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Donor-derived cell-free DNA (dd-cfDNA) is a biomarker for rejection after organ transplantation. We hypothesized that high release of cfDNA immediately after liver transplant also has a biologic role in inflammation in ischemia and reperfusion injury (IRI). To investigate this concept, C57BL/6 mice were subjected to 90 min in situ liver ischemia. After 6 h reperfusion, cfDNA was purified from serum and used to stimulate macrophages in vitro , which resulted in production of high levels of inflammatory cytokines TNFα and IL-6, and chemokine CXCL10. Enzymatic degradation of cfDNA by DNase I inhibited these inflammatory responses (e.g., TNFα: DNase I 48.1 ± 37.4 vs. untreated 1,030 ± 206 pg/mL, p = 0.0001). cfDNA from netosis-deficient PAD4KO mice was found to be equally pro-inflammatory compared to wild type cfDNA (TNFα: PAD4KO 1048 ± 199 vs. wild-type 1,162 ± 150 pg/mL, p = 0.64), indicating its mechanism is not dependent on neutrophils undergoing netosis. Next, a single dose of DNase I was added to the perfusate during rat liver normothermic machine perfusion (NMP) to significantly reduce perfusate cfDNA levels (384 ± 132 to 129 ± 18 ng/mL, p = 0.026). In conclusion, our data suggest that cfDNA can have pro-inflammatory effects during liver IRI beyond being a biomarker. DNase I may be a promising therapeutic intervention during NMP to reduce the graft’s inflammatory propensity prior to implantation.

A series of intramolecular fluorescent FlAsH BRET reporters is used to monitor conformational changes in β-arrestin2 following activation of seven G-protein-coupled receptors (GPCRs), showing that different GPCRs produce distinct β-arrestin2 conformational signatures that correlate with the stability of the receptor–arrestin complex and the role of β-arrestin2 in activating or dampening downstream signalling events, which explains how different GPCRs can use a common effector for different purposes. Arrestin behaviour and GCPR function Much has been learnt about the structure of G-protein-coupled receptors (GPCRs), ubiquitous mediators of the actions of many hormones, neurotransmitters and drugs, but little is known about how the conformations of these membrane proteins change when they interact with proteins that regulate their function. Susanne Nuber et al . used a series of FRET-based β-arrestin2 biosensors to study the dynamics of the conformational changes that take place in β-arrestin2 when it binds to and eventually dissociates from the β-adrenergic receptor in living cells. They find that after dissociation, β-arrestin2 remains at the cell membrane in an active conformation at least initially, indicating that β-arrestin is able to signal in a GPCR-free state. Mi-Hye Lee et al . used a series of intramolecular FlAsH BRET reporters to monitor conformational changes in β-arrestin2 in the presence of six different GPCRs. They found that different GPCRs and ligands produce β-arrestin2 conformational signatures that correlate with the stability of the receptor–arrestin complex; this explains how different GPCRs can use a common effector for different purposes. Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling 1 , 2 . Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits 3 , 4 . Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based ‘signalsomes’ 5 , 6 . Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters 7 to monitor conformational changes in β-arrestin2, here we show that GPCRs impose distinctive arrestin ‘conformational signatures’ that reflect the stability of the receptor–arrestin complex and role of β-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in β-arrestin2 conformation. Our findings demonstrate that information about ligand–receptor conformation is encoded within the population average β-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands 8 , 9 and in identifying factors that dictate arrestin conformation and function.

Unopposed angiotensin (Ang) II-mediated cellular effects may lead to progressive glomerulosclerosis. While Ang-II can be locally generated in the kidneys, we previously showed that glomerular podocytes primarily convert Ang-I, the precursor of Ang-II, to Ang-(1-7) and Ang-(2-10), peptides that have been independently implicated in biological actions opposing those of Ang-II. Therefore, we hypothesized that Ang-(1-7) and Ang-(2-10) could be renoprotective in the fawn-hooded hypertensive rat, a model of focal segmental glomerulosclerosis. We evaluated the ability of 8-12 week-long intravenous administration of either Ang-(1-7) or Ang-(2-10) (100-400 ng/kg/min) to reduce glomerular injury in uni-nephrectomized fawn-hooded hypertensive rats, early or late in the disease. Vehicle-treated rats developed hypertension and lesions of focal segmental glomerulosclerosis. No reduction in glomerular damage was observed, as measured by either 24-hour urinary protein excretion or histological examination of glomerulosclerosis, upon Ang-(1-7) or Ang-(2-10) administration, regardless of peptide dose or disease stage. On the contrary, when given at 400 ng/kg/min, both peptides induced a further increase in systolic blood pressure. Content of Ang peptides was measured by parallel reaction monitoring in kidneys harvested at sacrifice. Exogenous administration of Ang-(1-7) and Ang-(2-10) did not lead to a significant increase in their corresponding intrarenal levels. However, the relative abundance of Ang-(1-7) with respect to Ang-II was increased in kidney homogenates of Ang-(1-7)-treated rats. We conclude that chronic intravenous administration of Ang-(1-7) or Ang-(2-10) does not ameliorate glomerular damage in a rat model of focal segmental glomerulosclerosis and may induce a further rise in blood pressure, potentially aggravating glomerular injury.

