Exploiting open source omics data to advance pancreas research.

The "omics" revolution has transformed the biomedical research landscape by equipping scientists with the ability to interrogate complex biological phenomenon and disease processes at an unprecedented level. The volume of "big" data generated by the different omics studies such as genomics, transcriptomics, proteomics, and metabolomics has led to the concurrent development of computational tools to enable in silico analysis and aid data deconvolution. Considering the intensive resources and high costs required to generate and analyze big data, there has been centralized, collaborative efforts to make the data and analysis tools freely available as "Open Source," to benefit the wider research community. Pancreatology research studies have contributed to this "big data rush" and have additionally benefitted from utilizing the open source data as evidenced by the increasing number of new research findings and publications that stem from such data. In this review, we briefly introduce the evolution of open source omics data, data types, the "FAIR" guiding principles for data management and reuse, and centralized platforms that enable free and fair data accessibility, availability, and provide tools for omics data analysis. We illustrate, through the case study of our own experience in mining pancreatitis omics data, the power of repurposing open source data to answer translationally relevant questions in pancreas research.

Single-Cell Imaging Maps Inflammatory Cell Subsets to Pulmonary Arterial Hypertension Vasculopathy.

Rationale: Unraveling immune-driven vascular pathology in pulmonary arterial hypertension (PAH) requires a comprehensive understanding of the immune cell landscape. Although patients with hereditary (H)PAH and bone morphogenetic protein receptor type 2 (BMPR2) mutations have more severe pulmonary vascular pathology, it is not known whether this is related to specific immune cell subsets. Objectives: This study aims to elucidate immune-driven vascular pathology by identifying immune cell subtypes linked to severity of pulmonary arterial lesions in PAH. Methods: We used cutting-edge multiplexed ion beam imaging by time of flight to compare pulmonary arteries (PAs) and adjacent tissue in PAH lungs (idiopathic [I]PAH and HPAH) with unused donor lungs, as controls. Measurements and Main Results: We quantified immune cells' proximity and abundance, focusing on those features linked to vascular pathology, and evaluated their impact on pulmonary arterial smooth muscle cells (SMCs) and endothelial cells. Distinct immune infiltration patterns emerged between PAH subtypes, with intramural involvement independently linked to PA occlusive changes. Notably, we identified monocyte-derived dendritic cells within PA subendothelial and adventitial regions, influencing vascular remodeling by promoting SMC proliferation and suppressing endothelial gene expression across PAH subtypes. In patients with HPAH, pronounced immune dysregulation encircled PA walls, characterized by heightened perivascular inflammation involving T cell immunoglobulin and mucin domain-3 (TIM-3)+ T cells. This correlated with an expanded DC subset expressing indoleamine 2,3-dioxygenase 1, TIM-3, and SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1, alongside increased neutrophils, SMCs, and alpha-smooth muscle actin (ACTA2)+ endothelial cells, reinforcing the heightened severity of pulmonary vascular lesions. Conclusions: This study presents the first architectural map of PAH lungs, connecting immune subsets not only with specific PA lesions but also with heightened severity in HPAH compared with IPAH. Our findings emphasize the therapeutic potential of targeting monocyte-derived dendritic cells, neutrophils, cellular interactions, and immune responses to alleviate severe vascular pathology in IPAH and HPAH.

A systems approach points to a therapeutic role for retinoids in asparaginase-associated pancreatitis.

