Debra F. Higgins

Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition

Hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis; however, the underlying mechanisms are not well defined. To examine the role of hypoxia-inducible factor-1 (HIF-1), a key mediator of cellular adaptation to hypoxia, in the development of fibrosis in mice, we inactivated Hif-1alpha in primary renal epithelial cells and in proximal tubules of kidneys subjected to unilateral ureteral obstruction (UUO) using Cre-loxP-mediated gene targeting. We found that Hif-1alpha enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1alpha inhibited the development of tubulointerstitial fibrosis in UUO kidneys, which was associated with decreased interstitial collagen deposition, decreased inflammatory cell infiltration, and a reduction in the number of fibroblast-specific protein-1-expressing (FSP-1-expressing) interstitial cells. Furthermore, we demonstrate that increased renal HIF-1alpha expression is associated with tubulointerstitial injury in patients with chronic kidney disease. Thus, we provide clinical and genetic evidence that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis by increasing expression of extracellular matrix-modifying factors and lysyl oxidase genes and by facilitating EMT.

Hypoxia-inducible factor signaling in the development of tissue fibrosis.

Capillary rarefaction is a hallmark of fibrotic diseases and results in reduced blood perfusion and oxygen delivery. In the kidney, tubulointerstitial fibrosis, which leads to the destruction of renal tissue and the irreversible loss of kidney function, is associated with hypoxia and the activation of Hypoxia-Inducible-Factor (HIF) signaling. HIF-1 and HIF-2 are basic-helix-loop-helix transcription factors that allow cells to survive in a low oxygen environment by regulating energy metabolism, vascular remodeling, erythropoiesis, cellular proliferation and apoptosis. Recent studies suggest that HIF activation promotes epithelial to mesenchymal transition (EMT) and renal fibrogenesis. These findings raise the possibility that the spectrum of HIF activated biological responses to hypoxic stress may differ under conditions of acute and chronic hypoxia. Here we discuss the role of HIF signaling in the pathogenesis and progression of chronic kidney disease.

Inactivation of the Arylhydrocarbon Receptor Nuclear Translocator (Arnt) Suppresses von Hippel-Lindau Disease-Associated Vascular Tumors in Mice

ABSTRACT Patients with germ line mutations in the VHL tumor suppressor gene are predisposed to the development of highly vascularized tumors within multiple tissues. Loss of pVHL results in constitutive activation of the transcription factors HIF-1 and HIF-2, whose relative contributions to the pathogenesis of the VHL phenotype have yet to be defined. In order to examine the role of HIF in von Hippel-Lindau (VHL)-associated vascular tumorigenesis, we utilized Cre-loxP-mediated recombination to inactivate hypoxia-inducible factor-1α (Hif-1α) and arylhydrocarbon receptor nuclear translocator (Arnt) genes in a VHL mouse model of cavernous liver hemangiomas and polycythemia. Deletion of Hif-1α did not affect the development of vascular tumors and polycythemia, nor did it suppress the increased expression of vascular endothelial growth factor (Vegf) and erythropoietin (Epo). In contrast, phosphoglycerokinase (Pgk) expression was substantially decreased, providing evidence for target gene-dependent functional redundancy between different Hif transcription factors. Inactivation of Arnt completely suppressed the development of hemangiomas, polycythemia, and Hif-induced gene expression. Here, we demonstrate genetically that the development of VHL-associated vascular tumors in the liver depends on functional ARNT. Furthermore, we provide evidence that individual HIF transcription factors may play distinct roles in the development of specific VHL disease manifestations.

Lipoxins Attenuate Renal Fibrosis by Inducing let-7c and Suppressing TGFβR1

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-β1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-β1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-β1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-β1 signaling pathway, including the TGF-β receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-β receptor type 1 and the response to TGF-β1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-β1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

