Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform.

In the field of quantitative proteomics, the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology has demonstrated efficacy for proteome monitoring despite its lack of a consensus for data handling. In the present study, after peptide and protein identification, we compared the widespread quantitation method based on the calculation of MS/MS reporter ion peaks areas ratios (ProteinPilot) to the alternative method based on the calculation of ratios of the sum of peak intensities (jTRAQx [Quant]) and we processed output data with the in-house Customizable iTRAQ Ratios Calculator (CiR-C) algorithm. Quantitation based on peak area ratios displayed no significant linear correlation with Western blot quantitation. In contrast, quantitation based on the sum of peak intensities displayed a significant linear association with Western blot quantitation (non-zero slope; Pearson correlation coefficient test, r = 0.296, P=0.010**) with an average bias of 0.087 ± 0.500 and 95% Limits of Agreement from -0.893 to 1.068. We proposed the Mascot-jTRAQx-CiR-C strategy as a simple yet powerful data processing adjunct to the iTRAQ technology.

Kinin B1 receptor antagonism is equally efficient as angiotensin receptor 1 antagonism in reducing renal fibrosis in experimental obstructive nephropathy, but is not additive.

BACKGROUND: Renal tubulointerstitial fibrosis is the pathological hallmark of chronic kidney disease (CKD). Currently, inhibitors of the renin-angiotensin system (RAS) remain the sole therapy in human displaying antifibrotic properties. Further antifibrotic molecules are needed. We have recently reported that the delayed blockade of the bradykinin B1 receptor (B1R) reduced the development of fibrosis in two animal models of renal fibrosis. The usefulness of new drugs also resides in outperforming the gold standards and eventually being additive or complementary to existing therapies. METHODS: In this study we compared the efficacy of a B1R antagonist (B1Ra) with that of an angiotensin type 1 receptor antagonist (AT1a) in the unilateral ureteral obstruction (UUO) model of renal fibrosis and determined whether bi-therapy presented higher efficacy than any of the drugs alone. RESULTS: B1R antagonism was as efficient as the gold-standard AT1a treatment. However, bitherapy did not improve the antifibrotic effects at the protein level. We sought for the reason of the absence of this additive effect by studying the expression of a panel of genes involved in the fibrotic process. Interestingly, at the molecular level the different drugs targeted different players of fibrosis that, however, in this severe model did not result in improved reduction of fibrosis at the protein level. CONCLUSIONS: As the B1R is induced specifically in the diseased organ and thus potentially displays low side effects it might be an interesting alternative in cases of poor tolerability to RAS inhibitors.

Dual effect of chemokine CCL7/MCP-3 in the development of renal tubulointerstitial fibrosis.

Most end-stage renal disease kidneys display accumulation of extracellular matrix (ECM) in the renal tubular compartment (tubular interstitial fibrosis - TIF) which is strongly correlated with the future loss of renal function. Although inflammation is a key event in the development of TIF, it can also have a beneficial anti-fibrotic role depending in particular on the stage of the pathology. Chemokines play an important role in monocyte extravasation in the inflammatory process. CCL2 has already been shown to be involved in the development of TIF but CCL7, a close relative of CCL2 and able to bind to similar receptors, has not been studied in renal disease. We therefore studied chemokine CCL7 in a model of unilateral ureteral obstruction (UUO)-induced TIF. We observed that the role of CCL7 differs depending on the stage of the pathology. In early stages (0-8 days), CCL7 deficient (CCL7-KO) mice displayed attenuated TIF potentially involving two mechanisms: an early (0-3 days) decrease of inflammatory cell infiltration followed (3-8 days) by a decrease in tubular ECM production independent of inflammation. In contrast, during later stages of obstruction (10-14 days), CCL7-KO mice displayed increased TIF which was again associated with reduced inflammation. Interestingly, the switch between this anti- to profibrotic effect was accompanied by an increased influx of immunosuppressive regulatory T cells. In conclusion, these results highlight for the first time a dual role for CCL7 in the development of renal TIF, deleterious in early stages but beneficial during later stages.

