Opioid Preconditioning Modulates Repair Responses to Prevent Renal Ischemia-Reperfusion Injury.

Progression to renal damage by ischemia-reperfusion injury (IRI) is the result of the dysregulation of various tissue damage repair mechanisms. Anesthetic preconditioning with opioids has been shown to be beneficial in myocardial IRI models. Our main objective was to analyze the influence of pharmacological preconditioning with opioids in renal function and expression of molecules involved in tissue repair and angiogenesis. Experimental protocol includes male rats with 45 min ischemia occluding the left renal hilum followed by 24 h of reperfusion with or without 60 min preconditioning with morphine/fentanyl. We analyzed serum creatinine and renal KIM-1 expression. We measured circulating and intrarenal VEGF. Immunohistochemistry for HIF-1 and Cathepsin D (CTD) and real-time PCR for angiogenic genes HIF-1α, VEGF, VEGF Receptor 2 (VEGF-R2), CTD, CD31 and IL-6 were performed. These molecules are considered important effectors of tissue repair responses mediated by the development of new blood vessels. We observed a decrease in acute renal injury mediated by pharmacological preconditioning with opioids. Renal function in opioid preconditioning groups was like in the sham control group. Both anesthetics modulated the expression of HIF-1, VEGF, VEGF-R2 and CD31. Preconditioning negatively regulated CTD. Opioid preconditioning decreased injury through modulation of angiogenic molecule expression. These are factors to consider when establishing strategies in pathophysiological and surgical processes.

Anesthetic preconditioning increases sirtuin 2 gene expression in a renal ischemia reperfusion injury model.

BACKGROUND: Renal transplant surgical proceedings are known to elicit periods of hypoxia and consequent blood flow reestablishment triggering ischemia-reperfusion (I-R) injury. Kidney damage induced by I-R injury associates with a higher risk of graft dysfunction and rejection. Anesthetic preconditioning exerts a beneficial effect on I-R injury by reducing oxidative stress, inflammation and apoptosis. However, the degree of renoprotection stimulated by commonly used anesthetics, as well as their mechanisms of action, are largely unknown. Sirtuins are class III histone deacetylases that reduce cellular stress, promote genome stability and regulate senescence. So far, the relationship between sirtuins and anesthetic preconditioning in the context of renal I-R has not been studied. The main objective of the present work was to determine the renal expression of sirtuins after I-R damage in rats under different anesthetic preconditioning treatments. METHODS: Unilateral ischemia was performed via occlusion of the left renal hilum for 45 min and followed by 24 hours of reperfusion. Anesthetic preconditioning schemes (morphine 0.5 mg/kg, fentanyl 10 µg/kg, propofol 7.5 mg/kg, or dexmedetomidine 25 µg/kg) were administered 1 hour before ischemia. Creatinine levels were determined in serum, and expression of kidney injury molecule 1 and sirtuin 1, 2, 3 and 7 in kidney tissue was quantified by RT-PCR. RESULTS: Anesthetic preconditioning with morphine, fentanyl, propofol and dexmedetomidine reduced kidney injury markers after I-R and modulated sirtuin gene expression. Opioids or dexmedetomidine administration before ischemia increased sirtuin 2 expression and correlated with improved renal function. CONCLUSIONS: Anesthetic preconditioning is a promising strategy to prevent I-R injury associated with transplantation. Our results suggest that sirtuin 2 is involved in the protective mechanisms of some commonly used anesthetics against I-R damage.

Opioids Preconditioning Upon Renal Function and Ischemia-Reperfusion Injury: A Narrative Review.

Kidneys have an important role in regulating water volume, blood pressure, secretion of hormones and acid-base and electrolyte balance. Kidney dysfunction derived from acute injury can, under certain conditions, progress to chronic kidney disease. In the late stages of kidney disease, treatment is limited to replacement therapy: Dialysis and transplantation. After renal transplant, grafts suffer from activation of immune cells and generation of oxidant molecules. Anesthetic preconditioning has emerged as a promising strategy to ameliorate ischemia reperfusion injury. This review compiles some significant aspects of renal physiology and discusses current understanding of the effects of anesthetic preconditioning upon renal function and ischemia reperfusion injury, focusing on opioids and its properties ameliorating renal injury. According to the available evidence, opioid preconditioning appears to reduce inflammation and reactive oxygen species generation after ischemia reperfusion. Therefore, opioid preconditioning represents a promising strategy to reduce renal ischemia reperfusion injury and, its application on current clinical practice could be beneficial in events such as acute renal injury and kidney transplantation.

