Joachim Jankowski

The Antioxidant Acetylcysteine Reduces Cardiovascular Events in Patients With End-Stage Renal Failure A Randomized, Controlled Trial

Background—Patients with end-stage renal failure have increased oxidative stress and show elevated cardiovascular mortality. Whether increased cardiovascular events can be prevented by the administration of antioxidants is unknown. Methods and Results—We evaluated the effects of acetylcysteine, a thiol-containing antioxidant, on cardiovascular events in patients undergoing hemodialysis. A prospective, randomized, placebo-controlled trial was conducted between October 1, 1999, and September 30, 2001, in 134 patients (76 male and 58 female) with a mean age of 62±16 years (mean±SD) who had been undergoing maintenance hemodialysis for a minimum of 3 months 3 times weekly in an ambulatory center. Median (range) follow-up was 14.5 (1 to 24) months. Patients were randomly assigned either to receive acetylcysteine (600 mg BID) or placebo. The primary end point was a composite variable consisting of cardiac events including fatal and nonfatal myocardial infarction, cardiovascular disease death, need for coronary angioplasty or coronary bypass surgery, ischemic stroke, peripheral vascular disease with amputation, or need for angioplasty. Secondary end points included each of the component outcomes, total mortality, and cardiovascular mortality. A total of 18 (28%) of the 64 hemodialysis patients assigned to acetylcysteine group and 33 (47%) of the 70 hemodialysis patients assigned to control group had a primary end point (relative risk, 0.60 [95% CI, 0.38 to 0.95], P =0.03). No significant differences in secondary end points or total mortality were detected. Conclusions—In hemodialysis patients, treatment with acetylcysteine (600 mg BID) reduces composite cardiovascular end points.

Normal and Pathologic Concentrations of Uremic Toxins

An updated review of the existing knowledge regarding uremic toxins facilitates the design of experimental studies. We performed a literature search and found 621 articles about uremic toxicity published after a 2003 review of this topic. Eighty-seven records provided serum or blood measurements of one or more solutes in patients with CKD. These records described 32 previously known uremic toxins and 56 newly reported solutes. The articles most frequently reported concentrations of β2-microglobulin, indoxyl sulfate, homocysteine, uric acid, and parathyroid hormone. We found most solutes (59%) in only one report. Compared with previous results, more recent articles reported higher uremic concentrations of many solutes, including carboxymethyllysine, cystatin C, and parathyroid hormone. However, five solutes had uremic concentrations less than 10% of the originally reported values. Furthermore, the uremic concentrations of four solutes did not exceed their respective normal concentrations, although they had been previously described as uremic retention solutes. In summary, this review extends the classification of uremic retention solutes and their normal and uremic concentrations, and it should aid the design of experiments to study the biologic effects of these solutes in CKD.

Clinical proteomics: A need to define the field and to begin to set adequate standards

The aim of this manuscript is to initiate a constructive discussion about the definition of clinical proteomics, study requirements, pitfalls and (potential) use. Furthermore, we hope to stimulate proposals for the optimal use of future opportunities and seek unification of the approaches in clinical proteomic studies. We have outlined our collective views about the basic principles that should be considered in clinical proteomic studies, including sample selection, choice of technology and appropriate quality control, and the need for collaborative interdisciplinary efforts involving clinicians and scientists. Furthermore, we propose guidelines for the critical aspects that should be included in published reports. Our hope is that, as a result of stimulating discussion, a consensus will be reached amongst the scientific community leading to guidelines for the studies, similar to those already published for mass spectrometric sequencing data. We contend that clinical proteomics is not just a collection of studies dealing with analysis of clinical samples. Rather, the essence of clinical proteomics should be to address clinically relevant questions and to improve the state‐of‐the‐art, both in diagnosis and in therapy of diseases.

Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

Advances in Urinary Proteome Analysis and Biomarker Discovery

Noninvasive diagnosis of kidney diseases and assessment of the prognosis are still challenges in clinical nephrology. Definition of biomarkers on the basis of proteome analysis, especially of the urine, has advanced recently and may provide new tools to solve those challenges. This article highlights the most promising technological approaches toward deciphering the human proteome and applications of the knowledge in clinical nephrology, with emphasis on the urinary proteome. The data in the current literature indicate that although a thorough investigation of the entire urinary proteome is still a distant goal, clinical applications are already available. Progress in the analysis of human proteome in health and disease will depend more on the standardization of data and availability of suitable bioinformatics and software solutions than on new technological advances. It is predicted that proteomics will play an important role in clinical nephrology in the very near future and that this progress will require interactive dialogue and collaboration between clinicians and analytical specialists.

