Minimum active structure of insulin-like peptide 5.

Insulin-like peptide 5 (INSL5) is a complex two-chain peptide hormone constrained by three disulfide bonds in a pattern identical to insulin. High expression of INSL5 in the colon suggests roles in activation of colon motility and appetite control. A more recent study indicates it may have significant roles in the regulation of insulin secretion and ß-cell homeostasis. This peptide thus has considerable potential for the treatment of eating disorders, obesity, and/or diabetes. However, the synthesis of INSL5 is extremely challenging either by chemical or recombinant means. The A-chain is very poorly soluble and the B-chain is highly aggregating in nature which, together, makes their postsynthesis handling and purification very difficult. Given these difficulties, we have developed a highly active INSL5 analogue that has a much simpler structure with two disulfide bonds and is thus easier to assemble compared to native INSL5. This minimized peptide represents an attractive new mimetic for investigating the functional role of INSL5.

Research performance evaluation: the experience of an independent medical research institute.

BACKGROUND: Evaluation of the social and economic outcomes of health research funding is an area of intense interest and debate. Typically, approaches have sought to assess the impact of research funding by medical charities or regional government bodies. Independent research institutes have a similar need for accountability in investment decisions but have different objectives and funding, thus the existing approaches are not appropriate. METHODS: An evaluation methodology using eight indicators was developed to assess research performance across three broad categories: knowledge creation; inputs to research; and commercial, clinical and public health outcomes. The evaluation approach was designed to provide a balanced assessment across laboratory, clinical and public health research. RESULTS AND DISCUSSION: With a diverse research agenda supported by a large number of researchers, the Research Performance Evaluation process at the Murdoch Childrens Research Institute has, by necessity, been iterative and responsive to the needs of the Institute and its staff. Since its inception 5 years ago, data collection systems have been refined, the methodology has been adjusted to capture appropriate data, staff awareness and participation has increased, and issues regarding the methodology and scoring have been resolved. CONCLUSIONS: The Research Performance Evaluation methodology described here provides a fair and transparent means of disbursing internal funding. It is also a powerful tool for evaluating the Institute's progress towards achieving its strategic goals, and is therefore a key driver for research excellence.

Structure and function relationship of murine insulin-like peptide 5 (INSL5): free C-terminus is essential for RXFP4 receptor binding and activation.

Insulin-like peptide 5 (INSL5) is a member of insulin/relaxin superfamily of peptides. It has recently been identified as the cognate ligand for the G-protein-coupled receptor, RXFP4. Although the complete physiological role of this naturally occurring peptide is still under investigation, there is evidence that it acts to both stimulate appetite and activate colon motility. This suggests that both agonists and antagonists of the peptide may have potential therapeutic applications. To further investigate the physiological role of this peptide and because of the ready availability of the mouse as an experimental animal, the preparation of mouse INSL5 was undertaken. Because of its complex structure and the intractable nature of the two constituent chains, different solid phase synthesis strategies were investigated, including the use of a temporary B-chain solubilizing tag. Unfortunately, none provided significantly improved yield of purified mouse INSL5 which reflects the complexity of this peptide. In addition to the native peptide, two mouse INSL5 analogues were also prepared. One had its two chains as C-terminal amides, and the other contained a europium chelate monolabel for use in RXFP4 receptor assays. It was found that the INSL5 amide was substantially less potent than the native acid form. A similar observation was made for the human peptide acid and amide, highlighting the necessity for free C-terminal carboxylates for function. Two additional human INSL5 analogues were prepared to further investigate the necessity of a free C-terminal. The results together provide a first insight into the mechanism whereby INSL5 binds to and activates RXFP4.

Relaxin remodels fibrotic healing following myocardial infarction.

In the setting of myocardial infarction (MI), implanted stem cell viability is low and scar formation limits stem cell homing, viability, and integration. Thus, interventions that favorably remodel fibrotic healing may benefit stem cell therapies. However, it remains unclear whether it is feasible and safe to remodel fibrotic healing post-MI without compromising ventricular remodeling and dysfunction. This study, therefore, determined the anti-fibrotic and other effects of the hormone, relaxin in a mouse model of MI. Adult male mice underwent left coronary artery ligation-induced MI and were immediately treated with recombinant human relaxin (MI+RLX) or vehicle (MI+VEH) over 7 or 30 days, representing time points of early and mature fibrotic healing. Cardiac function was assessed by echocardiography and catheterization, while comprehensive immunohistochemistry, morphometry, and western blotting were performed to explore the relaxin-induced mechanisms of action post-MI. RLX significantly inhibited the MI-induced progression of cardiac fibrosis over 7 and 30 days, which was associated with a reduction in TGF-ß1 expression, myofibroblast differentiation, and cardiomyocyte apoptosis in addition to a promotion of matrix metalloproteinase-13 levels and de novo blood vessel growth (all P<0.05 vs respective measurements from MI+VEH mice). Despite the evident fibrotic healing post-MI, relaxin did not adversely affect the incidence of ventricular free-wall rupture or the extent of LV remodeling and dysfunction. These combined findings demonstrate that RLX favorably remodels the process of fibrotic healing post-infarction by lowering the density of mature scar tissue in the infarcted myocardium, border zone, and non-infarcted myocardium, and may, therefore, facilitate cell-based therapies in the setting of ischemic heart disease.

