NUAK1 promotes organ fibrosis via YAP and TGF-ß/SMAD signaling.

Fibrosis is a central pathway that drives progression of multiple chronic diseases, yet few safe and effective clinical antifibrotic therapies exist. In most fibrotic disorders, transforming growth factor-ß (TGF-ß)-driven scarring is an important pathologic feature and a key contributor to disease progression. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two closely related transcription cofactors that are important for coordinating fibrogenesis after organ injury, but how they are activated in response to tissue injury has, so far, remained unclear. Here, we describe NUAK family kinase 1 (NUAK1) as a TGF-ß-inducible profibrotic kinase that is up-regulated in multiple fibrotic organs in mice and humans. Mechanistically, we show that TGF-ß induces a rapid increase in NUAK1 in fibroblasts. NUAK1, in turn, can promote profibrotic YAP and TGF-ß/SMAD signaling, ultimately leading to organ scarring. Moreover, activated YAP and TAZ can induce further NUAK1 expression, creating a profibrotic positive feedback loop that enables persistent fibrosis. Using mouse models of kidney, lung, and liver fibrosis, we demonstrate that this fibrogenic signaling loop can be interrupted via fibroblast-specific loss of NUAK1 expression, leading to marked attenuation of fibrosis. Pharmacologic NUAK1 inhibition also reduced scarring, either when initiated immediately after injury or when initiated after fibrosis was already established. Together, our data suggest that NUAK1 plays a critical, previously unrecognized role in fibrogenesis and represents an attractive target for strategies that aim to slow fibrotic disease progression.

Myofibroblast YAP/TAZ activation is a key step in organ fibrogenesis.

Fibrotic diseases account for nearly half of all deaths in the developed world. Despite its importance, the pathogenesis of fibrosis remains poorly understood. Recently, the two mechanosensitive transcription cofactors YAP and TAZ have emerged as important profibrotic regulators in multiple murine tissues. Despite this growing recognition, a number of important questions remain unanswered, including which cell types require YAP/TAZ activation for fibrosis to occur and the time course of this activation. Here, we present a detailed analysis of the role that myofibroblast YAP and TAZ play in organ fibrosis and the kinetics of their activation. Using analyses of cells, as well as multiple murine and human tissues, we demonstrated that myofibroblast YAP and TAZ were activated early after organ injury and that this activation was sustained. We further demonstrated the critical importance of myofibroblast YAP/TAZ in driving progressive scarring in the kidney, lung, and liver, using multiple transgenic models in which YAP and TAZ were either deleted or hyperactivated. Taken together, these data establish the importance of early injury-induced myofibroblast YAP and TAZ activation as a key event driving fibrosis in multiple organs. This information should help guide the development of new antifibrotic YAP/TAZ inhibition strategies.

A new, easily generated mouse model of diabetic kidney fibrosis.

Our understanding of diabetic kidney disease pathogenesis has been hampered by the lack of easily generated pre-clinical animal models that faithfully recapitulate critical features of human disease. While most standard animal models develop manifestations of early stage diabetic injury such as hyperfiltration and mesangial matrix expansion, only a select few develop key late stage features such as interstitial fibrosis and reduced glomerular filtration rate. An underlying theme in these late stage disease models has been the addition of renin-angiotensin system hyperactivation, an important contributor to human disease pathogenesis. Widespread use of these models has been limited, however, as they are either labour intensive to generate, or have been developed in the rat, preventing the use of the many powerful genetic tools developed for mice. Here we describe the Akita+/- Ren+/- mouse, a new, easily generated murine model of diabetic kidney disease that develops many features of late stage human injury, including not only hyperglycemia, hypertension, and albuminuria, but also reduced glomerular filtration rate, glomerulosclerosis, and interstitial fibrosis.

Novel reno-protective mechanism of Aspirin involves H2AK119 monoubiquitination and Set7 in preventing type 1 diabetic nephropathy.

