ERK/RSK-mediated phosphorylation of Y-box binding protein-1 aggravates diabetic cardiomyopathy by suppressing its interaction with deubiquitinase OTUB1.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, but its underlying mechanisms still remain unclear. The multifunctional protein Y-box binding protein-1 (YB-1) plays an important role in cardiac pathogenesis by regulating cardiac apoptosis, cardiac fibrosis, and pathological remodeling, whereas its role in chronic DCM requires further investigation. Here, we report that the phosphorylation of YB-1 at serine102 (S102) was markedly elevated in streptozotocin-induced diabetic mouse hearts and in high glucose-treated cardiomyocytes, whereas total YB-1 protein levels were significantly reduced. Coimmunoprecipitation experiments showed that YB-1 interacts with the deubiquitinase otubain-1, but hyperglycemia-induced phosphorylation of YB-1 at S102 diminished this homeostatic interaction, resulting in ubiquitination and degradation of YB-1. Mechanistically, the high glucose-induced phosphorylation of YB-1 at S102 is dependent on the upstream extracellular signal-regulated kinase (ERK)/Ras/mitogen-activated protein kinase (p90 ribosomal S6 kinase [RSK]) signaling pathway. Accordingly, pharmacological inhibition of the ERK pathway using the upstream kinase inhibitor U0126 ameliorated features of DCM compared with vehicle-treated diabetic mice. We demonstrate that ERK inhibition with U0126 also suppressed the phosphorylation of the downstream RSK and YB-1 (S102), which stabilized the interaction between YB-1 and otubain-1 and thereby preserved YB-1 protein expression in diabetic hearts. Taken together, we propose that targeting the ERK/RSK/YB-1 pathway could be a potential therapeutic approach for treating DCM.

Regulation of the Homeostatic Unfolded Protein Response in Diabetic Nephropathy.

A growing body of scientific evidence indicates that protein homeostasis, also designated as proteostasis, is causatively linked to chronic diabetic nephropathy (DN). Experimental studies have demonstrated that the insulin signaling in podocytes maintain the homeostatic unfolded protein response (UPR). Insulin signaling via the insulin receptor non-canonically activates the spliced X-box binding protein-1 (sXBP1), a highly conserved endoplasmic reticulum (ER) transcription factor, which regulates the expression of genes that control proteostasis. Defective insulin signaling in mouse models of diabetes or the genetic disruption of the insulin signaling pathway in podocytes propagates hyperglycemia induced maladaptive UPR and DN. Insulin resistance in podocytes specifically promotes activating transcription factor 6 (ATF6) dependent pathogenic UPR. Akin to insulin, recent studies have identified that the cytoprotective effect of anticoagulant serine protease-activated protein C (aPC) in DN is mediated by sXBP1. In mouse models of DN, treatment with chemical chaperones that improve protein folding provides an additional benefit on top of currently used ACE inhibitors. Understanding the molecular mechanisms that transmute renal cell specific adaptive responses and that deteriorate renal function in diabetes will enable researchers to develop new therapeutic regimens for DN. Within this review, we focus on the current understanding of homeostatic mechanisms by which UPR is regulated in DN.

Pharmacologic IRE1α kinase inhibition alleviates aortic dissection by decreasing vascular smooth muscle cells apoptosis.

Aortic dissection (AD) is a rare but catastrophic disorder, and associated with significant morbidity among survivors. This study aimed to target IRE1α-XBP1s pathway pharmacologically, and evaluate its therapeutic potential in the occurrence and progression of AD. Western Blot and immunohistochemistry results showed that expression of XBP1s was significantly increased in the human aorta samples of AD group in compared with the control group, and exclusively in aortic vascular smooth muscle cells (VSMCs). Further in vitro study revealed that Angiotensin II (Ang II) could increase the expression of XBP1s and promote its nuclear translocation in cultured VSMCs, which leads to numerous gene transcription, including gp91phox, Chop, Cleaved-caspase 3, Bax, and Bcl-2. These genes contribute to the production of reactive oxygen species (ROS), VMSCs phenotypic switch and apoptosis. Whereas an IRE1α endoribonuclease domain inhibitor MKC-3946 could reverse it. Finally, the efficacy of MKC-3946 was tested in a mouse AD model. As shown in vitro, MKC-3946 could reduce the expression of XBP1s and protect against AD by suppressing XBP1s associated ROS production and apoptosis in VSMCs in vivo. The current study revealed the relevant role of IRE1α-XBP1s signaling pathway in AD occurrence and progression. MKC-3946 could be of great potential in clinical application.