The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist.

Relaxin is a pleiotropic hormone which exerts its biological functions through its G-protein coupled receptor, RXFP1. While relaxin is well known for its reproductive and antifibrotic roles, recent studies suggest that it is produced by cancer cells and acts on RXFP1 to induce growth and metastasis. Furthermore, more recently Silvertown et al. demonstrated that lentiviral production of a human gene-2 (H2) relaxin analog reduced the growth of prostate xenograft tumors. The authors proposed that the lentivirally produced peptide was an RXFP1 antagonist; however, the processed form of the peptide produced was not demonstrated. In this study, we have chemically synthesized the H2 relaxin analog, B-R13/17K H2 relaxin, and subjected it to detailed chemical characterization by HPLC, MALDI-TOF mass spectrometry, and amino acid analysis. The biological activity of the synthetic peptide was then tested in three different cell lines. It was found to bind with 500-fold lower affinity than H2 relaxin to RXFP1 receptors over-expressed in HEK-293T cells where it acted as a partial agonist. However, in cells which natively express the RXFP1 receptor, rat renal myofibroblasts and MCF-7 cancer cells, it acted as a full antagonist. Importantly, it was able to significantly inhibit cell invasion induced by H2 relaxin in MCF-7 cells consistent with the results of the lentiviral-driven expression in prostate cancer cells. The relaxin analog, B-R13/17K H2, can now be used as a tool to further understand RXFP1 function, and serve as a template for drug design for a therapeutic to treat prostate and other cancers.

Effect of helix-promoting strategies on the biological activity of novel analogues of the B-chain of INSL3.

Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central alpha-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the alpha-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher alpha-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.

Use of a temporary "solubilizing" peptide tag for the Fmoc solid-phase synthesis of human insulin glargine via use of regioselective disulfide bond formation.

Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However, the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains--including the notoriously poorly soluble A-chain--followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.

Structure and activity in the relaxin family of peptides.

The availability of improved peptide synthesis procedures, convenient and sensitive assays for receptor binding and activation, together with advances in methods for structural characterization, has enabled the key structural features of the relaxin family of peptides responsible for biological activity to be defined. Not surprisingly, despite the similarities in primary amino acid sequences, different structural domains and residues are involved in the binding and activation at the four known relaxin family peptide receptors (RXFP1 to -4). Most of our knowledge on structure and function relates to the relaxin-RXFP1, insulin-like peptide 3 (INSL3)-RXFP2, and relaxin-3-RXFP3 systems, with information accumulating not only on the critical ligand structures but also the domains and residues on the receptor itself that are required for specificity and activation. These studies provide the framework for the design of small-molecule mimetics. While the B-chain cassette R-X-X-X-R-X-X-I, defined by Büllesbach and Schwabe, is essential for binding and activation of RXFP1, it is now recognized that the A chain, particularly the N-terminal domain, is also critical for receptor specificity. Studies of the various endogenous ligand-receptor pairs have led to the design of potent and specific agonists and antagonists. The relaxin-3 A chain-INSL5 B chain chimeric peptide and analogs with C-terminal truncations of the B chain, developed by Liu and colleagues at Johnson & Johnson, have provided selective agonist and antagonist peptides that are proving invaluable for in vivo studies of the relaxin-3-RXFP3 system.

The chemical synthesis of relaxin and related peptides.

Successful methods for the chemical assembly of insulin-like peptides allow the detailed study of their structure and function relationships. However, the two-chain, three-disulfide bond structure of this family of peptides, which includes relaxin, has long represented a significant challenge with respect to their chemical synthesis. Early efforts involved the random combination of the two synthetic S-reduced chains under oxidizing conditions to spontaneously form the three disulfide bonds. Such an approach, while generally effective for native sequences, is critically dependent upon the presence of intact secondary structures within the individual chains which guide the subsequent folding and oxidation pathway. This limitation prevents the use of this approach for the preparation of analogs in which these secondary elements are either absent or modified. Nowadays, the use of highly efficient solid-phase peptide synthesis methodologies together with selective S-thiol-protecting groups allows the acquisition of individual chains that can be combined by effective sequential chemically directed formation of each of the three disulfide bonds. These approaches have allowed the high-yield assembly of an array of insulin-like peptides which, in turn, has provided considerable and valuable structural and biological information.

