapt and optimize a suitable method for repetitive measurements of intrarenal oxygen tension during the course of disease development. Electron paramagnetic resonance (EPR) oximetry had previously been used in tumor biology and was now adapted and optimized for measurements of kidney oxygenation in our diabetic mouse model. EPR oximetry in normoglycemic control mice recorded cortical oxygen tension values similar to previous reports using invasive techniques. Surprisingly, intrarenal hypoxia developed already within the first 72h after induction of hyperglycemia and persisted throughout the two-week study period. Importantly, this was well before albuminuria developed.

The final part of this thesis was to investigate the role of endothelin signaling for the intrarenal hypoxia in a diabetic rat model. Endothelin 1 signals via two distinctly different receptor-mediated pathways. In normal physiology, endothelin 1 binding to endothelin receptor type A (ETA) induces vasoconstriction, which can be blocked by the specific ETA antagonist BQ123, whereas endothelin 1 binding to endothelin receptor type B (ETB) induces nitric oxide-dependent vasodilation. ETB receptors can be selectively activated by Sarafotoxin 6c. The results from blocking ETA and activating ETB receptors demonstrated that endothelin 1 signaling via ETA receptors contributes to intrarenal hypoxia in the rat diabetic kidney, and that ETB stimulation significantly reduces the diabetes-induced intrarenal hypoxia. The beneficial effects on kidney oxygen availability in diabetes by ETA blockade or ETB stimulation were mainly linked to hemodynamic improvements rather than direct effects on kidney oxygen consumption or oxidative stress status.

In conclusion, by applying EPR oximetry in a mouse model of insulinopenic diabetes mimicking the human disease, we demonstrated intrarenal hypoxia already within the first couple of days after the onset of hyperglycemia, which is well before detectable signs of kidney disease development. Furthermore, blockade of ETA or activation of ETB receptors significantly reduced intrarenal hypoxia in the diabetic kidney. These results demonstrate involvement of ETA receptor signaling in diabetes-induced intrarenal hypoxia and ETA blockade or ETB activation might provide new therapeutical targets to reduce kidney hypoxia and disease progression in diabetes.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , 53 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1510
Keyword [en]
nephropathy, diabetes, hypoxia, EPR
National Category
Physiology
Identifiers
URN: urn:nbn:se:liu:diva-125522DOI: 10.3384/diss.diva-125522ISBN: 978-91-7685-825-7 (print)OAI: oai:DiVA.org:liu-125522DiVA: diva2:907280
Public defence
2016-04-29, Berzelius, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Heart Lung FoundationSwedish Research CouncilSwedish Diabetes Association
Available from: 2016-03-09 Created: 2016-02-25 Last updated: 2016-03-09Bibliographically approved
List of papers
1. Differences in susceptibility to develop parameters of diabetic nephropathy in four mouse strains with type 1 diabetes
Open this publication in new window or tab >>Differences in susceptibility to develop parameters of diabetic nephropathy in four mouse strains with type 1 diabetes
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2014 (English)In: AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY, ISSN 1931-857X, Vol. 306, no 10, F1171-F1178 p.Article in journal (Refereed) Published
Abstract [en]

One-third of diabetes mellitus patients develop diabetic nephropathy, and with underlying mechanisms unknown it is imperative that diabetic animal models resemble human disease. The present study investigated the susceptibility to develop diabetic nephropathy in four commonly used and commercially available mouse strains with type 1 diabetes to determine the suitability of each strain. Type 1 diabetes was induced in C57Bl/6, NMRI, BALB/c, and 129Sv mice by alloxan, and conscious glomerular filtration rate, proteinuria, and oxidative stress levels were measured in control and diabetic animals at baseline and after 5 and 10 wk. Histological alterations were analyzed using periodic acid-Schiff staining. Diabetic C57Bl/6 displayed increased glomerular filtration rate, i.e., hyperfiltration, whereas all other parameters remained unchanged. Diabetic NMRI developed the most pronounced hyperfiltration as well as increased oxidative stress and proteinuria but without glomerular damage. Diabetic BALB/c did not develop hyperfiltration but presented with pronounced proteinuria, increased oxidative stress, and glomerular damage. Diabetic 129Sv displayed proteinuria and increased oxidative stress without glomerular hyperfiltration or damage. However, all strains displayed intras-train correlation between oxidative stress and proteinuria. In conclusion, diabetic C57Bl/6 and NMRI both developed glomerular hyperfiltration but neither presented with histological damage, although NMRI developed low-degree proteinuria. Thus these strains may be suitable when investigating the mechanism causing hyperfiltration. Neither BALB/c nor 129Sv developed hyperfiltration although both developed pronounced proteinuria. However, only BALB/c developed detectable histological damage. Thus BALB/c may be suitable when studying the roles of proteinuria and histological alterations for the progression of diabetic nephropathy.

