The Genomic Response to TGF-ß1 Dictates Failed Repair and Progression of Fibrotic Disease in the Obstructed Kidney.

Tubulointerstitial fibrosis is a common and diagnostic hallmark of a spectrum of chronic renal disorders. While the etiology varies as to the causative nature of the underlying pathology, persistent TGF-ß1 signaling drives the relentless progression of renal fibrotic disease. TGF-ß1 orchestrates the multifaceted program of kidney fibrogenesis involving proximal tubular dysfunction, failed epithelial recovery or re-differentiation, capillary collapse and subsequent interstitial fibrosis eventually leading to chronic and ultimately end-stage disease. An increasing complement of non-canonical elements function as co-factors in TGF-ß1 signaling. p53 is a particularly prominent transcriptional co-regulator of several TGF-ß1 fibrotic-response genes by complexing with TGF-ß1 receptor-activated SMADs. This cooperative p53/TGF-ß1 genomic cluster includes genes involved in cellular proliferative control, survival, apoptosis, senescence, and ECM remodeling. While the molecular basis for this co-dependency remains to be determined, a subset of TGF-ß1-regulated genes possess both p53- and SMAD-binding motifs. Increases in p53 expression and phosphorylation, moreover, are evident in various forms of renal injury as well as kidney allograft rejection. Targeted reduction of p53 levels by pharmacologic and genetic approaches attenuates expression of the involved genes and mitigates the fibrotic response confirming a key role for p53 in renal disorders. This review focuses on mechanisms underlying TGF-ß1-induced renal fibrosis largely in the context of ureteral obstruction, which mimics the pathophysiology of pediatric unilateral ureteropelvic junction obstruction, and the role of p53 as a transcriptional regulator within the TGF-ß1 repertoire of fibrosis-promoting genes.

PAI-1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF-ß1-receptor signaling.

Renal fibrosis leads to chronic kidney disease, which affects over 15% of the U.S. population. PAI-1 is highly upregulated in the tubulointerstitial compartment in several common nephropathies and PAI-1 global ablation affords protection from fibrogenesis in mice. The precise contribution of renal tubular PAI-1 induction to disease progression, however, is unknown and surprisingly, appears to be independent of uPA inhibition. Human renal epithelial (HK-2) cells engineered to stably overexpress PAI-1 underwent dedifferentiation (E-cadherin loss, gain of vimentin), G2/M growth arrest (increased p-Histone3, p21), and robust induction of fibronectin, collagen-1, and CCN2. These cells are also susceptible to apoptosis (elevated cleaved caspase-3, annexin-V positivity) compared to vector controls, demonstrating a previously unknown role for PAI-1 in tubular dysfunction. Persistent PAI-1 expression results in a loss of klotho expression, p53 upregulation, and increases in TGF-ßRI/II levels and SMAD3 phosphorylation. Ectopic restoration of klotho in PAI-1-transductants attenuated fibrogenesis and reversed the proliferative defects, implicating PAI-1 in klotho loss in renal disease. Genetic suppression of p53 reversed the PA1-1-driven maladaptive repair, moreover, confirming a pathogenic role for p53 upregulation in this context and uncovering a novel role for PAI-1 in promoting renal p53 signaling. TGF-ßRI inhibition also attenuated PAI-1-initiated epithelial dysfunction, independent of TGF-ß1 ligand synthesis. Thus, PAI-1 promotes tubular dysfunction via klotho reduction, p53 upregulation, and activation of the TGF-ßRI-SMAD3 axis. Since klotho is an upstream regulator of both PAI-1-mediated p53 induction and SMAD3 signaling, targeting tubular PAI-1 expression may provide a novel, multi-level approach to the therapy of CKD.

Negative regulators of TGF-ß1 signaling in renal fibrosis; pathological mechanisms and novel therapeutic opportunities.