Among drug-induced adverse events, pancreatitis is life-threatening and results in substantial morbidity. A prototype example is the pancreatitis caused by asparaginase, a crucial drug used to treat acute lymphoblastic leukemia (ALL). Here, we used a systems approach to identify the factors affecting asparaginase-associated pancreatitis (AAP). Connectivity Map analysis of the transcriptomic data showed that asparaginase-induced gene signatures were potentially reversed by retinoids (vitamin A and its analogs). Analysis of a large electronic health record database (TriNetX) and the U.S. Federal Drug Administration Adverse Events Reporting System demonstrated a reduction in AAP risk with concomitant exposure to vitamin A. Furthermore, we performed a global metabolomic screening of plasma samples from 24 individuals with ALL who developed pancreatitis (cases) and 26 individuals with ALL who did not develop pancreatitis (controls), before and after a single exposure to asparaginase. Screening from this discovery cohort revealed that plasma carotenoids were lower in the cases than in controls. This finding was validated in a larger external cohort. A 30-day dietary recall showed that the cases received less dietary vitamin A than the controls did. In mice, asparaginase administration alone was sufficient to reduce circulating and hepatic retinol. Based on these data, we propose that circulating retinoids protect against pancreatic inflammation and that asparaginase reduces circulating retinoids. Moreover, we show that AAP is more likely to develop with reduced dietary vitamin A intake. The systems approach taken for AAP provides an impetus to examine the role of dietary vitamin A supplementation in preventing or treating AAP.

Impact of acute lymphoblastic leukemia induction therapy: findings from metabolomics on non-fasted plasma samples from a biorepository.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is among the most common cancers in children. With improvements in combination chemotherapy regimens, the overall survival has increased to over 90%. However, the current challenge is to mitigate adverse events resulting from the complex therapy. Several chemotherapies intercept cancer metabolism, but little is known about their collective role in altering host metabolism. OBJECTIVES: We profiled the metabolomic changes in plasma of ALL patients initial- and post- induction therapy. METHODS: We exploited a biorepository of non-fasted plasma samples derived from the Dana Farber Cancer Institute ALL Consortium; these samples were obtained from 50 ALL patients initial- and post-induction therapy. Plasma metabolites and complex lipids were analyzed by high resolution tandem mass spectrometry and differential mobility tandem mass spectrometry. Data were analyzed using a covariate-adjusted regression model with multiplicity adjustment. Pathway enrichment analysis and co-expression network analysis were performed to identify unique clusters of molecules. RESULTS: More than 1200 metabolites and complex lipids were identified in the total of global metabolomics and lipidomics platforms. Over 20% of those molecules were significantly altered. In the pathway enrichment analysis, lipids, particularly phosphatidylethanolamines (PEs), were identified. Network analysis indicated that the bioactive fatty acids, docosahexaenoic acid (DHA)-containing (22:6) triacylglycerols (TAGs), were decreased in the post-induction therapy. CONCLUSION: Metabolomic profiling in ALL patients revealed a large number of alterations following induction chemotherapy. In particular, lipid metabolism was substantially altered. The changes in metabolites and complex lipids following induction therapy could provide insight into the adverse events experienced by ALL patients.

Monocyte-released HERV-K dUTPase engages TLR4 and MCAM causing endothelial mesenchymal transition.

We previously reported heightened expression of the human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of patients with PA hypertension (PAH). Furthermore, recombinant HERV-K dUTPase increased IL-6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase, as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG and proinflammatory molecules IL-6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response-88 to increase IL-6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to p-pSMAD3 required for SNAIL and pSTAT1 for IL-6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor-2 (ATF2) to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated proinflammatory molecules.

iPSC-endothelial cell phenotypic drug screening and in silico analyses identify tyrphostin-AG1296 for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disorder leading to occlusive vascular remodeling. Current PAH therapies improve quality of life but do not reverse structural abnormalities in the pulmonary vasculature. Here, we used high-throughput drug screening combined with in silico analyses of existing transcriptomic datasets to identify a promising lead compound to reverse PAH. Induced pluripotent stem cell-derived endothelial cells generated from six patients with PAH were exposed to 4500 compounds and assayed for improved cell survival after serum withdrawal using a chemiluminescent caspase assay. Subsequent validation of caspase activity and improved angiogenesis combined with data analyses using the Gene Expression Omnibus and Library of Integrated Network-Based Cellular Signatures databases revealed that the lead compound AG1296 was positively associated with an anti-PAH gene signature. AG1296 increased abundance of bone morphogenetic protein receptors, downstream signaling, and gene expression and suppressed PAH smooth muscle cell proliferation. AG1296 induced regression of PA neointimal lesions in lung organ culture and PA occlusive changes in the Sugen/hypoxia rat model and reduced right ventricular systolic pressure. Moreover, AG1296 improved vascular function and BMPR2 signaling and showed better correlation with the anti-PAH gene signature than other tyrosine kinase inhibitors. Specifically, AG1296 up-regulated small mothers against decapentaplegic (SMAD) 1/5 coactivators, cAMP response element-binding protein 3 (CREB3), and CREB5: CREB3 induced inhibitor of DNA binding 1 and downstream genes that improved vascular function. Thus, drug discovery for PAH can be accelerated by combining phenotypic screening with in silico analyses of publicly available datasets.

