Emerging role of tumor suppressor p53 in acute and chronic kidney diseases.

p53 is a major regulator of cell cycle arrest, apoptosis, and senescence. While involvement of p53 in tumorigenesis is well established, recent studies implicate p53 in the initiation and progression of several renal diseases, which is the focus of this review. Ischemic-, aristolochic acid (AA) -, diabetic-, HIV-associated-, obstructive- and podocyte-induced nephropathies are accompanied by activation and/or elevated expression of p53. Studies utilizing chemical or renal-specific inhibition of p53 in mice confirm the pathogenic role of this transcription factor in acute kidney injury and chronic kidney disease. TGF-ß1, NOX, ATM/ATR kinases, Cyclin G, HIPK, MDM2 and certain micro-RNAs are important determinants of renal p53 function in response to trauma. AA, cisplatin or TGF-ß1-mediated ROS generation via NOXs promotes p53 phosphorylation and subsequent tubular dysfunction. p53-SMAD3 transcriptional cooperation downstream of TGF-ß1 orchestrates induction of fibrotic factors, extracellular matrix accumulation and pathogenic renal cell communication. TGF-ß1-induced micro-RNAs (such as mir-192) could facilitate p53 activation, leading to renal hypertrophy and matrix expansion in response to diabetic insults while AA-mediated mir-192 induction regulates p53 dependent epithelial G2/M arrest. The widespread involvement of p53 in tubular maladaptive repair, interstitial fibrosis, and podocyte injury indicate that p53 clinical targeting may hold promise as a novel therapeutic strategy for halting progression of certain acute and chronic renal diseases, which affect hundreds of million people worldwide.

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.

TGF-ß1-p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications.

Chronic kidney disease affects >15% of the U.S. population and >850 million individuals worldwide. Fibrosis is the common outcome of many chronic renal disorders and, although the etiology varies (i.e., diabetes, hypertension, ischemia, acute injury, and urologic obstructive disorders), persistently elevated renal TGF-ß1 levels result in the relentless progression of fibrotic disease. TGF-ß1 orchestrates the multifaceted program of renal fibrogenesis involving proximal tubular dysfunction, failed epithelial recovery and redifferentiation, and subsequent tubulointerstitial fibrosis, eventually leading to chronic renal disease. Recent findings implicate p53 as a cofactor in the TGF-ß1-induced signaling pathway and a transcriptional coregulator of several TGF-ß1 profibrotic response genes by complexing with receptor-activated SMADs, which are homologous to the small worms (SMA) and Drosophilia mothers against decapentaplegic (MAD) gene families. The cooperative p53-TGF-ß1 genomic cluster includes genes involved in cell growth control and extracellular matrix remodeling [e.g., plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor, clade E, member 1), connective tissue growth factor, and collagen I]. Although the molecular basis for this codependency is unclear, many TGF-ß1-responsive genes possess p53 binding motifs. p53 up-regulation and increased p53 phosphorylation; moreover, they are evident in nephrotoxin- and ischemia/reperfusion-induced injury, diabetic nephropathy, ureteral obstructive disease, and kidney allograft rejection. Pharmacologic and genetic approaches that target p53 attenuate expression of the involved genes and mitigate the fibrotic response, confirming a key role for p53 in renal disorders. This review focuses on mechanisms whereby p53 functions as a transcriptional regulator within the TGF-ß1 cluster with an emphasis on the potent fibrosis-promoting PAI-1 gene.-Higgins, C. E., Tang, J., Mian, B. M., Higgins, S. P., Gifford, C. C., Conti, D. J., Meldrum, K. K., Samarakoon, R., Higgins, P. J. TGF-ß1-p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications.

Autophagy as a Therapeutic Target for Chronic Kidney Disease and the Roles of TGF-ß1 in Autophagy and Kidney Fibrosis.

