Publications by authors named "Detlef Schlondorff"

111 Publications

Podocyte and endothelial-specific elimination of BAMBI identifies differential transforming growth factor-β pathways contributing to diabetic glomerulopathy.

Kidney Int 2020 09 26;98(3):601-614. Epub 2020 Apr 26.

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

Transforming growth factor-β (TGF-β) is a central mediator of diabetic nephropathy. The effect of TGF-β, mediated by the type I TGF-β receptor, ALK5, and subsequent Smad2/3 activation results in podocyte apoptosis and loss. Previously, we demonstrated that the genetic deletion of the BMP and Activin Membrane-Bound Inhibitor (BAMBI), a negative modulator TGF-β signaling, accelerates diabetic nephropathy in mice. This was associated with heightened ALK1-mediated activation of Smad1/5 in the glomerular endothelial cells (ECs). Therefore, to evaluate the glomerular cell-specific effects of TGF-β in diabetic nephropathy we examined the effects of the podocyte- or EC-specific loss of Bambi (Pod-Bambi-/- or EC-Bambi-/-) in streptozotocin-induced diabetic mice with endothelial nitric oxide synthase deficiency. Interestingly, although hyperglycemia and body weight loss were similar in all groups of diabetic mice, significant hypertension was present only in the diabetic EC-Bambi-/- mice. While the podocyte or EC-specific loss of BAMBI both accelerated the progression of diabetic nephropathy, the worsened podocyte injury and loss observed in the diabetic Pod-Bambi-/- mice were associated with enhanced Smad3 activation. Increased Smad1/5 activation and EC proliferation were apparent only in the glomeruli of diabetic EC-Bambi-/- mice. The enhanced Smad1/5 activation in diabetic EC-Bambi-/- mice was associated with increased glomerular expression of plasmalemma vesicle-associated protein, pointing to the involvement of immature or dedifferentiated glomerular ECs in diabetic nephropathy. Notably, diabetic EC-Bambi-/- mice displayed podocyte injury and loss that were comparable to diabetic Pod-Bambi-/- mice. Thus, our results highlight the glomerular cell-specific contribution of TGF-β signaling and the intricate cross-talk between injured glomerular cells in the progression of diabetic nephropathy.
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http://dx.doi.org/10.1016/j.kint.2020.03.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484013PMC
September 2020

Role of CD8+ T cells in crescentic glomerulonephritis.

Nephrol Dial Transplant 2020 04;35(4):564-572

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

Crescentic glomerulonephritis (cGN) comprises three main types according to the pathogenesis and immunofluorescence patterns: anti-glomerular basement membrane antibody cGN, vasculitis-associated cGN and post-infectious immune complex cGN. In this brief review of the immune-pathogenesis of cGN, the focus is mainly on the role of CD8+ T cells in the progression of cGN. Under control conditions, Bowman's capsule (BC) provides a protected immunological niche by preventing access of cytotoxic CD8+ T cells to Bowman's space and thereby podocytes. Even in experimental nephrotoxic nephritis, leukocytes accumulate around the glomeruli, but remain outside of BC, as long as the latter remains intact. However, when and where breaches in BC occur, the inflammatory cells can gain access to and destroy podocytes, thus converting cGN into rapidly progressive glomerulonephritis (RPGN). These conclusions also apply to human cGN, where biopsies show that loss of BC integrity is associated with RPGN and progression to end-stage kidney disease. We propose a two-hit hypothesis for the role of cytotoxic CD8+ T cells in the progression of cGN. The initial insult occurs in response to the immune complex formation or deposition, resulting in local capillary and podocyte injury (first hit). The injured podocytes release neo-epitopes, eventually causing T-cell activation and migration to the glomerulus. Upon generation of breaches in BC, macrophages and CD8+ T cells can now gain access to the glomerular space and destroy neo-epitope expressing podocytes (second hit), resulting in RPGN. While further investigation will be required to test this hypothesis, future therapeutic trials should consider targeting of CD8+ T cells in the therapy of progressive cGN.
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http://dx.doi.org/10.1093/ndt/gfz043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139212PMC
April 2020

LRG1 Promotes Diabetic Kidney Disease Progression by Enhancing TGF--Induced Angiogenesis.

J Am Soc Nephrol 2019 04 11;30(4):546-562. Epub 2019 Mar 11.