A Bench to Bedside View of Uremic Toxins

Reviewing the current picture of uremic toxicity reveals its complexity. Focusing on cardiovascular damage as a model of uremic effects resulting in substantial morbidity and mortality, most molecules with potential to affect the function of a variety of cell types within the vascular system are difficult to remove by dialysis. Examples are the larger middle molecular weight molecules and protein-bound molecules. Recent clinical studies suggest that enhancing the removal of these compounds is beneficial for survival. Future therapeutic options are discussed, including improved removal of toxins and the search for pharmacologic strategies blocking responsible pathophysiologic pathways.

Discovery and Characterization of Alamandine, a Novel Component of the Renin-Angiotensin System

Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand β-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/β-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders. # Novelty and Significance {#article-title-32}Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand &bgr;-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/&bgr;-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.

Uridine adenosine tetraphosphate: a novel endothelium- derived vasoconstrictive factor

Beyond serving as a mechanical barrier, the endothelium has important regulatory functions. The discovery of nitric oxide revolutionized our understanding of vasoregulation. In contrast, the identity of endothelium-derived vasoconstrictive factors (EDCFs) remains unclear. The supernatant obtained from mechanically stimulated human endothelial cells obtained from dermal vessels elicited a vasoconstrictive response in an isolated perfused rat kidney. A purinoceptor blocker had a greater effect than an endothelin receptor blocker in decreasing endothelially derived vasoconstriction in the isolated perfused rat kidney. The nucleotide uridine adenosine tetraphosphate (Up4A) was isolated from the supernatant of stimulated human endothelium and identified by mass spectrometry. Up4A is likely to exert vasoconstriction predominantly through P2X1 receptors, and probably also through P2Y2 and P2Y4 receptors. Plasma concentrations of Up4A that cause vasoconstriction are found in healthy subjects. Stimulation with adenosine 5′-triphosphate (ATP), uridine 5′-triphosphate (UTP), acetylcholine, endothelin, A23187 and mechanical stress releases Up4A from endothelium, suggesting that Up4A contributes to vascular autoregulation. To our knowledge, Up4A is the first dinucleotide isolated from living organisms that contains both purine and pyrimidine moieties. We conclude that Up4A is a novel potent nonpeptidic EDCF. Its vasoactive effects, plasma concentrations and its release upon endothelial stimulation strongly suggest that Up4A has a functional vasoregulatory role.

Implementation of proteomic biomarkers: Making it work

Eur J Clin Invest 2012; 42 (9): 1027–1036

Immunomodulator FTY720 Induces eNOS-Dependent Arterial Vasodilatation via the Lysophospholipid Receptor S1P3

The novel immunomodulator FTY720 is effective in experimental models of transplantation and autoimmunity, and is currently undergoing Phase III clinical trials for prevention of kidney graft rejection. FTY720 is a structural analogue of sphingosine-1-phosphate (S1P) and activates several of the S1P receptors. We show that FTY720 induces endothelium-dependent arterial vasodilation in phenylephrine precontracted mouse aortae. Vasodilation did not occur in thoracic aortic rings from eNOS-deficient mice, implicating and effect dependent of activation of the eNOS/NO pathway. Accordingly, FTY720 induced NO release, Akt-dependent eNOS phosphorylation and activation in human endothelial cells. For biological efficacy, FTY720 required endogenous phosphorylation, since addition of the sphingosine kinase antagonist N′,N-dimethylsphingosine (DMS) prevented activation of eNOS in vitro and inhibited vasodilation in isolated arteries. The endothelial phosphorylation of FTY720 was extremely rapid with almost complete conversion after 10 minutes as determined by mass spectrometry. Finally, we identified the lysophospholipid receptor S1P3 as the S1P receptor responsible for arterial vasodilation by FTY720, as the effect was completely abolished in arteries from S1P3-deficient mice. In summary, we have identified FTY720 as the first immunomodulator for prevention of organ graft rejection in clinical development that, in addition, positively affects the endothelium by stimulating NO production, and thus potentially displaying beneficial effects on transplant survival beyond classical T cell immunosuppression.