Modeling the primary hormone-binding site of RXFP1 and RXFP2.

The primary binding sites of the relaxin and insulin-like peptide 3 (INSL3) receptors, RXFP1 and RXFP2, are found within the leucine-rich repeats (LRRs) of the ectodomains. Specific B-chain residues in the peptides interact with residues in the inner beta-sheets of the LRRs of the receptors. Relaxin binds to RXFP2 with high affinity, although INSL3 has a very poor affinity for RXFP1. In this paper we present evidence that relaxin binds to the LRRs of RXFP2 in a manner similar to INSL3 binding to its receptor. Additionally, we introduce a model of this binding interaction and compare it to an alternate model for relaxin-RXFP1 binding.

Relaxin family peptides and receptors in mammalian brain.

As a foundation for regulatory and functional studies of central relaxin family peptide receptor systems, we are mapping the distribution of the different receptors in the brain of rat, mouse, and nonhuman primates, attempting to identify the nature of the receptor-positive neurons in key circuits and establish the complementary distribution of the respective ligands in these species. Here we review progress in mapping RXFP1, RXFP2, and RXFP3 (mRNAs and proteins) and their respective ligands and discuss some of the putative functions for these peptides and receptors that are being explored using receptor-selective agonist and antagonist peptides and receptor and peptide gene deletion mouse strains. Comparative studies reveal an association of RXFP1 and RXFP2 with excitatory neurons but a differential regional or cellular distribution, in contrast to the association of RXFP3 with inhibitory neurons. These studies also reveal differences in the distribution of RXFP1 and RXFP2 in rat and mouse brain, whereas the distribution of RXFP3 is more conserved across these species. Enrichment of RXFP1/2/3 in olfactory, cortical, thalamic, limbic, hypothalamic, midbrain, and pontine circuits suggests a diverse range of modulatory actions for these receptors. For example, experimental evidence in the rat reveals that RXFP1 activation in the amygdala inhibits memory consolidation, RXFP2 activation in striatum produces sniffing behavior, and RXFP3 modulation has effects on feeding and metabolism, the activity of the septohippocampal pathway, and spatial memory. Further studies are now required to reveal additional details of these and other functions linked to relaxin family peptide receptor signaling in mammalian brain and the precise mechanisms involved.

Reversal of cardiac fibrosis and related dysfunction by relaxin.

As a hallmark of heart disease, cardiac fibrosis contributes to the development of heart failure and arrhythmias and forms a key therapeutic target. There is a major unmet need for selective, potent, and safe antifibrotic drugs. Earlier studies revealed a cardiac fibrosis phenotype in relaxin-1-deficient mice. Recent studies in several rodent models of cardiac fibrosis have documented reversal of fibrosis by treatment with relaxin peptide or virally mediated relaxin gene delivery. In mice with surgically induced transmural myocardial infarction, relaxin therapy inhibited scar density. In these studies, however, functional benefits achieved by relaxin therapy were limited or less explored. Collectively, there is good experimental evidence that relaxin is able to reverse cardiac fibrosis due to distinct mechanisms. Future research needs to explore functional improvement following fibrosis reversal by relaxin and the usefulness of relaxin in antiarrhythmic or stem cell-based therapy.

Investigations into the inhibitory effects of relaxin on renal myofibroblast differentiation.

Derived from fibroblasts, myofibroblasts are the principal cells that are responsible for the synthesis and reorganization of excess matrix in renal interstitial fibrosis. Recognized from their de novo expression of alpha-smooth muscle actin, myofibroblast differentiation and activity can be influenced by several factors, including a combination of growth factors and other soluble mediators, extracellular matrix components, and mechanical stress. Relaxin has previously been shown to inhibit renal myofibroblast differentiation in vitro, an effect partly mediated through its ability to interfere with the transforming growth factor-beta1 (TGF-beta1) pathway via inhibition of Smad2 phosphorylation and translocation. Furthermore, endogenous relaxin has been shown to protect the kidney from a myofibroblast-mediated model of injury in vivo. However, the pathways involved in the interaction between relaxin and TGF-beta1 remain unknown. In this report, the inhibitory actions of relaxin on TGF-beta1-induced renal myofibroblast differentiation are summarized to date, and the potential signaling pathways that are implicated in relaxin's inhibitory actions are discussed.