Autophagy is a lysosomal protein degradation system that eliminates cytoplasmic components such as protein aggregates, damaged organelles, and even invading pathogens. Autophagy is an evolutionarily conserved homoeostatic strategy for cell survival in stressful conditions and has been linked to a variety of biological processes and disorders. It is vital for the homeostasis and survival of renal cells such as podocytes and tubular epithelial cells, as well as immune cells in the healthy kidney. Autophagy activation protects renal cells under stressed conditions, whereas autophagy deficiency increases the vulnerability of the kidney to injury, resulting in several aberrant processes that ultimately lead to renal failure. Renal fibrosis is a condition that, if chronic, will progress to end-stage kidney disease, which at this point is incurable. Chronic Kidney Disease (CKD) is linked to significant alterations in cell signaling such as the activation of the pleiotropic cytokine transforming growth factor-ß1 (TGF-ß1). While the expression of TGF-ß1 can promote fibrogenesis, it can also activate autophagy, which suppresses renal tubulointerstitial fibrosis. Autophagy has a complex variety of impacts depending on the context, cell types, and pathological circumstances, and can be profibrotic or antifibrotic. Induction of autophagy in tubular cells, particularly in the proximal tubular epithelial cells (PTECs) protects cells against stresses such as proteinuria-induced apoptosis and ischemia-induced acute kidney injury (AKI), whereas the loss of autophagy in renal cells scores a significant increase in sensitivity to several renal diseases. In this review, we discuss new findings that emphasize the various functions of TGF-ß1 in producing not just renal fibrosis but also the beneficial TGF-ß1 signaling mechanisms in autophagy.

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.

Heat Shock Protein 27, a Novel Downstream Target of Collagen Type XI alpha 1, Synergizes with Fatty Acid Oxidation to Confer Cisplatin Resistance in Ovarian Cancer Cells.

Collagen type XI alpha 1 (COL11A1) is a novel biomarker associated with cisplatin resistance in ovarian cancer. We have previously reported that COL11A1 activates Src-Akt signaling through the collagen receptors discoidin domain receptor 2 (DDR2) and integrin α1ß1 to confer cisplatin resistance to ovarian cancer cells. To identify the potential signaling molecules downstream of COL11A1 signaling, we performed protein kinase arrays and identified heat shock protein 27 (HSP27) as a potential mediator of COL11A1-induced cisplatin resistance. Through receptor knockdown and inhibitor experiments, we demonstrated that COL11A1 significantly upregulates HSP27 phosphorylation and expression via DDR2/integrin α1ß1 and Src/Akt signaling in ovarian cancer cells. Furthermore, genetic knockdown and pharmacological inhibition of HSP27, via ivermectin treatment, significantly sensitizes ovarian cancer cells cultured on COL11A1 to cisplatin treatment. HSP27 knockdown or inhibition also decreases NFκB activity as well as the expression of inhibitors of apoptosis proteins (IAPs), which are known downstream effector molecules of COL11A1 that promote cisplatin resistance. Interestingly, HSP27 knockdown or inhibition stimulates ovarian cancer cells to upregulate fatty acid oxidation (FAO) for survival and cisplatin resistance, and dual inhibition of HSP27 and FAO synergistically kills ovarian cancer cells that are cultured on COL11A1. Collectively, this study identifies HSP27 as a novel and druggable COL11A1 downstream effector molecule that may be targeted to overcome cisplatin resistance in recurrent ovarian cancer, which often overexpress COL11A1.

Deregulation of Negative Controls on TGF-ß1 Signaling in Tumor Progression.

The multi-functional cytokine transforming growth factor-ß1 (TGF-ß1) has growth inhibitory and anti-inflammatory roles during homeostasis and the early stages of cancer. Aberrant TGF-ß activation in the late-stages of tumorigenesis, however, promotes development of aggressive growth characteristics and metastatic spread. Given the critical importance of this growth factor in fibrotic and neoplastic disorders, the TGF-ß1 network is subject to extensive, multi-level negative controls that impact receptor function, mothers against decapentaplegic homolog 2/3 (SMAD2/3) activation, intracellular signal bifurcation into canonical and non-canonical pathways and target gene promotor engagement. Such negative regulators include phosphatase and tensin homologue (PTEN), protein phosphatase magnesium 1A (PPM1A), Klotho, bone morphogenic protein 7 (BMP7), SMAD7, Sloan-Kettering Institute proto-oncogene/ Ski related novel gene (Ski/SnoN), and bone morphogenetic protein and activin membrane-bound Inhibitor (BAMBI). The progression of certain cancers is accompanied by loss of expression, overexpression, mislocalization, mutation or deletion of several endogenous repressors of the TGF-ß1 cascade, further modulating signal duration/intensity and phenotypic reprogramming. This review addresses how their aberrant regulation contributes to cellular plasticity, tumor progression/metastasis and reversal of cell cycle arrest and discusses the unexplored therapeutic value of restoring the expression and/or function of these factors as a novel approach to cancer treatment.