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

Background: Glomerular endothelial dysfunction and neoangiogenesis have long been implicated in the pathogenesis of diabetic kidney disease (DKD). However, the specific molecular pathways contributing to these processes in the early stages of DKD are not well understood. Our recent transcriptomic profiling of glomerular endothelial cells identified a number of proangiogenic genes that were upregulated in diabetic mice, including leucine-rich -2-glycoprotein 1 (LRG1). LRG1 was previously shown to promote neovascularization in mouse models of ocular disease by potentiating endothelial TGF-/activin receptor-like kinase 1 (ALK1) signaling. However, LRG1's role in the kidney, particularly in the setting of DKD, has been unclear.

Methods: We analyzed expression of LRG1 mRNA in glomeruli of diabetic kidneys and assessed its localization by RNA hybridization. We examined the effects of genetic ablation of on DKD progression in unilaterally nephrectomized, streptozotocin-induced diabetic mice at 12 and 20 weeks after diabetes induction. We also assessed whether plasma LRG1 was associated with renal outcome in patients with type 2 diabetes.

Results: LRG1 localized predominantly to glomerular endothelial cells, and its expression was elevated in the diabetic kidneys. LRG1 ablation markedly attenuated diabetes-induced glomerular angiogenesis, podocyte loss, and the development of diabetic glomerulopathy. These improvements were associated with reduced ALK1-Smad1/5/8 activation in glomeruli of diabetic mice. Moreover, increased plasma LRG1 was associated with worse renal outcome in patients with type 2 diabetes.

Conclusions: These findings identify LRG1 as a potential novel pathogenic mediator of diabetic glomerular neoangiogenesis and a risk factor in DKD progression.
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http://dx.doi.org/10.1681/ASN.2018060599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442349PMC
April 2019

Single-Cell RNA Profiling of Glomerular Cells Shows Dynamic Changes in Experimental Diabetic Kidney Disease.

J Am Soc Nephrol 2019 04 7;30(4):533-545. Epub 2019 Mar 7.

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

Background: Recent single-cell RNA sequencing (scRNA-seq) analyses have offered much insight into cell-specific gene expression profiles in normal kidneys. However, in diseased kidneys, understanding of changes in specific cells, particularly glomerular cells, remains limited.

Methods: To elucidate the glomerular cell-specific gene expression changes in diabetic kidney disease, we performed scRNA-seq analysis of isolated glomerular cells from streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)-deficient (eNOS) mice and control eNOS mice.

Results: We identified five distinct cell populations, including glomerular endothelial cells, mesangial cells, podocytes, immune cells, and tubular cells. Using scRNA-seq analysis, we confirmed the expression of glomerular cell-specific markers and also identified several new potential markers of glomerular cells. The number of immune cells was significantly higher in diabetic glomeruli compared with control glomeruli, and further cluster analysis showed that these immune cells were predominantly macrophages. Analysis of differential gene expression in endothelial and mesangial cells of diabetic and control mice showed dynamic changes in the pattern of expressed genes, many of which are known to be involved in diabetic kidney disease. Moreover, gene expression analysis showed variable responses of individual cells to diabetic injury.

Conclusions: Our findings demonstrate the ability of scRNA-seq analysis in isolated glomerular cells from diabetic and control mice to reveal dynamic changes in gene expression in diabetic kidneys, with variable responses of individual cells. Such changes, which might not be apparent in bulk transcriptomic analysis of glomerular cells, may help identify important pathophysiologic factors contributing to the progression of diabetic kidney disease.
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http://dx.doi.org/10.1681/ASN.2018090896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442341PMC
April 2019

Bowman's capsule provides a protective niche for podocytes from cytotoxic CD8+ T cells.

J Clin Invest 2018 08 9;128(8):3413-3424. Epub 2018 Jul 9.

Institute of Precision Immunology, Icahn School of Medicine at Mount Sinai, New York New York, USA.