Vasoinhibin Suppresses Nerve Growth Factor-Induced Differentiation and Survival of PC12 Pheochromocytoma Cells.

BACKGROUND: Vasoinhibin, a protein derived from prolactin, regulates various vascular functions including endothelial cell survival. Of note, vasoinhibin is present in the central nervous system, where it triggers neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin compromises nerve growth factor (NGF)-induced neurite outgrowth in primary sensory neurons of the peripheral nervous system. Nonetheless, information on the functions of vasoinhibin in developing neurons remains limited. The present study explored whether vasoinhibin affects the neurotrophic actions of NGF by measuring the cell differentiation and survival of PC12 pheochromocytoma cells. METHODS: The effects of recombinant or lentiviral vector-transduced human vasoinhibin were tested on differentiating PC12 cells. Neurite outgrowth was quantified by measuring their length and density. The MTT assay was employed to assess cell viability, and ELISA was used to quantify DNA fragmentation as an index of apoptosis. Phosphorylated Akt and ERK1/2 were analyzed by Western blotting. RESULTS: The addition of a human recombinant vasoinhibin, and the transduction of a lentiviral vector carrying a human vasoinhibin sequence, significantly reduced NGF-induced neurite outgrowth, cell survival, and phosphorylation of Akt and ERK1/2, and increased DNA fragmentation and caspase 3 activation in PC12 cells. CONCLUSIONS: Vasoinhibin downregulates NGF-induced differentiation and survival of PC12 cells, blocking tropomyosin receptor kinase A-triggered signaling pathways and increasing apoptosis. These results establish that vasoinhibin interaction with NGF and other neurotrophins may be critical in mediating pathways involved in neuronal survival and differentiation.

Pharmacological blockade of the P2X7 receptor reverses retinal damage in a rat model of type 1 diabetes.

AIMS: Retinopathy is a leading cause of vision impairment in diabetes. Its pathogenesis involves inflammation, pathological angiogenesis, neuronal and glial dysfunction. The purinergic P2X7 receptor (P2X7R) has a leading role in inflammation and angiogenesis. Potent and selective P2X7R blockers have been synthesized and tested in Phase I/II clinical studies. We hypothesize that P2X7R blockade will ameliorate diabetes-related pathological retinal changes. METHODS: Streptozotocin (STZ)-treated rats were intraperitoneally inoculated with either of two small molecule P2X7R receptor inhibitors, A740003 and AZ10606120, and after blood glucose levels increased to above 400 mg/dL, retinae were analyzed for P2X7R expression, vascular permeability, VEGF, and IL-6 expression. RESULTS: STZ administration caused a near fourfold increase in blood glucose, a large increase in retinal microvasculature permeability, as well as in retinal P2X7R, VEGF, and IL-6 expression. P2X7R blockade fully reversed retinal vascular permeability increase, VEGF accumulation, and IL-6 expression, with no effect on blood glucose. CONCLUSION: P2X7R blockade might be promising strategy for the treatment of microvascular changes observed in the early phases of diabetic retinopathy.

Diabetes enhances the efficacy of AAV2 vectors in the retina: therapeutic effect of AAV2 encoding vasoinhibin and soluble VEGF receptor 1.

Adeno-associated virus (AAV) vector-mediated delivery of inhibitors of blood-retinal barrier breakdown (BRBB) offers promise for the treatment of diabetic macular edema. Here, we demonstrated a reversal of blood-retinal barrier pathology mediated by AAV type 2 (AAV2) vectors encoding vasoinhibin or soluble VEGF receptor 1 (sFlt-1) when administered intravitreally to diabetic rats. Efficacy and safety of the AAV2 vasoinhibin vector were tested by monitoring its effect on diabetes-induced changes in the retinal vascular bed and thickness, and in the electroretinogram (ERG). Also, the transduction of AAV2 vectors and expression of AAV2 receptors and co-receptors were compared between the diabetic and the non-diabetic rat retinas. AAV2 vasoinhibin or AAV2 sFlt-1 vectors were injected intravitreally before or after enhanced BRBB due to diabetes induced by streptozotocin. The BRBB was examined by the Evans blue method, the vascular bed by fluorescein angiography, expression of the AAV2 EGFP reporter vector by confocal microscopy, and the AAV2 genome, expression of transgenes, receptors, and co-receptors by quantitative PCR. AAV2 vasoinhibin and sFlt-1 vectors inhibited the diabetes-mediated increase in BRBB when injected after, but not before, diabetes was induced. The AAV2 vasoinhibin vector decreased retinal microvascular abnormalities and the diabetes-induced reduction of the B-wave of the ERG, but it had no effect in non-diabetic controls. Also, retinal thickness was not altered by diabetes or by the AAV2 vasoinhibin vector. The AAV2 genome, vasoinhibin and sFlt-1 transgenes, and EGFP levels were higher in the retinas from diabetic rats and were associated with an elevated expression of AAV2 receptors (syndecan, glypican, and perlecan) and co-receptors (fibroblast growth factor receptor 1, αvß5 integrin, and hepatocyte growth factor receptor). We conclude that retinal transduction and efficacy of AAV2 vectors are enhanced in diabetes, possibly due to their elevated cell entry. AAV2 vectors encoding vasoinhibin and sFlt-1 may be desirable gene therapeutics to target diabetic retinopathy and macular edema.