Activated Protein C Ameliorates Diabetic Cardiomyopathy via Modulating OTUB1/YB-1/MEF2B Axis.

Aims: The pathogenesis of diabetic cardiomyopathy (DCM) is complex and the detailed mechanism remains unclear. Coagulation protease activated Protein C (aPC) has been reported to have a protective effect in diabetic microvascular disease. Here, we investigated whether aPC could play a protective role in the occurrence and development of major diabetic complication DCM, and its underlying molecular mechanism. Methods and Results: In a mouse model of streptozotocin (STZ) induced DCM, endogenous aPC levels were reduced. Restoring aPC levels by exogenous administration of zymogen protein C (PC) improved cardiac function of diabetic mice measured by echocardiography and invasive hemodynamics. The cytoprotective effect of aPC in DCM is mediated by transcription factor Y-box binding protein-1 (YB-1). Mechanistically, MEF2B lies downstream of YB-1 and YB-1/MEF2B interaction restrains deleterious MEF2B promoter activity in DCM. The regulation of YB-1 on MEF2B transcription was analyzed by dual-luciferase and chromatin immunoprecipitation assays. In diabetic mice, aPC ameliorated YB-1 degradation via reducing its K48 ubiquitination through deubiquitinating enzyme otubain-1 (OTUB1) and improving the interaction between YB-1 and OTUB1. Using specific agonists and blocking antibodies, PAR1 and EPCR were identified as crucial receptors for aPC's dependent cytoprotective signaling. Conclusion: These data identify that the cytoprotective aPC signaling via PAR1/EPCR maintains YB-1 levels by preventing the ubiquitination and subsequent proteasomal degradation of YB-1 via OTUB1. By suppressing MEF2B transcription, YB-1 can protect against DCM. Collectively, the current study uncovered the important role of OTUB1/YB-1/MEF2B axis in DCM and targeting this pathway might offer a new therapeutic strategy for DCM. Translational Perspective: DCM is emerging at epidemic rate recently and the underlying mechanism remains unclear. This study explored the protective cell signaling mechanisms of aPC in mouse models of DCM. As a former FDA approved anti-sepsis drug, aPC along with its derivatives can be applied from bench to bed and can be explored as a new strategy for personalized treatment for DCM. Mechanistically, OTUB1/YB-1/MEF2B axis plays a critical role in the occurrence and development of DCM and offers a potential avenue for therapeutic targeting of DCM.

The MEK inhibitor U0126 ameliorates diabetic cardiomyopathy by restricting XBP1's phosphorylation dependent SUMOylation.