T cells play a key role in immune-mediated glomerulonephritis, but how cytotoxic T cells interact with podocytes remains unclear. To address this, we injected EGFP-specific CD8+ T cells from just EGFP death inducing (Jedi) mice into transgenic mice with podocyte-specific expression of EGFP. In healthy mice, Jedi T cells could not access EGFP+ podocytes. Conversely, when we induced nephrotoxic serum nephritis (NTSN) and injected Jedi T cells, EGFP+ podocyte transgenic mice showed enhanced proteinuria and higher blood urea levels. Morphometric analysis showed greater loss of EGFP+ podocytes, which was associated with severe crescentic and necrotizing glomerulonephritis. Notably, only glomeruli with disrupted Bowman's capsule displayed massive CD8+ T cell infiltrates that were in direct contact with EGFP+ podocytes, causing their apoptosis. Thus, under control conditions with intact Bowman's capsule, podocytes are not accessible to CD8+ T cells. However, breaches in Bowman's capsule, as also noted in human crescentic glomerulonephritis, allow access of CD8+ T cells to the glomerular tuft and podocytes, resulting in their destruction. Through these mechanisms, a potentially reversible glomerulonephritis undergoes an augmentation process to a rapidly progressive glomerulonephritis, leading to end-stage kidney disease. Translating these mechanistic insights to human crescentic nephritis should direct future therapeutic interventions at blocking CD8+ T cells, especially in progressive stages of rapidly progressive glomerulonephritis.
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http://dx.doi.org/10.1172/JCI97879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063505PMC
August 2018

Gene expression profiles of glomerular endothelial cells support their role in the glomerulopathy of diabetic mice.

Kidney Int 2018 08 31;94(2):326-345. Epub 2018 May 31.

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

Endothelial dysfunction promotes the pathogenesis of diabetic nephropathy (DN), which is considered to be an early event in disease progression. However, the molecular changes associated with glomerular endothelial cell (GEC) injury in early DN are not well defined. Most gene expression studies have relied on the indirect assessment of GEC injury from isolated glomeruli or renal cortices. Here, we present transcriptomic analysis of isolated GECs, using streptozotocin-induced diabetic wildtype (STZ-WT) and diabetic eNOS-null (STZ-eNOS) mice as models of mild and advanced DN, respectively. GECs of both models in comparison to their respective nondiabetic controls showed significant alterations in the regulation of apoptosis, oxidative stress, and proliferation. The extent of these changes was greater in STZ-eNOS than in STZ-WT GECs. Additionally, genes in STZ-eNOS GECs indicated further dysregulation in angiogenesis and epigenetic regulation. Moreover, a biphasic change in the number of GECs, characterized by an initial increase and subsequent decrease over time, was observed only in STZ-eNOS mice. This is consistent with an early compensatory angiogenic process followed by increased apoptosis, leading to an overall decrease in GEC survival in DN progression. From the genes altered in angiogenesis in STZ-eNOS GECs, we identified potential candidate genes, Lrg1 and Gpr56, whose function may augment diabetes-induced angiogenesis. Thus, our results support a role for GEC in DN by providing direct evidence for alterations of GEC gene expression and molecular pathways. Candidate genes of specific pathways, such as Lrg1 and Gpr56, can be further explored for potential therapeutic targeting to mitigate the initiation and progression of DN.
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http://dx.doi.org/10.1016/j.kint.2018.02.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054896PMC
August 2018

The changing of the guard: the past, the present, and the future of Kidney International.

Kidney Int 2017 12;92(6):1296-1298

Mount Sinai Medical Center, New York, New York, USA. Electronic address:

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http://dx.doi.org/10.1016/j.kint.2017.10.003DOI Listing
December 2017

Revisiting the determinants of the glomerular filtration barrier: what goes round must come round.

Kidney Int 2017 09;92(3):533-536

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

The glomerular filtration barrier (GFB) is characterized by a very high hydraulic permeability, combined with a marked permselectivity that excludes macromolecules such as albumin. Thus, the GFB retains most of the plasma proteins, with only 0.06% of albumin getting across the basement membrane. The GFB consists of 3 layers: fenestrated endothelial cells, the glomerular basement membrane, and podocytes. Injury to any of these components can result in the development of proteinuria. The contribution of the major components of the GFB has recently been reexamined and is discussed in the context of our past and present understanding.
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http://dx.doi.org/10.1016/j.kint.2017.06.003DOI Listing
September 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

Precision medicine comes of age in nephrology: identification of novel biomarkers and therapeutic targets for chronic kidney disease.

Kidney Int 2016 Apr;89(4):734-7

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

The goal of "precision medicine" is to characterize diseases based on the underlying molecular biology, in order to identify specific biomarkers and therapeutic targets that will ultimately improve clinical outcomes. The nephrology research community has developed a strong foundation for precision medicine, and recent publications demonstrate the feasibility of this approach to identify potential biomarkers and therapeutic targets in chronic kidney disease.
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http://dx.doi.org/10.1016/j.kint.2016.02.009DOI Listing
April 2016

BAMBI elimination enhances alternative TGF-β signaling and glomerular dysfunction in diabetic mice.