Prolactin-derived vasoinhibins increase anxiety- and depression-related behaviors.

The hormone prolactin (PRL) regulates neuroendocrine and emotional stress responses. It is found in the hypothalamus, where the protein is partially cleaved to vasoinhibins, a family of N-terminal antiangiogenic PRL fragments ranging from 14 to 18kDa molecular masses, with unknown effects on the stress response. Here, we show that the intracerebroventricular administration of a recombinant vasoinhibin, containing the first 123 amino acids of human PRL that correspond to a 14kDa PRL, exerts anxiogenic and depressive-like effects detected in the elevated plus-maze, the open field, and the forced swimming tests. To investigate whether stressor exposure affects the generation of vasoinhibins in the hypothalamus, the concentrations of PRL mRNA, PRL, and vasoinhibins were evaluated in hypothalamic extracts of virgin female rats immobilized for 30min at different time points after stress onset. The hypothalamic levels of PRL mRNA and protein were higher at 60min but declined at 360min to levels seen in non-stressed animals. The elevation of hypothalamic PRL did not correlate with the stress-induced increase in circulating PRL levels, nor was it modified by blocking adenohypophyseal PRL secretion with bromocriptine. A vasoinhibin having an electrophoretic migration rate corresponding to 17kDa was detected in the hypothalamus. Despite the elevation in hypothalamic PRL, the levels of this hypothalamic vasoinhibin were similar in stressed and non-stressed rats. Stress reduced the rate of cleavage of PRL to this vasoinhibin as shown by the incubation of recombinant PRL with hypothalamic extracts from stressed rats. These results suggest that vasoinhibins are potent anxiogenic and depressive factors and that stress increases PRL levels in the hypothalamus partly by reducing its conversion to vasoinhibins. The reciprocal interplay between PRL and vasoinhibins may represent an effective mechanism to regulate anxiety and depression.

Vasoinhibin gene transfer by adenoassociated virus type 2 protects against VEGF- and diabetes-induced retinal vasopermeability.

PURPOSE: Specific proteolytic cleavages of the hormone prolactin (PRL) generate vasoinhibins, a family of peptides (including 16-kDa PRL) that are able to inhibit the pathologic increase in retinal vasopermeability (RVP) associated with diabetes. Here the authors tested the ability of an adenoassociated virus type 2 (AAV2) vasoinhibin vector to inhibit vascular endothelial growth factor (VEGF)- and diabetes-induced RVP. METHODS: AAV2 vectors encoding vasoinhibin, PRL, or soluble VEGF receptor 1 (soluble FMS-like tyrosine kinase-1 [sFlt-1]) were injected intravitreally into the eyes of rats. Four weeks later, either VEGF was injected intravitreally or diabetes was induced with streptozotocin. Tracer accumulation was evaluated as an index of RVP using fluorescein angiography or the Evans blue dye method. RT-PCR verified transgene expression in the retina, and the intravitreal injection of an AAV2 vector encoding green fluorescent protein revealed transduced cells in the retinal ganglion cell layer. In addition, Western blot analysis of AAV2-transduced HEK293 cells confirmed the expression and secretion of the vector-encoded proteins. RESULTS: The AAV2-vasoinhibin vector prevented the increase in tracer accumulation that occurs 24 hours after the intravitreal injection of VEGF. Diabetes induced a significant increase in tracer accumulation compared with nondiabetic controls. This increase was blocked by the AAV2-vasoinhibin vector and reduced by the AAV2-sFlt-1 vector. The AAV2-PRL vector had no effect. CONCLUSIONS: These results show that an AAV2-vasoinhibin vector prevents pathologic RVP and suggest it could have therapeutic value in patients with diabetic retinopathy.