[Renal urinary shear stress: a novel actor in nephropathies]. / Quand l'urine cisaille les cellules rénales - Un nouveau rôle pour le shear stress dans les néphropathies.

The role of fluid shear stress is well established in vascular pathophysiology. However, urinary shear stress now also appears as a key mechanism in the regulation of renal function. In addition, there is a growing body of evidence showing that modified urinary shear stress is involved in the development of nephropathies. Therefore we review here the state-of-the-art on the pathophysiological roles of urinary shear stress.

Urinary proteomics based on capillary electrophoresis coupled to mass spectrometry in kidney disease.

Urine is an excellent sample source in the proteomic study of diseases. It is available in large quantities, is relatively stable, is not contaminated by cells or lipids, and has shown to provide information not only on the organs in contact with the urinary tract but also of more remote organs and tissues. In addition to these qualities, it can be collected by untrained personnel. For these reasons, urinary proteomic studies have escalated in recent years with the aim of identifying biomarkers that could be use for diagnosis or to predict the outcome of renal pathologies. In this chapter, we present one of the analytical platforms that has been successfully used in a number of studies for the identification and validation of biomarkers in kidney diseases. This technique is capillary electrophoresis coupled online to an electrospray ionization time-of-flight mass spectrometer (CE-MS). This technology has proven to be highly reproducible, sensitive with a quick analysis time, important features when analytical platforms have to be used in a clinical setting.

Renal tubular fluid shear stress facilitates monocyte activation toward inflammatory macrophages.

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. Recently, we reported that renal tubular FSS promotes endothelial cell activation and subsequent adhesion of human monocytes, thereby suggesting that changes in urinary FSS can induce the development of inflammation (Miravète M, Klein J, Besse-Patin A, Gonzalez J, Pecher C, Bascands JL, Mercier-Bonin M, Schanstra JP, Buffin-Meyer B, BBRC 407: 813-817, 2011). Here, we evaluated the influence of tubular FSS on monocytes as they play an important role in the progression of inflammation in nephropathies. Human renal tubular cells (HK-2) were exposed to FSS 0.01 Pa for 30 min or 5 h. Treatment of human THP-1 monocytes with the resulting conditioned medium (FSS-CM) modified the expression of macrophage differentiation markers, suggesting differentiation toward the inflammatory M1-type macrophage. The effect was confirmed in freshly isolated human monocytes. In contrast to endothelial cells, the activation of monocytes by FSS-CM did not require TNF-α. Cytokine array analysis of FSS-CM showed that FSS modified secretion of cytokines by HK-2 cells, particularly by increasing secretion of TGF-ß and by decreasing secretion of C-C chemokine ligand 2 (CCL2). Neutralization of TGF-ß or CCL2 supplementation attenuated the effect of FSS-CM on macrophage differentiation. Finally, FSS-injured HK-2 cells expressed and secreted early biomarkers of tubular damage such as kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. In conclusion, changes in urinary FSS should now also be considered as potential insults for tubular cells that initiate/perpetuate interstitial inflammation.

Performance of different separation methods interfaced in the same MS-reflection TOF detector: a comparison of performance between CE versus HPLC for biomarker analysis.

One of the aims in the field of proteomics is the identification of a protein or polypeptide, or a range of these compounds, that could provide pre-symptomatic indication of the onset of a disease. A number of analytical techniques have been employed to try and achieve this end. These techniques have been applied to the complete range of body fluids and tissues that are readily available from clinical studies. Of these sample sources, the urinary low molecular weight peptidome has been shown to reflect changes in the health status of the individual. The alterations that occur in the polypeptide make up of urine, which reflect changes in biological status, are known as biomarkers. To be able to determine these changes no single technique has emerged that can cope with detecting the large number of peptides present and quantifying them over the wide concentration range they exist in. In this investigation, we made use of a single reflectron time of flight (RTOF)-MS analyser to which we first connected a CE system and then a nanoflow HPLC. Two pooled male and female standard urine samples were compared on these systems. Both techniques had similar results in terms of number of peptides detected and the mass range the peptides were detected over. The major differences in terms of biomarker research were the ability in CE to calibrate the migration time of the peptides to allow comparison between samples. In addition, CE was shown not to suffer from carry over from previous samples as was seen in the LC analysis.