Diabetes 2015 Jun 9;64(6):2220-33. Epub 2015 Jan 9.

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

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-β type I receptor family and a negative modulator of TGF-β kinase signaling, and BAMBI(-/-) mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-β overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI(-/-) mice developed more albuminuria, with a widening of foot processes, than BAMBI(+/+) mice, along with increased activation of alternative TGF-β pathways such as extracellular signal-related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI(-/-) mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI(-/-) mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI(+/+) or BAMBI(-/-) mice with TGF-β resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI(-/-) mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-β signaling may therefore be of interest in diabetic nephropathy.
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http://dx.doi.org/10.2337/db14-1397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439561PMC
June 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

Are serum suPAR determinations by current ELISA methodology reliable diagnostic biomarkers for FSGS?

Kidney Int 2014 Mar;85(3):499-501

Mount Sinai School of Medicine, New York, New York, USA.

A soluble proteinuric substance has been postulated as a cause of primary focal segmental glomerulosclerosis (FSGS), and one candidate soluble urokinase receptor (suPAR). Evaluation of published results shows that serum suPAR determinations by current methodology do not reliably distinguish FSGS from other proteinuric glomerular diseases and serum suPAR cannot currently be considered a valid biomarker for primary or secondary FSGS. However, this should not discourage further research on potential roles of suPAR in proteinuric renal disease, including FSGS.
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http://dx.doi.org/10.1038/ki.2013.549DOI Listing
March 2014

Kidney international web focus on transplantation.

Kidney Int 2014 Feb;85(2):227-9

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

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http://dx.doi.org/10.1038/ki.2013.550DOI Listing
February 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

Accelerated reendothelialization, increased neovascularization and erythrocyte extravasation after arterial injury in BAMBI-/- mice.

PLoS One 2013 1;8(3):e58550. Epub 2013 Mar 1.

Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America.

Background: Intimal injury rapidly activates TGFβ and enhances vascular repair by the growth of endothelial (EC) and vascular smooth muscle cells (VSMC). The response to the TGFβ family of growth factors can be modified by BAMBI (BMP, Activin, Membrane Bound Inhibitor) acting as a non-signaling, competitive antagonist of TGFβ type I receptors such as ALK 1 and 5. In vivo the effect of BAMBI will depend on its cell-specific expression and of that of the ALK type receptors. We recently reported EC restricted BAMBI expression and genetic elimination of BAMBI resulting in an in vitro and in vivo phenotype characterized by endothelial activation and proliferation involving alternative pathway activation by TGFβ through ALK 1.

Methodology/principal Findings: To test the hypothesis that BAMBI modulates arterial response to injury via its effects on endothelial repair and arterial wall neovascularization we used a model of femoral arterial denudation injury in wild type (WT) and BAMBI(-/-) mice. Arterial response was evaluated at 2 and 4 weeks after luminal endothelial denudation of femoral arteries. The BAMBI(-/-) genotype mice showed accelerated luminal endothelial repair at 2 weeks and a highly unusual increase in arterial wall neovascularization compared to WT mice. The exuberant intimal and medial neovessel formation with BAMBI(-/-) genotype was also associated with significant red blood cell extravasation. The bleeding into the neointima at 2 weeks transiently increased it's area in the BAMBI(-/-)genotype despite the faster luminal endothelial repair in this group. Vascular smooth muscle cells were decreased at 2 weeks in BAMBI(-/-) mice, but comparable to wild type at 4 weeks.

Conclusions/significance: The absence of BAMBI results in a highly unusual surge in arterial wall neovascularization that surprisingly mimiks features of intra-plaque hemorrhage of advanced atheroma in a mechanical injury model. This suggests important effects of BAMBI on arterial EC homeostasis that need to be further studied in a model of inflammatory atherosclerosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058550PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585719PMC
August 2013

Up, up and away….

Kidney Int 2013 Jan;83(1)

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http://dx.doi.org/10.1038/ki.2012.377DOI Listing
January 2013

BAMBI regulates angiogenesis and endothelial homeostasis through modulation of alternative TGFβ signaling.

PLoS One 2012 25;7(6):e39406. Epub 2012 Jun 25.

Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America.