BACKGROUND: Even after several novel therapeutic approaches, the number of people with diabetic nephropathy (DN) still continues to increase globally, this suggest to find novel therapeutic strategies to prevent it completely. Recent reports, are indicating the ubiquitin proteasome system alterations in DN. Recently, we also showed that, histone H2AK119 mono-ubiquitination (H2AK119-Ub) found to regulate Set7, a key epigenetic enzyme in the development of renal fibrosis under type 1 diabetic condition. Hence, we aimed to study the role of a known 20s proteasome inhibitor Aspirin, on histone ubiquitination in the progression of DN. METHODS: Male Wistar rats were rendered diabetic using a single dose of Streptozotocin (55mgkg-1, ip). After 4 weeks, diabetic animals were grouped into respective groups and the drug, aspirin, low dose (25mgkg-1day-1), high dose (50mgkg-1day-1) was administered through po route. At the end of the study, kidneys from all the groups were collected and processed separately for glomerular isolation, protein isolation, and for histopathological studies. RESULTS: Aspirin administration, reduced the protein expression of Mysm1, increased the protein expression of H2AK119-Ub and thereby reduced the Set7 protein expression in glomeruli isolated from diabetic animals and prevented renal fibrosis. CONCLUSIONS: In conclusion, our results are clearly indicating that, aspirin prevents renal fibrosis in diabetic animals through decreasing the expression of Mysm1, increasing the expression of H2AK119-Ub and thereby decreasing the protein expression of Set7, which is a novel mechanism. Moreover, this mechanism may lay down a novel strategy to prevent DN completely in future.

Esculetin ameliorates vascular perturbation by intervening in the occupancy of H2BK120Ub at At1, At2, Tgfß1 and Mcp1 promoter gene in thoracic aorta of IR and T2D rats.

Micro and macro vascular complications under diabetic condition are the responses to pathological stimuli exerted by up regulated renin angiotensin system (RAS) via deteriorating vascular physiology. Up-regulated RAS could influence in the adaptive mechanisms of target tissues to alter the abundance of angiotensin II type 1 receptor (AT1) and angiotensin II type 2 receptor (AT2). Such differential regulation of AT1 and AT2 have been reported to be associated with post-translational histone modifications (PTHMs). Additionally, recent evidences provide, esculetin (6,7-dihydroxycoumarin) reverses post-translational histone modifications (PTHMs) in diabetic cardiomyopathy and nephropathy. On account of these evidences, we further pursued this study to investigate the effects of esculetin on PTHMs in progressive vascular complications under insulin resistance (IR) and type 2 diabetic (T2D) conditions. Esculetin treatment in both IR and T2D conditions substantially improved vascular reactivity, increased eNos and decreased Vcam1 mRNA levels, and reduced collagen deposition in rat thoracic aorta. Further, the fold changes in At1 and At2 receptor mRNA in IR and T2D were reversed by esculetin treatment. Modifications in histone H2B lysine 120 monoubiquitination (H2BK120Ub) were also reversed in esculetin treatment group. Further, modification in the occupancy of H2BK120Ub at At1a, At2, Tgfß and Mcp1 promoter genes was evidenced by treatment with esculetin. Taken together, our investigation concluded with the involvement of esculetin in the amelioration of vascular perturbation by modifying H2BK120Ub along with occupancy at At1, At2, Tgfß1 and Mcp1 promoter gene.

Diminazene aceturate prevents nephropathy by increasing glomerular ACE2 and AT2 receptor expression in a rat model of type1 diabetes.

BACKGROUND AND PURPOSE: One of the protective actions of angiotensin converting enzyme-2 (ACE2) is the inactivation of angiotensin II. Expression and activity of ACE2 was reduced in glomeruli of diabetic patients and in animal models of diabetes. Recently the potential role of recombinant ACE2 administration in preventing diabetic nephropathy (DN) has been shown. Here we have tested the effects of the ACE2 activator, diminazene aceturate (DIZE), in a model of DN. EXPERIMENTAL APPROACH: Male Wistar rats were rendered diabetic using a single dose of streptozotocin (55 mg·kg-1 , i.p.). After 4 weeks, diabetic animals were divided into experimental groups and treated with DIZE, at a low dose (5 mg·kg-1 ·day-1 ), a high dose (15 mg·kg-1 ·day-1 ) and the high dose with of the AT2 receptor antagonist PD123319 (10 mg·kg-1 ·day-1 ). At the end of the treatment , kidneys from all the groups were collected and processed separately for glomerular isolation, protein isolation, mRNA extraction and for immunohistochemical studies. KEY RESULTS: Treatment with DIZE restored ACE2 expression in glomeruli and increased expression of AT2 receptors in whole kidney and isolated glomeruli of diabetic animals. DIZE administration reduced angiotensin II levels and increased angiotensin-(1-7) levels in diabetic kidney. However, PD123319 treatment reversed all these actions of DIZE. CONCLUSIONS AND IMPLICATIONS: DIZE treatment reduced diabetes-induced renal damage as shown by reduction of fibrosis and apoptosis. These protective actions of DIZE were blocked by the AT2 receptor antagonist. Taken together, these results suggest that DIZE protected against DN through the ACE2/angiotensin-(1-7)/ AT2 receptor axis.

