Impact of recently identified angiotensin peptides and their cofactors on vascular hemodynamics in AKI patients
Nephrology
Final Report Abstract
The first part of the project focused on the isolation, identification and the physiological and pathophysiological characterization of a new cofactor of Angiotensin-II on intrarenal hemodynamics in the context of AKI. The peptide modulates the vasoregulatory effects of Angiotensin-II. The effects of this Angiotensin-II cofactor were evaluated in-vitro and, in-vivo and the receptor affinity was analysed. The cofactor concentration was quantified in plasma isolated from renal failure patients and healthy controls. The peptide is a degradation product of Chromogranin-A and its amino acid sequence is HSGFEDELSE VLENQSSQAE LKEAVEEPSS KDVME. The peptide diminished significantly the vasoconstrictive effect of Ang-II in-vitro. Therefore, we named the peptide "vasoconstriction inhibiting factor" (VIF). The vasoregulatory effects of VIF are mediated by the AT2-receptor. VIF impairs Ang-II-induced phosphorylation of the p38MAPK-pathway but not of ERK1/2. The vasodilatory effects were confirmed in-vivo. The plasma concentration is significantly increased in renal failure patients. It is likely that the increase in VIF may serve as a counter-regulatory effect to defend against increased pressure of the vessels. The identification of this target may help us to understand the pathophysiology of renal failure and forms a basis for the development of new strategies for the prevention and treatment of cardiovascular disease. Since clinical data of the project clearly demonstrate increased VIF plasma concentrations in patients suffering from heart failure compared to healthy controls too, VIF obviously serve as a counterregulator of end-organ damage. We were able to demonstrate that VIF protects the end-organ tissue during ischemic events. We analyzed the protective effects of VIF on myocardial infarction. We were treated mice with VIF via a minipump for 2 days before and 2 weeks after inducing the myocardial infarction. This treatment significantly decreased the infarcted area of the mice and in parallel improved the ejection fraction compared with the control mice. The identification of VIF may help to clarify the pathophysiology of ischemic events like in the context of AKI and myocardial infarction and, therefore, could form the basis for the development of a strategy for prevention and treatment of end-organ tissue during ischemic events. Since VIF might have additional protective effects in the context of myocardial infarction, a patent is currently being prepared by an external patent attorney. Since there was no reliable, high throughput quantification method for Angiotensin peptides like Angiotensin-II, Angiotensin-A and/or Angioprotectin available at the beginning of the project, we developed and validated a sensitive, selective and reliable LC-ESI-MS/MS-based method for absolute quantification of Angiotensin peptide concentration in human plasma. Plasma samples were extracted by solid-phase chromatography and were subjected to a further immunoaffinity-purification using immobilized anti-angiotensin antibodies in order to isolate endogenous angiotensin peptides. Stable isotope angiotensin peptides were used as internal standards. The fractionated samples were analysed using mass-spectrometric analyses. The developed and validated method was successfully applied for quantification of endogenous Angiotensin peptides in human plasma of healthy volunteers and renal failure patients. The mass-spectrometric based method for quantification of Angiotensin peptide levels in human plasma was successfully evaluated. This method was applied for a clinical study aiming at the validation of the impact of physiologically and pathophysiologically relevant effects of Angiotensin peptide in acute kidney injury. In cooperation with another project, the Angiotensin peptides Angiotensin-II, Angiotensin-A, Angioprotectin, Almandine were quantified in the plasma of patients before and after acute renal failure in order to test the extent to which the peptides concerned contribute to the genesis and progression of acute renal failure. Since the hemodynamic situation of the kidney is significantly changed in the context of acute renal failure, the involvement of these peptides in the context of acute renal failure seems to be likely. Therefore, the concentrations of peptides in the blood of patients with acute renal failure were determined in a clinical study of patients undergoing a surgery. A total of four parameters were tested. Therefore, a significance level of 1.25% was assumed for the single test. The power analysis indicates that in a sample size of 30 patients with acute renal failure and 90 healthy controls, an effect size of 0.71 could be demonstrated with a power of 80% with a t-test for unrelated samples. This effect size for all parameters appeared to be appropriate and sufficient. Acute