Background: Chronic diabetes accelerates vascular dysfunction often resulting in cardiomyopathy but underlying mechanisms remain unclear. Recent studies have shown that the deregulated unfolded protein response (UPR) dependent on highly conserved IRE1α-spliced X-box- binding protein (XBP1s) and the resulting endoplasmic reticulum stress (ER-Stress) plays a crucial role in the occurrence and development of diabetic cardiomyopathy (DCM). In the present study, we determined whether targeting MAPK/ERK pathway using MEK inhibitor U0126 could ameliorate DCM by regulating IRE1α-XBP1s pathway. Method: Three groups of 8-week-old C57/BL6J mice were studied: one group received saline injection as control (n=8) and two groups were made diabetic by streptozotocin (STZ) (n=10 each). 18 weeks after STZ injection and stable hyperglycemia, one group had saline treatment while the second group was treated with U0126 (1mg/kg/day), 8 weeks later, all groups were sacrificed. Cardiac function/histopathological changes were determined by echocardiogram examination, Millar catheter system, hematoxylin-eosin staining and western blot analysis. H9C2 cardiomyocytes were employed for in vitro studies. Results: Echocardiographic, hemodynamic and histological data showed overt myocardial hypertrophy and worsened cardiac function in diabetic mice. Chronic diabetic milieu enhanced SUMOylation and impaired nuclear translocation of XBP1s. Intriguingly, U0126 treatment significantly ameliorated progression of DCM, and this protective effect was achieved through enriching XBP1s' nuclear accumulation. Mechanistically, U0126 inhibited XBP1s' phosphorylation on S348 and SUMOylation on K276 promoting XBP1s' nuclear translocation. Collectively, these results identify that MEK inhibition restores XBP1s-dependent UPR and protects against diabetes-induced cardiac remodeling. Conclusion: The current study identifies previously unknown function of MEK/ERK pathway in regulation of ER-stress in DCM. U0126 could be a therapeutic target for the treatment of DCM.

Chronic Obstructive Pulmonary Disease and the Cardiovascular System: Vascular Repair and Regeneration as a Therapeutic Target.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide and encompasses chronic bronchitis and emphysema. It has been shown that vascular wall remodeling and pulmonary hypertension (PH) can occur not only in patients with COPD but also in smokers with normal lung function, suggesting a causal role for vascular alterations in the development of emphysema. Mechanistically, abnormalities in the vasculature, such as inflammation, endothelial dysfunction, imbalances in cellular apoptosis/proliferation, and increased oxidative/nitrosative stress promote development of PH, cor pulmonale, and most probably pulmonary emphysema. Hypoxemia in the pulmonary chamber modulates the activation of key transcription factors and signaling cascades, which propagates inflammation and infiltration of neutrophils, resulting in vascular remodeling. Endothelial progenitor cells have angiogenesis capabilities, resulting in transdifferentiation of the smooth muscle cells via aberrant activation of several cytokines, growth factors, and chemokines. The vascular endothelium influences the balance between vaso-constriction and -dilation in the heart. Targeting key players affecting the vasculature might help in the development of new treatment strategies for both PH and COPD. The present review aims to summarize current knowledge about vascular alterations and production of reactive oxygen species in COPD. The present review emphasizes on the importance of the vasculature for the usually parenchyma-focused view of the pathobiology of COPD.

Lipid presentation by the protein C receptor links coagulation with autoimmunity.

Antiphospholipid antibodies (aPLs) cause severe autoimmune disease characterized by vascular pathologies and pregnancy complications. Here, we identify endosomal lysobisphosphatidic acid (LBPA) presented by the CD1d-like endothelial protein C receptor (EPCR) as a pathogenic cell surface antigen recognized by aPLs for induction of thrombosis and endosomal inflammatory signaling. The engagement of aPLs with EPCR-LBPA expressed on innate immune cells sustains interferon- and toll-like receptor 7-dependent B1a cell expansion and autoantibody production. Specific pharmacological interruption of EPCR-LBPA signaling attenuates major aPL-elicited pathologies and the development of autoimmunity in a mouse model of systemic lupus erythematosus. Thus, aPLs recognize a single cell surface lipid-protein receptor complex to perpetuate a self-amplifying autoimmune signaling loop dependent on the cooperation with the innate immune complement and coagulation pathways.

Podocyte Integrin-ß3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-ß3, a subunit of integrin-αvß3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvß3 induces transient binding of integrin-ß3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-ß3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-ß3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-ß3 interaction by specifically deleting podocyte integrin-ß3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvß3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvß3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

Coagulation Signalling and Metabolic Disorders: Lessons Learned from Animal Models.