Esculetin ameliorates hepatic fibrosis in high fat diet induced non-alcoholic fatty liver disease by regulation of FoxO1 mediated pathway.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD), a chronic metabolic disorder is associated with oxidative stress, inflammation and fibrotic cascades. In this study, we aimed to examine the effects of Esculetin, a well-known anti-oxidant on TGF-ß1 mediated liver fibrosis and FoxO1 activity. METHODS: A non-genetic murine model for NAFLD was developed by chronic high fat diet (HFD) (58% calories from fats) feeding in Wistar rats. The plasma biochemical parameters, liver function tests, oxidative stress, and histopathological alterations were assessed. The alterations in extracellular matrix (ECM) deposition and FoxO1 activity were assessed by immunohistochemistry. RESULTS: The aberrations in plasma parameters, liver functioning, morphometric and microscopic changes in liver structure of HFD fed rats were significantly improved by treatment with Esculetin. Liver fibrosis, identified in the form of collagen deposition and expression of fibrotic proteins like TGF-ß1 and fibronectin was also markedly controlled by Esculetin. The expression of phospho-FoxO1 was found to be reduced in HFD fed rats' liver, showing an increase in activation of FoxO1 under insulin resistant and hyperglycemic states. Esculetin treatment could improve phospho-FoxO1 expression, thus showing its ability to act on Akt/PI3K/FoxO1 pathway. CONCLUSIONS: As per the previous studies, a potential therapy for NAFLD may be the one with multi-faceted actions on insulin resistance, oxidative stress, inflammation and fibrosis. This study demonstrates the efficiency of Esculetin in improving liver fibrosis in HFD induced NAFLD.

Hidden targets of ubiquitin proteasome system: To prevent diabetic nephropathy.

Diabetic nephropathy (DN) is the major cause of end stage renal failure. Although, several therapeutic targets have emerged to prevent the progression of DN, the number of people with DN still continues to rise worldwide, suggesting an urgent need of novel targets to prevent DN completely. Currently, the role of ubiquitin proteasome system (UPS) has been highlighted in the pathogenesis and progression of various diseases like obesity, insulin resistance, atherosclerosis, cancers, neurodegerative disorders and including secondary complications of diabetes. UPS mainly involves in protein homeostatis through ubiquitination (post translational modification) and proteasomal degradation of various proteins. Ubiquitination, not only involves in proteasomal degradation, but also directs the substrate proteins to participate in multitude of cell signalling pathways. However, very little is known about ubiquitination and UPS in the progression of DN. This review mainly focuses on UPS and its components including E2 conjugating enzymes, E3 ligases and deubiquitinases (DUBs) in the development of DN and thus may help us to find novel therapeutic targets with in UPS to prevent DN completely in future.

Telmisartan and esculetin combination ameliorates type 2 diabetic cardiomyopathy by reversal of H3, H2A, and H2B histone modifications.

OBJECTIVES: Although cardioprotective effects of telmisartan are well explored, its effects on epigenetic alterations associated with type 2 diabetic (T2D) cardiomyopathy remain unmapped. Thus, the present study was designed to evaluate the potential of esculetin and telmisartan combination to reverse histone posttranslational modifications (PTMs) in curbing T2D cardiomyopathy. MATERIALS AND METHODS: T2D was induced by high-fat diet feeding along with low dose of streptozotocin (35 mg/kg, I.P) in male Wistar rats. T2D rats were treated with either telmisartan (10 mg/kg/day, P.O) or esculetin (50 mg/kg/day doses, P.O) or their combination for 2 weeks. Biochemical estimations, vascular reactivity, immunohistochemistry, and western blotting experiments were performed to evaluate the effects of the treatment in T2D cardiomyopathy. RESULTS: Esculetin and telmisartan combination alleviated the pathological features of T2D cardiomyopathy including metabolic perturbations, morphometric alterations, altered vascular reactivity, increased Keap1 and fibronectin expression more effectively than their respective monotherapy. This is the first report showing that telmisartan attenuates increased level of histone PTMs such as H3K9me2, H3K9Ac, H2AK119Ub, and H2BK120Ub in heart of T2D rats. The combination regimen showed a more significant reduction in augmented histone PTMs associated with T2D cardiomyopathy than their independent treatments. CONCLUSIONS: The present study demonstrates that esculetin and telmisartan combination can be an advanced pharmacological approach to ameliorate T2D cardiomyopathy which could be partially attributed to its ability to reverse the epigenetic alterations.

H2AK119 monoubiquitination regulates Angiotensin II receptor mediated macrophage infiltration and renal fibrosis in type 2 diabetic rats.