Smooth Muscle Contact Drives Endothelial Regeneration by BMPR2-Notch1-Mediated Metabolic and Epigenetic Changes.

RATIONALE: Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as atherosclerosis and pulmonary arterial hypertension. OBJECTIVE: We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity. METHODS AND RESULTS: We found that in SMC and EC contact cocultures, BMPR2 (bone morphogenetic protein receptor 2) is required by both cell types to produce collagen IV to activate ILK (integrin-linked kinase). This enzyme directs p-JNK (phospho-c-Jun N-terminal kinase) to the EC membrane, where it stabilizes presenilin1 and releases N1ICD (Notch1 intracellular domain) to promote EC proliferation. This response is necessary for EC regeneration after carotid artery injury. It is deficient in EC-SMC Bmpr2 double heterozygous mice in association with reduced collagen IV production, decreased N1ICD, and attenuated EC proliferation, but can be rescued by targeting N1ICD to EC. Deletion of EC- Notch1 in transgenic mice worsens hypoxia-induced pulmonary hypertension, in association with impaired EC regenerative function associated with loss of precapillary arteries. We further determined that N1ICD maintains EC proliferative capacity by increasing mitochondrial mass and by inducing the phosphofructokinase PFKFB3 (fructose-2,6-bisphosphatase 3). Chromatin immunoprecipitation sequencing analyses showed that PFKFB3 is required for citrate-dependent H3K27 acetylation at enhancer sites of genes regulated by the acetyl transferase p300 and by N1ICD or the N1ICD target MYC and necessary for EC proliferation and homeostasis. CONCLUSIONS: Thus, SMC-EC contact is required for activation of Notch1 by BMPR2, to coordinate metabolism with chromatin remodeling of genes that enable EC regeneration, and to maintain monolayer integrity and vascular homeostasis in response to injury.

Dominant Role for Regulatory T Cells in Protecting Females Against Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension (PH) is a life-threatening condition associated with immune dysregulation and abnormal regulatory T cell (Treg) activity, but it is currently unknown whether and how abnormal Treg function differentially affects males and females. OBJECTIVE: To evaluate whether and how Treg deficiency differentially affects male and female rats in experimental PH. METHODS AND RESULTS: Male and female athymic rnu/rnu rats, lacking Tregs, were treated with the VEGFR2 (vascular endothelial growth factor receptor 2) inhibitor SU5416 or chronic hypoxia and evaluated for PH; some animals underwent Treg immune reconstitution before SU5416 administration. Plasma PGI2 (prostacyclin) levels were measured. Lung and right ventricles were assessed for the expression of the vasoprotective proteins COX-2 (cyclooxygenase 2), PTGIS (prostacyclin synthase), PDL-1 (programmed death ligand 1), and HO-1 (heme oxygenase 1). Inhibitors of these pathways were administered to athymic rats undergoing Treg immune reconstitution. Finally, human cardiac microvascular endothelial cells cocultured with Tregs were evaluated for COX-2, PDL-1, HO-1, and ER (estrogen receptor) expression, and culture supernatants were assayed for PGI2 and IL (interleukin)-10. SU5416-treatment and chronic hypoxia produced more severe PH in female than male athymic rats. Females were distinguished by greater pulmonary inflammation, augmented right ventricular fibrosis, lower plasma PGI2 levels, decreased lung COX-2, PTGIS, HO-1, and PDL-1 expression and reduced right ventricular PDL-1 levels. In both sexes, Treg immune reconstitution protected against PH development and raised levels of plasma PGI2 and cardiopulmonary COX-2, PTGIS, PDL-1, and HO-1. Inhibiting COX-2, HO-1, and PD-1 (programmed death 1)/PDL-1 pathways abrogated Treg protection. In vitro, human Tregs directly upregulated endothelial COX-2, PDL-1, HO-1, ERs and increased supernatant levels of PGI2 and IL-10. CONCLUSIONS: In 2 animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on the normal Treg function to counteract the effects of pulmonary vascular injury leading to PH.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

