Lajos Markó

Neutrophil gelatinase‐associated lipocalin: pathophysiology and clinical applications

Neutrophil gelatinase‐associated lipocalin (NGAL), a 25 kDa protein produced by injured nephron epithelia, is one of the most promising new markers of renal epithelial injury. In contrast to serum creatinine and urinary output, which are the measures of kidney function, NGAL is specifically induced in the damaged nephron and then released into blood and urine, where it can be readily measured. Careful proof‐of‐concept studies using defined animal models have uncovered the sources and trafficking of NGAL in acute kidney injury (AKI) and have addressed the contributions of renal and non‐renal sources. Clinical studies indicate that NGAL, unlike creatinine, is a marker responsive to tissue stress and nephron injury, but less so to adaptive hemodynamic responses. In certain clinical settings, NGAL is an earlier marker compared with serum creatinine. In addition, clinical studies have shown that NGAL is a powerful predictor of poor clinical outcomes, which can be used to risk stratify patients when combined with serum creatinine. NGAL has important limitations, including its responsiveness in systemic inflammation, which is partially uncoupled from its response to kidney injury and which needs to be considered when interpreting NGAL results clinically. This review covers the biology and pathophysiology of NGAL and summarizes the results of the growing body of clinical studies that have addressed the utility of NGAL in the early diagnosis of AKI, in the distinction of intrinsic AKI and in the prognostic assessment of broad patient populations.

Interferon-γ Signaling Inhibition Ameliorates Angiotensin II–Induced Cardiac Damage

Angiotensin (Ang) II induces vascular injury in part by activating innate and adaptive immunity; however, the mechanisms are unclear. We investigated the role of interferon (IFN)-&ggr; and interleukin (IL)-23 signaling. We infused Ang II into IFN-&ggr; receptor (IFN-&ggr;R) knockout mice and wild-type controls, as well as into mice treated with neutralizing antibodies against IL-23 receptor and IL-17A. Ang II–treated IFN-&ggr;R knockout mice exhibited reduced cardiac hypertrophy, reduced cardiac macrophage and T-cell infiltration, less fibrosis, and less arrhythmogenic electric remodeling independent of blood pressure changes. In contrast, IL-23 receptor antibody treatment did not reduce cardiac hypertrophy, fibrosis, or electric remodeling despite mildly reduced inflammation. IL-17A antibody treatment behaved similarly. In the kidney, IFN-&ggr;R deficiency reduced inflammation and tubulointerstitial damage and improved glomerular filtration rate. Nonetheless, albuminuria was increased compared with Ang II–treated wild-type controls. The glomeruli of Ang II–treated IFN-&ggr;R knockout mice exhibited fewer podocytes, less nephrin and synaptopodin staining, and impaired podocyte autophagy. Thus, IFN-&ggr; blockade, but not IL-23 receptor antibody treatment, protects from Ang II–induced cardiac damage and electric remodeling. In the kidney, IFN-&ggr; signaling acts in a cell type–specific manner. Glomerular filtration rate is preserved in the absence of the IFN-&ggr;R, whereas podocytes may require the IFN-&ggr;R in the presence of Ang II for normal integrity and function.

Salt-responsive gut commensal modulates TH17 axis and disease

A Western lifestyle with high salt consumption can lead to hypertension and cardiovascular disease. High salt may additionally drive autoimmunity by inducing T helper 17 (TH17) cells, which can also contribute to hypertension. Induction of TH17 cells depends on gut microbiota; however, the effect of salt on the gut microbiome is unknown. Here we show that high salt intake affects the gut microbiome in mice, particularly by depleting Lactobacillus murinus. Consequently, treatment of mice with L. murinus prevented salt-induced aggravation of actively induced experimental autoimmune encephalomyelitis and salt-sensitive hypertension by modulating TH17 cells. In line with these findings, a moderate high-salt challenge in a pilot study in humans reduced intestinal survival of Lactobacillus spp., increased TH17 cells and increased blood pressure. Our results connect high salt intake to the gut–immune axis and highlight the gut microbiome as a potential therapeutic target to counteract salt-sensitive conditions.

Differential Effects of Cystathionine-γ-lyase–Dependent Vasodilatory H2S in Periadventitial Vasoregulation of Rat and Mouse Aortas

Background Hydrogen sulfide (H2S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H2S are not fully understood. We tested the hypotheses that (1) H2S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (Kv) K+ channels and (2) that H2S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway. Methodology/Principal Findings Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H2S donors. KCNQ-type Kv channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H2S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition. Conclusions/Signficance Our results demonstrate potent vasorelaxant effects of H2S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H2S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H2S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H2S/KCNQ pathways to influence states of vascular dysfunction may be possible.