Background: BAMBI is a type I TGFβ receptor antagonist, whose in vivo function remains unclear, as BAMBI(-/-) mice lack an obvious phenotype.

Methodology/principal Findings: Identifying BAMBI's functions requires identification of cell-specific expression of BAMBI. By immunohistology we found BAMBI expression restricted to endothelial cells and by electron microscopy BAMBI(-/-) mice showed prominent and swollen endothelial cells in myocardial and glomerular capillaries. In endothelial cells over-expression of BAMBI reduced, whereas knock-down enhanced capillary growth and migration in response to TGFβ. In vivo angiogenesis was enhanced in matrigel implants and in glomerular hypertrophy after unilateral nephrectomy in BAMBI(-/-) compared to BAMBI(+/+) mice consistent with an endothelial phenotype for BAMBI(-/-) mice. BAMBI's mechanism of action in endothelial cells was examined by canonical and alternative TGFβ signaling in HUVEC with over-expression or knock-down of BAMBI. BAMBI knockdown enhanced basal and TGFβ stimulated SMAD1/5 and ERK1/2 phosphorylation, while over-expression prevented both.

Conclusions/significance: Thus we provide a first description of a vascular phenotype for BAMBI(-/-) mice, and provide in vitro and in vivo evidence that BAMBI contributes to endothelial and vascular homeostasis. Further, we demonstrate that in endothelial cells BAMBI interferes with alternative TGFβ signaling, most likely through the ALK 1 receptor, which may explain the phenotype observed in BAMBI(-/-) mice. This newly described role for BAMBI in regulating endothelial function has potential implications for understanding and treating vascular disease and tumor neo-angiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039406PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382616PMC
March 2013

Now we must work even harder.

Kidney Int 2012 Jun;81(11):1049-50

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http://dx.doi.org/10.1038/ki.2012.81DOI Listing
June 2012

Myeloid cell-derived hypoxia-inducible factor attenuates inflammation in unilateral ureteral obstruction-induced kidney injury.

J Immunol 2012 May 6;188(10):5106-15. Epub 2012 Apr 6.

Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University, Nashville, TN 37232, USA.

Renal fibrosis and inflammation are associated with hypoxia, and tissue pO(2) plays a central role in modulating the progression of chronic kidney disease. Key mediators of cellular adaptation to hypoxia are hypoxia-inducible factor (HIF)-1 and -2. In the kidney, they are expressed in a cell type-specific manner; to what degree activation of each homolog modulates renal fibrogenesis and inflammation has not been established. To address this issue, we used Cre-loxP recombination to activate or to delete both Hif-1 and Hif-2 either globally or cell type specifically in myeloid cells. Global activation of Hif suppressed inflammation and fibrogenesis in mice subjected to unilateral ureteral obstruction, whereas activation of Hif in myeloid cells suppressed inflammation only. Suppression of inflammatory cell infiltration was associated with downregulation of CC chemokine receptors in renal macrophages. Conversely, global deletion or myeloid-specific inactivation of Hif promoted inflammation. Furthermore, prolonged hypoxia suppressed the expression of multiple inflammatory molecules in noninjured kidneys. Collectively, we provide experimental evidence that hypoxia and/or myeloid cell-specific HIF activation attenuates renal inflammation associated with chronic kidney injury.
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http://dx.doi.org/10.4049/jimmunol.1103377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345098PMC
May 2012

Activation of innate immune defense mechanisms contributes to polyomavirus BK-associated nephropathy.

Kidney Int 2012 Jan 14;81(1):100-11. Epub 2011 Sep 14.

Medizinische Poliklinik Campus Innenstadt, Klinikum der LMU, Munich, Germany.