Monocyte chemoattractant protein (MCP-1) and transforming growth factor-ß (TGF-ß1)-markers of inflammation and fibrosis, are central to type 2 diabetic nephropathy (T2DN) progression. The epigenetic basis of their expression has also been explored to certain extent. H2A lysine 119 monoubiquitination (H2AK119Ub), a repressive chromatin mark regulates progression of hyperglycaemia induced fibrosis in glomerular mesangial cells. However, how H2AK119Ub affects the expression of MCP-1 and TGF-ß1 and their regulation by Angiotensin II receptor subtypes remains unknown. In the current study, we aimed to study the effect of Angiotensin II receptors' blockade on the macrophage infiltration and histone modifications occurring at the promoter region of Mcp1 and Tgfb1in high fat diet fed and low dose streptozotocin treated male Wistar rats. Hereby, we present the first report delineating a distinct link between H2AK119Ub and macrophage infiltration and fibrosis i.e. the enrichment of H2AUb at Mcp1 and Tgfb1 promoter region was found to reduce drastically in the T2DN which could be significantly reversed by Telmisartan and was further elevated by PD123319. We could conclude that the Angiotensin II mediated macrophage infiltration in T2DN is regulated at least partially by H2AK119Ub through both AT1 and AT2 receptors, which to the best of our knowledge, presents the first report for the regulation of Mcp1 by H2AK119Ub. Thus an approach targeting AT1R blockade and AT2R activation accompanied by an epigenetic modulator may be more suitable to ameliorate the macrophage infiltration and fibrosis associated with T2DN.

Histone H2AK119 and H2BK120 mono-ubiquitination modulate SET7/9 and SUV39H1 in type 1 diabetes-induced renal fibrosis.

Hyperglycaemia-induced expression of extracellular matrix (ECM) components plays a major role in the development of diabetic nephropathy (DN). The epigenetic mechanisms that modulate ECM gene expression in DN remain unclear. Therefore, we examined the role of histone H2A and H2B monoubiquitination on epigenetic chromatin marks, such as histone H3 lysine dimethylation (H3K4Me2, H3K9Me2 and H3K79Me2) in type 1 diabetic rat kidney. Hyperglycaemia increased collagen deposition and Col1a1 gene expression. In whole kidney of diabetic animals, both H2AK119 mono-ubiquitination (H2AK119Ub) and H2BK120 mono-ubiquitination (H2BK120Ub) were found to be increased, whereas, in glomeruli of diabetic animals, expression of both H2AK119Ub and H2BK120Ub was reduced. Changes in ubiquitin proteasome system components like increased Rnf2 (H2A-specific E3 ligase) and decreased H2A- and H2B-specific deubiquitinases (ubiquitin-specific proteases 7, 16, 21 and 22) were also observed. Globally increased levels of chromatin marks associated with active genes (H3K4Me2 and H3K79Me2) and decreased levels of repressive marks (H3K9Me2) were also observed. Hyperglycaemia also increased the protein expression of SET7/9 and decreased the expression of SUV39H1. We also showed the decreased occupancy of H2AK119Ub and H2BK120Ub on the promoters of Set7/9 and Suv39h1 in diabetic kidney. In addition, methylation marks regulated by H2AK119Ub (H3K27Me2 and H3K36Me2) and H2BK120Ub (H3K4Me2 and H3K79Me2) were also found to be altered on the promoters of Set7/9 and Suv39h1 Taken together, these results show the functional role of H2AK119Ub and H2BK120Ub in regulating histone H3K4Me2 and H3K9Me2 through modulating the expression of SET7/9 and SUV39H1 in the development of diabetic renal fibrosis.

E3 ubiquitin ligases as novel targets for inflammatory diseases.

Ubiquitination is one of the post translational modifications which decide the fate of various proteins in the cells, by either directing them towards proteasomal degradation or participation in several cell signalling pathways. Recently, the role of ubiquitination has been unravelled in pathogenesis and progression of various diseases, where inflammation is critical, like obesity, insulin resistance, atherosclerosis, angiotensin-II induced cardiac inflammation and asthma. E3 ligases are known to be instrumental in regulation of the inflammatory cascade. This review focuses on the role of different E3 ligases in the development of inflammatory diseases and thus may help us to target these E3 ligases in future drug discovery to prevent inflammation.

Insulin sensitizing and cardioprotective effects of Esculetin and Telmisartan combination by attenuating Ang II mediated vascular reactivity and cardiac fibrosis.