Apolipoprotein A5 T-1131C Variant Confers Risk for Metabolic Syndrome

The −1131C is a naturally occurring variant of the apolipoprotein A5 (ApoA5) gene, which has been shown to associate with increased triglyceride levels. This variant has also been shown to confer risk for development of ischemic heart disease and stroke. The gene is in linkage disequilibrium with factors known to correlate with impaired glucose homeostasis. These observations prompted us to study the prevalence of the ApoA5 –1131C allele in patients with metabolic syndrome. A total of 201 metabolic syndrome patients and 210 controls were studied. In both groups the triglyceride levels of patients with −1131C allele were significantly increased compared to the subjects with −1131T allele (3.22 ±0.43 mmol/1 vs. 2.24 ±0.12 mmol/1, p<0.01 in the metabolic syndrome patients; 2.10 ±0.19 mmol/1 vs. 1.22 ±0.05 mmol/1, p<0.01 in the controls). In metabolic syndrome patients the prevalence of the ApoA5 –1131C variant was increased compared to the healthy controls (11% vs. 6.20%). Multiplex regression analysis model adjusted for age, gender, serum total cholesterol levels, acute myocardial infarction and stroke events revealed that the examined ApoA5 variant confers risk for the development of metabolic syndrome: the odds ratio at 95% confidence interval was 3.622 (1.200–10.936), p=0.02. Our findings strongly suggest that this variant is a risk factor for the development of hypertriglyceridemia and metabolic syndrome.

Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography

Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammonium chloride/ammonia buffer with ionic strength of 150 mM, exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It was also demonstrated that elution with sorbitol at 1.2 M is superior to acidic media. Comparison of results was based on the numbers of detected peptides and their glycated sites. Using sorbitol for elution requires desalting prior to analysis. Therefore, three different sorbents were tested: fullerene-derivatized silica, ZipTip (C18), and C18 silica. Fullerene-derivatized silica and ZipTip showed the same performance regarding the numbers of glycated peptides, and sites were better than C18 silica. The elaborated off-line method was compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of human serum albumin (HSA) from sera of healthy and diabetic individuals. A peptide with a mass of 1783.9 Da could be detected only in samples of diabetic patients and, therefore, could be a very interesting biomarker candidate.

Haplotype analysis of the apolipoprotein A5 gene in patients with the metabolic syndrome

BACKGROUND AND AIMS In recent studies, the T-1131C variant of apolipoprotein A5 (APOA5) gene was found to confer a risk for metabolic syndrome (MS). Here we determined four haplotype-tagging polymorphisms (T-1131C, IVS3+G476A, T1259C, and C56G), and studied the distribution of the naturally occurring major haplotype profiles in MS. METHODS AND RESULTS A total of 343 MS patients and 284 controls were genotyped using PCR-RFLP methods. Both in MS and control groups, we confirmed the already known association of -1131C, IVS3+473A and 1259C minor alleles with elevated triglyceride levels. The prevalence of the APOA5*2 haplotype (the combination of T-1131C, IVS3+G476A and T1259C SNPs) was 13.1% in MS patients, and 4.9% in controls (p<0.001); multiple logistic regression analysis revealed that this haplotype confers risk for the development of MS (OR=2.880; 95% CI: 1.567-5.292; p=0.001). We also observed a gender effect: in males a more prominent degree of susceptibility was found. Contrary to the APOA5*2 haplotype, the prevalence rate of APOA5*4 (determined by the T-1131C SNP alone) did not differ between MS patients and controls. We identified a novel haplotype, designated here as APOA5*5 (1259C allele alone); which appears to be protective against MS. CONCLUSION Our results refined the role of SNP T-1131C in the development of MS. The susceptibility nature of this SNP is limited to the APOA5*2 haplotype, while in APOA5*4 haplotype it did not confer a risk for the disease. In addition, as our current data suggest, the novel APOA5*5 haplotype can confer protection against MS.

Novel signalling mechanisms and targets in renal ischaemia and reperfusion injury.