Nutrient excess in obesity drives metabolic reprogramming in multiple tissues involving extensive interorgan and intercellular crosstalk. Experimental and clinical studies show that prolonged nutrient excess often compromises metabolic adaptation propagating proobesogenic and proinflammatory responses. Chronic inflammation further promotes insulin resistance and associated comorbidities. Obesity and type 2 diabetes are characterized by a hypercoagulable state and clinical studies show a strong correlation of markers of coagulation activation in metabolic disorders. Coagulation protease-dependent signalling via protease-activated receptors is intimately associated with inflammation. The experimental evidence supports roles of tissue factor and G protein coupled protease-activated receptor-2 signalling in the regulation of insulin resistance and metabolic inflammation in diet-induced obesity. Likewise, increases in plasminogen activator inhibitor-1 levels and fibrin-driven inflammation promote insulin resistance in obesity. Additionally, impaired thrombomodulin-dependent protein C activation is mechanistically linked to diabetic kidney disease. Given the increased usage of direct oral anticoagulants, understanding the role of specific coagulation proteases in regulation of metabolic inflammation is highly relevant and might provide insights into the design of novel treatment regimens for patients suffering from thromboinflammatory and cardiometabolic disorders.

Homeostatic nuclear RAGE-ATM interaction is essential for efficient DNA repair.

The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE-/-), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis.

Farnesoid X Receptor Agonism Protects against Diabetic Tubulopathy: Potential Add-On Therapy for Diabetic Nephropathy.

Established therapies for diabetic nephropathy (dNP) delay but do not prevent its progression. The shortage of established therapies may reflect the inability to target the tubular compartment. The chemical chaperone tauroursodeoxycholic acid (TUDCA) ameliorates maladaptive endoplasmic reticulum (ER) stress signaling and experimental dNP. Additionally, TUDCA activates the farnesoid X receptor (FXR), which is highly expressed in tubular cells. We hypothesized that TUDCA ameliorates maladaptive ER signaling via FXR agonism specifically in tubular cells. Indeed, TUDCA induced expression of FXR-dependent genes (SOCS3 and DDAH1) in tubular cells but not in other renal cells. In vivo, TUDCA reduced glomerular and tubular injury in db/db and diabetic endothelial nitric oxide synthase-deficient mice. FXR inhibition with Z-guggulsterone or vivo-morpholino targeting of FXR diminished the ER-stabilizing and renoprotective effects of TUDCA. Notably, these in vivo approaches abolished tubular but not glomerular protection by TUDCA. Combined intervention with TUDCA and the angiotensin-converting enzyme inhibitor enalapril in 16-week-old db/db mice reduced albuminuria more efficiently than did either treatment alone. Although both therapies reduced glomerular damage, only TUDCA ameliorated tubular damage. Thus, interventions that specifically protect the tubular compartment in dNP, such as FXR agonism, may provide renoprotective effects on top of those achieved by inhibiting angiotensin-converting enzyme.

Signal integration at the PI3K-p85-XBP1 hub endows coagulation protease activated protein C with insulin-like function.

Coagulation proteases have increasingly recognized functions beyond hemostasis and thrombosis. Disruption of activated protein C (aPC) or insulin signaling impair function of podocytes and ultimately cause dysfunction of the glomerular filtration barrier and diabetic kidney disease (DKD). We here show that insulin and aPC converge on a common spliced-X-box binding protein-1 (sXBP1) signaling pathway to maintain endoplasmic reticulum (ER) homeostasis. Analogous to insulin, physiological levels of aPC maintain ER proteostasis in DKD. Accordingly, genetically impaired protein C activation exacerbates maladaptive ER response, whereas genetic or pharmacological restoration of aPC maintains ER proteostasis in DKD models. Importantly, in mice with podocyte-specific deficiency of insulin receptor (INSR), aPC selectively restores the activity of the cytoprotective ER-transcription factor sXBP1 by temporally targeting INSR downstream signaling intermediates, the regulatory subunits of PI3Kinase, p85α and p85ß. Genome-wide mapping of condition-specific XBP1-transcriptional regulatory patterns confirmed that concordant unfolded protein response target genes are involved in maintenance of ER proteostasis by both insulin and aPC. Thus, aPC efficiently employs disengaged insulin signaling components to reconfigure ER signaling and restore proteostasis. These results identify ER reprogramming as a novel hormonelike function of coagulation proteases and demonstrate that targeting insulin signaling intermediates may be a feasible therapeutic approach ameliorating defective insulin signaling.