renal insufficiency was the consequence of a coronary artery surgery, heart valves surgery or other types of cardiac surgeries. The Angiotensin peptides were quantified in the plasma of the patients before and after the surgery. An increase in the plasma concentration of Angioprotectin and almandine in the plasma of patients with acute renal failure was observed; however, did not reach the significance level of 0.05 compared to the corresponding control group without acute renal failure. In addition to the known vasoregulatory effect of the Angiotensin peptides identified by the principal investigators of Project 10, the effect of the Angiotensin peptides on the proliferation rate of smooth vascular muscle cells and endothelial cells compared to the respective effect of Angiotensin-II was also determined. Angioprotectin and Angiotensin-A significantly increase the proliferation rate of smooth vascular muscle cells, while Angiotensin-A significantly decreases the proliferation rate of endothelial cells. In addition, Angioprotectin and Angiotensin-A increase the vascular calcification rate of isolated aortic rings. Due to the pathophysiological significance of these effects, these Angiotensin peptides are currently being further investigated in the context of a dissertation. Iohexol is a frequently used drug for the quantification the glomerular filtration rate. However, high iohexol amounts might lead to adverse effects in organism, like acute kidney injury. As part of the translation of the project, a sensitive quantification method for iohexol was developed in order to minimize the iohexol dosage. A linear correlation of the iohexol amount and mass-signal intensity was found in the therapeutic relevant range. The lowest limit of quantification, intra- and inter-day accuracies, intra- and inter-day precisions and recovery rate of iohexol of this mass-spectrometric based method demonstrated to be highly sensitive, selective and suitable for the quantification of iohexol at low concentrations in serum. The gene expressions test revealed that iohexol induce a group of genes in thyroidal tissue that comprises transcription factors and genes of cellular stress response. Namely, Ptgs2, Smad7, Jun, Srf, Dusp1, Timp1 and others are up-regulated after iohexol i.v. application. The iohexol application dose as well as the sampling time in the clinical routine could be reduced in the future in order to further minimize side effects in humans due to high sensitivity of this novel method. In cooperation with another project, we were able to demonstrate that besides Angiotensin peptides diadenosine polyphosphates are released by in contrast-induced acute kidney injury by tubular cells and act on glomerular arterioles and reduce glomerular filtration rate. We were able to show that iodixanol treatment of tubules significantly increased the concentration of diadenosine polyphosphates. Acute kidney injury patient shows higher concentrations of diadenosine polyphosphates compared to healthy. Application of diadenosine polyphosphates significantly reduced the glomerular filtration rate in conscious mice. Diadenosine polyphosphates reduced afferent arteriolar diameters, but did not influence efferent arterioles. The constrictor effect on afferent arterioles was strong immediately after application, but weakened with time. Then, non-selective P2 inhibitor suramin blocked the diadenosine polyphosphates -induced constriction. The data suggest that diadenosine polyphosphates play a role in the pathophysiology of contrast-induced acute kidney injury. We show a contrast media-induced release of diadenosine polyphosphates from tubules, which might increase afferent arteriolar resistance and reduce the GFR.
Publications
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A highly sensitive method for quantification of iohexol. Anal Methods 6: 3706-3712, 2014
Schulz A, Boeringer F, Swifka J, Kretschmer A, Schaefer M, Jankowski V, van der Giet M, Schuchardt M, Toelle M, Tepel M, Schlieper G, Zidek W, Jankowski J
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Absolute quantification of endogenous angiotensin II levels in human plasma using ESI-LC-MS/MS. Clin Proteomics 11: 37, 2014
Schulz A, Jankowski J, Zidek W, Jankowski V
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Diadenosine pentaphosphate modulates glomerular arteriolar tone and glomerular filtration rate. Acta Physiol (Oxf) 213: 285-293, 2015
Patzak A, Carlstrom M, Sendeski MM, Lai EY, Liu ZZ, Sallstrom J, Floge J, Heintz B, Jankowski J, Jankowski V
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Identification of the “vasoconstriction inhibiting factor” (VIF), a potent endogenous cofactor of angiotensin II acting on the AT2 receptor. Circulation 131: 1426-1434, 2015
Salem S, Jankowski V, Asare Y, Liehn E, Welker P, Raya-Bermudez A, Pineda-Martos C, Rodriguez M, Munoz-Castaneda JR, Bruck H, Marx N, Machado FB, Staudt M, Heinze G, Zidek W, Jankowski J
