Publications by authors named "Ilse Daehn"

25 Publications

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Molecular Mechanisms in Early Diabetic Kidney Disease: Glomerular Endothelial Cell Dysfunction.

Int J Mol Sci 2020 Dec 11;21(24). Epub 2020 Dec 11.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD), with prevalence increasing at an alarming rate worldwide and today, there are no known cures. The pathogenesis of DKD is complex, influenced by genetics and the environment. However, the underlying molecular mechanisms that contribute to DKD risk in about one-third of diabetics are still poorly understood. The early stage of DKD is characterized by glomerular hyperfiltration, hypertrophy, podocyte injury and depletion. Recent evidence of glomerular endothelial cell injury at the early stage of DKD has been suggested to be critical in the pathological process and has highlighted the importance of glomerular intercellular crosstalk. A potential mechanism may include reactive oxygen species (ROS), which play a direct role in diabetes and its complications. In this review, we discuss different cellular sources of ROS in diabetes and a new emerging paradigm of endothelial cell dysfunction as a key event in the pathogenesis of DKD.
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http://dx.doi.org/10.3390/ijms21249456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764016PMC
December 2020

The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes.

Cell Commun Signal 2020 07 8;18(1):105. Epub 2020 Jul 8.

Division of Nephrology, Department of Medicine, The Icahn School of Medicine at Mount Sinai, One Gustave Levy Place, Box 1003, New York, NY, 10029, USA.

Background: In the setting of diabetes mellitus, mitochondrial dysfunction and oxidative stress are important pathogenic mechanisms causing end organ damage, including diabetic kidney disease (DKD), but mechanistic understanding at a cellular level remains obscure. In mouse models of DKD, glomerular endothelial cell (GEC) dysfunction precedes albuminuria and contributes to neighboring podocyte dysfunction, implicating GECs in breakdown of the glomerular filtration barrier. In the following studies we wished to explore the cellular mechanisms by which GECs become dysfunctional in the diabetic milieu, and the impact to neighboring podocytes.

Methods: Mouse GECs were exposed to high glucose media (HG) or 2.5% v/v serum from diabetic mice or serum from non-diabetic controls, and evaluated for mitochondrial function (oxygen consumption), structure (electron microscopy), morphology (mitotracker), mitochondrial superoxide (mitoSOX), as well as accumulation of oxidized products (DNA lesion frequency (8-oxoG, endo-G), double strand breaks (γ-H2AX), endothelial function (NOS activity), autophagy (LC3) and apoptotic cell death (Annexin/PI; caspase 3). Supernatant transfer experiments from GECs to podocytes were performed to establish the effects on podocyte survival and transwell experiments were performed to determine the effects in co-culture.

Results: Diabetic serum specifically causes mitochondrial dysfunction and mitochondrial superoxide release in GECs. There is a rapid oxidation of mitochondrial DNA and loss of mitochondrial biogenesis without cell death. Many of these effects are blocked by mitoTEMPO a selective mitochondrial anti-oxidant. Secreted factors from dysfunctional GECs were sufficient to cause podocyte apoptosis in supernatant transfer experiments, or in co-culture but this did not occur when GECs had been previously treated with mitoTEMPO.

Conclusion: Dissecting the impact of the diabetic environment on individual cell-types from the kidney glomerulus indicates that GECs become dysfunctional and pathological to neighboring podocytes by increased levels of mitochondrial superoxide in GEC. These studies indicate that GEC-signaling to podocytes contributes to the loss of the glomerular filtration barrier in DKD. Video abstract.
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http://dx.doi.org/10.1186/s12964-020-00605-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341607PMC
July 2020

Mitochondria Matter: A Critical Role of in Stabilizing the CoQ Complex in Podocytes in Steroid-Resistant Nephrotic Syndrome.

Authors:
Ilse S Daehn

J Am Soc Nephrol 2020 06 7;31(6):1167-1169. Epub 2020 May 7.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York City, New York

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http://dx.doi.org/10.1681/ASN.2020040467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269363PMC
June 2020

Endothelin receptor-A mediates degradation of the glomerular endothelial surface layer via pathologic crosstalk between activated podocytes and glomerular endothelial cells.