Caspase-1, but Not Caspase-3, Promotes Diabetic Nephropathy.

Glomerular apoptosis may contribute to diabetic nephropathy (dNP), but the pathophysiologic relevance of this process remains obscure. Here, we administered two partially disjunct polycaspase inhibitors in 8-week-old diabetic (db/db) mice: M-920 (inhibiting caspase-1, -3, -4, -5, -6, -7, and -8) and CIX (inhibiting caspase-3, -6, -7, -8, and -10). Notably, despite reduction in glomerular cell death and caspase-3 activity by both inhibitors, only M-920 ameliorated dNP. Nephroprotection by M-920 was associated with reduced renal caspase-1 and inflammasome activity. Accordingly, analysis of gene expression data in the Nephromine database revealed persistently elevated glomerular expression of inflammasome markers (NLRP3, CASP1, PYCARD, IL-18, IL-1ß), but not of apoptosis markers (CASP3, CASP7, PARP1), in patients with and murine models of dNP. In vitro, increased levels of markers of inflammasome activation (Nlrp3, caspase-1 cleavage) preceded those of markers of apoptosis activation (caspase-3 and -7, PARP1 cleavage) in glucose-stressed podocytes. Finally, caspase-3 deficiency did not protect mice from dNP, whereas both homozygous and hemizygous caspase-1 deficiency did. Hence, these results suggest caspase-3-dependent cell death has a negligible effect, whereas caspase-1-dependent inflammasome activation has a crucial function in the establishment of dNP. Furthermore, small molecules targeting caspase-1 or inflammasome activation may be a feasible therapeutic approach in dNP.

The emerging role of coagulation proteases in kidney disease.

A role of coagulation proteases in kidney disease beyond their function in normal haemostasis and thrombosis has long been suspected, and studies performed in the past 15 years have provided novel insights into the mechanisms involved. The expression of protease-activated receptors (PARs) in renal cells provides a molecular link between coagulation proteases and renal cell function and revitalizes research evaluating the role of haemostasis regulators in renal disease. Renal cell-specific expression and activity of coagulation proteases, their regulators and their receptors are dynamically altered during disease processes. Furthermore, renal inflammation and tissue remodelling are not only associated, but are causally linked with altered coagulation activation and protease-dependent signalling. Intriguingly, coagulation proteases signal through more than one receptor or induce formation of receptor complexes in a cell-specific manner, emphasizing context specificity. Understanding these cell-specific signalosomes and their regulation in kidney disease is crucial to unravelling the pathophysiological relevance of coagulation regulators in renal disease. In addition, the clinical availability of small molecule targeted anticoagulants as well as the development of PAR antagonists increases the need for in-depth knowledge of the mechanisms through which coagulation proteases might regulate renal physiology.

Activated Protein C Ameliorates Renal Ischemia-Reperfusion Injury by Restricting Y-Box Binding Protein-1 Ubiquitination.