The combination of the angiotensin receptor blockers (ARBs) with other synthetic and natural molecules has been reported to have better safety profile and therapeutic efficacy in prevention of diabetes and its associated complications than their monotherapy. Driven by the aforementioned facts, this study was conceived to evaluate the potential additive effect of combination of Telmisartan and Esculetin in prevention of insulin resistance and associated cardiac fibrosis. Recently, we have reported that Esculetin prevented cardiovascular dysfunction associated with insulin resistance (IR) and type 2 diabetes. Insulin resistance was developed by high fat diet (HFD) feeding to Wistar rats. Telmisartan and Esculetin were administered at 10 mg/kg/day and 50 mg/kg/day doses (P.O, 2 weeks), respectively either alone or in combination. Plasma biochemical analyses, vascular reactivity and immunohistochemical experiments were performed to assess the beneficial effect of Telmisartan, Esculetin and their combination on insulin resistance and associated cardiac fibrosis. The study results showed that, co-administered Telmisartan and Esculetin ameliorated the pathological features like metabolic perturbation, morphometric alterations, vascular hyper responsiveness, extracellular matrix accumulation and the expression of fibronectin and TGF-ß more effectively than monotherapy in HFD fed rats. Hence, the study urges us to conclude that the solution to IR and associated cardiovascular dysfunction may lie in the Telmisartan and Esculetin combination therapy.

Differential regulation of angiotensin converting enzyme 2 and nuclear factor-κB by angiotensin II receptor subtypes in type 2 diabetic kidney.

Angiotensin II (Ang II) acts through Angiotensin Converting Enzyme (ACE)/Ang II type 1 receptor (AT1R) axis to promote renal failure whereas the Ang II type 2 receptor (AT2R)/Angiotensin Converting Enzyme 2 (ACE2)/Ang1-7/Mas axis constitutes the protective arm of Renin Angiotensin System (RAS). Though Ang II has been known to activate the Nuclear Factor-κB (NF-κB) signalling pathway through different receptor subtype(s) in different tissues under various diseases, the subtype orchestrating this stimulation in type 2 diabetic kidney remains elusive. ACE2, a protective monocarboxypeptidase, responsible for conversion of Ang II to Ang1-7, opposes the deleterious effects of RAS pathway but how its expression is altered with blockade of AT1R and AT2R is not yet known. Hence, the present study was conceived to understand the regulation of NF-κB and ACE2 by using specific AT1 and AT2 receptor antagonists in non-genetic model of type 2 diabetic nephropathy. Our results show that the AT1R and AT2R antagonists lead to the repression and activation of NF-κB signalling pathway, respectively which suggests the role of AT1R in NF-κB activation. The blockade of AT2R led to an increase in ACE2 expression, which may be a compensatory response to the drastically increased inflammatory mediators and oxidative stress in the diabetic kidney. To the best of our knowledge, this is the first study showing the differential regulation of NF-κB and ACE2 by Ang II receptor subtypes and thus this study improves our understanding regarding regulation of inflammatory cascade and ACE2 by AT1R and AT2R in type 2 diabetic kidney, which may help in designing novel strategies to combat the disease in future.

Esculetin attenuates alterations in Ang II and acetylcholine mediated vascular reactivity associated with hyperinsulinemia and hyperglycemia.

Esculetin (6, 7- dihydroxycoumarin) was found to be protective against hepatic and renal damage associated with Streptozotocin (STZ) induced type 1 diabetes, because of its radical scavenging property. However, there are no reports regarding its effect on vascular dysfunction under hyperinsulinemic and hyperglycemic conditions. Hence, the present study aimed to investigate the effect of esculetin on vascular dysfunction under these conditions. Non-genetic model of hyperinsulinemia and hyperglycemia were developed by high fat diet (HFD) feeding and HFD + Streptozotocin (STZ, 35 mg/kg, I.P) treatment in Wistar rats, respectively. Esculetin was administered at 50 and 100 mg/kg/day (P.O, 2 weeks) doses and biochemical, vascular reactivity and immunohistochemical experiments were performed to assess the effect of esculetin on vascular dysfunctions. Esculetin treatment significantly attenuates metabolic perturbations, alleviates insulin levels in hyperinsulinemic condition. Thoracic aorta of hyperinsulinemic and hyperglycemic rats showed hyper-responsiveness to Ang II mediated contraction and impaired acetylcholine mediated relaxation, and esculetin attenuates alterations in vascular reactivity to Ang II and acetylcholine challenges. In addition, immunohistochemical evaluations revealed that esculetin prevents increase in AT1R, AT2R, Keap1, TGF-ß, and decrease in ACE2 expression in aorta of hyperinsulinemic and hyperglycemic rats.