Kidney Int 2019 10 22;96(4):957-970. Epub 2019 May 22.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address:

Emerging evidence of crosstalk between glomerular cells in pathological settings provides opportunities for novel therapeutic discovery. Here we investigated underlying mechanisms of early events leading to filtration barrier defects of podocyte and glomerular endothelial cell crosstalk in the mouse models of primary podocytopathy (podocyte specific transforming growth factor-β receptor 1 signaling activation) or Adriamycin nephropathy. We found that glomerular endothelial surface layer degradation and albuminuria preceded podocyte foot process effacement. These abnormalities were prevented by endothelin receptor-A antagonism and mitochondrial reactive oxygen species scavenging. Additional studies confirmed increased heparanase and hyaluronoglucosaminidase gene expression in glomerular endothelial cells in response to podocyte-released factors and to endothelin-1. Atomic force microscopy measurements showed a significant reduction in the endothelial surface layer by endothelin-1 and podocyte-released factors, which could be prevented by endothelin receptor-A but not endothelin receptor-B antagonism. Thus, our studies provide evidence of early crosstalk between activated podocytes and glomerular endothelial cells resulting in loss of endothelial surface layer, glomerular endothelial cell injury and albuminuria. Hence, activation of endothelin-1-endothelin receptor-A and mitochondrial reactive oxygen species contribute to the pathogenesis of primary podocytopathies in experimental focal segmental glomerulosclerosis.
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http://dx.doi.org/10.1016/j.kint.2019.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200072PMC
October 2019

Corrigendum: Glomerular Endothelial Cell Stress and Cross-Talk With Podocytes in Early Diabetic Kidney Disease.

Authors:
Ilse Sofia Daehn

Front Med (Lausanne) 2018 26;5:113. Epub 2018 Apr 26.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, The Charles Bronfman Institute for Personalized Medicine, New York City, NY, United States.

[This corrects the article on p. 76 in vol. 5, PMID: 29629372.].
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http://dx.doi.org/10.3389/fmed.2018.00113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932145PMC
April 2018

Glomerular Endothelial Cell Stress and Cross-Talk With Podocytes in Early [corrected] Diabetic Kidney Disease.

Authors:
Ilse Sofia Daehn

Front Med (Lausanne) 2018 23;5:76. Epub 2018 Mar 23.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, The Charles Bronfman Institute for Personalized Medicine, New York City, NY, United States.

Diabetic kidney disease (DKD) is one of the major causes of morbidity and mortality in diabetic patients and also the leading single cause of end-stage renal disease in the United States. A large proportion of diabetic patients develop DKD and others don't, even with comparable blood glucose levels, indicating a significant genetic component of disease susceptibility. The glomerulus is the primary site of diabetic injury in the kidney, glomerular hypertrophy and podocyte depletion are glomerular hallmarks of progressive DKD, and the degree of podocyte loss correlates with severity of the disease. We know that chronic hyperglycemia contributes to both microvascular and macrovascular complications, as well as podocyte injury. We are beginning to understand the role of glomerular endothelial injury, as well as the involvement of reactive oxygen species and mitochondrial stress, which play a direct role in DKD and in other diabetic complications. There is, however, a gap in our knowledge that links genetic susceptibility to early molecular mechanisms and proteinuria in DKD. Emerging research that explores glomerular cell's specific responses to diabetes and cell cross-talk will provide mechanistic clues that underlie DKD and provide novel avenues for therapeutic intervention.
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http://dx.doi.org/10.3389/fmed.2018.00076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876248PMC
March 2018

WT1 Is Necessary for the Proliferation and Migration of Cells of Renin Lineage Following Kidney Podocyte Depletion.

Stem Cell Reports 2017 10 28;9(4):1152-1166. Epub 2017 Sep 28.