Polyomavirus-associated nephropathy (PVAN) is a significant complication after kidney transplantation, often leading to premature graft loss. In order to identify antiviral responses of the renal tubular epithelium, we studied activation of the viral DNA and the double-stranded RNA (dsRNA) sensors Toll-like receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I) in allograft biopsy samples of patients with PVAN, and in human collecting duct cells in culture after stimulation by the dsRNA mimic polyriboinosinic:polyribocytidylic acid (poly(I:C)), cytokines, or infection with BK virus. Double staining using immunofluorescence for BK virus and TLR3 showed strong signals in epithelial cells of distal cortical tubules and the collecting duct. In biopsies microdissected to isolate tubulointerstitial lesions, TLR3 but not RIG-I mRNA expression was found to be increased in PVAN. Collecting duct cells in culture expressed TLR3 intracellularly, and activation of TLR3 and RIG-I by poly(I:C) enhanced expression of cytokine, chemokine, and IFN-β mRNA. This inflammatory response could be specifically blocked by siRNA to TLR3. Finally, infection of the collecting duct cells with BK virus enhanced the expression of cytokines and chemokines. This led to an efficient antiviral immune response with TLR3 and RIG-I upregulation without activation of IL-1β or components of the inflammasome pathway. Thus, PVAN activation of innate immune defense mechanisms through TLR3 is involved in the antiviral and anti-inflammatory response leading to the expression of proinflammatory cytokines and chemokines.
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http://dx.doi.org/10.1038/ki.2011.311DOI Listing
January 2012

BAMBI is expressed in endothelial cells and is regulated by lysosomal/autolysosomal degradation.

PLoS One 2010 Sep 24;5(9):e12995. Epub 2010 Sep 24.

Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America.

Background: BAMBI (BMP and Activin Membrane Bound Inhibitor) is considered to influence TGFβ and Wnt signaling, and thereby fibrosis. Surprisingly data on cell type-specific expression of BAMBI are not available. We therefore examined the localization, gene regulation, and protein turnover of BAMBI in kidneys.

Methodology/principal Findings: By immunofluorescence microscopy and by mRNA expression, BAMBI is restricted to endothelial cells of the glomerular and some peritubular capillaries and of arteries and veins in both murine and human kidneys. TGFβ upregulated mRNA of BAMBI in murine glomerular endothelial cells (mGEC). LPS did not downregulate mRNA for BAMBI in mGEC or in HUVECs. BAMBI mRNA had a half-life of only 60 minutes and was stabilized by cycloheximide, indicating post-transcriptional regulation due to AU-rich elements, which we identified in the 3' untranslated sequence of both the human and murine BAMBI gene. BAMBI protein turnover was studied in HUVECs with BAMBI overexpression using a lentiviral system. Serum starvation as an inducer of autophagy caused marked BAMBI degradation, which could be totally prevented by inhibition of lysosomal and autolysosomal degradation with bafilomycin, and partially by inhibition of autophagy with 3-methyladenine, but not by proteasomal inhibitors. Rapamycin activates autophagy by inhibiting TOR, and resulted in BAMBI protein degradation. Both serum starvation and rapamycin increased the conversion of the autophagy marker LC3 from LC3-I to LC3-II and also enhanced co-staining for BAMBI and LC3 in autolysosomal vesicles.

Conclusions/significance: 1. BAMBI localizes to endothelial cells in the kidney and to HUVECs. 2. BAMBI mRNA is regulated by post-transcriptional mechanisms. 3. BAMBI protein is regulated by lysosomal and autolysosomal degradation. The endothelial localization and the quick turnover of BAMBI may indicate novel, yet to be defined functions of this modulator for TGFβ and Wnt protein actions in the renal vascular endothelium in health and disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012995PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945319PMC
September 2010

Innate immune receptors and autophagy: implications for autoimmune kidney injury.

Kidney Int 2010 Jul 28;78(1):29-37. Epub 2010 Apr 28.

Department of Nephrology, University of Munich, Munich, Germany.

Inflammation is the immune system's response to infectious or noninfectious sources of danger. Danger recognition is facilitated by various innate immune receptor families including the Toll-like receptors (TLRs), which detect danger signals in extracellular and intracellular compartments. It is an evolving concept that renal damage triggers intrarenal inflammation by immune recognition of molecules that are being released by dying cells. Such danger-associated molecules act as immunostimulatory agonists to TLRs and other innate immune receptors and induce cytokine and chemokine secretion, leukocyte recruitment, and tissue remodeling. As a new entry to this concept, autophagy allows stressed cells to reduce intracellular microorganisms, protein aggregates, and cellular organelles by moving and subsequently digesting them in autophagolysosomes. Within the autophagolysosome, endogenous molecules and danger-associated molecules may be presented to TLRs or loaded onto the major histocompatibility complex and presented as autoantigens. Here we discuss the current evidence for the danger signaling concept in autoimmune kidney injury and propose that autophagy-related processing of self-proteins provides a source of immunostimulatory molecules and autoantigens. A better understanding of danger signaling should enable us to unravel yet unknown triggers for renal immunopathology and progressive kidney disease.
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http://dx.doi.org/10.1038/ki.2010.111DOI Listing
July 2010

Human nephrosclerosis triggers a hypoxia-related glomerulopathy.