BACKGROUND: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. METHODS: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. RESULTS: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1ß, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. CONCLUSIONS: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

Increased cerebrospinal fluid osteopontin levels and its involvement in macrophage infiltration in neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system that predominantly affects the optic nerves and spinal cord. Although NMO has long been considered a subtype of multiple sclerosis (MS), the effects of interferon-ß treatment are different between NMO and MS. Recent findings of NMO-IgG suggest that NMO could be a distinct disease rather than a subtype of MS. However, the underlying molecular mechanism of NMO pathology remains poorly understood. METHODS: OPN in the cerebrospinal fluid and brain of patients with NMO and with MS, as well as of patients with other neurologic disease/idiopathic other neurologic disease was examined using Western blotting, ELISA, immunohistochemistry and Boyden chamber. RESULTS: Here we show that osteopontin is significantly increased in the cerebrospinal fluid of NMO patients compared with MS patients. Immunohistochemical analyses revealed that osteopontin was markedly elevated in the cerebral white matter of NMO patients and produced by astrocytes, neurons, and oligodendroglia as well as infiltrating macrophages. We also demonstrate that the interaction of the cerebrospinal fluid osteopontin in NMO patients with integrin αvß3 promoted macrophage chemotaxis by activating phosphoinositide 3-kinase and MEK1/2 signaling pathways. CONCLUSION: These results indicate that osteopontin is involved in NMO pathology. GENERAL SIGNIFICANCE: Thus therapeutic strategies that target osteopontin signaling may be useful to treat NMO.

In situ visualization of a glycoform of transferrin: localization of α2,6-sialylated transferrin in the liver.

We previously found that a lectin, Sambucus sieboldiana agglutinin (SSA), bound to α2,6-sialylated glycan epitopes on transferrin and inhibited anti-transferrin antibody binding to the antigen in ELISA (SSA inhibition). Here we report that SSA inhibition is applicable to immunohistochemistry, localizing α2,6-sialylated transferrin in the liver. Immunohistochemistry using anti-transferrin polyclonal antibody revealed that transferrin was detected in hepatocytes near interlobular veins. Addition of SSA lectin markedly attenuated the staining. Sialidase treatment of a liver section abolished SSA binding and concomitantly cancelled SSA inhibition, suggesting that SSA binding to glycan epitopes on the section was essential for the inhibition. To examine the importance of proximity between antigen epitopes and SSA-binding (glycosylation) sites, we prepared two anti-peptide antibodies against partial amino acid sequences of transferrin. One antibody (Tf-596Ab) is against a peptide sequence, Cys596-Ala614, which is proximal to N-glycosylation sites (Asn-432 and Asn-630). The other (Tf-120Ab) is against a peptide sequence, Val120-Cys137, distal to the sites. The staining signals of Tf-596Ab were reduced by the addition of SSA, whereas those of Tf-120Ab were reduced only a little. This result suggests that proximity of the antigen epitope to SSA binding sites is critical for SSA inhibition in immunohistochemistry.

Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension.

Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34-PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)-positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

High Throughput ELISAs to Measure a Unique Glycan on Transferrin in Cerebrospinal Fluid: A Possible Extension toward Alzheimer's Disease Biomarker Development.