Lipoxin A4 attenuates adipose inflammation

Aging and adiposity are associated with chronic low‐grade inflammation, which underlies the development of obesity‐associated complications, including type 2 diabetes mellitus (T2DM). The mechanisms underlying adipose inflammation may include macrophage infiltration and activation, which, in turn, affect insulin sensitivity of adipocytes. There is a growing appreciation that specific lipid mediators (including lipoxins, resolvins, and protectins) can promote the resolution of inflammation. Here, we investigated the effect of lipoxin A4 (LXA4), the predominant endogenously generated lipoxin, on adipose tissue inflammation. Using adipose tissue explants from perigonadal depots of aging female C57BL/6J mice (Animalia, Chordata, Mus musculus) as a model of age‐associated adipose inflammation, we report that LXA4 (1 nM) attenuates adipose inflammation, decreasing IL‐6 and increasing IL‐10 expression (P<0.05). The altered cytokine milieu correlated with increased GLUT‐4 and IRS‐1 expression, suggesting improved insulin sensitivity. Further investigations revealed the ability of LXA4 to rescue macrophage‐induced desensitization to insulin‐stimulated signaling and glucose uptake in cultured adipocytes, using vehicle‐stimulated cells as controls. This was associated with preservation of Akt activation and reduced secretion of proinflammatory cytokines, including TNF‐α. We therefore propose that LXA4 may represent a potentially useful and novel therapeutic strategy to subvert adipose inflammation and insulin resistance, key components of T2DM.—Börgeson, E., McGillicuddy, F. C., Harford, K. A., Corrigan, N., Higgins, D. F., Maderna, P., Roche, H. M., Godson C. Lipoxin A4 attenuates adipose inflammation. FASEB J. 26, 4287–4294 (2012). www.fasebj.org

Wnt6 regulates epithelial cell differentiation and is dysregulated in renal fibrosis

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3β (Ser9), nuclear accumulation of β-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-β (TGF-β); Wnt6 reversed TGF-β-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-β-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/β(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis.

Hypoxia promotes production of neural crest cells in the embryonic head.

ABSTRACT Hypoxia is encountered in either pathological or physiological conditions, the latter of which is seen in amniote embryos prior to the commencement of a functional blood circulation. During the hypoxic stage, a large number of neural crest cells arise from the head neural tube by epithelial-to-mesenchymal transition (EMT). As EMT-like cancer dissemination can be promoted by hypoxia, we investigated whether hypoxia contributes to embryonic EMT. Using chick embryos, we show that the hypoxic cellular response, mediated by hypoxia-inducible factor (HIF)-1α, is required to produce a sufficient number of neural crest cells. Among the genes that are involved in neural crest cell development, some genes are more sensitive to hypoxia than others, demonstrating that the effect of hypoxia is gene specific. Once blood circulation becomes fully functional, the embryonic head no longer produces neural crest cells in vivo, despite the capability to do so in a hypoxia-mimicking condition in vitro, suggesting that the oxygen supply helps to stop emigration of neural crest cells in the head. These results highlight the importance of hypoxia in normal embryonic development. Highlighted article: Naturally occurring hypoxia and HIF pathway activation at an early stage of chick development is required to produce sufficient cranial neural crest cells through EMT.