Evaluation of relaxin's antifibrotic action by SELDI-TOF mass spectrometry-based profiling of relaxin knockout mice, a model of progressive fibrosis.

Surface-enhanced laser desorption ionization-time-of-flight mass spectrometry was employed to identify potential biomarkers for early-onset fibrosis. These biomarkers were then used to evaluate the efficacy of relaxin to reverse or ameliorate the development of the condition.

Solid phase synthesis and structural analysis of novel A-chain dicarba analogs of human relaxin-3 (INSL7) that exhibit full biological activity.

Replacement of disulfide bonds with non-reducible isosteres can be a useful means of increasing the in vivo stability of a protein. We describe the replacement of the A-chain intramolecular disulfide bond of human relaxin-3 (H3 relaxin, INSL7), an insulin-like peptide that has potential applications in the treatment of stress and obesity, with the physiologically stable dicarba bond. Solid phase peptide synthesis was used to prepare an A-chain analogue in which the two cysteine residues that form the intramolecular bond were replaced with allylglycine. On-resin microwave-mediated ring closing metathesis was then employed to generate the dicarba bridge. Subsequent cleavage of the peptide from the solid support, purification of two isomers and their combination with the B-chain via two intermolecular disulfide bonds, then furnished two isomers of dicarba-H3 relaxin. These were characterized by CD spectroscopy, which suggested a structural similarity to the native peptide. Additional analysis by solution NMR spectroscopy also identified the likely cis/trans form of the analogs. Both peptides demonstrated binding affinities that were equivalent to native H3 relaxin on RXFP1 and RXFP3 expressing cells. However, although the cAMP activity of the analogs on RXFP3 expressing cells was similar to the native peptide, the potency on RXFP1 expressing cells was slightly lower. The data confirmed the use of a dicarba bond as a useful isosteric replacement of the disulfide bond.

Relaxin reverses airway remodeling and airway dysfunction in allergic airways disease.

Mice deficient in the antifibrotic hormone relaxin develop structural changes in the airway that resemble airway remodeling, and demonstrate exaggerated remodeling changes in models of allergic airways disease (AAD). Relaxin expression in asthma has not been previously studied. We evaluated the efficacy of relaxin in the treatment of established airway remodeling in a mouse model of AAD. Relaxin expression in mouse AAD was also examined by immunohistochemistry and real-time PCR. BALB/c mice with established AAD were treated with relaxin or vehicle control (sc for 14 d), and effects on airway remodeling, airway inflammation, and airway hyperresponsiveness (AHR) were assessed. Relaxin expression was significantly reduced in the airways of mice with AAD compared with controls. Recombinant relaxin treatment in a mouse model of AAD reversed collagen deposition and epithelial thickening, and significantly improved AHR (all P < 0.05 vs. vehicle control), but did not influence airway inflammation or goblet cell hyperplasia. Relaxin treatment was associated with increased matrix metalloproteinase-2 levels, suggesting a possible mechanism for its antifibrotic effects. Endogenous relaxin expression is decreased in murine AAD, whereas exogenous relaxin represents a novel treatment capable of reversing established airway remodeling and AHR.

The structural and functional role of the B-chain C-terminal arginine in the relaxin-3 peptide antagonist, R3(BDelta23-27)R/I5.

Relaxin-3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin-3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin-3/RXFP3 pair. The analog R3(BDelta23-27)R/I5, in which a C-terminally truncated human relaxin-3 (H3) B-chain is combined with the INSL5 A-chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B-chain C-terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(BDelta23-27)R/I5 and R3(BDelta23-27)R containing the B-chain C-terminal Arg as well as R3(BDelta23-27)/I5 and R3(BDelta23-27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(BDelta23-27)R and R3(BDelta23-27)R/I5, the peptide R3(BDelta23-27) is a weak agonist. This suggests that the C-terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(BDelta23-27)R/I5 its potent antagonistic activity.

Relaxin family peptide receptor-1 protects against airway fibrosis during homeostasis but not against fibrosis associated with chronic allergic airways disease.