We have established high-throughput lectin-antibody ELISAs to measure different glycans on transferrin (Tf) in cerebrospinal fluid (CSF) using lectins and an anti-transferrin antibody (TfAb). Lectin blot and precipitation analysis of CSF revealed that PVL (Psathyrella velutina lectin) bound an unique N-acetylglucosamine-terminated N-glycans on "CSF-type" Tf whereas SSA (Sambucus sieboldiana agglutinin) bound α2,6-N-acetylneuraminic acid-terminated N-glycans on "serum-type" Tf. PVL-TfAb ELISA of 0.5 µL CSF samples detected "CSF-type" Tf but not "serum-type" Tf whereas SSA-TfAb ELISA detected "serum-type" Tf but not "CSF-type" Tf, demonstrating the specificity of the lectin-TfAb ELISAs. In idiopathic normal pressure hydrocephalus (iNPH), a senile dementia associated with ventriculomegaly, amounts of the SSA-reactive Tf were significantly higher than in non-iNPH patients, indicating that Tf glycan analysis by the high-throughput lectin-TfAb ELISAs could become practical diagnostic tools for iNPH. The lectin-antibody ELISAs of CSF proteins might be useful for diagnosis of the other neurological diseases.

Increased urinary angiotensinogen is precedent to increased urinary albumin in patients with type 1 diabetes.

BACKGROUND: We previously reported that kidney and urinary angiotensinogen levels were significantly increased before the development of diabetic nephropathy in diabetic rats. To address this system in humans, we have developed an enzyme-linked immunosorbent assay for human angiotensinogen and reported that urinary excretion of angiotensinogen levels is enhanced in patients with chronic kidney disease, including patients with type 2 diabetes. On the basis of these findings, this study was performed to demonstrate that urinary angiotensinogen levels increased before the onset of microalbuminuria and that urinary angiotensinogen can be an early biomarker of intrarenal renin-angiotensin system status in normoalbuminuric patients with type 1 diabetes compared with age- and sex-matched control subjects. METHODS: The study included 28 patients with type 1 diabetes and 21 control subjects. No subject received renin-angiotensin system blockades. Random spot urine samples as well as blood samples were obtained and analyzed. RESULTS: Urinary albumin:creatinine ratio or urinary protein:creatinine ratio did not increase in patients compared with control subjects, suggesting that these patients were in their premicroalbuminuric phase of diabetic nephropathy. However, the urinary angiotensinogen:creatinine ratio was significantly higher in patients than in control subjects (12.1 +/- 3.2 microg/g versus 4.2 +/- 0.7 microg/g). Importantly, an increase in plasma angiotensinogen levels was not observed (26.3 +/- 1.3 microg/mL versus 29.5 +/- 3.3 microg/mL). CONCLUSIONS: Thus, in patients, an increase in urinary angiotensinogen levels is observed, and this increase is precedent to an increase in urinary albumin levels, suggesting that urinary angiotensinogen may function as an early marker of diabetic nephropathy.

Activation of reactive oxygen species and the renin-angiotensin system in IgA nephropathy model mice.

1. Although IgA nephropathy is the most common form of primary glomerulopathy, the detailed mechanisms underlying its development remain uncertain. 2. In the present study, we used male high IgA strain of ddY (HIGA) mice as the IgA nephropathy model and age-matched male BALB/c mice as the control. Recent studies have demonstrated that reactive oxygen species (ROS)-dependent enhancement of the renin-angiotensin system (RAS) plays a potential role in the development and progression of renal injury. Therefore, in the present study we periodically measured the systolic blood pressure (SBP) of mice over the period 21-25 weeks of age and estimated markers for ROS, RAS and renal damage after mice had been killed at 25 weeks of age. 3. Markers for ROS (urinary 8-isoprostane excretion and renal 4-hydroxy-2-nonenal accumulation), RAS (renal angiotensinogen protein expression, urinary angiotensinogen excretion and renal angiotensin II) and renal damage (desmin-positive area and urinary protein excretion), as well as SBP, were significantly increased in HIGA mice compared with control BALB/c mice. 4. The data suggest that both ROS and the RAS are activated at an early phase in IgA nephropathy model mice.