Lysophosphatidic acid-1-receptor targeting agents for fibrosis.

INTRODUCTION: The presence of fibrosis is associated with alterations in organ architecture and is responsible for the morbidity of diseases including pneumopathies, systemic sclerosis, liver cirrhosis, chronic cardiovascular diseases, progressive kidney diseases and diabetes. Although a growing number of pro-fibrotic molecules, mediators and other pathways have been reported, there are currently very few antifibrotic molecules being evaluated in clinical trials. AREAS COVERED: Current knowledge about the contribution of lysophosphatidic acid (LPA), a bioactive mediator acting via specific G-protein coupled receptors (LPAR), in the etiology of fibrosis. In a number of organs, fibrosis is associated with increased LPA production as well as with increased expression of some LPAR subtypes (mainly LPA1R). LPAR(-/-) knockout mice and treatment of animal models with specific antagonists clearly demonstrate the contribution of LPA1R subtype to the development of kidney, lung, vascular and dermal fibrosis. The involvement of LPA in liver fibrosis is also strongly suspected but still unproven. EXPERT OPINION: Experiments in animal models clearly demonstrate that LPA1R antagonists have interesting anti-fibrotic potencies. This reveals promising perspectives for the design of new therapeutic approaches to prevent fibrosis-associated diseases. Nevertheless, the number of efficient LPA1R antagonists currently available is still low, and none of them has been used in clinical trials so far.

Renal tubular fluid shear stress promotes endothelial cell activation.

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. In this study, we hypothesized that changes in urinary FSS represent a tubular aggression that contributes to the development of inflammation, a key event in progression of nephropathies. Human renal tubular cells (HK-2) were exposed to FSS for 30 min at 0.01 Pa. Treatment of human endothelial cells (HMEC-1) with the resulting conditioned medium (FSS-CM) increased C-C chemokine ligand 2 (CCL2) and tumor necrosis factor (TNF)-α protein secretion, increased endothelial vascular adhesion molecule-1 (VCAM-1) mRNA expression and stimulated adhesion of human (THP-1) monocytes to the endothelial monolayer. These effects were TNF-α dependent as they were abolished by neutralization of TNF-α. Interestingly, the origin of TNF-α was not epithelial, but resulted from autocrine endothelial production. However, in contrast to short term FSS, long term FSS (5h) induced the release of the key inflammatory proteins CCL2 and TNF-α directly from tubular cells. In conclusion, these results suggest for the first time that urinary FSS can contribute to the inflammatory state involved in initiation/perpetuation of renal diseases.

Congenital ureteropelvic junction obstruction: human disease and animal models.

Ureteropelvic junction (UPJ) obstruction is the most frequently observed cause of obstructive nephropathy in children. Neonatal and foetal animal models have been developed that mimic closely what is observed in human disease. The purpose of this review is to discuss how obstructive nephropathy alters kidney histology and function and describe the molecular mechanisms involved in the progression of the lesions, including inflammation, proliferation/apoptosis, renin-angiotensin system activation and fibrosis, based on both human and animal data. Also we propose that during obstructive nephropathy, hydrodynamic modifications are early inducers of the tubular lesions, which are potentially at the origin of the pathology. Finally, an important observation in animal models is that relief of obstruction during kidney development has important effects on renal function later in adult life. A major short-coming is the absence of data on the impact of UPJ obstruction on long-term adult renal function to elucidate whether these animal data are also valid in humans.

Blockade of the kinin B1 receptor ameloriates glomerulonephritis.