Am J Pathol 2010 Feb 17;176(2):594-607. Epub 2009 Dec 17.

Clinic and Policlinic for Internal Medicine, University Hospital Zurich, 8057 Zurich, Switzerland.

In the kidney, hypoxia contributes to tubulointerstitial fibrosis, but little is known about its implications for glomerular damage and glomerulosclerosis. Chronic hypoxia was hypothesized to be involved in nephrosclerosis (NSC) or "hypertensive nephropathy." In the present study genome-wide expression data from microdissected glomeruli were studied to examine the role of hypoxia in glomerulosclerosis of human NSC. Functional annotation analysis revealed prominent regulation of hypoxia-associated biological processes in NSC, including angiogenesis, fibrosis, and inflammation. Glomerular expression levels of a majority of genes regulated by the hypoxia-inducible factors (HIFs) were significantly altered in NSC. Among these HIF targets, chemokine C-X-C motif receptor 4 (CXCR4) was prominently induced. Glomerular CXCR4 mRNA induction was confirmed by quantitative RT-PCR in an independent cohort with NSC but not in those with other glomerulopathies. By immunohistological analysis, CXCR4 showed enhanced positivity in podocytes in NSC biopsy specimens. This CXCR4 positivity was associated with nuclear localization of HIF1alpha only in podocytes of NSC, indicating transcriptional activity of HIF. As the CXCR4 ligand CXCL12/SDF-1 is constitutively expressed in podocytes, autocrine signaling may contribute to NSC. In addition, a blocking CXCR4 antibody caused significant inhibition of wound closure by podocytes in an in vitro scratch assay. These data support a role for CXCR4/CXCL12 in human NSC and indicate that hypoxia not only is involved in tubulointerstitial fibrosis but also contributes to glomerular damage in NSC.
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http://dx.doi.org/10.2353/ajpath.2010.090268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808068PMC
February 2010

Chemokine receptor Ccr5 deficiency induces alternative macrophage activation and improves long-term renal allograft outcome.

Eur J Immunol 2010 Jan;40(1):267-78

Klinikum der Universität München, Medizinische Poliklinik-Innenstadt, München, Germany.

The chemokine (C-C motif) receptor 5 (CCR5) has been implicated in experimental and clinical allograft rejection. To dissect the function of CCR5 in acute and chronic renal allograft rejection, bilaterally nephrectomized WT and Ccr5-/- C57BL/6 mice were used as recipients of WT BALB/c renal allografts and analyzed 7 and 42 days after transplantation. Lesion scores (glomerular damage, vascular rejection, tubulointerstitial inflammation) and numbers of CD4+, CD8+, CD11c+ and alpha smooth muscle actin (alphaSMA)+ cells were reduced in allografts from Ccr5-/- recipients during the chronic phase. Increasing creatinine levels indicated deterioration of allograft function over time. While mRNA expression of Th1-associated markers decreased between 7 and 42 days, Th2-associated markers increased. Markers for alternatively activated macrophages (arginase 1, chitinase 3-like 3, resistin-like alpha, mannose receptor, C type 1), were strongly upregulated (mRNA and/or protein level) only in allografts from Ccr5-/- recipients at 42 days. Ccr5 deficiency shifted intragraft immune responses during the chronic phase towards the Th2 type and led to accumulation of alternatively activated macrophages. Additionally, splenocytes from unchallenged Ccr5-/- mice showed significantly increased arginase 1 and mannose receptor 1 mRNA levels, suggesting constitutive alternative activation of splenic macrophages. We conclude that Ccr5 deficiency favors alternative macrophage activation. This finding may be relevant for other inflammatory diseases that involve macrophage activation and may also influence future therapeutic strategies targeting CCR5.
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http://dx.doi.org/10.1002/eji.200939652DOI Listing
January 2010

Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice.

Genesis 2009 Aug;47(8):545-58

Klinikum der Universität München, Campus Innenstadt, Medizinische Poliklinik, Arbeitsgruppe Klinische Biochemie, D-80336 Munich, Germany.

The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double-deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double-deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose-binding protein and Cre (MBP-Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks.
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http://dx.doi.org/10.1002/dvg.20531DOI Listing
August 2009
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