Role of activated intrarenal reactive oxygen species and renin-angiotensin system in IgA nephropathy model mice.

1. Using HIGA (high IgA of ddY) mice as an IgA nephropathy model and BALB/c mice as controls, we demonstrated that reactive oxygen species (ROS) and the renin-angiotensin system (RAS) were activated in kidneys of HIGA mice. However, it was difficult to establish an association between renal damage and changes in ROS and the RAS. Therefore, the present study was performed to determine whether renal injury is associated with changes in ROS and the RAS in HIGA mice. 2. Male HIGA mice were divided into four groups of 10 each: (i) untreated mice (HIGA + null); (ii) mice treated with the angiotensin AT(1) receptor antagonist olmesartan (5 mg/kg per day; HIGA + OLM); (iii) mice treated with the superoxide dismutase mimetic tempol (50 mg/kg per day; HIGA + Tempol); and (iv) mice treated with RAS-independent antihypertensive drugs (30 mg/kg per day hydralazine, 0.6 mg/kg per day reserpine and 12 mg/kg per day hydrochlorothiazide; HIGA + HRH). Mice were treated for 5 weeks. 3. Systolic blood pressure decreased significantly in the HIGA + OLM and HIGA + HRH groups, but not in the HIGA + Tempol group, compared with HIGA + null mice. The expression of two ROS markers (4-hydroxy-2-nonenal and heme oxygenase-1) and angiotensin II as a marker of the RAS decreased significantly in HIGA + OLM and HIGA + Tempol mice, but not in HIGA + HRH mice, compared with HIGA + null mice. As a marker of renal damage, mesangial matrix expansion and the desmin-positive area decreased significantly in the HIGA + OLM and HIGA + Tempol groups, but not in HIGA + HRH group, compared with the HIGA + null group. 4. These data suggest that intrarenal ROS and RAS activation play a pivotal role in the development of IgA nephropathy model mice, from the early phase, independent of blood pressure.

Urinary angiotensinogen as a novel biomarker of the intrarenal renin-angiotensin system status in hypertensive patients.

We reported previously that urinary angiotensinogen (UAGT) levels provide a specific index of the intrarenal renin-angiotensin system (RAS) status in angiotensin II-dependent hypertensive rats. To study this system in humans, we recently developed a human angiotensinogen ELISA. To test the hypothesis that UAGT is increased in hypertensive patients, we recruited 110 adults. Four subjects with estimated glomerular filtration levels <30 mL/min per 1.73 m(2) were excluded because previous studies have already shown that UAGT is highly correlated with estimated glomerular filtration in this stage of chronic kidney disease. Consequently, 106 paired samples of urine and plasma were analyzed from 70 hypertensive patients (39 treated with RAS blockers [angiotensin-converting enzyme inhibitors or angiotensin II type 1 receptor blockers; systolic blood pressure: 139+/-3 mm Hg] and 31 not treated with RAS blockers [systolic blood pressure: 151+/-4 mm Hg]) and 36 normotensive subjects (systolic blood pressure: 122+/-2 mm Hg). UAGT, normalized by urinary concentrations of creatinine, were not correlated with race, gender, age, height, body weight, body mass index, fractional excretion of sodium, plasma angiotensinogen levels, or estimated glomerular filtration. However, UAGT/urinary concentration of creatinine was significantly positively correlated with systolic blood pressure, diastolic blood pressure, urinary albumin:creatinine ratio (r=0.5994), and urinary protein:creatinine ratio (r=0.4597). UAGT/urinary concentration of creatinine was significantly greater in hypertensive patients not treated with RAS blockers (25.00+/-4.96 microg/g) compared with normotensive subjects (13.70+/-2.33 microg/g). Importantly, patients treated with RAS blockers exhibited a marked attenuation of this augmentation (13.26+/-2.60 microg/g). These data indicate that UAGT is increased in hypertensive patients, and treatment with RAS blockers suppresses UAGT, suggesting that the efficacy of RAS blockade to reduce the intrarenal RAS activity can be assessed by measurements of UAGT.

