Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

Tubular reabsorption of filtered sodium is quantitatively the main contribution of kidneys to salt and water homeostasis. The transcellular reabsorption of sodium proceeds by a two-step mechanism: Na(+)-K(+)-ATPase-energized basolateral active extrusion of sodium permits passive apical entry through various sodium transport systems. In the past 15 years, most of the renal sodium transport systems (Na(+)-K(+)-ATPase, channels, cotransporters, and exchangers) have been characterized at a molecular level. Coupled to the methods developed during the 1965-1985 decades to circumvent kidney heterogeneity and analyze sodium transport at the level of single nephron segments, cloning of the transporters allowed us to move our understanding of hormone regulation of sodium transport from a cellular to a molecular level. The main purpose of this review is to analyze how molecular events at the transporter level account for the physiological changes in tubular handling of sodium promoted by hormones. In recent years, it also became obvious that intracellular signaling pathways interacted with each other, leading to synergisms or antagonisms. A second aim of this review is therefore to analyze the integrated network of signaling pathways underlying hormone action. Given the central role of Na(+)-K(+)-ATPase in sodium reabsorption, the first part of this review focuses on its structural and functional properties, with a special mention of the specificity of Na(+)-K(+)-ATPase expressed in renal tubule. In a second part, the general mechanisms of hormone signaling are briefly introduced before a more detailed discussion of the nephron segment-specific expression of hormone receptors and signaling pathways. The three following parts integrate the molecular and physiological aspects of the hormonal regulation of sodium transport processes in three nephron segments: the proximal tubule, the thick ascending limb of Henle's loop, and the collecting duct.

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.

Pseudohypoaldosteronism type II is a salt-sensitive form of hypertension with hyperkalemia in humans caused by mutations in the with-no-lysine kinase 4 (WNK4). Several studies have shown that WNK4 modulates the activity of the renal Na + Cl − cotransporter, NCC. Because the renal consequences of WNK4 carrying pseudoaldosteronism type II mutations resemble the response to intravascular volume depletion (promotion of salt reabsorption without K + secretion), a condition that is associated with high angiotensin II (AngII) levels, it has been proposed that AngII signaling might affect WNK4 modulation of the NCC. In Xenopus laevis oocytes, WNK4 is required for modulation of NCC activity by AngII. To demonstrate that WNK4 is required in the AngII-mediated regulation of NCC in vivo, we used a total WNK4-knockout mouse strain (WNK4 −/− ). WNK4 mRNA and protein expression were absent in WNK4 −/− mice, which exhibited a mild Gitelman-like syndrome, with normal blood pressure, increased plasma renin activity, and reduced NCC expression and phosphorylation at T-58. Immunohistochemistry revealed normal morphology of the distal convoluted tubule with reduced NCC expression. Low-salt diet or infusion of AngII for 4 d induced phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and of NCC at S-383 and T-58, respectively, in WNK4 +/+ but not WNK4 −/− mice. Thus, the absence of WNK4 in vivo precludes NCC and SPAK phosphorylation promoted by a low-salt diet or AngII infusion, suggesting that AngII action on the NCC occurs via a WNK4-SPAK-dependent signaling pathway. Additionally, stimulation of aldosterone secretion by AngII, but not by a high-K + diet, was impaired in WNK4 −/− mice. distal tubule | diuretics | thiazide | renin-angiotensin-aldosterone system

Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloridesensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na + and Cl -in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na + -driven Cl -/HCO 3 -exchanger (NDCBE/SLC4A8) and the Na + -independent Cl -/HCO 3 -exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na + reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.