Epigenetic Unsilencing Reverses Renal Fibrosis

In CKD, the development of fibrosis is directly related to progressive loss of renal function. Several recent reports indicate a critical role for epigenetics in the development of fibrotic pathways leading to CKD,1–3 opening new avenues for the identification of biomarkers and novel therapeutics. Epigenetic changes are essential for kidney development and function, yet little is known about the epigenome of renal disease or indeed of any human disease other than cancer. It is now becoming apparent that epigenetic silencing of gene expression may be an important contributing factor to fibrogenesis in CKD. This concept is consistent with findings from epidemiologic studies indicating that adverse environments early in life and in adulthood (e.g., metabolic memory) have a major effect on CKD development.4 Epigenetic modification (methylation) by DNA methyltransferases at the 5-position of cytosine (5-methylcytosine [5mC]), sometimes called the “fifth base,” is associated with transcriptional silencing. 5mC-associated transcriptional silencing may be maintained in the long term, such as in genomic imprinting and X-chromosome inactivation.5 Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in disease, primarily in cancer, but more recently in the development of fibrosis in the kidney and other organs. Whereas CpG island promoter methylation causes transcriptional silencing, active demethylation resulting in formation of 5-hydroxymethylcytosine (5hmC) is associated with increased gene expression.6 In the elegant study by Tampe et al.7 in this issue of JASN, endogenous DNA demethylation mechanisms and their role in the homeostasis of epigenetic modifications are described for the first time in the adult kidney. Identification of demethylation processes of specific genes and subsequent reversal of fibrogenesis opens the attractive possibilities of using this information both to identify specific pharmacologic agents to transiently target these processes and to identify the patients that will benefit from such therapies. This research group previously established hypermethylation of the Rasal1 promoter and consequential gene silencing of Rasal1 as a critical component of experimental renal fibrogenesis after AKI leading to CKD.3 In the study by Tampe et al.,7 Rasal1 hypermethylation is found in experimental renal fibrosis, irrespective of the underlying disease model. Interestingly, treatment with the well characterized antifibrotic bone morphogenetic protein 7 (BMP7) resulted in hydroxymethylation of the Rasal1 promoter, indicating that BMP7 may mediate its antifibrotic actions in the kidney at least in part through enhancing active demethylation mechanisms. The most prominent active demethylation mechanism involves enzymatic oxidation (hydroxymethylation) of 5mC to 5hmC.8 5hmC accumulates in most cell types, suggesting the possibility that this “sixth base”9 in the genome may have a distinctive epigenetic role. The recently discovered ten-eleven translocation (Tet) proteins catalyze 5mC oxidation and generate 5mC derivatives, including 5hmC. Tet enzymes are thought to regulate the rapid disappearance of 5mC and the resulting increase in 5hmC.10 This concept is supported by studies showing that Tet-mediated hydroxylation is critical for resetting methylation at imprinted domains.10 Interestingly, hydroxylation of 5mC to 5hmC catalyzed by Tet enzymes not only reverses gene silencing, but also enhances gene expression from covalently modified promoters even compared with unmethylated promoters.6 Tampe et al.7 for the first time describe the role of Tet-mediated endogenous DNA demethylation mechanisms in kidney fibrosis and show antifibrotic BMP7-induced Rasal1 hydroxymethylation in mice that had been challenged with experimental renal fibrosis via a variety of independent models, including unilateral ureteral obstruction, streptozotocin-induced diabetic nephropathy in CD-1 mice, Col4A3-deficient Alport mice, and 5/6 nephrectomy, further supporting their data reported in the folic-acid induced nephropathy model and human fibroblast cultures in vitro.3 Because physiologic hydroxymethylation is mediated by Tet1, Tet2, and Tet3, Tampe et al.7 examined the expression of these genes in disease models and found reduced expression of Tet3, but not Tet1 or Tet2, to be associated with experimental renal fibrosis. The impact of this discovery is considerably strengthened with the finding that Tet3 expression is also reduced in human CKD (i.e., patients with diabetic nephropathy, hypertensive nephrosclerosis, IgA nephropathy, or lupus nephritis). The critical role of Tet3 in renal fibrosis is further strengthened by the observation that BMP7 reduced experimental renal fibrosis by normalizing Tet3 expression. Indeed, the antifibrotic action of BMP7 was considerably hampered when Tet3 was no longer expressed. Interestingly, Tet3 is also the most abundant Tet enzyme in zygotes and is critical in embryogenesis, whereas BMP7 is critical in kidney development regulating the differentiation of metanephric mesenchymal cells required for ureteric bud branching.11–13 It is tempting to speculate that BMP7 regulation of Tet3 expression and the resulting effect on the epigenome play an important role in nephrogenesis. The study by Tampe et al.7 indicates that Rasal1 promoter hypermethylation and reduced Tet3 expression are features of renal fibrosis. BMP7 treatment resulted in normalization of Tet3 expression and consequently hydroxymethylation of the Rasal1 promoter. BMP7-mediated hydroxymethylation of the Rasal1 promoter was impaired when Tet3 expression was lost. Electrophoretic mobility shift assays confirmed binding of Tet3 to its CXXC binding motif flanking the Rasal1 CpG island promoter, further indicating the role of Tet3 in Rasal1 hydroxymethylation. Preclinical data from this study indicate that aberrant methylation or perhaps active demethylation processes catalyzed by Tet3 determine the balance between health and CKD. These findings suggest that transient therapies targeted at amplifying or restoring Tet3 activity may prove beneficial to patients with CKD. Furthermore, analysis of methylation status (e.g., using RASAL1 as a methylation biomarker) to determine the individual risk of a patient to develop fibrosis or suitability for demethylation-based therapy appears both plausible and attractive. However, further studies in larger patient cohorts are warranted to establish these targets for both diagnosis and therapy. Demethylation agents, such as 5-azacytidine, have already been described to rescue kidney fibrosis in experimental models3 and demethylation agents are in clinical trials for treatment of cancers; however, these drugs are cytotoxic and the long-term effects of genome-wide demethylation are unknown. It therefore seems more attractive to transiently and specifically reactivate an endogenous demethylation process and thereby readdress the imbalance that results in CKD. The identification of Tet3 as a critical player in both the development and reversal of renal fibrogenesis described in the study by Tampe et al.7 may be the first step toward achieving this attractive possibility.