Endogenous relaxin has recently been demonstrated to protect the airway/lung against age-related fibrosis and against inflammation-associated airway fibrosis in animal models of allergic airways disease (AAD). In the current study, we examined the contribution of the primary relaxin receptor, relaxin family peptide receptor-1 (RXFP1), in mediating these effects of relaxin. Lung tissues from healthy aging RXFP1 gene-knockout (Rxfp1(-/-)) and wild-type (Rxfp1(+/+)) mice and from 8- to 10-wk-old Rxfp1(-/-) and Rxfp1(+/+) mice subjected to a mouse model of AAD were assessed for various markers of airway fibrosis and remodeling. Male and female Rxfp1(-/-) mice demonstrated an age-related progression of airway/lung fibrosis. Saline-treated Rxfp1(-/-) mice had significantly increased myofibroblast differentiation and lung collagen deposition (both P < 0.05), decreased matrix metalloproteinase (MMP)-9 expression and activity (P < 0.05), but equivalent levels of MMP-2 and tissue inhibitor of metalloproteinases (TIMPs) to that measured in saline-treated Rxfp1(+/+) mice. As expected, ovalbumin (OVA)-treated Rxfp1(+/+) mice developed markedly increased lung myofibroblast differentiation and collagen deposition (both P < 0.01 vs saline-treated Rxfp1(+/+) mice), significantly decreased lung MMP-2 and MMP-9 expression and activity and increased TIMP-1 expression (all P < 0.05 vs. respective measurements from saline-treated Rxfp1(+/+) mice). Surprisingly, however, OVA-treated Rxfp1(-/-) animals had equivalent levels of airway fibrosis and gelatinase activity but increased TIMP-1 expression (P < 0.05) compared with OVA-treated Rxfp1(+/+) mice. These combined findings demonstrate that RXFP1 is involved in mediating relaxin's effects on airway fibrosis during homeostasis but not during inflammation-induced fibrosis associated with chronic AAD.

Relaxin inhibits renal myofibroblast differentiation via RXFP1, the nitric oxide pathway, and Smad2.

The hormone relaxin inhibits renal myofibroblast differentiation by interfering with TGF-beta1/Smad2 signaling. However, the pathways involved in the relaxin-TGF-beta1/Smad2 interaction remain unknown. This study investigated the signaling mechanisms by which human gene-2 (H2) relaxin regulates myofibroblast differentiation in vitro by examining its effects on mixed populations of fibroblasts and myofibroblasts propagated from injured rat kidneys. Cultures containing approximately 60-70% myofibroblasts were used to determine which relaxin receptors, G-proteins, and signaling pathways were involved in the H2 relaxin-mediated regulation of alpha-smooth muscle actin (alpha-SMA; a marker of myofibroblast differentiation). H2 relaxin only inhibited alpha-SMA immunostaining and collagen concentration in the presence of relaxin family peptide receptor 1 (RXFP1). H2 relaxin also induced a transient rise in cAMP in the presence of G(i/o) inhibition, and a sustained increase in extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. Furthermore, inhibition of neuronal nitric oxide synthase (nNOS), NO, and cGMP significantly blocked the inhibitory effects of relaxin on alpha-SMA and Smad2 phosphorylation, while the NO inhibitor, L-nitroarginine methyl ester (hydrochloride) (L-NAME) significantly blocked the inhibitory actions of relaxin on collagen concentration in vivo. These findings suggest that relaxin signals through RXFP1, and a nNOS-NO-cGMP-dependent pathway to inhibit Smad2 phosphorylation and interfere with TGF-beta1-mediated renal myofibroblast differentiation and collagen production.

Aberrant protein expression in plasma and kidney tissue during experimental obstructive nephropathy.

Kidney failure is a major health problem worldwide. Patients with end-stage renal disease require intensive medical support by dialysis or kidney transplantation. Current methods for diagnosis of kidney disease are either invasive or insensitive, and renal function may decline by as much as 50% before it can be detected using current techniques. The goal of this study was, therefore, to identify biomarkers of kidney disease (associated with renal fibrosis) that can be used for the development of a non-invasive clinical test for early disease detection. We utilized two protein-profiling technologies (SELDI-TOF MS and 2-D) to screen the plasma and kidney proteome for aberrantly expressed proteins in an experimental mouse model of unilateral uretric obstruction, which mimics the pathology of human renal disease. Several differentially regulated proteins were detected at the plasma level of day-3-obstructed animals, which included serum amyloid A1, fibrinogen α, haptoglobin precursor protein, haptoglobin and major urinary proteins 11 and 8. Differentially expressed proteins detected at the tissue level included ras-like activator protein 2, haptoglobin precursor protein, malate dehydrogenase, α enolase and murine urinary protein (all p<0.05 versus controls). Immunohistochemistry was used to confirm the up-regulation of fibrinogen. Interestingly, these proteins are largely separated into four major classes: (i) acute-phase reactants (ii) cell-signaling molecules (iii) molecules involved in cell growth and metabolism and (iv) urinary proteins. These results provide new insights into the pathology of obstructive nephropathy and may facilitate the development of specific assay(s) to detect and monitor renal fibrosis.