Place, publisher, year, edition, pages
American Physiological Society, 2014
Keyword
C57Bl/6; NMRI; BALB/c; 129Sv; diabetic nephropathy; kidney function
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-108808 (URN)10.1152/ajprenal.00595.2013 (DOI)000336846400007 ()
Available from: 2014-07-07 Created: 2014-07-06 Last updated: 2016-03-09
2. Repetitive Measurements of Intrarenal Oxygenation In Vivo Using L Band Electron Paramagnetic Resonance
Open this publication in new window or tab >>Repetitive Measurements of Intrarenal Oxygenation In Vivo Using L Band Electron Paramagnetic Resonance
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2014 (English)In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 812, 135-141 p.Article in journal (Refereed) Published
Abstract [en]

Intrarenal oxygenation is heterogeneous with oxygen levels normally being highest in the superficial cortex and lowest in the inner medulla. Reduced intrarenal oxygenation has been implied in the pathology of several kidney diseases. However, there is currently no method available to repetitively monitor regional renal oxygenation using minimally invasive procedures. We therefore evaluated implantable lithium phthalocyanine (LiPc) probes, which display a close correlation between EPR line width and oxygen availability. LiPc probes were implanted in the kidney cortex and medulla in the same mouse and sEPR spectra were acquired using a L band scanner during inhalation of air (21 % oxygen) or a mixture of air and nitrogen (10 % oxygen). In order to separate the signals from the two probes, a 1 G/cm gradient was applied and the signals were derived from 40 consecutive sweeps. Peak-to-peak comparison of the EPR line was used to convert the signal to an approximate oxygen tension in MATLAB. Kidney cortex as well as medullary oxygenation was stable over the 45 day period (cortex 56 +/- 7 mmHg and medulla 43 +/- 6 mmHg). However, 10 % oxygen inhalation significantly reduced oxygenation in both cortex (56 +/- 6 to 34 +/- 2 mmHg n = 15 p less than 0.05) and medulla (42 +/- 5 to 29 +/- 3 mmHg n = 7 p less than 0.05). In conclusion, L band EPR using LiPc probes implanted in discrete intrarenal structures can be used to repetitively monitor regional renal oxygenation. This minimally invasive method is especially well suited for conditions of reduced intrarenal oxygenation since this increases the signal intensity which facilitates the quantification of the EPR signal to absolute oxygenation values.

Place, publisher, year, edition, pages
Kluwer Academic Publishers, 2014
Keyword
Kidney; LiPc; L-Band EPR; NMRI mice; Oxygenation
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-113035 (URN)10.1007/978-1-4939-0620-8_18 (DOI)000345121200019 ()24729225 (PubMedID)978-1-4939-0620-8; 978-1-4939-0583-6 (ISBN)
Conference
41st Annual Meeting of the International-Society-on-Oxygen-Transport-to-Tissue (ISOTT)
Available from: 2015-01-09 Created: 2015-01-08 Last updated: 2017-12-05
3. Pronounced kidney hypoxia precedes albuminuria in type 1 diabetic mice
Open this publication in new window or tab >>Pronounced kidney hypoxia precedes albuminuria in type 1 diabetic mice
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2016 (English)In: American Journal of Physiology, ISSN 0002-9513, E-ISSN 2163-5773, Vol. 310, no 9, F807-F809 p.Article in journal (Refereed) Published
Abstract [en]

Intrarenal tissue hypoxia has been proposed as a unifying mechanism for the development of chronic kidney disease, including diabetic nephropathy. However, hypoxia has to be present before the onset of kidney disease in order to be the causal mechanism. In order to establish if hypoxia precedes the onset of diabetic nephropathy, we implemented a minimally invasive electron paramagnetic resonance oximetry technique using implanted oxygen sensing probes for repetitive measurements of in vivo kidney tissue oxygen tensions in mice. Kidney cortex oxygen tensions were measured before and up to 15 days after the induction of insulinopenic diabetes in male mice and compared to normoglycemic controls. On day 16, urinary albumin excretions and conscious glomerular filtration rates were determined in order to define the temporal relationship between intrarenal hypoxia and disease development. Diabetic mice developed pronounced intrarenal hypoxia three days after the induction of diabetes, which persisted throughout the study period. On day 16, diabetic mice had glomerular hyperfiltration, but normal urinary albumin excretion. In conclusion, intrarenal tissue hypoxia in diabetes precedes albuminuria thereby being a plausible cause for the onset and progression of diabetic nephropathy.

Place, publisher, year, edition, pages
American Physiological Society Journals, 2016
Keyword
nephropathy, diabetes, hypoxia, EPR
National Category
Physiology
Identifiers
urn:nbn:se:liu:diva-125526 (URN)10.1152/ajprenal.00049.2016 (DOI)000375115700001 ()
Note

The status of this article was previous Manuscript.

