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Studies on the Role of Apoptosis in Kidney Diseases
KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis is one of the most common types of cell death. Under physiological conditions, it plays an essential role in removal of damaged and potentially harmful cells. Excessive apoptosis has however been linked to a number of diseases including proteinuric kidney disease and DKD, and is believed to enhance the disease progression. Albuminuria and hyperglycemia are common symptoms of these diseases and albumin and high glucose have been seen to trigger intrinsic apoptosis in renal cells. Ouabain, a cardiotonic steroid, has previously been identified as an antiapoptotic agent that in subsaturating concentrations protect from intrinsic apoptosis. The mechanism of the protective effect of ouabain is still not fully understood and it remains to be concluded whether ouabain can protect from albumin and/or glucotoxic-triggered apoptosis.

In study I we investigated the protective effects of ouabain in albumin-exposed primary rat PTC and podocytes and in the proteinuric kidney disease animal model passive Heymann nephritis. By reestablishing the balance between the proapoptotic protein BAX and the antiapoptotic protein BCL-XL, ouabain averted the albumin-triggered apoptosis in vitro and in vivo and protected from podocytes loss and glomerular-tubular disconnection.

In study II we investigated the relationship between the glucose transporters renal cells express and their susceptibility of glucotoxic-triggered apoptosis. We identified the SGLT expressing cells, PTC and MC, to be more susceptible to high glucose-induced apoptosis than cells without SGLT. The apoptosis was mediated by BAX and BCL-XL imbalance and mitochondrial dysfunction, and was abolished when treated with ouabain or SGLT inhibitors. Podocytes, which lack SGLT, did not respond to short-term high glucose exposure.

In study III we used super-resolution microscopy to investigate at which stage of the apoptotic process ouabain start to intervene. Ouabain interfered early in the apoptotic process, where it prevented activation of the sensitizer protein BAD. This allowed BCL-XL to avert BAX activation and translocation to mitochondria and thereby protected from mitochondrial dysfunction and apoptosis.

In study IV we investigated differentially expressed genes between renal cortex and primary short-term PTC cultures and between PTC exposed to control and high glucose. The mRNA expression level of most genes was significantly up- or downregulated in PTC compared to renal cortex, with the biggest differences in mitochondria and metabolism related genes. Early state glucotoxicity did not significantly alter mRNA expression levels in PTC.

Abstract [sv]

Apoptos, en av de vanligaste typerna av celldöd, är under normala förhållanden en viktig process för att kroppen på ett kontrollerat sätt ska kunna ta bort skadade och potentiellt farliga celler. Överdriven apoptos har dock observerats som en del av processen i flera sjukdomar som exempelvis proteinurisk njursjukdom, där stora mängder protein följer med urinen ut ur kroppen, och diabetisk njursjukdom, som orsakas av diabetes. Albuminuri, dvs albumin i urinen, och hyperglykemi är vanliga symptom på dessa sjukdomar. Tidigare studier har visat att albumin och höga glukoskoncentrationer kan aktivera apoptos i njurceller.

Ouabain är en hjärtglykosid, dvs. en substans som påverkar hjärtats rytm och slagkraft. I omättade koncentrationer har ouabain identifierats vara antiapoptotisk, dvs. den motverkar aktivering av apoptos. Mekanismen av den skyddande effekten hos ouabain är dock inte helt klarlagd och det är fortfarande inte känt huruvida ouabain kan skydda mot apoptos i njurceller utsatta för albumin och/eller hög glukos.

I studie I undersökte vi om ouabain skyddar mot aktivering av apoptos i proximala tubuli celler och podocyter utsatta för albumin, samt i en djurmodell av proteinurisk njursjukdom. Albumin orsakade en obalans mellan det proapoptotiska proteinet BAX och det antiapoptotiska proteinet BCL-XL, vilket ledde till mitokondriell dysfunktion och apoptos. Genom att återställa balansen mellan BAX och BCL-XL, avvärjde ouabain albumin från att aktivera den apoptotiska processen i vitro och i vivo, vilket förhindrade förlust av podocyter och dissociering av glomeruli från tubuli.

I studie II undersökte vi sambandet mellan njurcellers uttryck av glukostransportörer och deras benägenhet att genomgå apoptos under höga glukoskoncentrationer. Vi identifierade proximala tubuli och mesangial celler, som uttrycker SGLT, dvs. natriumberoende glukostransportörer, som särskilt utsatta för hög glukosinducerad apoptos. Apoptosen orsakades av en obalans mellan BAX och BCL-XL, vilket ledde till mitokondriell dysfunktion och slutligen apoptos. Behandling med ouabain eller SGLT-hämmare förhindrade aktivering av apoptos i proximala tubuli och mesangial celler utsatta för hög glukos. Podocyter, som saknar SGLT, påverkades inte av kortvarig exponering av hög glukos.

I studie III använde vi superupplösningsmikroskopi för att studera var i den apoptotiska processen som ouabain verkar. Ouabain förhindrade aktivering av den apoptotiska processen i ett tidigt skede genom att förebygga aktivering av sensibiliseringsproteinet BAD, vars huvudsakliga uppgift är att aktivera BAX. Inhiberingen av BAD möjliggjorde att BCL-XL kunde förhindra BAX från att aktiveras och förflytta sig från cytosol till mitokondrier och skyddade därmed mot mitokondriell dysfunktion och apoptos.