Division of Nephrology, University of Washington School of Medicine, 750 Republican Street, Seattle, WA 98109, USA. Electronic address:

Wilms' tumor suppressor 1 (WT1) plays an important role in cell proliferation and mesenchymal-epithelial balance in normal development and disease. Here, we show that following podocyte depletion in three experimental models, and in patients with focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, WT1 increased significantly in cells of renin lineage (CoRL). In an animal model of FSGS in RenWt1 reporter mice with inducible deletion of WT1 in CoRL, CoRL proliferation and migration to the glomerulus was reduced, and glomerular disease was worse compared with wild-type mice. To become podocytes, CoRL undergo mesenchymal-to-epithelial transformation (MET), typified by reduced staining for mesenchymal markers (MYH11, SM22, αSMA) and de novo expression of epithelial markers (E-cadherin and cytokeratin18). Evidence for changes in MET markers was barely detected in RenWt1 mice. Our results show that following podocyte depletion, WT1 plays essential roles in CoRL proliferation and migration toward an adult podocyte fate.
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http://dx.doi.org/10.1016/j.stemcr.2017.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639431PMC
October 2017

Glomerular Endothelial Mitochondrial Dysfunction Is Essential and Characteristic of Diabetic Kidney Disease Susceptibility.

Diabetes 2017 03 29;66(3):763-778. Epub 2016 Nov 29.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY

The molecular signaling mechanisms between glomerular cell types during initiation/progression of diabetic kidney disease (DKD) remain poorly understood. We compared the early transcriptome profile between DKD-resistant C57BL/6J and DKD-susceptible DBA/2J (D2) glomeruli and demonstrated a significant downregulation of essential mitochondrial genes in glomeruli from diabetic D2 mice, but not in C57BL/6J, with comparable hyperglycemia. Diabetic D2 mice manifested increased mitochondrial DNA lesions (8-oxoguanine) exclusively localized to glomerular endothelial cells after 3 weeks of diabetes, and these accumulated over time in addition to increased urine secretion of 8-oxo-deoxyguanosine. Detailed assessment of glomerular capillaries from diabetic D2 mice demonstrated early signs of endothelial injury and loss of fenestrae. Glomerular endothelial mitochondrial dysfunction was associated with increased glomerular endothelin-1 receptor type A (Ednra) expression and increased circulating endothelin-1 (Edn1). Selective Ednra blockade or mitochondrial-targeted reactive oxygen species scavenging prevented mitochondrial oxidative stress of endothelial cells and ameliorated diabetes-induced endothelial injury, podocyte loss, albuminuria, and glomerulosclerosis. In human DKD, increased urine 8-oxo-deoxyguanosine was associated with rapid DKD progression, and biopsies from patients with DKD showed increased mitochondrial DNA damage associated with glomerular endothelial EDNRA expression. Our studies show that DKD susceptibility was linked to mitochondrial dysfunction, mediated largely by Edn1-Ednra in glomerular endothelial cells representing an early event in DKD progression, and suggest that cross talk between glomerular endothelial injury and podocytes leads to defects and depletion, albuminuria, and glomerulosclerosis.
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http://dx.doi.org/10.2337/db16-0695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5319717PMC
March 2017

Designer Thiopurine-analogues for Optimised Immunosuppression in Inflammatory Bowel Diseases.

J Crohns Colitis 2016 Oct 25;10(10):1132-43. Epub 2016 Apr 25.

Department of Medicine 1, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany

Background And Aims: The clinical use of azathioprine and 6-mercaptopurine is limited by their delayed onset of action and potential side effects such as myelosuppression and hepatotoxicity. As these drugs specifically target the Vav1/Rac1 signalling pathway in T lamina propria lymphocytes via their metabolite 6-thio-GTP, we studied expression and optimised suppression of this pathway in inflammatory bowel diseases [IBD].

Methods: Rac1 and Vav1 expressions were analysed in mucosal immune cells in IBD patients. Targeted molecular modelling of the 6-thio-GTP molecule was performed to optimise Rac1 blockade; 44 modified designer thiopurine-analogues were tested for apoptosis induction, potential toxicity, and immunosuppression. Activation of the Vav1/Rac1 pathway in lymphocytes was studied in IBD patients and in lamina propria immune cells in the presence or absence of thiopurine-analogues.