Ischemia-reperfusion injury (IRI) is the leading cause of ARF. A pathophysiologic role of the coagulation system in renal IRI has been established, but the functional relevance of thrombomodulin (TM)-dependent activated protein C (aPC) generation and the intracellular targets of aPC remain undefined. Here, we investigated the role of TM-dependent aPC generation and therapeutic aPC application in a murine renal IRI model and in an in vitro hypoxia and reoxygenation (HR) model using proximal tubular cells. In renal IRI, endogenous aPC levels were reduced. Genetic or therapeutic reconstitution of aPC efficiently ameliorated renal IRI independently of its anticoagulant properties. In tubular cells, cytoprotective aPC signaling was mediated through protease activated receptor-1- and endothelial protein C receptor-dependent regulation of the cold-shock protein Y-box binding protein-1 (YB-1). The mature 50 kD form of YB-1 was required for the nephro- and cytoprotective effects of aPC in vivo and in vitro, respectively. Reduction of mature YB-1 and K48-linked ubiquitination of YB-1 was prevented by aPC after renal IRI or tubular HR injury. aPC preserved the interaction of YB-1 with the deubiquitinating enzyme otubain-1 and maintained expression of otubain-1, which was required to reduce K48-linked YB-1 ubiquitination and to stabilize the 50 kD form of YB-1 after renal IRI and tubular HR injury. These data link the cyto- and nephroprotective effects of aPC with the ubiquitin-proteasome system and identify YB-1 as a novel intracellular target of aPC. These insights may provide new impetus for translational efforts aiming to restrict renal IRI.

Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy.

Endoplasmic reticulum (ER) stress is associated with diabetic nephropathy (DN), but its pathophysiological relevance and the mechanisms that compromise adaptive ER signalling in podocytes remain unknown. Here we show that nuclear translocation of the transcription factor spliced X-box binding protein-1 (sXBP1) is selectively impaired in DN, inducing activating transcription factor-6 (ATF6) and C/EBP homology protein (CHOP). Podocyte-specific genetic ablation of XBP1 or inducible expression of ATF6 in mice aggravates DN. sXBP1 lies downstream of insulin signalling and attenuating podocyte insulin signalling by genetic ablation of the insulin receptor or the regulatory subunits phosphatidylinositol 3-kinase (PI3K) p85α or p85ß impairs sXBP1 nuclear translocation and exacerbates DN. Corroborating our findings from murine DN, the interaction of sXBP1 with p85α and p85ß is markedly impaired in the glomerular compartment of human DN. Thus, signalling via the insulin receptor, p85, and XBP1 maintains podocyte homeostasis, while disruption of this pathway impairs podocyte function in DN.

Nlrp3-inflammasome activation in non-myeloid-derived cells aggravates diabetic nephropathy.

Diabetic nephropathy is a growing health concern with characteristic sterile inflammation. As the underlying mechanisms of this inflammation remain poorly defined, specific therapies targeting sterile inflammation in diabetic nephropathy are lacking. Intriguingly, an association of diabetic nephropathy with inflammasome activation has recently been shown, but the pathophysiological relevance of this finding remains unknown. Within glomeruli, inflammasome activation was detected in endothelial cells and podocytes in diabetic humans and mice and in glucose-stressed glomerular endothelial cells and podocytes in vitro. Abolishing Nlrp3 or caspase-1 expression in bone marrow-derived cells fails to protect mice against diabetic nephropathy. Conversely, Nlrp3-deficient mice are protected against diabetic nephropathy despite transplantation of wild-type bone marrow. Pharmacological IL-1R antagonism prevented or even reversed diabetic nephropathy in mice. Mitochondrial reactive oxygen species (ROS) activate the Nlrp3 inflammasome in glucose or advanced glycation end product stressed podocytes. Inhibition of mitochondrial ROS prevents glomerular inflammasome activation and nephropathy in diabetic mice. Thus, mitochondrial ROS and Nlrp3-inflammasome activation in non-myeloid-derived cells aggravate diabetic nephropathy. Targeting the inflammasome may be a potential therapeutic approach to diabetic nephropathy.

Negative interactions and feedback regulations are required for transient cellular response.