Secreted frizzled-related protein 2 (SFRP2) is a pro-angiogenic factor expressed in the vasculature of a wide variety of human tumors, and modulates angiogenesis via the calcineurin-dependent nuclear factor of activated T-cells cytoplasmic 3 (NFATc3) pathway in endothelial cells. However, until now, SFRP2 receptor for this pathway was unknown. In the present study, we first used amino acid alignments and molecular modeling to demonstrate that SFRP2 interaction with frizzled-5 (FZD5) is typical of Wnt/FZD family members. To confirm this interaction, we performed co-immunofluorescence, co-immunoprecipitation, and ELISA binding assays, which demonstrated SFRP2/FZD5 binding. Functional knock-down studies further revealed that FZD5 is necessary for SFRP2-induced tube formation and intracellular calcium flux in endothelial cells. Using protein analysis on endothelial cell nuclear extracts, we also discovered that FZD5 is required for SFRP2-induced activation of NFATc3. Our novel findings reveal that FZD5 is a receptor for SFRP2 and mediates SFRP2-induced angiogenesis via calcineurin/NFATc3 pathway in endothelial cells.

Background: The vasoactive peptide bradykinin (BK) acts as a potent growth factor for normal kidney cells, but there have been few studies on the role of BK in renal cell carcinomas. Purpose: In this study, we tested the hypothesis that BK also acts as a mitogen in kidney carcinomas, and explored the effects of BK in human renal carcinoma A498 cells. Methods: The presence of mRNAs for BK B1 and BK B2 receptors in A498 cells was demonstrated by reverse transcription–polymerase chain reaction. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) with a Cytosensor microphysiometer, and assessed extracellular signal-regulated kinase (ERK) activation by Western blotting. Results: Exposure to 100 nM of BK resulted in the rapid elevation of intracellular Ca2+, caused a ≥30% increase in NHE activity, and a ≥300% increase in ERK phosphorylation. All BK signals were blocked by HOE140, a BK B2 receptor antagonist, but not by a B1 receptor antagonist. Inhibitor studies suggest that BK-induced ERK activation requires phospholipase C and protein kinase C activities, and is Ca2+/calmodulin-dependent. The amiloride analog 5-(N-methyl-N-isobutyl)-amiloride (MIA) blocked short-term NHE activation and inhibited ERK phosphorylation, suggesting that NHE is critical for ERK activation by BK. BK induced an approximately 40% increase in the proliferation of A498 cells as assessed by bromodeoxyuridine uptake. This effect was blocked by the ERK inhibitor PD98059, and was dependent on NHE activity. Conclusion: We conclude that BK exerts mitogenic effects in A498 cells via the BK B2 receptor activation of growth-associated NHE and ERK.

A series of intramolecular fluorescent FlAsH BRET reporters is used to monitor conformational changes in [beta]-arrestin2 following activation of seven G-protein-coupled receptors (GPCRs), showing that different GPCRs produce distinct [beta]-arrestin2 conformational signatures that correlate with the stability of the receptor-arrestin complex and the role of [beta]-arrestin2 in activating or dampening downstream signalling events, which explains how different GPCRs can use a common effector for different purposes.

Objective: Since peritoneal dialysis causes peritoneal fibrosis, we examined how glucose (osmotic factor), mannitol (osmotic control), and angiotensin II (AngII) regulate proinflammatory cyclooxygenase 2 (COX-2) in primary rat peritoneal mesothelial cells. Materials and Methods: For this study, we used the following material (n = 4-8 cell lines): cells, passages 1-2; 125I-AngII receptor surface binding (AT1R antagonist losartan, AT2R antagonist PD123319; both 10 µM ); intracellular calcium probe calcium-5; COX-2 immunoblotting (β-actin normalized); real-time PCR of COX-2 gene PTGS2, and NF-κB inhibitor Ro-1069920 (5 µM ). Results: AngII surface receptors were predominantly AT1R (minimally AT2R). AngII and glucose increased COX-2 protein expression concentration dependently; mannitol also increased COX-2 expression. Maximal COX-2 protein expression was observed after 6 h (AngII) and 24 h (glucose, mannitol). The time course of increases in PTGS2 mRNA levels reflected that of COX-2 protein expression. At optimal exposure conditions (time/concentration), glucose was 5-fold more efficacious in stimulating COX-2 protein expression than AngII or mannitol. Losartan fully inhibited COX-2 protein responses to AngII and mannitol, but minimally inhibited responses to glucose. Ro-1069920 fully inhibited COX-2 protein responses to each effector. Conclusion: AngII, glucose, and osmotic stress (mannitol) activate COX-2; NF-κB may be an ideal site for COX-2 blockade, and COX-2 activation by osmotic stress requires AT1R, but activation by glucose is more robust and mechanistically complex. © 2014 S. Karger AG, Basel