Results: Several thiopurine-analogues induced significantly higher T cell apoptosis than 6-mercaptopurine. We identified a compound, denoted B-0N, based on its capacity to mediate earlier and stronger induction of T cell apoptosis than 6-mercaptopurine. B-0N-treatment resulted in accelerated inhibition of Rac1 activity in primary peripheral blood T cells as well as in intestinal lamina propria immune cells. Compared with 6-thio-GTP and 6-mercaptopurine, B-0N-treatment was associated with decreased myelo- and hepatotoxicity.

Conclusions: The Vav1/Rac1 pathway is activated in mucosal immune cells in IBD. The designer thiopurine-analogue B-0N induces immunosuppression more potently than 6-mercaptopurine.
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http://dx.doi.org/10.1093/ecco-jcc/jjw091DOI Listing
October 2016

Shift in Focus-To Explore the Role of the Endothelium in Kidney Disease.

Authors:
Ilse Daehn

HSOA J Nephrol Ren Ther 2016 10;2(1). Epub 2016 Mar 10.

Department of Medicine, The Icahn School of Medicine at Mount Sinai, Madison Avenue, New York, USA.

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http://dx.doi.org/10.24966/NRT-7313/100004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608090PMC
March 2016

TGFβ-Induced Actin Cytoskeleton Rearrangement in Podocytes Is Associated with Compensatory Adaptation of Mitochondrial Energy Metabolism.

Nephron 2015 28;131(4):278-84. Epub 2015 Nov 28.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, N.Y., USA.

Background/aims: In podocytes, the overexpression of TGFβ ligands and receptors during glomerulosclerosis could be a causal factor for injury induction and perpetuation in glomerular tufts. Mitochondrial dysfunction and oxidative stress are emerging as potential therapeutic targets in glomerular injury, and TGFβ has been shown to modulate mitochondrial metabolism in different cell types. This study aims at investigating the role of TGFβ in podocyte energy metabolism and cytoskeleton dynamics.

Methods: Mitochondrial function and cytoskeleton dynamics were analyzed in TGFβ-treated WT and Smad2/3 double KO podocytes.

Results: TGFβ treatment in podocytes induced a significant Smad-dependent increase of mitochondrial oxygen consumption rate (OCR). ATP content was unchanged and increased respiration was not associated with increased mitochondrial mass. Increased cellular reactive oxygen species induced by Smad-mediated TGFβ signaling were reverted by NADPH oxidase inhibitor apocynin. TGFβ treatment did not induce mitochondrial oxidative stress, and Smad2/3-dependent TGFβ signaling and increased mitochondrial OCR were found to be associated with actin cytoskeleton dynamics. The role of motor proteins myosin II and dynamin in TGFβ-induced actin polymerization was demonstrated by specific inhibition, resulting in actin stabilization and normalization of mitochondrial OCR.

Conclusion: TGFβ-induced rearrangements of actin cytoskeleton are controlled by Smad2/3 signaling pathways and coupled with the activation of mitochondrial ATP synthesis as bioenergetic adaptation to ATP consumption by ATP- and GTP-dependent motor proteins, myosin II and dynamin.
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http://dx.doi.org/10.1159/000442051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687741PMC
October 2016

Dendrin ablation prolongs life span by delaying kidney failure.

Am J Pathol 2015 Aug 12;185(8):2143-57. Epub 2015 Jun 12.

Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address:

Podocyte loss is central to the progression of proteinuric kidney diseases leading to end-stage kidney disease (ESKD), requiring renal replacement therapy, such as dialysis. Despite modern tools and techniques, the 5-year mortality of some patients requiring dialysis remains at about 70% to 80%. Thus, there is a great unmet need for podocyte-specific treatments aimed at preventing podocyte loss and the ensuing development of ESKD. Here, we show that ablation of the podocyte death-promoting protein dendrin delays the onset of ESKD, thereby expanding the life span of mice lacking the adapter protein CD2AP. Ablation of dendrin delays onset and severity of proteinuria and podocyte loss. In addition, dendrin ablation ameliorates mesangial volume expansion and up-regulation of mesangial fibronectin expression, which is mediated by a podocyte-secreted factor. In conclusion, onset of ESKD and death can be markedly delayed by blocking the function of dendrin.
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http://dx.doi.org/10.1016/j.ajpath.2015.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530132PMC
August 2015

Krüppel-like factor 6 regulates mitochondrial function in the kidney.