Signal transduction is a process required to conduct information from a receptor to the nucleus. This process is vital for the control of cellular function and fate. The dynamics of signaling activation and inhibition determine processes such as apoptosis, proliferation, and differentiation. Thus, it is important to understand the factors modulating transient and sustained response. To address this question, by applying mathematical approach we have studied the factors which can alter the activation nature of downstream signaling molecules. The factors which we have investigated are loops (feed forward and feedback loops), cross-talk of signal transduction pathways, and the change in the concentration of the signaling molecules. Based on our results we conclude that among these factors feedback loop and the cross-talks which directly inhibit the target protein dominantly controls the transient cellular response.

Activated protein C ameliorates diabetic nephropathy by epigenetically inhibiting the redox enzyme p66Shc.

The coagulation protease activated protein C (aPC) confers cytoprotective effects in various in vitro and in vivo disease models, including diabetic nephropathy. The nephroprotective effect may be related to antioxidant effects of aPC. However, the mechanism through which aPC may convey these antioxidant effects and the functional relevance of these properties remain unknown. Here, we show that endogenous and exogenous aPC prevents glomerular accumulation of oxidative stress markers and of the redox-regulating protein p66(Shc) in experimental diabetic nephropathy. These effects were predominately observed in podocytes. In vitro, aPC inhibited glucose-induced expression of p66(Shc) mRNA and protein in podocytes (via PAR-1 and PAR-3) and various endothelial cell lines, but not in glomerular endothelial cells. Treatment with aPC reversed glucose-induced hypomethylation and hyperacetylation of the p66(Shc) promoter in podocytes. The hyperacetylating agent sodium butyrate abolished the suppressive effect of aPC on p66(Shc) expression both in vitro and in vivo. Moreover, sodium butyrate abolished the beneficial effects of aPC in experimental diabetic nephropathy. Inhibition of p66(Shc) expression and mitochondrial translocation by aPC normalized mitochondrial ROS production and the mitochondrial membrane potential in glucose-treated podocytes. Genetic ablation of p66(Shc) compensated for the loss of protein C activation in vivo, normalizing markers of diabetic nephropathy and oxidative stress. These studies identify a unique mechanism underlying the cytoprotective effect of aPC. Activated PC epigenetically controls expression of the redox-regulating protein p66(Shc), thus linking the extracellular protease aPC to mitochondrial function in diabetic nephropathy.

The lectin-like domain of thrombomodulin ameliorates diabetic glomerulopathy via complement inhibition.

Coagulation and complement regulators belong to two interactive systems constituting emerging mechanisms of diabetic nephropathy. Thrombomodulin (TM) regulates both coagulation and complement activation, in part through discrete domains. TM's lectin like domain dampens complement activation, while its EGF-like domains independently enhance activation of the anti-coagulant and cytoprotective serine protease protein C (PC). A protective effect of activated PC in diabetic nephropathy is established. We hypothesised that TM controls diabetic nephropathy independent of PC through its lectin-like domain by regulating complement. Diabetic nephropathy was analysed in mice lacking TM's lectin-like domain (TMLeD/LeD) and controls (TMwt/wt). Albuminuria (290 µg/mg vs. 166 µg/mg, p=0.03) and other indices of experimental diabetic nephropathy were aggravated in diabetic TMLeD/LeD mice. Complement deposition (C3 and C5b-9) was markedly increased in glomeruli of diabetic TMLeD/LeD mice. Complement inhibition with enoxaparin ameliorated diabetic nephropathy in TMLeD/LeD mice (e.g. albuminuria 85 µg/mg vs. 290 µg/mg, p<0.001). In vitro TM's lectin-like domain cell-autonomously prevented glucose-induced complement activation on endothelial cells and - notably - on podocytes. Podocyte injury, which was enhanced in diabetic TMLeD/LeD mice, was reduced following complement inhibition with enoxaparin. The current study identifies a novel mechanism regulating complement activation in diabetic nephropathy. TM's lectin-like domain constrains glucose-induced complement activation on endothelial cells and podocytes and ameliorates albuminuria and glomerular damage in mice.