Urinary angiotensinogen as a potential biomarker of severity of chronic kidney diseases.

We previously reported that urinary excretion rates of angiotensinogen (AGT) provide a specific index of the activity of the intrarenal renin-angiotensin system in angiotensin II-dependent hypertensive rats. Meanwhile, we have recently developed direct enzyme-linked immunosorbent assays (ELISAs) to measure plasma and urinary AGT in humans. This study was performed to test a hypothesis that urinary AGT levels are enhanced in chronic kidney disease (CKD) patients and correlated with some clinical parameters. Eighty patients with CKD (37 women and 43 men, from 18 to 94 years old) and seven healthy volunteers (two women and five men, from 27 to 43 years old) were included. Plasma AGT levels showed a normal distribution; however, urinary AGT-creatinine ratios (UAGT/UCre) deviated from the normal distribution. When a logarithmic transformation was executed, Log(UAGT/UCre) levels showed a normal distribution. Therefore, Log(UAGT/UCre) levels were used for further analyses. Log(UAGT/UCre) levels were not correlated with age, gender, height, body weight, body mass index, systolic blood pressure, diastolic blood pressure, serum sodium levels, serum potassium levels, urinary sodium-creatinine ratios, plasma renin activity, or plasma AGT levels. However, Log(UAGT/UCre) levels were significantly correlated positively with urinary albumin-creatinine ratios, fractional excretion of sodium, urinary protein-creatinine ratios, and serum creatinine, and correlated negatively with estimated glomerular filtration rate. Log(UAGT/UCre) levels were significantly increased in CKD patients compared with control subjects (1.8801 +/- 0.0885 vs. 0.9417 +/- 0.1048; P = .0024). These data confirmed our earlier report and showed that a new ELISA assay is a valid approach for measuring urinary AGT.

Determination of plasma and urinary angiotensinogen levels in rodents by newly developed ELISA.

We recently reported that urinary excretion rates of angiotensinogen provide a specific index of the intrarenal renin-angiotensin system status in angiotensin II-dependent hypertensive rats. Angiotensinogen concentrations in mouse plasma are thought to be much lower than those in rat plasma; however, detailed information is deficient due to lack of direct quantitative measurements of rodent angiotensinogen. To elucidate this issue, we have developed a quantitative method for measurement of rodent angiotensinogen using a sandwich-type ELISA. The standard curve for mouse and rat angiotensinogen exhibited a high linearity at 0.16-10 and 0.08-5 ng/ml, respectively, with correlation coefficients >0.99. While plasma angiotensinogen concentrations of male high serum IgA (HIGA) mice (IgA nephritis model animals, 1,308 +/- 47 ng/ml; n = 10) were lower than those of control BALB/c mice (1,620 +/- 384; n = 12), urinary angiotensinogen concentrations of HIGA mice (14.6 +/- 1.5 ng/ml; n = 34) were higher than those of BALB/c mice (4.6 +/- 0.1; n = 2). In a similar manner, while plasma angiotensinogen concentrations of Zucker diabetic fatty (ZDF) obese rats (type 2 diabetic model animals, 1,789 +/- 50 ng/ml; n = 5) were lower than those of control ZDF lean rats (2,296 +/- 47; n = 5), urinary angiotensinogen concentrations of ZDF obese rats (88.2 +/- 11.4 ng/ml; n = 15) were higher than those of ZDF lean rats (31.3 +/- 1.9; n = 15). These data indicate that plasma and urinary angiotensinogen concentrations are less in mice than rats. However, these data suggest that urinary angiotensinogen levels are different from plasma angiotensinogen levels in rodents. The development of rodent angiotensinogen ELISA allows quantitative comparisons in mouse and rat angiotensinogen levels in models of hypertension and cardiovascular and kidney diseases.