J Clin Invest 2015 Mar 17;125(3):1347-61. Epub 2015 Feb 17.

Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.
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http://dx.doi.org/10.1172/JCI77084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362257PMC
March 2015

Microvascular endothelial cells poised to take center stage in experimental renal fibrosis.

J Am Soc Nephrol 2015 Apr 22;26(4):767-9. Epub 2014 Dec 22.

Charles Bronfman Institute for Personalized Medicine, and Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York.

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http://dx.doi.org/10.1681/ASN.2014121170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378115PMC
April 2015

Mpv17 in mitochondria protects podocytes against mitochondrial dysfunction and apoptosis in vivo and in vitro.

Am J Physiol Renal Physiol 2014 Jun 5;306(11):F1372-80. Epub 2014 Mar 5.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, New York;

Mitochondrial dysfunction is increasingly recognized as contributing to glomerular diseases, including those secondary to mitochondrial DNA (mtDNA) mutations and deletions. Mitochondria maintain cellular redox and energy homeostasis and are a major source of intracellular reactive oxygen species (ROS) production. Mitochondrial ROS accumulation may contribute to stress-induced mitochondrial dysfunction and apoptosis and thereby to glomerulosclerosis. In mice, deletion of the gene encoding Mpv17 is associated with glomerulosclerosis, but the underlying mechanism remains poorly defined. Here we report that Mpv17 localizes to mitochondria of podocytes and its expression is reduced in several glomerular injury models and in human focal segmental glomerulosclerosis (FSGS) but not in minimal change disease. Using models of mild or severe nephrotoxic serum nephritis (NTSN) in Mpv17(+/+) wild-type (WT) and Mpv17(-/-) knockout mice, we found that Mpv17 deficiency resulted in increased proteinuria (mild NTSN) and renal insufficiency (severe NTSN) compared with WT. These lesions were associated with increased mitochondrial ROS generation and mitochondrial injury such as oxidative DNA damage. In vitro, podocytes with loss of Mpv17 function were characterized by increased susceptibility to apoptosis and ROS injury including decreased mitochondrial function, loss of mtDNA content, and change in mitochondrial configuration. In summary, the inner mitochondrial membrane protein Mpv17 in podocytes is essential for the maintenance of mitochondrial homeostasis and protects podocytes against oxidative stress-induced injury both in vitro and in vivo.
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http://dx.doi.org/10.1152/ajprenal.00608.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042102PMC
June 2014

Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.

J Clin Invest 2014 Apr 3;124(4):1608-21. Epub 2014 Mar 3.

Focal segmental glomerular sclerosis (FSGS) is a primary kidney disease that is commonly associated with proteinuria and progressive loss of glomerular function, leading to development of chronic kidney disease (CKD). FSGS is characterized by podocyte injury and depletion and collapse of glomerular capillary segments. Progression of FSGS is associated with TGF-β activation in podocytes; however, it is not clear how TGF-β signaling promotes disease. Here, we determined that podocyte-specific activation of TGF-β signaling in transgenic mice and BALB/c mice with Adriamycin-induced glomerulosclerosis is associated with endothelin-1 (EDN1) release by podocytes, which mediates mitochondrial oxidative stress and dysfunction in adjacent endothelial cells via paracrine EDN1 receptor type A (EDNRA) activation. Endothelial dysfunction promoted podocyte apoptosis, and inhibition of EDNRA or scavenging of mitochondrial-targeted ROS prevented podocyte loss, albuminuria, glomerulosclerosis, and renal failure. We confirmed reciprocal crosstalk between podocytes and endothelial cells in a coculture system. Biopsies from patients with FSGS exhibited increased mitochondrial DNA damage, consistent with EDNRA-mediated glomerular endothelial mitochondrial oxidative stress. Our studies indicate that segmental glomerulosclerosis develops as a result of podocyte-endothelial crosstalk mediated by EDN1/EDNRA-dependent mitochondrial dysfunction and suggest that targeting the reciprocal interaction between podocytes and endothelia may provide opportunities for therapeutic intervention in FSGS.
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http://dx.doi.org/10.1172/JCI71195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973074PMC
April 2014