I studie IV undersökte vi skillnader i mRNA nivåer mellan färsk vävnad från njurcortex och primära proximala tubuli celler odlade i tre dagar, samt mellan proximala tubuli celler odlade i normal och hög glukos. De flesta geners mRNA nivåer var signifikant förändrade i PTC jämfört med njurcortex. Störst förändringar observerades i mitokondrie- och metabolismrelaterade gener. Kortvarig hyperglykemi förändrade inte mRNA nivåerna i PTC.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. , p. 83
Series
TRITA-SCI-FOU ; 2019:26
Keywords [en]
Apoptosis, diabetic kidney disease, hyperglycemia, mesangial cells, microscopy, ouabain, podocytes, proximal tubule cells, RNA-seq, SGLT
Keywords [sv]
Apoptos, diabetisk njursjukdom, hyperglykemi, mesangiala celler, mikroskopi, ouabain, podocyter, proximala tubuli celler, RNA-seq, SGLT
National Category
Biophysics Cell Biology Bioinformatics and Systems Biology Biochemistry and Molecular Biology Cell and Molecular Biology Physiology
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-250264ISBN: 978-91-7873-207-4 (print)OAI: oai:DiVA.org:kth-250264DiVA, id: diva2:1307413
Public defence
2019-05-24, Air & Fire, Science for Life Laboratory, Tomtebodavägen 23A, Solna, 13:00 (English)
Opponent
Supervisors
Note

QC 20190428

Available from: 2019-04-29 Created: 2019-04-26 Last updated: 2019-04-29Bibliographically approved
List of papers
1. Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease
Open this publication in new window or tab >>Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease
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2016 (English)In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 90, no 1, p. 135-148Article in journal (Refereed) Published
Abstract [en]

There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis. Our group has described an ouabain-activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time-and dose-dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the antiapoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptosis in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an antiapoptotic and kidney-protective therapeutic tool was then tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserved renal function, protected from renal cortical apoptosis, up-regulated Bax, down-regulated Bcl-xL, and rescued from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
Keywords
albuminuria, apoptosis, cell signaling, chronic kidney disease, ouabain, podocyte, proximal tubule, sodium potassium adenosine tri-phosphatase, a-tubular glomeruli
National Category
Urology and Nephrology
Identifiers
urn:nbn:se:kth:diva-189657 (URN)10.1016/j.kint.2016.03.026 (DOI)000377929400022 ()27217195 (PubMedID)2-s2.0-84988335957 (Scopus ID)
Funder
Swedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20160715

Available from: 2016-07-15 Created: 2016-07-11 Last updated: 2019-04-26Bibliographically approved
2. Prompt apoptotic response to high glucose in SGLT expressing cells
Open this publication in new window or tab >>Prompt apoptotic response to high glucose in SGLT expressing cells
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2019 (English)In: American Journal of Physiology - Renal Physiology, ISSN 1931-857X, E-ISSN 1522-1466Article in journal (Refereed) Epub ahead of print
Abstract [en]

It is generally believed that cells that are unable to downregulate glucose transport are particularly vulnerable to hyperglycemia. Yet little is known about the relation between expression of glucose transporters and acute toxic effects of high glucose exposure.Here we have, in an ex vivo study on rat renal cells, compared the apoptotic response to a moderate increase in glucose concentration. We have studied the cell types that commonly are targeted in diabetic kidney disease (DKD): proximal tubule cells (PTC) that express SGLT2, mesangial cells (MC) that express SGLT1, and podocytes that lack SGLT and take up glucose via the insulin dependent GLUT4.PTC and MC responded within 4-8 h exposure to 15 mM glucose with translocation of the apoptotic protein Bax to mitochondria and increased apoptotic index. SGLT down-regulation and exposure to SGLT inhibitors abolished the apoptotic response. Onset of overt DKD generally coincides with onset of albuminuria. Albumin had an additive effect on the apoptotic response. Ouabain, which interferes with apoptotic onset, rescued from the apoptotic response. Insulin supplemented podocytes remained resistant to 15 and 30 mM glucose for at least 24 h.Our study points to a previously unappreciated role of SGLT dependent glucose uptake as a risk-factor for diabetic complications and highlights the importance of therapeutic approaches that specifically target the different cell types in DKD.

National Category
Biophysics Cell Biology Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-249971 (URN)10.1152/ajprenal.00615.2018 (DOI)
Note

QC 20190429

Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-04-29Bibliographically approved
3. Ouabain intervenes early in the apoptotic process by preventing BAD activation
Open this publication in new window or tab >>Ouabain intervenes early in the apoptotic process by preventing BAD activation
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(English)Manuscript (preprint) (Other academic)
National Category
Biophysics Biochemistry and Molecular Biology Cell Biology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-250259 (URN)
Note

QC 20190428

Available from: 2019-04-26 Created: 2019-04-26 Last updated: 2019-04-29Bibliographically approved
4. RNA-seq reveals altered gene expression levels in PTC cultures compared to renal cortex but not in early state glucotoxic PTC
Open this publication in new window or tab >>RNA-seq reveals altered gene expression levels in PTC cultures compared to renal cortex but not in early state glucotoxic PTC
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics Bioinformatics and Systems Biology Cell Biology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-250260 (URN)
Note

QC 20190428

Available from: 2019-04-26 Created: 2019-04-26 Last updated: 2019-04-29Bibliographically approved

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