Shrey Kohli

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.

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 p85β impairs sXBP1 nuclear translocation and exacerbates DN. Corroborating our findings from murine DN, the interaction of sXBP1 with p85α and p85β 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.

Tea and human health: the dark shadows.

Tea is one of the most popularly consumed beverage. Depending on the manufacturing process, different varieties of tea can be produced. The antioxidative and antimutagenic potential of tea in cardiovascular diseases, cancer and obesity have long been studied. These therapeutic and nutritional benefits of tea can be attributed to the presence of flavanoids. However, these flavanoids also have certain detrimental effects on human health when their consumption exceeds certain limits. The toxicity of these flavanoids can be attributed to the formation of reactive oxygen species in the body which causes damage to the DNA, lipid membranes etc. The aim of this review is to summarize briefly, the less studied evidences of various forms of toxicity associated with tea and its harmful effects on human health.

Transcription Factors in Heart: Promising Therapeutic Targets in Cardiac Hypertrophy

Regulation of gene expression is central to cell growth, differentiation and diseases. Context specific and signal dependent regulation of gene expression is achieved to a large part by transcription factors. Cardiac transcription factors regulate heart development and are also involved in stress regulation of the adult heart, which may lead to cardiac hypertrophy. Hypertrophy of cardiac myocytes is an outcome of the imbalance between prohypertrophic factors and anti-hypertrophic factors. This is initially a compensatory mechanism but sustained hypertrophy may lead to heart failure. The growing knowledge of transcriptional control mechanisms is helpful in the development of novel therapies. This review summarizes the role of cardiac transcription factors in cardiac hypertrophy, emphasizing their potential as attractive therapeutic targets to prevent the onset of heart failure and sudden death as they can be converging targets for current therapy.

Stabilization of endogenous Nrf2 by minocycline protects against Nlrp3-inflammasome induced diabetic nephropathy.

While a plethora of studies support a therapeutic benefit of Nrf2 activation and ROS inhibition in diabetic nephropathy (dNP), the Nrf2 activator bardoxolone failed in clinical studies in type 2 diabetic patients due to cardiovascular side effects. Hence, alternative approaches to target Nrf2 are required. Intriguingly, the tetracycline antibiotic minocycline, which has been in clinical use for decades, has been shown to convey anti-inflammatory effects in diabetic patients and nephroprotection in rodent models of dNP. However, the mechanism underlying the nephroprotection remains unknown. Here we show that minocycline protects against dNP in mouse models of type 1 and type 2 diabetes, while caspase -3,-6,-7,-8 and -10 inhibition is insufficient, indicating a function of minocycline independent of apoptosis inhibition. Minocycline stabilizes endogenous Nrf2 in kidneys of db/db mice, thus dampening ROS-induced inflammasome activation in the kidney. Indeed, minocycline exerts antioxidant effects in vitro and in vivo, reducing glomerular markers of oxidative stress. Minocycline reduces ubiquitination of the redox-sensitive transcription factor Nrf2 and increases its protein levels. Accordingly, minocycline mediated Nlrp3 inflammasome inhibition and amelioration of dNP are abolished in diabetic Nrf2−/− mice. Taken together, we uncover a new function of minocycline, which stabilizes the redox-sensitive transcription factor Nrf2, thus protecting from dNP.

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.

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-1β), 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.

Curcumin Suppresses Gelatinase B Mediated Norepinephrine Induced Stress in H9c2 Cardiomyocytes

Background Extracellular matrix (ECM) remodeling facilitates biomechanical signals in response to abnormal physiological conditions. This process is witnessed as one of the major effects of the stress imposed by catecholamines, such as epinephrine and norepinephrine (NE), on cardiac muscle cells. Matrix metalloproteinases (MMPs) are the key proteases involved in degradation of the ECM in heart. Objectives The present study focuses on studying the effect of curcumin on Gelatinase B (MMP-9), an ECM remodeling regulatory enzyme, in NE-induced cardiac stress. Curcumin, a bioactive polyphenol found in the spice turmeric, has been studied for its multi-fold beneficial properties. This study focuses on investigating the role of curcumin as a cardio-protectant. Methods H9c2 cardiomyocytes were subjected to NE and curcumin treatments to study the response in stress conditions. Effect on total collagen content was studied using Picrosirus red staining. Gelatinase B activity was assessed through Gel-Diffusion Assay and Zymographic techniques. RT-PCR, Western Blotting and Immunocytochemistry were performed to study effect on expression of gelatinase B. Further, the effect of curcumin on the localization of NF-κB, known to regulate gelatinase B, was also examined. Results Curcumin suppressed the increase in the total collagen content under hypertrophic stress and was found to inhibit the in-gel and in-situ gelatinolytic activity of gelatinase B. Moreover, it was found to suppress the mRNA and protein expression of gelatinase B. Conclusions The study provides an evidence for an overall inhibitory effect of curcumin on Gelatinase B in NE-induced hypertrophic stress in H9c2 cardiomyocytes which may contribute in the prevention of ECM remodeling.

Curcumin: a potential therapeutic polyphenol, prevents noradrenaline-induced hypertrophy in rat cardiac myocytes.

Objectives  This study was designed to evaluate the effect of curcumin on H9c2 cardiac cell line and primary rat cardiac myocytes, using purified noradrenaline as a hypertrophy‐inducing agent.

An assessment of norepinephrine mediated hypertrophy to apoptosis transition in cardiac cells: A signal for cell death

OBJECTIVES Heart is an organ which is under a constant work load that generates numerous stress responses. Heart failure is associated with increased plasma norepinephrine (NE) and hypertrophic cell death. Within the current study we try to understand the concentration dependent molecular switch from hypertrophy to apoptosis under stress. METHODS The effect of increasing concentration of NE on cell death was studied using MTT assay based on which further experimental conditions were decided. Trypan Blue staining and TUNEL assay were done at selected concentrations of NE. Cellular and nuclear morphology at these concentrations was studied using Haematoxylin-Eosin, DAPI and PI stains. The molecular switch between hypertrophy and cell death was studied by expression analysis of β-MyHC and TNF-α. Rhodamine and DCFH-DA staining were done to evaluate the role of mitochondria and ROS under these conditions. Role of caspases under these transitions was also evaluated. RESULT NE shows steep falls in cell viability at 50 μM and 100 μM concentrations. The cellular and nuclear morphology is altered at these concentrations along with alterations at molecular level showing a shift from hypertrophy towards cell death. Altered mitochondrial membrane potential and increase in ROS support this which leads to caspase dependent activation of cell death. CONCLUSION We show that at 50 μM NE, there occurs a transition from cellular hypertrophy towards death. This could be beneficial to prevent hypertrophy induced cardiac cell death and evaluating cardio protective therapeutic targets in vitro.