Epithelial cell TGFβ signaling induces acute tubular injury and interstitial inflammation.

J Am Soc Nephrol 2013 Apr 28;24(5):787-99. Epub 2013 Mar 28.

Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA.

TGFβ signaling plays a central role in the development of acute and chronic kidney diseases. Previous in vivo studies involved systemic alteration of TGFβ signaling, however, limiting conclusions about the direct role of TGFβ in tubular cell injury. Here, we generated a double transgenic mouse that inducibly expresses a ligand-independent constitutively active TGFβ receptor type 1 (TβR1) kinase specifically in tubular epithelial cells, with expression restricted by the Pax8 promoter. In this model, activation of TGFβ signaling in the tubular epithelium alone was sufficient to cause AKI characterized by marked tubular cell apoptosis and necrosis, oxidative stress, dedifferentiation and regenerative cell proliferation, reduced renal function, and interstitial accumulation of inflammatory cells. This tubular injury was associated with mitochondrial-derived generation of reactive oxygen species (ROS), but cell damage and apoptosis were partially independent of mitochondrial-derived ROS. TβR1 signaling-induced tubular injury also associated with significant leukocyte infiltration consisting of F4/80(+) macrophages, CD11c(+) F4/80(+) dendritic cells, CD11c(+) F4/80(-) Ly6C(high) dendritic cells/monocytes, and T cells. Inhibition of mitochondrial-derived ROS significantly reduced accumulation of CD11c(+) F4/80(+) dendritic cells and T cells, suggesting a role for ROS in the activation and recruitment of the adaptive immune response to tubular injury. Taken together, these results suggest that TGFβ signaling in the tubular epithelium alone is sufficient to cause acute tubular injury and inflammation; therefore, TGFβ may be a mechanistic link between acute injury and chronic progression of kidney disease.
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http://dx.doi.org/10.1681/ASN.2012101024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636798PMC
April 2013

Transforming growth factor-β, bioenergetics, and mitochondria in renal disease.

Semin Nephrol 2012 May;32(3):295-303

Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

The transforming growth factor-β (TGF-β) family comprises more than 30 family members that are structurally related secreted dimeric cytokines, including TGF-β, activins, and bone morphogenetic proteins/growth and differentiation factors. TGF-β are pluripotent regulators of cell proliferation, differentiation, apoptosis, migration, and adhesion of many different cell types. TGF-β pathways are highly evolutionarily conserved and control embryogenesis, tissue repair, and tissue homeostasis in invertebrates and vertebrates. Aberrations in TGF-β activity and signaling underlie a broad spectrum of developmental disorders and major pathologies in human beings, including cancer, fibrosis, and autoimmune diseases. Recent observations have indicated an emerging role for TGF-β in the regulation of mitochondrial bioenergetics and oxidative stress responses characteristic of chronic degenerative diseases and aging. Conversely, energy and metabolic sensory pathways cross-regulate mediators of TGF-β signaling. Here, we review TGF-β and regulation of bioenergetic and mitochondrial functions, including energy and oxidant metabolism and apoptotic cell death, as well as their emerging relevance in renal biology and disease.
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http://dx.doi.org/10.1016/j.semnephrol.2012.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444292PMC
May 2012

6-Thioguanine damages mitochondrial DNA and causes mitochondrial dysfunction in human cells.

FEBS Lett 2011 Dec 2;585(24):3941-6. Epub 2011 Nov 2.

Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK.