Unopposed angiotensin (Ang) II-mediated cellular effects may lead to progressive glomerulosclerosis. While Ang-II can be locally generated in the kidneys, we previously showed that glomerular podocytes primarily convert Ang-I, the precursor of Ang-II, to Ang-(1-7) and Ang-(2-10), peptides that have been independently implicated in biological actions opposing those of Ang-II. Therefore, we hypothesized that Ang-(1-7) and Ang-(2-10) could be renoprotective in the fawn-hooded hypertensive rat, a model of focal segmental glomerulosclerosis. We evaluated the ability of 8-12 week-long intravenous administration of either Ang-(1-7) or Ang-(2-10) (100-400 ng/kg/min) to reduce glomerular injury in uni-nephrectomized fawn-hooded hypertensive rats, early or late in the disease. Vehicle-treated rats developed hypertension and lesions of focal segmental glomerulosclerosis. No reduction in glomerular damage was observed, as measured by either 24-hour urinary protein excretion or histological examination of glomerulosclerosis, upon Ang-(1-7) or Ang-(2-10) administration, regardless of peptide dose or disease stage. On the contrary, when given at 400 ng/kg/min, both peptides induced a further increase in systolic blood pressure. Content of Ang peptides was measured by parallel reaction monitoring in kidneys harvested at sacrifice. Exogenous administration of Ang-(1-7) and Ang-(2-10) did not lead to a significant increase in their corresponding intrarenal levels. However, the relative abundance of Ang-(1-7) with respect to Ang-II was increased in kidney homogenates of Ang-(1-7)-treated rats. We conclude that chronic intravenous administration of Ang-(1-7) or Ang-(2-10) does not ameliorate glomerular damage in a rat model of focal segmental glomerulosclerosis and may induce a further rise in blood pressure, potentially aggravating glomerular injury.

Background We compared the effects of the sulfhydryl-containing (thiol) antioxidant dithiothreitol (DTT), which disrupts disulfide bonds, on cell signaling through angiotensin II (AngII) Type 1 receptors (AT1Rs) and arginine vasopressin (AVP) V1 receptors (V1Rs). The AT1R contains two extracellular disulfides bonds but its ligand contains none, whereas the V1R contains no extracellular disufides bonds but its ligand contains 1. Methods We measured radioligand binding, intracellular calcium responses, and extracellular signal-regulated kinase phosphorylation in cultured rat aortic vascular smooth muscle cells and alterations in urine osmolality in intact rats. Results Preincubation of cells with DTT, a maneuver designed to target receptor disulfides, resulted in concentration-dependent decreases in specific 125I-AngII binding to AT1Rs and acute angiotensin-stimulated intracellular calcium mobilization but no decreases in specific 125I-AVP binding to V1Rs or AVP-stimulated intracellular calcium mobilization. In contrast, preincubation of the ligands with DTT followed by acute exposure to the cells, a maneuver designed to target ligand disulfides, blunted calcium mobilization to AVP robustly but to AngII only minimally. In intact rats, the increase in urine osmolality caused by subcutaneous injection with the AVP analogue desmopressin was significantly diminished when the analogue was preincubated with an excess of DTT. Conclusion DTT inhibits cell signaling to AngII AT1Rs and AVP V1Rs, at least in part through disruption of disulfide linkages, but the pattern of response depends upon whether disulfides of ligand or receptor are targeted. American Journal of Hypertension 2009; 22:221–227 © 2009 American Journal of Hypertension, Ltd.

Arrestins are cytosolic proteins that regulate G protein-coupled receptor (GPCR) desensitization, internalization, trafficking, and signaling1,2. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets them for internalization via clathrin-coated pits3,4. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G protein effectors into GPCR-based ‘signalsomes’5,6. While the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify divergent arrestin functions is not understood. Using a panel of intramolecular FlAsH-BRET reporters7 to monitor conformational changes in arrestin3, we show here that GPCRs impose distinctive arrestin ‘conformational signatures’ that reflect the stability of the receptor-arrestin complex and role of arrestin3 in activating or dampening downstream signaling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in arrestin3 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average arrestin3 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands8,9 and in identifying factors that dictate arrestin conformation and function.