The More Complex Renin-Angiotensin System: New Insights Into An Old System

Since the first description of renin by Tigerstedt and Bergmann in 1898,1 the reninangiotensin system (RAS) has been extensively studied. In the last decades, many investigations have demonstrated the importance of the RAS for maintenance and regulation of many physiological processes. One of the most prominent functions of the RAS is the regulation of blood

New Function for an Old Enzyme: NEP Deficient Mice Develop Late-Onset Obesity

BACKGROUND: According to the World Health Organization (WHO) there is a pandemic of obesity with approximately 300 million people being obese. Typically, human obesity has a polygenetic causation. Neutral endopeptidase (NEP), also known as neprilysin, is considered to be one of the key enzymes in the metabolism of many active peptide hormones. METHODOLOGY/PRINCIPAL FINDINGS: An incidental observation in NEP-deficient mice was a late-onset excessive gain in body weight exclusively from a ubiquitous accumulation of fat tissue. In accord with polygenetic human obesity, mice were characterized by deregulation of lipid metabolism, higher blood glucose levels, with impaired glucose tolerance. The key role of NEP in determining body mass was confirmed by the use of the NEP inhibitor candoxatril in wild-type mice that increased body weight due to increased food intake. This is a peripheral and not a central NEP action on the switch for appetite control, since candoxatril cannot cross the blood-brain barrier. Furthermore, we demonstrated that inhibition of NEP in mice with cachexia delayed rapid body weight loss. Thus, lack in NEP activity, genetically or pharmacologically, leads to a gain in body fat. CONCLUSIONS/SIGNIFICANCE: In the present study, we have identified NEP to be a crucial player in the development of obesity. NEP-deficient mice start to become obese under a normocaloric diet in an age of 6-7 months and thus are an ideal model for the typical human late-onset obesity. Therefore, the described obesity model is an ideal tool for research on development, molecular mechanisms, diagnosis, and therapy of the pandemic obesity

A new analysis approach for single nephron GFR in intravital microscopy of mice [version 3; peer review: 2 approved, 2 not approved]

Background: Intravital microscopy is an emerging technique in life science with applications in kidney research. Longitudinal observation of (patho-)physiological processes in living mice is possible in the smallest functional unit of the kidney, a single nephron (sn). In particular, effects on glomerular filtration rate (GFR) - a key parameter of renal function - can be assessed. Methods: After intravenous injection of a freely filtered, non-resorbable, fluorescent dye in C57BL/6 mice, a time series was captured by multiphoton microsopy. Filtration was observed from the glomerular capillaries to the proximal tubule (PT) and the tubular signal intensity shift was analyzed to calculate the snGFR. Results: Previously described methods for snGFR analysis relied on two manually defined measurement points in the PT and the tubular volume was merely estimated in 2D images. We present an extended image processing workflow by adding continuous measurement of intensity along the PT in every frame of the time series using ImageJ. Automatic modelling of actual PT volume in a 3D dataset replaced 2D volume estimation. Subsequent data analysis in R, with a calculation of intensity shifts in every frame and normalization against tubular volume, allowed exact assessment of snGFR by linear regression. Repeated analysis of image data obtained in healthy mice showed a striking increase of reproducibility by reduction of user interaction. Conclusions: These improvements in image processing and data analysis maximize the reliability of a sophisticated intravital microscopy technique for the precise assessment of snGFR, a highly relevant predictor of kidney function

Gasdermin D-deficient mice are hypersensitive to acute kidney injury

Signaling pathways of regulated necrosis, such as necroptosis and ferroptosis, contribute to acute kidney injury (AKI), but the role of pyroptosis is unclear. Pyroptosis is mediated by the pore-forming protein gasdermin D (GSDMD). Here, we report a specific pattern of GSDMD-protein expression in the peritubular compartment of mice that underwent bilateral ischemia and reperfusion injury (IRI). Along similar lines, the GSDMD-protein expression in whole kidney lysates increased during the first 84 h following cisplatin-induced AKI. Importantly, unlike whole kidney lysates, no GSDMD-protein expression was detectable in isolated kidney tubules. In IRI and cisplatin-induced AKI, GSDMD-deficient mice exhibited hypersensitivity to injury as assessed by tubular damage, elevated markers of serum urea, and serum creatinine. This hypersensitivity was reversed by a combined deficiency of GSDMD and the necroptosis mediator mixed lineage kinase domain-like (MLKL). In conclusion, we demonstrate a non-cell autonomous role for GSDMD in protecting the tubular compartment from necroptosis-mediated damage in IRI

vPIF-1 is an insulin-like antiferroptotic viral peptide

Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin-like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone-releasing hormone antagonist-induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms

Developmental endothelial locus-1 protects from hypertension-induced cardiovascular remodeling via immunomodulation

The causative role of inflammation in hypertension-related cardiovascular diseases is evident and calls for development of specific immunomodulatory therapies. We tested the therapeutic efficacy and mechanisms of action of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor, in angiotensin-II (ANGII)- and DOCA (deoxycorticosterone acetate)-salt-induced cardiovascular organ damage and hypertension. By using mice with endothelial overexpression of DEL-1 (EC-Del1) and performing preventive and interventional studies by injecting recombinant DEL-1 in mice, we showed that DEL-1 improved endothelial function and abrogated aortic adventitial fibrosis, medial thickening and loss of elastin. DEL-1 also protected the mice from cardiac concentric hypertrophy, interstitial and perivascular coronary fibrosis and improved left-ventricular function and myocardial coronary perfusion. DEL-1 prevented aortic stiffness and abolished the progression of hypertension. Mechanistically, DEL-1 acted by inhibiting αvβ3-integrin dependent activation of pro-MMP2 in mice and in human isolated aorta. Moreover, DEL-1 stabilized αvβ3-integrin dependent CD25+FoxP3+ Treg numbers and IL-10 levels, which were associated with decreased pro-inflammatory cell recruitment of inflammatory cells and reduced production of pro-inflammatory cytokines in cardiovascular organs. The demonstrated effects and immune-modulating mechanisms of DEL-1 in abrogation of cardiovascular remodeling and progression of hypertension identify DEL-1 as a potential therapeutic factor

Characterization of the brain-specific non-AT(1), non-AT(2) angiotensin binding site in the mouse

In the present study the existence of a non-AT(1), non-AT(2) angiotensin (Ang) binding site unmasked by the organomercurial protease inhibitor p-chloromercuribenzoate (PCMB) was demonstrated in mouse brain membranes, consistent with observations previously reported in the rat (Karamyan and Speth, 2007b). The pharmacological specificity of the non-AT(1), non-AT(2) angiotensin binding site was similar to the rat brain: Sar(1)-Ile(8)-Ang II \u3e Ang III \u3eor= Ang II \u3e Ang I\u3e p-aminophenylalanine(6) Ang II\u3e CGP42112 \u3e\u3e Ang IV \u3e Ang 1-7 congruent with shorter angiotensin fragments. Neurotensin, bradykinin, and luteinizing hormone-releasing hormone showed K(i) values \u3e10 microM, while substance P and VIP had K(i) values of approximately 2 microM. The non-AT(1), non-AT(2) angiotensin binding site was not present in adrenal, liver or kidney. Subcellular fractionation showed a higher density of [(125)I]Ang II binding in plasma membrane (P2) fractions of cerebral cortex and hypothalamus relative to debris (P1) fractions. The binding site is present in the brains of mice in which the AT(1a), AT(1b), AT(2), Mas, and neprilysin (EC 3.4.24.11, neutral endopeptidase) was knocked out confirming that the binding site is not a heretofore described angiotensin receptor or neprilysin. These observations confirm that this novel Ang binding site is distinct from classical AT(1), AT(2), AT(4) and Ang 1-7 receptors while retaining a high specificity for angiotensins that act on the known angiotensin receptors. Whether this binding site functions as a novel receptor for angiotensins or a specific angiotensinase with variable functionality at different redox states will require further study

Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1–7)

The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells

The clinical relevance of necroinflammation-highlighting the importance of acute kidney injury and the adrenal glands

Necroinflammation is defined as the inflammatory response to necrotic cell death. Different necrotic cell death pathways exhibit different immune reponses, despite a comparable level of intracellular content release (referred to as damage associated molecular patterns or DAMPs). In addition to DAMP release, which is inevitably associated with necrotic cell death, the active production of pro/anti-inflammatory cytokines characterizes certain necrotic pathways. Necroptosis, ferroptosis and pyroptosis, therefore, are immunogenic to a different extent. In this review, we discuss the clinical relevance of necroinflammation highlighting potential human serum markers. We focus on the role of the adrenal glands and the lungs as central organs affected by systemic and/or local DAMP release and underline their role in intensive care medicine. In addition, data from models of acute kidney injury (AKI) and kidney transplantation have significantly shaped the field of necroinflammation and may be helpful for the understanding of the potential role of dialysis and plasma exchange to treat ongoing necroinflammation upon intensive care unit (ICU) conditions. In conclusion, we are only beginning to understand the importance of necroinflammation in diseases and transplantation, including xenotransplantation. However, given the existing efforts to develop inhibitors of necrotic cell death (ferrostatins, necrostatins, etc), we consider it likely that interference with necroinflammation reaches clinical routine in the near future