The anticancer and immunosuppressant thiopurines cause 6-thioguanine (6-TG) to accumulate in nuclear DNA. We report that 6-TG is also readily incorporated into mitochondrial DNA (mtDNA) where it is rapidly oxidized. The oxidized forms of mtDNA 6-TG inhibit replication by DNA Pol-γ. Accumulation of oxidized 6-TG is associated with reduced mtDNA transcription, a decline in mitochondrial protein levels, and loss of mitochondrial function. Ultraviolet A radiation (UVA) also oxidizes mtDNA 6-TG. Cells without mtDNA are less sensitive to killing by a combination of 6-TG and UVA than their mtDNA-containing counterparts, indicating that photochemical mtDNA 6-TG oxidation contributes to 6-TG-mediated UVA photosensitization.
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http://dx.doi.org/10.1016/j.febslet.2011.10.040DOI Listing
December 2011

Efficient DNA interstrand crosslinking by 6-thioguanine and UVA radiation.

DNA Repair (Amst) 2011 Aug 30;10(8):869-76. Epub 2011 Jun 30.

Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK.

Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.
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http://dx.doi.org/10.1016/j.dnarep.2011.05.010DOI Listing
August 2011

T-lymphocyte-induced, Fas-mediated apoptosis is associated with early keratinocyte differentiation.

Exp Dermatol 2010 Apr 23;19(4):372-80. Epub 2009 Jul 23.

Women's & Children's Health Research Institute, Women's and Children's Hospital, North Adelaide, SA, Australia.

The development of eczematous lesions is thought to be due in part to a breakdown in skin barrier function as a result of T lymphocytes (T cells) invading the skin causing epidermal keratinocyte apoptosis. In this study, we investigated the interaction of T cells and keratinocytes on apoptosis and terminal differentiation using an in vitro co-culture system. Experiments were performed using the HaCaT keratinocyte cell line or normal human epidermal keratinocytes. Activated human peripheral blood-derived T cells were found to induce Fas-dependent keratinocyte apoptosis by up to sixfold. Increased Fas was associated with increased IFN-gamma. The T-cell apoptotic signal was found to target preferentially keratinocytes in the very early stages of terminal differentiation, such as those with low levels of alpha 6-integrin expression, and result in subsequent increased caspase 3 activity. This observation was accompanied by a marked increase in keratinocyte ICAM-1 expression and its ligand LFA-1 on T cells. Our data suggest that T cells may initiate the onset of keratinocyte terminal differentiation making them more susceptible to Fas-dependent cell death signals delivered by the T cells.
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http://dx.doi.org/10.1111/j.1600-0625.2009.00917.xDOI Listing
April 2010

Immune effector cells produce lethal DNA damage in cells treated with a thiopurine.

Cancer Res 2009 Mar 24;69(6):2393-9. Epub 2009 Feb 24.

Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, United Kingdom.

Azathioprine, a widely used immunosuppressant, is also used in the control of inflammatory disorders. These are characterized by the local accumulation of immune effector cells that produce reactive oxygen species (ROS). The DNA of azathioprine-treated patients contains 6-thioguanine (6-TG), a base analogue that is particularly susceptible to oxidation. Here, we show that 6-TG is vulnerable to ROS produced by chemical oxidants and that cells containing DNA 6-TG are hypersensitive to these oxidants. We also show that 6-TG incorporated into the DNA of macrophages sensitizes them to killing by endogenously produced ROS. ROS generated by macrophages are also a hazard for cocultured nonmacrophage cells containing DNA 6-TG. This bystander vulnerability of cells containing DNA 6-TG to oxidation by ROS generated by immune effector cells has implications for the long-term use of azathioprine in the management of inflammatory disorders.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-4264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2658672PMC
March 2009

Sodium butyrate induced keratinocyte apoptosis.

Apoptosis 2006 Aug;11(8):1379-90

Child Health Research Institute, Women's and Children's Hospital, North Adelaide, SA, Australia.