Funding agencies: Swedish Research Council; Swedish Heart Lung Foundation; Swedish Diabetes Foundation

Available from: 2016-02-26 Created: 2016-02-25 Last updated: 2017-05-02Bibliographically approved
4. Endothelin type A receptor inhibition normalises intrarenal hypoxia in rats used as a model of type 1 diabetes by improving oxygen delivery
Open this publication in new window or tab >>Endothelin type A receptor inhibition normalises intrarenal hypoxia in rats used as a model of type 1 diabetes by improving oxygen delivery
2015 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 58, no 10, 2435-2442 p.Article in journal (Refereed) Published
Abstract [en]

Aims/hypothesis Intrarenal tissue hypoxia, secondary to increased oxygen consumption, has been suggested as a unifying mechanism for the development of diabetic nephropathy. Increased endothelin-1 signalling via the endothelin type A receptor (ETA-R) has been shown to contribute to the development of chronic kidney disease, but its role in kidney oxygen homeostasis is presently unknown. Methods The effects of acute ETA-R inhibition (8 nmol/l BQ-123 for 30-40 min directly into the left renal artery) on kidney function and oxygen metabolism were investigated in normoglycaemic control and insulinopenic male Sprague Dawley rats (55 mg/kg streptozotocin intravenously 2 weeks before the main experiment) used as a model of type 1 diabetes. Results Local inhibition of ETA-R in the left kidney did not affect BP in either the control or the diabetic rats. As previously reported, diabetic rats displayed increased kidney oxygen consumption resulting in tissue hypoxia in both the kidney cortex and medulla. The inhibition of ETA-Rs restored normal kidney tissue oxygen availability in the diabetic kidney by increasing renal blood flow, but did not affect oxygen consumption. Furthermore, ETA-R inhibition reduced the diabetes-induced glomerular hyperfiltration and increased the urinary sodium excretion. Kidney function in normoglycaemic control rats was largely unaffected by BQ-123 treatment, although it also increased renal blood flow and urinary sodium excretion in these animals. Conclusions/interpretation Acutely reduced intrarenal ETA-R signalling results in significantly improved oxygen availability in the diabetic kidney secondary to elevated renal perfusion. Thus, the beneficial effects of ETA-R inhibition on kidney function in diabetes may be due to improved intrarenal oxygen homeostasis.

Place, publisher, year, edition, pages
SPRINGER, 2015
Keyword
BQ-123; Diabetic nephropathy; Endothelin type; A receptor; Hypoxia; Kidney function; Rats
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-122101 (URN)10.1007/s00125-015-3690-9 (DOI)000361538600027 ()26173672 (PubMedID)
Note

Funding Agencies|Swedish Research Council; Swedish Diabetes Foundation; Family Ernfors Fund

Available from: 2015-10-19 Created: 2015-10-19 Last updated: 2017-12-01
5. Intrarenal activation of endothelin type B receptors improve intrarenal oxygenation in type 1 diabetic rats
Open this publication in new window or tab >>Intrarenal activation of endothelin type B receptors improve intrarenal oxygenation in type 1 diabetic rats
(English)Manuscript (preprint) (Other academic)
Keyword
nephropathy, diabetes, hypoxia, endothelin, sarafotoxin 6c
National Category
Physiology
Identifiers
urn:nbn:se:liu:diva-125525 (URN)
Note

About one third of patients with type 1 diabetes develop kidney damage. The mechanism is largely unknown, but intrarenal hypoxia has been proposed as a unifying mechanism for chronic kidney disease including diabetic nephropathy. The endothelin system has recently been demonstrated to regulate oxygen availability in the diabetic kidney via a pathway involving endothelin type A receptors (ETA-R). These receptors mainly mediate vasoconstriction and tubular sodium retention, and inhibition of ETA-R improves intrarenal oxygenation in the diabetic kidney. Endothelin type B receptors (ETB-R) have been reported to have opposite effects on vascular tone and tubular sodium handling. However, the role of ETB-R in kidney oxygen homeostasis is unknown.

The effects of acute intrarenal ETB-R activation (Sarafotoxin 6c for 30-40 minutes; 0.78 pmol h-1 directly into the renal artery) on kidney function and oxygen metabolism were investigated in normoglycemic control and insulinopenic male Sprague Dawley rats administered streptozotocin (55 mg kg-1) two weeks before the acute experiments.

Intrarenal activation of ETB-R improved oxygenation of the hypoxia diabetic kidney. However, neither effects on the diabetes-induced increased kidney oxygen consumption nor alterations in parameters related to tubular sodium transport could explain the improved oxygenation in the diabetic kidney after ETB-R activation. Rather, the improved kidney oxygenation was due to hemodynamic effects increasing oxygen delivery.

In conclusion, increased ETB-R signaling in the diabetic kidney improves tissue oxygenation due to increased oxygen delivery as a result of increased total renal blood flow.

Available from: 2016-02-25 Created: 2016-02-25 Last updated: 2016-03-09

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