Severe inflammation characterizes rapidly progressive glomerulonephritides, and expression of the kinin B1 receptor (B1R) associates with inflammation. Delayed B1R blockade reduces renal inflammation in a model of unilateral ureteral obstruction, but whether B1R modulates the pathophysiology of glomerulonephritides is unknown. Here, we observed an association of B1R protein expression and inflammation, in both glomeruli and the renal interstitium, in biopsies of patients with glomerulonephritides, Henoch-Schönlein purpura nephropathy, and ANCA-associated vasculitis. In the nephrotoxic serum-induced glomerulonephritis model, we observed upregulation of the B1R receptor; treatment with a B1R antagonist beginning 2 weeks after the onset of disease reduced both glomerular and tubular lesions and improved renal function. B1R blockade reduced renal chemokine expression and macrophage accumulation. Collectively, our data demonstrate that blockade of the kinin B1R has significant potential for the treatment of glomerulonephritis.

Delayed treatment with plasminogen activator inhibitor-1 decoys reduces tubulointerstitial fibrosis.

We examined the capacity of delayed inhibition of plasminogen activator inhibitor-1 (PAI-1) to reduce tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO) in mice. Small peptides mimicking parts of urokinase (uPA) and tissular plasminogen activator (tPA) and serving as decoy molecules for PAI-1 were administered daily during the late stages (3 to 8 days) of UUO. Treatment with PAI-1 decoys reduced interstitial deposition of fibronectin, collagen III and collagen IV without changes in macrophage and myofibroblast infiltration. Interestingly, while PAI-1 activity was reduced and the combined uPA and tPA activity was increased, the antifibrotic effect was obtained without modification of plasmin activity but with increased of hepatocyte growth factor (HGF) expression. We show for the first time that treatment with small PAI-1 decoy peptides reduces established tubulointerstitial fibrosis. This protective effect probably resulted from increased degradation of the extracellular matrix by an HGF dependent mechanism.

Critical role of tissue kallikrein in vessel formation and maturation: implications for therapeutic revascularization.

OBJECTIVE: Human Tissue Kallikrein (hKLK1) overexpression promotes an enduring neovascularization of ischemic tissue, yet the cellular mechanisms of hKLK1-induced arteriogenesis remain unknown. Furthermore, no previous study has compared the angiogenic potency of hKLK1, with its loss of function polymorphic variant, rs5515 (R53H), which possesses reduced kinin-forming activity. METHODS AND RESULTS: Here, we demonstrate that tissue kallikrein knockout mice (KLK1-/-) show impaired muscle neovascularization in response to hindlimb ischemia. Gene-transfer of wild-type Ad.hKLK1 but not Ad.R53H-hKLK1 was able to rescue this defect. Similarly, in the rat mesenteric assay, Ad.hKLK1 induced a mature neovasculature with increased vessel diameter through kinin-B2 receptor-mediated recruitment of pericytes and vascular smooth muscle cells, whereas Ad.R53H-hKLK1 was ineffective. Moreover, hKLK1 but not R53H-hKLK1 overexpression in the zebrafish induced endothelial precursor cell migration and vascular remodeling. Furthermore, Ad.hKLK1 activates metalloproteinase (MMP) activity in normoperfused muscle and fails to promote reparative neovascularization in ischemic MMP9-/- mice, whereas its proarteriogenic action was preserved in ApoE-/- mice, an atherosclerotic model of impaired angiogenesis. CONCLUSIONS: These results demonstrate the fundamental role of endogenous Tissue Kallikrein in vascular repair and provide novel information on the cellular and molecular mechanisms responsible for the robust arterialization induced by hKLK1 overexpression.

Lysophosphatidic acid and renal fibrosis.

The development of fibrosis involves a multitude of events and molecules. Until now the majority of these molecules were found to be proteins or peptides. But recent data show significant involvement of the phospholipid lysophosphatidic acid (LPA) in the development of pulmonary, liver and renal fibrosis. The latest data on the role of LPA and the G-protein-coupled LPA1 receptor in the development of renal fibrosis will be discussed. LPA1-receptor activation was found to be associated with increased vascular leakage and increased fibroblast recruitment in pulmonary fibrosis. Furthermore, in renal fibrosis LPA1-receptor activation stimulates macrophage recruitment and connective tissue growth factor expression. The observations make this receptor an interesting alternative and new therapeutic target in fibrotic diseases.