Apoptosis of keratinocytes is a key mechanism required for epidermal homeostasis and the renewal of damaged cells. Its dysregulation has been implicated in many skin diseases including cancer and hyperproliferative disorders. In the present study, the effect of sodium butyrate, a histone deacetylase inhibitor, on keratinocyte apoptosis was investigated using the HaCaT human keratinocyte cell line. Sodium butyrate induced morphological changes associated with apoptosis and nuclear fragmentation of HaCaTs. Annexin V staining demonstrated that sodium butyrate induced apoptosis in a dose and time-dependent manner with 50% of HaCaTs apoptotic after exposure to 0.8 mg/ml sodium butyrate for 24 h. Apoptosis was associated with upregulation of cell surface expression of the death receptor Fas and activation of the extrinsic caspase pathway, with induction of caspase 8 activity peaking after 8 h. Caspase 3 activity peaked after 24 h and was associated with cleavage of the caspase 3 substrate, poly (ADP-ribose) polymerase (PARP). The intrinsic caspase pathway was not activated as caspase 9 activity was not detected, and there was no change in the expression of terminal differentiation markers keratin 10 and involucrin following sodium butyrate treatment. Together these results indicate that sodium butyrate is a potent inducer of Fas associated apoptosis via caspase activation in HaCaT keratinocytes, an effect that is independent of the induction of terminal differentiation.
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http://dx.doi.org/10.1007/s10495-006-7960-3DOI Listing
August 2006

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin.

J Invest Dermatol 2004 Mar;122(3):791-804

Wound Healing and Injury Research Centre, The University of Adelaide Department of Surgery, The Queen Elizabeth Hospital, Woodville, South Australia.

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E(17)) in the rat such that embryonic day 19 (E(19)) wounds do not re-epithelialize. Moreover, wounds created in E(17) fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E(17) and E(19) skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E(17) and E(19) skin. c-fos and c-jun induction was transient in E(17) skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E(19) skin, AP-1 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E(17) skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.
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http://dx.doi.org/10.1111/j.0022-202X.2004.22319.xDOI Listing
March 2004

Bcl-xL overexpression protects from apoptosis induced by HMG-CoA reductase inhibitors in murine tubular cells.

Kidney Int 2003 Jul;64(1):181-91

Renal and Vascular Research Laboratory, Division of Nephrology-Hypertension, Fundación Jiménez Díaz,Universidad Autónoma Madrid, Madrid, Spain.

Background: Hyperplasia is attributed to enhanced tubular cell proliferation with unbalanced cell death. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors induce apoptosis in a variety of cell lines, including proximal tubular cells. However, the mechanisms by which statins induce apoptosis in tubular cells have not been fully addressed.

Methods: Apoptosis induced by simvastatin was measured in murine tubular cells with and without overexpressing Bcl-xL. Expression of genes implicated in cell death was studied by Northern and Western blot.

Results: The treatment of proliferating murine tubular cells (MCT) with simvastatin induced apoptosis in a time- and dose-dependent manner (0.1 to 1 micromol/L). Apoptosis was correlated with Bcl-xL mRNA and protein down-regulation. By contrast, the treatment with simvastatin did not modify the expression of the proapoptotic protein Bax. Simvastatin treatment was associated with cytochrome C release from the mitochondria to the cytosol. We also observed the presence of active caspase 9 and 3 during apoptosis induced by simvastatin. These effects were reversed by mevalonate, farnesylpyrophosphate (FPP), and geranylgeranylpyrophosphate (GGPP), suggesting the involvement of protein prenylation. Simvastatin appears to alter the balance between cell-life and death-promoting genes, as reflected by the decreased Bcl-xL/Bax ratio. Supporting this hypothesis, overexpression of Bcl-xL reduced the amount of apoptosis induced by simvastatin by 80% when compared with control vector-expressing cells. The overexpression of Bcl-xL also prevented the activation of caspase 9 and 3.

Conclusion: Our results indicate that down-regulation of Bcl-xL expression mediates apoptosis induced by statins in tubular cells. These results may be relevant to the treatment of disorders characterized by altered tubular proliferation.
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http://dx.doi.org/10.1046/j.1523-1755.2003.00080.xDOI Listing
July 2003