IntroductionAcute inflammation is the host response to tissue injury or infection that is characterized by the production of inflammatory mediators, culminating in the initial but transient recruitment of polymorphonuclear leukocytes (PMNs) that is followed by a prolonged macrophage accumulation. 1 However, the underlying, multifactorial mechanisms that shape the extent and kinetics of PMN and macrophage recruitment, apoptosis, and clearance remain unclear. Chemokines are an important class of chemoattractant cytokines produced locally in tissues that provide the directional cues for the movement of blood-derived leukocytes in development, homeostasis, and inflammation. 2 These potent chemoattractants are classified according to the position and spacing of their N-terminal cysteine residues, with 46 human chemokines currently divided into 4 families: C, CC, CXC, and CX 3 C. 3 The initial phase of inflammation involves a subset of CXC chemokines, which rapidly attract PMNs. 4 These PMN chemoattractants contain a conserved Glu-Leu-Arg (ELR) motif proximal to the CXC sequence, which is critical in cognate receptor binding and activation, 5 as well as PMN chemotaxis. 6 In humans, there are 7 ELR ϩ CXC chemokines: CXCL1, -2, and -3, also known as growth-related oncogenes ␣, ␤, and ␥, respectively; CXCL5/epithelial cell-derived neutrophil activating peptide-78 (ENA-78); CXCL6/granulocyte chemotactic protein-2 (GCP-2); CXCL7/neutrophil-activating peptide-2 (NAP-2); and CXCL8/interleukin-8 (IL-8).All bind the CXC-receptor (CXCR) 1; CXCL6 and -8 also signal through CXCR2. 7 Mice lack complete homologs of the 7 human ELR ϩ chemokines, having only 4: mCXCL1/ keratinocyte-derived chemokine (KC); mCXCL2/macrophage-inflammatory protein-2 (MIP-2); the more recently described mCXCL3/ dendritic cell inflammatory protein-1 (DCIP-1) 8 ; and mCXCL5/ lipopolysaccharide (LPS)-induced CXC chemokine (LIX). 9 All bind a single receptor that is homologous to human CXCR2. 10 After the initial PMN influx, the next stage of inflammation is directed in part by CC chemokines consisting of CCL2/monocyte chemoattractant protein (MCP)-1, CCL7/MCP-3, CCL8/MCP-2, and CCL13/MCP-4, which target multiple leukocyte subsets (monocytes, T lymphocytes, basophils, and eosinophils). 11 Monocytes can differentiate into macrophages whose role is to phagocytose and degrade microorganisms and foreign material and to present these antigens to initiate specific immune responses. In addition, macrophages ingest apoptotic PMNs from the inflamed site as a prelude to tissue resolution. 12 However, apoptosis alone cannot account for the entirety of the reduction in PMN numbers, because the potential for continued recruitment and replacement.Resident mast cells, macrophages, and epithelial cells have been proposed to produce the initial signals responsible for the accumulation of PMNs, eosinophils, and mononuclear cells in experimental models of inflammation by secretion of chemokines such as mCXCL1 and mCCL3. 13 Modulation of specific ELR ϩ chemokines by proteolytic ...

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To gain a molecular understanding of kidney functions, we established a high-resolution map of gene expression patterns in the human kidney. The glomerulus and seven different nephron segments were isolated by microdissection from fresh tissue specimens, and their transcriptome was characterized by using the serial analysis of gene expression (SAGE) method. More than 400,000 mRNA SAGE tags were sequenced, making it possible to detect in each structure transcripts present at 18 copies per cell with a 95% confidence level. Expression of genes responsible for nephron transport and permeability properties was evidenced through transcripts for 119 solute carriers, 84 channels, 43 ion-transport ATPases, and 12 claudins. Searching for differences between the transcriptomes, we found 998 transcripts greatly varying in abundance from one nephron portion to another. Clustering analysis of these transcripts evidenced different extents of similarity between the nephron portions. Approximately 75% of the differentially distributed transcripts corresponded to cDNAs of known or unknown function that are accurately mapped in the human genome. This systematic large-scale analysis of individual structures of a complex human tissue reveals sets of genes underlying the function of well-defined nephron portions. It also provides quantitative expression data for a variety of genes mutated in hereditary diseases and helps in sorting candidate genes for renal diseases that affect specific portions of the human nephron.