Acute kidney injury (AKI) induced by ischaemia and reperfusion (I/R) injury is a common and severe clinical problem. Vascular dysfunction, immune system activation and tubular epithelial cell injury contribute to functional and structural deterioration. The search for novel therapeutic interventions for I/R‐induced AKI is a dynamic area of experimental research. Pharmacological targeting of injury mediators and corresponding intracellular signalling in endothelial cells, inflammatory cells and the injured tubular epithelium could provide new opportunities yet may also pose great translational challenge. Here, we focus on signalling mediators, their receptors and intracellular signalling pathways which bear potential to abrogate cellular processes involved in the pathogenesis of I/R‐induced AKI. Sphingosine 1 phosphate (S1P) and its respective receptors, cytochrome P450 (CYP450)‐dependent vasoactive eicosanoids, NF‐κB‐ and protein kinase‐C (PKC)‐related pathways are representatives of such ‘druggable’ pleiotropic targets. For example, pharmacological agents targeting S1P and PKC isoforms are already in clinical use for treatment for autoimmune diseases and were previously subject of clinical trials in kidney transplantation where I/R‐induced AKI occurs as a common complication. We summarize recent in vitro and in vivo experimental studies using pharmacological and genomic targeting and highlight some of the challenges to clinical application of these advances.

Use of fullerene‐, octadecyl‐, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen

SPE plays a crucial role in bioanalytical research. In the present work a novel fullerene(C60)-derivatised silica material is compared with octadecyl(C18) - and triaconthyl(C30)-silicas regarding recoveries of peptides and sequence coverage of HSA and fibrinogen digests. C30- and C60(30 nm)-SPE materials were found to be the two most prominent SPE materials. At low peptide concentrations C60-material prepared from a silica gel with a pore size of 30 nm has proven to be the best material with regards to recoveries. By increasing the amount of loaded peptides recoveries decrease due to its relative low binding capacity in contrast to C30-silica particles, showing no changes. The best sequence coverages of Aalpha- and Bbeta-chains of 20 pmol fibrinogen digest can also be achieved using these two SPE materials, C60 (30 nm) demonstrates an outstanding value of sequence coverage (62.15%) achieved for the gamma-chain. After nonenzymatic glycation the digests of fibrinogen and HSA were also separated. This makes the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. For HSA, ten new sites of glycation at lysine and arginine residues have been explored. Using the detailed SPE/off-line MALDI method the glycation sites on fibrinogen are first described in this paper.

Cigarette Smoke-Induced Alterations in Endothelial Nitric Oxide Synthase Phosphorylation: Role of Protein Kinase C

Endothelial nitric oxide synthase (eNOS) is regulated by phosphorylation of Ser(1177) and Thr(495), which affects NO bioavailability. Cigarette smoke disturbs the eNOS-cGMP-NO pathway and causes decreased NO production. Here the authors investigated the acute effects of cigarette smoke on eNOS phosphorylation, focusing on protein kinases (PKs). Endothelial cell culture was concentration- and time-dependently treated first with cigarette smoke buffer (CSB), then with reduced glutathione (GSH) or various PK inhibitors (H-89, LY-294002, Ro-318425, and ruboxistaurin). eNOS, phospho-Ser(1177)-eNOS, phospho-Thr(495)-eNOS, Akt(PKB), and phospho-Akt protein levels were determined by Western blot. CSB increased the phosphorylation of eNOS at Ser(1177) and more at Thr(495) in a concentration- and time-dependent manner (p < .01, p < .05 versus control, respectively) and resulted in the dissociation of the active dimeric form of eNOS (p < .05). GSH decreased the phosphorylation of eNOS at both sites (p < .05 versus CSB without GSH) and prevented the decrease of dimer eNOS level. CSB treatment also decreased the level of phospho-Ser(473)-Akt (p < .05 versus control). Inhibition of PKA by H-89 did not affect CSB-induced phosphorylation, whereas the PKB inhibitor LY-294002 enhanced it at Ser(1117). The PKC blockers Ro-318425 and ruboxistaurin augmented the CSB-induced phosphorylation at Ser(1177) but decreased phosphorylation at Thr(495) (p < .05 versus CSB). Cigarette smoke causes a disruption of the enzymatically active eNOS dimers and shifts the eNOS phosphorylation to an inhibitory state. Both effects might lead to reduced NO bioavailability. The shift of the eNOS phosphorylation pattern to an inhibitory state seems to be independent of the PKA and phosphoinositol 3-kinase (PI3-K)/Akt pathways, whereas PKC appears to play a key role.