Elevated expression of the multifunctional cytokine transforming growth factor ß1 (TGF-ß1) is causatively linked to kidney fibrosis progression initiated by diabetic, hypertensive, obstructive, ischemic and toxin-induced injury. Therapeutically relevant approaches to directly target the TGF-ß1 pathway (e.g., neutralizing antibodies against TGF-ß1), however, remain elusive in humans. TGF-ß1 signaling is subjected to extensive negative control at the level of TGF-ß1 receptor, SMAD2/3 activation, complex assembly and promoter engagement due to its critical role in tissue homeostasis and numerous pathologies. Progressive kidney injury is accompanied by the deregulation (loss or gain of expression) of several negative regulators of the TGF-ß1 signaling cascade by mechanisms involving protein and mRNA stability or epigenetic silencing, further amplifying TGF-ß1/SMAD3 signaling and fibrosis. Expression of bone morphogenetic proteins 6 and 7 (BMP6/7), SMAD7, Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene (SnoN), phosphate tensin homolog on chromosome 10 (PTEN), protein phosphatase magnesium/manganese dependent 1A (PPM1A) and Klotho are dramatically decreased in various nephropathies in animals and humans albeit with different kinetics while the expression of Smurf1/2 E3 ligases are increased. Such deregulations frequently initiate maladaptive renal repair including renal epithelial cell dedifferentiation and growth arrest, fibrotic factor (connective tissue growth factor (CTGF/CCN2), plasminogen activator inhibitor type-1 (PAI-1), TGF-ß1) synthesis/secretion, fibroproliferative responses and inflammation. This review addresses how loss of these negative regulators of TGF-ß1 pathway exacerbates renal lesion formation and discusses the therapeutic value in restoring the expression of these molecules in ameliorating fibrosis, thus, presenting novel approaches to suppress TGF-ß1 hyperactivation during chronic kidney disease (CKD) progression.

Hematopoietic stem cell loss and hematopoietic failure in severe aplastic anemia is driven by macrophages and aberrant podoplanin expression.

Severe aplastic anemia (SAA) results from profound hematopoietic stem cell loss. T cells and interferon gamma (IFNγ) have long been associated with SAA, yet the underlying mechanisms driving hematopoietic stem cell loss remain unknown. Using a mouse model of SAA, we demonstrate that IFNγ-dependent hematopoietic stem cell loss required macrophages. IFNγ was necessary for bone marrow macrophage persistence, despite loss of other myeloid cells and hematopoietic stem cells. Depleting macrophages or abrogating IFNγ signaling specifically in macrophages did not impair T-cell activation or IFNγ production in the bone marrow but rescued hematopoietic stem cells and reduced mortality. Thus, macrophages are not required for induction of IFNγ in SAA and rather act as sensors of IFNγ. Macrophage depletion rescued thrombocytopenia, increased bone marrow megakaryocytes, preserved platelet-primed stem cells, and increased the platelet-repopulating capacity of transplanted hematopoietic stem cells. In addition to the hematopoietic effects, SAA induced loss of non-hematopoietic stromal populations, including podoplanin-positive stromal cells. However, a subset of podoplanin-positive macrophages was increased during disease, and blockade of podoplanin in mice was sufficient to rescue disease. Our data further our understanding of disease pathogenesis, demonstrating a novel role for macrophages as sensors of IFNγ, thus illustrating an important role for the microenvironment in the pathogenesis of SAA.

Cutting Edge: Notch Signaling Promotes the Plasticity of Group-2 Innate Lymphoid Cells.

The mechanisms underlying lymphocyte lineage stability and plasticity remain elusive. Recent work indicates that innate lymphoid cells (ILC) possess substantial plasticity. Whereas natural ILC2 (nILC2) produce type-2 cytokines, plastic inflammatory ILC2 (iILC2) can coproduce both type-2 cytokines and the ILC3-characteristic cytokine, IL-17. Mechanisms that elicit this lineage plasticity, and the importance in health and disease, remain unclear. In this study we show that iILC2 are potent inducers of airway inflammation in response to acute house dust mite challenge. We find that Notch signaling induces lineage plasticity of mature ILC2 and drives the conversion of nILC2 into iILC2. Acute blockade of Notch signaling abolished functional iILC2, but not nILC2, in vivo. Exposure of isolated nILC2 to Notch ligands induced Rorc expression and elicited dual IL-13/IL-17 production, converting nILC2 into iILC2. Together these results reveal a novel role for Notch signaling in eliciting ILC2 plasticity and driving the emergence of highly proinflammatory innate lymphocytes.