BMP7-induced-Pten inhibits Akt and prevents renal fibrosis

Bone morphogenetic protein-7 (BMP-7) counteracts pro-fibrotic effects of TGFβ1 in cultured renal cells and protects from fibrosis in acute and chronic renal injury models. Using the unilateral ureteral obstruction (UUO) model of chronic renal fibrosis, we investigated the effect of exogenous-rhBMP-7 on pro-fibrotic signaling pathways mediated by TGFβ1 and hypoxia. Mice undergoing UUO were treated with vehicle or rhBMP-7 (300μg/kg i.p.) every other day for eight days and kidneys analysed for markers of fibrosis and SMAD, MAPK, and PI3K signaling. In the kidney, collecting duct and tubular epithelial cells respond to BMP-7 via activation of SMAD1/5/8. Phosphorylation of SMAD1/5/8 was reduced in UUO kidneys from vehicle-treated animals yet maintained in UUO kidneys from BMP-7-treated animals, confirming renal bioactivity of exogenous rhBMP-7. BMP-7 inhibited Collagen Iα1 and Collagen IIIα1 gene expression and Collagen I protein accumulation, while increasing expression of Collagen IVα1 in UUO kidneys. Activation of SMAD2, SMAD3, ERK, p38 and PI3K/Akt signaling occurred during fibrogenesis and BMP-7 significantly attenuated SMAD3 and Akt signaling in vivo. Analysis of renal collecting duct (mIMCD) and tubular epithelial (HK-2) cells stimulated with TGFβ1 or hypoxia (1% oxygen) to activate Akt provided further evidence that BMP-7 specifically inhibited PI3K/Akt signaling. PTEN is a negative regulator of PI3K and BMP-7 increased PTEN expression in vivo and in vitro. These data demonstrate an important mechanism by which BMP-7 orchestrates renal protection through Akt inhibition and highlights Akt inhibitors as anti-fibrotic therapeutics.

Profibrotic IHG-1 complexes with renal disease associated HSPA5 and TRAP1 in Mitochondria.

The highly conserved mitochondrial protein induced in high glucose-1 (IHG-1) functions to maintain mitochondrial quality and is associated with the development of fibrosis in diabetic nephropathy. Towards identifying novel approaches to treating diabetic kidney disease, IHG-1-protein-protein interactions were investigated using epitope-tagged immunoprecipitation analyses followed by mass spectrometry. Here we show that IHG-1 is solely expressed in mitochondria and localised to the inner mitochondrial membrane, the region where mitochondrial reactive oxygen species are generated. Chaperones HSPA5 and TRAP1 and cold shock protein YBX1 were identified as IHG-1 binding partners. All three proteins are important in the cellular response to oxidative stress and play important roles in mitochondrial transcription and DNA repair. Both redox imbalance and IHG-1 stimulate TGF-β signalling. IHG-1, HSPA5 and YBX1 all show increased expression in diabetic nephropathy, chronic kidney disease and in the Unilateral Ureteral Obstruction model of kidney fibrosis. Increased IHG-1 expression in UUO correlated with loss of TRAP1 expression. IHG-1 may target TRAP1 for degradation. When IHG-1 is no longer localised to mitochondria, it retains the ability to interact with the cold shock protein YBX1, facilitating anti-fibrotic actions in the nucleus. Targeting these proteins may offer alternative treatments for fibrotic kidney disease.